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this article explores how these goals have been implemented through the research program of 1 small commodity board , the california dried plum board ( cdpb ) . the cdpb obtains funds for research through a grower 's check - off program , which supports a limited number of projects on dried plums ( also known as prunes ) based on sufficient resources . my role as a founding member of the nutrition advisory panel ( nap ) of cdpb may serve as an instructive example of responsible scientific partnership between academically based scientists ( public ) and commodity boards ( private ) . the nap was established in 1999 , in partial response to advice given to the cdpb who had joined the university of illinois functional foods for health program . * the cdpb was receiving proposals , in abstract form , from various university - based researchers to use prunes ( the fda approval to also use the term dried plums came in 2001 ) in their dietary fiber research . functional foods for health program professors quickly saw that the cdpb had no framework for making decisions as to what to fund . we suggested that they develop a panel of independent scientists that could evaluate research proposals , determine future direction for research efforts , and evaluate research results for efficacy and worthiness for public relations efforts . in return , these scientists would receive a small honorarium for their efforts . currently , the nap is composed of academic scientists with particular expertise in gastroenterology , oxidative stress , immunology , bone and energy metabolism , as well as health promotion to the public . these scientists are not now actively engaged in research using dried plums ; however , a few have received grant funding in the past. our work consists of determining the most productive avenues of inquiry , issuing requests for proposals in particular areas of interest , evaluating research proposals , and suggesting further measurements to researchers with the goal of publication in respected scientific journals . the vision is to establish promising areas where dried plums ( prunes ) may improve human health and to draw additional researchers and funding sources to the effort . the cdpb also provided dried plum powder for animal studies upon request . over the years , we have followed a number of leads : oxidative stress , cancer , heart disease , bone metabolism , and intestinal health . before launching into these findings , it is appropriate to share what we know about the chemical composition of dried plums . the nap advised the need for 2 extensive reviews of the literature ( 1 at the outset6 and 1 as an update7 ) of what was known of dried plum nutrient content and their health benefits . dried plum is made from a special plum ( prunus domestica l cv d'agen ) that can fully ripen on the tree without fermenting and has a sugar content that prevents it from molding during the drying process . the fresh prune plum or sugar plum is slightly smaller than the more common varieties of fresh plums and has a lovely purple skin . it is rarely found in supermarkets or is in limited supply at farmer 's markets . table 1 provides the highlights of dried plum nutrient content compared with their dietary reference intake ( dri ) , and table 2 lists notable phenolic compounds thought to provide antioxidant properties and may be bioactive in bone and intestinal health . major nutrients in dried plums and juice and percentage of dri / serving antioxidant compounds and major secondary metabolites in dried plums and prune juice dried plums have a high antioxidant capacity due to high phenolic content . a serving of dried plum ( ~5 plums/50 g ) offers a complex of nutrients and other compounds , many of which provide more than 10% of the dri . especially notable its complex sugar content with high amounts of sorbitol is probably the reason that its glycemic index ( 29 for a 60-g serving ) is so low for such a sweet food.7 dried plums have a high antioxidant capacity compared to other fruits , dried or not , depending upon the assay used . the low values of carotenoids , ascorbic acid , and vitamin e point to their high content of phenolic compounds as the contributing factor . yet the nap recommended to the cdpb that they focus on particular areas of research and seek to develop the science in those areas . in retrospect , 2 areas have received the most intense funding . gastrointestinal ( gi ) health , for obvious reasons , and bone health , which became an important area serendipitously . these studies are reviewed here with the objective that the reader sees the progression and type of studies funded , how other investigators not funded by cdpb become interested , and how paying close attention to the type of study needed as evidence leads to an official health claim . dried plums / prunes have been the subject of many a joke , especially in the united states . seeking to reposition the fruit away from the jokes and to younger consumers , the cdpb applied to the fda for a formal name change to dried plum in 2001 , which was approved . surprisingly little research has been published on the effect of dried plum or prune juice on gi function . there are 3 components of dried plum that could affect gi motility : sorbitol , dietary fiber , and phenolic compounds . the sorbitol content of dried plum ( 1012 g/100 g or 2 servings ) is uniquely high and is equivalent to consuming 12 sticks of sugarless gum or individual candies that can act as an osmotic laxative in high quantities.8 whether one is affected by consuming high levels of sorbitol is highly variable and seems to be related to the ability to absorb it . breath hydrogen studies indicated that 71% of healthy adults showed malabsorption after consuming 10 g of sorbitol and 20% reported symptoms of bloating , flatulence , and abdominal pain.9 however , a dose of pure sorbitol would enter the lower gi tract at a faster rate than found in an equivalent dose of whole dried plums. those with irritable bowel syndrome have a greater likelihood of having a lower absorption potential.8 phenolic compounds are also thought to have an effect on gi motility.6 high sorbitol content is likely reason for dried plum stool softening effect . the cdpb funded a number of human studies that evaluated bowel habit with dried plum supplementation at various doses . with these data in hand , the cdpb applied for a health claim to the european safety authority.10 after weighing the evidence on these studies and plausible mechanisms , the european food safety authority stated on the basis of the data presented , the panel concludes that a cause and effect relationship has been established between consumption of dried plums of prune cultivars ( prunus domestica l. ) and maintenance of normal bowel function . in order to obtain the claimed effect , about 100 g ( or about 2 us servings ) of dried plums ( prunes ) should be consumed daily . the target population is the general population.10 most of the following studies were funded by cdpb but not all of them . subjects ( 41 men with mild hypercholesterolemia or 40 chronically constipated men and women ) consumed 12 dried plums a day for 8 and 3 weeks , respectively , in the 2 crossover studies . there was a notable lack of adverse gi effects and no increase in the number of bowel movements or decreased transit time . however , stools were softer and there was an increase in fecal weight.11,12 the study by attaluri et al was the most impressive ( not funded by cdpb ) . forty constipated subjects consumed either 100 g ( 1012 ) dried plums or 22 g of psyllium for 3 weeks in a crossover design with a 1-week washout . the number of complete spontaneous bowel movements per week and stool consistency scores improved significantly compared with psyllium.12 a 3-month study of 58 postmenopausal women ( 38 completers ) randomized to consume 100 g ( 1012 ) of dried plum or 75 g of dried apple ( 1.2 g sorbitol/100 g fresh apple ) not on hormone therapy ( bone health the major outcome ) was also submitted to the european food safety authority . both groups experienced the same mean number of spontaneous bowel movements with the sensation of complete evacuation.13 another study of 29 women consumed 84 g of dried plum or low - fat cookies for 2 weeks each in a crossover design . notable was that during the dried plum intervention , subjects reported significantly softer stools.14 a more extensive study of bowel health was subsequently funded by the cdpb in the united kingdom . a total of 120 healthy subjects were randomized to control or 80 or 120 g of dried plum per day ; 14 gi symptoms were tracked over the 4-week period . of the 104 subjects completing the study , for both doses , dried plum significantly increased stool weight and bowel movements but did not affect transit time . there was no reported diarrhea , but incidence of flatulence and/or reflux was higher in the dried plum groups compared with controls , but symptoms were rare and rated mild.15 we are beginning to explore the question , could dried plum consumption lower the risk of colon cancer by promoting a microbial population favorable to its prevention ? we also would like to determine whether 1 serving ( 5 prunes ) would also contribute to bowel health . exploring the role of dried plum on bone health seems to be an improbable leap . as i took on the role as nap member for the cdpb , i noticed that dried plum had an unusually high boron content . i remembered that boron had been associated with strengthening bones in some early animal studies . a young faculty member , bahram arjmandi , in my department at the university of illinois at chicago had a model for overiectomized animals to simulate osteoporosis , so i suggested that he put in a proposal to study dried plums with his animal model . the nap advised the cdpb that the proposal was low cost and scientifically worthwhile and cdpb funded the animal project although there was no previous evidence of a dried plum bone connection . to arjmandi 's surprise and ours , the dried plum treated ovariectomized rats ( a good model for postmenopausal osteoporosis ) experienced less bone loss than their counterparts.16 since that first experiment , a number of animal studies using various models of bone loss have been performed funded by the cdpb ( as reviewed17 ) . previous bone loss was somewhat restored in the femur and tibia at a low dose of 5% dried plum powder ( w / w ) , but it took a 25% dose to improve lumbar vertebra . immobilization leads to bone loss and a dried plum diet performed as well as parathyroid hormone injections to restore bone during the reambulation period in bone mineral density ( bmd ) , bone strength and bone structure . a study in a rat male model of osteoporosis found that feeding 15% and 25% of the diet as dried plum powder ( w / w ) for 90 days postcastration prevented loss of bmd , whereas the 5% diet was effective only for the lumbar vertebra and femur . again , male rats allowed to lose bone after castration showed reversal of that loss with either 25% dried plum or parathyroid hormone . most interesting was a long - term study where adult ( 6 months ) and old ( 18 months ) mice were fed calorically adjusted diets of 15% and 25% dried plum ( w / w ) for 6 months . on the 25% diet , bone volume increased 50% in the adults and 40% in the old mice but the control groups lost 24% and 28% of their bone mass , respectively . the most recent study used lower doses ( 5% , 15% , and 25% w / w ) of dried plum in growing mice and found increased cancellous bone of 12% , 36% , and 64% , respectively.18 these investigators observed the dried plum appears to disrupt the balance between resorption and formation with a net effect of more bone and this might be due to a suppression of proinflammatory cytokines . some of these studies have been funded by the cdpb and others have not . almost all of them have used dried plum powder fed at 25% of the diet ( w / w ) , a much higher dose than would be reasonable for human beings . since these initial studies , efforts to find mechanism and the components of dried plum that are bioactive have led to understanding that dried plum phenolics are not the sole component19 and that dried plum has unique properties compared with other dried fruits.20 three human trials have been performed . three months of 100 g of dried plums a day ( 1012 dried plums ) improved the biomarkers of bone formation in postmenopausal women.21 a 12-month study showed that the same dose could prevent the normal loss of bmd in postmenopausal women . dried apple had little effect . both groups were supplemented with calcium and vitamin d.22 the third study assigned 48 osteopenic women to 6 months of either 50 , 100 , or 0 g of dried plums per day . all were supplemented with calcium and vitamin d. both doses of dried plum preserved bmd compared with the normal loss experienced by the control group . bone resorption appeared to be inhibited.23 the gi complaints of subjects were minimal in these studies . because we want to be sure of the dried plum effect in humans , we have decided to fund a larger clinical trial by a different set of investigators , using a standard serving size ( 45 dried plums ) and higher dose ( 1012 dried plums ) to characterize the effects on bone mass in this older population of women . bone loss caused by ionizing radiation is a health concern for radiotherapy patients , radiation workers , and astronauts . animals exposed to such radiation experience skeletal damage and an imbalance between bone building and bone loss . mice were divided into groups and given a selection of antioxidant and anti - inflammatory candidates ( antioxidant cocktail , dihydropipoic acid , ibuprofen , or dried plum 25% diet w / w ) . dried plum was the most effective in reducing expression of genes related to bone resorption and the prevention of cancellous bone loss . the authors concluded that dietary supplementation with dried plum may prevent the skeletal effects of radiation exposures either in space or on earth.24 the effect of whole dried plum on various dynamics of bone growth and resorption is certainly intriguing and worthy of further research and holds great promise as a dietary component that may promote bone health , not only in the elderly but throughout the life cycle . dried plums / prunes have been the subject of many a joke , especially in the united states . seeking to reposition the fruit away from the jokes and to younger consumers , the cdpb applied to the fda for a formal name change to dried plum in 2001 , which was approved . surprisingly little research has been published on the effect of dried plum or prune juice on gi function . there are 3 components of dried plum that could affect gi motility : sorbitol , dietary fiber , and phenolic compounds . the sorbitol content of dried plum ( 1012 g/100 g or 2 servings ) is uniquely high and is equivalent to consuming 12 sticks of sugarless gum or individual candies that can act as an osmotic laxative in high quantities.8 whether one is affected by consuming high levels of sorbitol is highly variable and seems to be related to the ability to absorb it . breath hydrogen studies indicated that 71% of healthy adults showed malabsorption after consuming 10 g of sorbitol and 20% reported symptoms of bloating , flatulence , and abdominal pain.9 however , a dose of pure sorbitol would enter the lower gi tract at a faster rate than found in an equivalent dose of whole dried plums. those with irritable bowel syndrome have a greater likelihood of having a lower absorption potential.8 phenolic compounds are also thought to have an effect on gi motility.6 high sorbitol content is likely reason for dried plum stool softening effect . the cdpb funded a number of human studies that evaluated bowel habit with dried plum supplementation at various doses . with these data in hand , the cdpb applied for a health claim to the european safety authority.10 after weighing the evidence on these studies and plausible mechanisms , the european food safety authority stated on the basis of the data presented , the panel concludes that a cause and effect relationship has been established between consumption of dried plums of prune cultivars ( prunus domestica l. ) and maintenance of normal bowel function . in order to obtain the claimed effect , about 100 g ( or about 2 us servings ) of dried plums ( prunes ) should be consumed daily . the target population is the general population.10 most of the following studies were funded by cdpb but not all of them . subjects ( 41 men with mild hypercholesterolemia or 40 chronically constipated men and women ) consumed 12 dried plums a day for 8 and 3 weeks , respectively , in the 2 crossover studies . there was a notable lack of adverse gi effects and no increase in the number of bowel movements or decreased transit time . however , stools were softer and there was an increase in fecal weight.11,12 the study by attaluri et al was the most impressive ( not funded by cdpb ) . forty constipated subjects consumed either 100 g ( 1012 ) dried plums or 22 g of psyllium for 3 weeks in a crossover design with a 1-week washout . the number of complete spontaneous bowel movements per week and stool consistency scores improved significantly compared with psyllium.12 a 3-month study of 58 postmenopausal women ( 38 completers ) randomized to consume 100 g ( 1012 ) of dried plum or 75 g of dried apple ( 1.2 g sorbitol/100 g fresh apple ) not on hormone therapy ( bone health the major outcome ) was also submitted to the european food safety authority . both groups experienced the same mean number of spontaneous bowel movements with the sensation of complete evacuation.13 another study of 29 women consumed 84 g of dried plum or low - fat cookies for 2 weeks each in a crossover design . notable was that during the dried plum intervention , subjects reported significantly softer stools.14 a more extensive study of bowel health was subsequently funded by the cdpb in the united kingdom . a total of 120 healthy subjects were randomized to control or 80 or 120 g of dried plum per day ; 14 gi symptoms were tracked over the 4-week period . of the 104 subjects completing the study , for both doses , dried plum significantly increased stool weight and bowel movements but did not affect transit time . there was no reported diarrhea , but incidence of flatulence and/or reflux was higher in the dried plum groups compared with controls , but symptoms were rare and rated mild.15 we are beginning to explore the question , could dried plum consumption lower the risk of colon cancer by promoting a microbial population favorable to its prevention ? we also would like to determine whether 1 serving ( 5 prunes ) would also contribute to bowel health . most of the following studies were funded by cdpb but not all of them . subjects ( 41 men with mild hypercholesterolemia or 40 chronically constipated men and women ) consumed 12 dried plums a day for 8 and 3 weeks , respectively , in the 2 crossover studies . there was a notable lack of adverse gi effects and no increase in the number of bowel movements or decreased transit time . however , stools were softer and there was an increase in fecal weight.11,12 the study by attaluri et al was the most impressive ( not funded by cdpb ) . forty constipated subjects consumed either 100 g ( 1012 ) dried plums or 22 g of psyllium for 3 weeks in a crossover design with a 1-week washout . the number of complete spontaneous bowel movements per week and stool consistency scores improved significantly compared with psyllium.12 a 3-month study of 58 postmenopausal women ( 38 completers ) randomized to consume 100 g ( 1012 ) of dried plum or 75 g of dried apple ( 1.2 g sorbitol/100 g fresh apple ) not on hormone therapy ( bone health the major outcome ) was also submitted to the european food safety authority . both groups experienced the same mean number of spontaneous bowel movements with the sensation of complete evacuation.13 another study of 29 women consumed 84 g of dried plum or low - fat cookies for 2 weeks each in a crossover design . notable was that during the dried plum intervention , subjects reported significantly softer stools.14 a more extensive study of bowel health was subsequently funded by the cdpb in the united kingdom . a total of 120 healthy subjects were randomized to control or 80 or 120 g of dried plum per day ; 14 gi symptoms were tracked over the 4-week period . of the 104 subjects completing the study , for both doses , dried plum significantly increased stool weight and bowel movements but did not affect transit time . there was no reported diarrhea , but incidence of flatulence and/or reflux was higher in the dried plum groups compared with controls , but symptoms were rare and rated mild.15 we are beginning to explore the question , could dried plum consumption lower the risk of colon cancer by promoting a microbial population favorable to its prevention ? we also would like to determine whether 1 serving ( 5 prunes ) would also contribute to bowel health . exploring the role of dried plum on bone health seems to be an improbable leap . as i took on the role as nap member for the cdpb , i noticed that dried plum had an unusually high boron content . i remembered that boron had been associated with strengthening bones in some early animal studies . a young faculty member , bahram arjmandi , in my department at the university of illinois at chicago had a model for overiectomized animals to simulate osteoporosis , so i suggested that he put in a proposal to study dried plums with his animal model . the nap advised the cdpb that the proposal was low cost and scientifically worthwhile and cdpb funded the animal project although there was no previous evidence of a dried plum bone connection . treated ovariectomized rats ( a good model for postmenopausal osteoporosis ) experienced less bone loss than their counterparts.16 since that first experiment , a number of animal studies using various models of bone loss have been performed funded by the cdpb ( as reviewed17 ) . previous bone loss was somewhat restored in the femur and tibia at a low dose of 5% dried plum powder ( w / w ) , but it took a 25% dose to improve lumbar vertebra . immobilization leads to bone loss and a dried plum diet performed as well as parathyroid hormone injections to restore bone during the reambulation period in bone mineral density ( bmd ) , bone strength and bone structure . a study in a rat male model of osteoporosis found that feeding 15% and 25% of the diet as dried plum powder ( w / w ) for 90 days postcastration prevented loss of bmd , whereas the 5% diet was effective only for the lumbar vertebra and femur . again , male rats allowed to lose bone after castration showed reversal of that loss with either 25% dried plum or parathyroid hormone . most interesting was a long - term study where adult ( 6 months ) and old ( 18 months ) mice were fed calorically adjusted diets of 15% and 25% dried plum ( w / w ) for 6 months . on the 25% diet , bone volume increased 50% in the adults and 40% in the old mice but the control groups lost 24% and 28% of their bone mass , respectively . the most recent study used lower doses ( 5% , 15% , and 25% w / w ) of dried plum in growing mice and found increased cancellous bone of 12% , 36% , and 64% , respectively.18 these investigators observed the dried plum appears to disrupt the balance between resorption and formation with a net effect of more bone and this might be due to a suppression of proinflammatory cytokines . some of these studies have been funded by the cdpb and others have not . almost all of them have used dried plum powder fed at 25% of the diet ( w / w ) , a much higher dose than would be reasonable for human beings . since these initial studies , efforts to find mechanism and the components of dried plum that are bioactive have led to understanding that dried plum phenolics are not the sole component19 and that dried plum has unique properties compared with other dried fruits.20 three human trials have been performed . three months of 100 g of dried plums a day ( 1012 dried plums ) improved the biomarkers of bone formation in postmenopausal women.21 a 12-month study showed that the same dose could prevent the normal loss of bmd in postmenopausal women . dried apple had little effect . both groups were supplemented with calcium and vitamin d.22 the third study assigned 48 osteopenic women to 6 months of either 50 , 100 , or 0 g of dried plums per day . all were supplemented with calcium and vitamin d. both doses of dried plum preserved bmd compared with the normal loss experienced by the control group . bone resorption appeared to be inhibited.23 the gi complaints of subjects were minimal in these studies . because we want to be sure of the dried plum effect in humans , we have decided to fund a larger clinical trial by a different set of investigators , using a standard serving size ( 45 dried plums ) and higher dose ( 1012 dried plums ) to characterize the effects on bone mass in this older population of women . bone loss caused by ionizing radiation is a health concern for radiotherapy patients , radiation workers , and astronauts . animals exposed to such radiation experience skeletal damage and an imbalance between bone building and bone loss . mice were divided into groups and given a selection of antioxidant and anti - inflammatory candidates ( antioxidant cocktail , dihydropipoic acid , ibuprofen , or dried plum 25% diet w / w ) . dried plum was the most effective in reducing expression of genes related to bone resorption and the prevention of cancellous bone loss . the authors concluded that dietary supplementation with dried plum may prevent the skeletal effects of radiation exposures either in space or on earth.24 the effect of whole dried plum on various dynamics of bone growth and resorption is certainly intriguing and worthy of further research and holds great promise as a dietary component that may promote bone health , not only in the elderly but throughout the life cycle . the experience of scientists with the cdpb may be informative in how such commodity boards listen to scientific panels and determine how to fund and promote scientific studies . although there are grounds for concern when commodity boards and self - interested groups fund and then promote research that benefits the marketing of their products , great gains can be made with industry / academic partnerships that aid health and well - being . ( 1 ) young faculty may find financial support for their first projects which then form the preliminary studies that aid in successful applications for national institutes of health or usda grants . ( 2 ) studies using whole foods are more difficult to control because of their variable and complex composition . commodity board funding is essential to clarify the epidemiological findings that point to single foods or food groups as beneficial . ( 3 ) all academic institutions decline to enter into financial obligations with industry sponsors that do not allow their faculty to publish results from industry - sponsored research . ( 4 ) the existence of independent scientific panels who direct the research programs of commodity boards is essential to guide commodity boards in the efficient and ethical use of funds set aside for research .

food and agriculture commodity boards have become important funders of nutrition research . there are benefits and cautions ( biases toward health benefits , failure to publish negative results , and aggressive promotion of single studies ) for this activity . the california dried plum board , along with other commodity boards , have developed independent scientific nutrition advisory panels to guide and evaluate the research they fund . in the case of the california dried plum board , this has resulted in research that has distinguished the nature and dose of dried plum and juice to maintain bowel health and opened up a surprising new function for dried plum in the prevention of age - related bone loss .

CALIFORNIA DRIED PLUM BOARD AS AN EXAMPLE OF INDUSTRY/ACADEMIC PARTNERSHIP NUTRIENT AND BIOACTIVE COMPOUNDS IN DRIED PLUMS AND PRUNE JUICE TWO AREAS OF INVESTIGATION THAT HAVE BEEN A FOCUS OF CDPB FUNDING GI Effects Details of Studies Bone Health CONCLUSION

this article explores how these goals have been implemented through the research program of 1 small commodity board , the california dried plum board ( cdpb ) . my role as a founding member of the nutrition advisory panel ( nap ) of cdpb may serve as an instructive example of responsible scientific partnership between academically based scientists ( public ) and commodity boards ( private ) . we suggested that they develop a panel of independent scientists that could evaluate research proposals , determine future direction for research efforts , and evaluate research results for efficacy and worthiness for public relations efforts . these scientists are not now actively engaged in research using dried plums ; however , a few have received grant funding in the past. our work consists of determining the most productive avenues of inquiry , issuing requests for proposals in particular areas of interest , evaluating research proposals , and suggesting further measurements to researchers with the goal of publication in respected scientific journals . the vision is to establish promising areas where dried plums ( prunes ) may improve human health and to draw additional researchers and funding sources to the effort . before launching into these findings , it is appropriate to share what we know about the chemical composition of dried plums . the nap advised the need for 2 extensive reviews of the literature ( 1 at the outset6 and 1 as an update7 ) of what was known of dried plum nutrient content and their health benefits . table 1 provides the highlights of dried plum nutrient content compared with their dietary reference intake ( dri ) , and table 2 lists notable phenolic compounds thought to provide antioxidant properties and may be bioactive in bone and intestinal health . a serving of dried plum ( ~5 plums/50 g ) offers a complex of nutrients and other compounds , many of which provide more than 10% of the dri . the low values of carotenoids , ascorbic acid , and vitamin e point to their high content of phenolic compounds as the contributing factor . these studies are reviewed here with the objective that the reader sees the progression and type of studies funded , how other investigators not funded by cdpb become interested , and how paying close attention to the type of study needed as evidence leads to an official health claim . seeking to reposition the fruit away from the jokes and to younger consumers , the cdpb applied to the fda for a formal name change to dried plum in 2001 , which was approved . surprisingly little research has been published on the effect of dried plum or prune juice on gi function . there are 3 components of dried plum that could affect gi motility : sorbitol , dietary fiber , and phenolic compounds . the sorbitol content of dried plum ( 1012 g/100 g or 2 servings ) is uniquely high and is equivalent to consuming 12 sticks of sugarless gum or individual candies that can act as an osmotic laxative in high quantities.8 whether one is affected by consuming high levels of sorbitol is highly variable and seems to be related to the ability to absorb it . breath hydrogen studies indicated that 71% of healthy adults showed malabsorption after consuming 10 g of sorbitol and 20% reported symptoms of bloating , flatulence , and abdominal pain.9 however , a dose of pure sorbitol would enter the lower gi tract at a faster rate than found in an equivalent dose of whole dried plums. those with irritable bowel syndrome have a greater likelihood of having a lower absorption potential.8 phenolic compounds are also thought to have an effect on gi motility.6 high sorbitol content is likely reason for dried plum stool softening effect . the cdpb funded a number of human studies that evaluated bowel habit with dried plum supplementation at various doses . with these data in hand , the cdpb applied for a health claim to the european safety authority.10 after weighing the evidence on these studies and plausible mechanisms , the european food safety authority stated on the basis of the data presented , the panel concludes that a cause and effect relationship has been established between consumption of dried plums of prune cultivars ( prunus domestica l. ) and maintenance of normal bowel function . the target population is the general population.10 most of the following studies were funded by cdpb but not all of them . subjects ( 41 men with mild hypercholesterolemia or 40 chronically constipated men and women ) consumed 12 dried plums a day for 8 and 3 weeks , respectively , in the 2 crossover studies . there was a notable lack of adverse gi effects and no increase in the number of bowel movements or decreased transit time . the number of complete spontaneous bowel movements per week and stool consistency scores improved significantly compared with psyllium.12 a 3-month study of 58 postmenopausal women ( 38 completers ) randomized to consume 100 g ( 1012 ) of dried plum or 75 g of dried apple ( 1.2 g sorbitol/100 g fresh apple ) not on hormone therapy ( bone health the major outcome ) was also submitted to the european food safety authority . both groups experienced the same mean number of spontaneous bowel movements with the sensation of complete evacuation.13 another study of 29 women consumed 84 g of dried plum or low - fat cookies for 2 weeks each in a crossover design . notable was that during the dried plum intervention , subjects reported significantly softer stools.14 a more extensive study of bowel health was subsequently funded by the cdpb in the united kingdom . a total of 120 healthy subjects were randomized to control or 80 or 120 g of dried plum per day ; 14 gi symptoms were tracked over the 4-week period . of the 104 subjects completing the study , for both doses , dried plum significantly increased stool weight and bowel movements but did not affect transit time . there was no reported diarrhea , but incidence of flatulence and/or reflux was higher in the dried plum groups compared with controls , but symptoms were rare and rated mild.15 we are beginning to explore the question , could dried plum consumption lower the risk of colon cancer by promoting a microbial population favorable to its prevention ? we also would like to determine whether 1 serving ( 5 prunes ) would also contribute to bowel health . exploring the role of dried plum on bone health seems to be an improbable leap . as i took on the role as nap member for the cdpb , i noticed that dried plum had an unusually high boron content . the nap advised the cdpb that the proposal was low cost and scientifically worthwhile and cdpb funded the animal project although there was no previous evidence of a dried plum bone connection . to arjmandi 's surprise and ours , the dried plum treated ovariectomized rats ( a good model for postmenopausal osteoporosis ) experienced less bone loss than their counterparts.16 since that first experiment , a number of animal studies using various models of bone loss have been performed funded by the cdpb ( as reviewed17 ) . previous bone loss was somewhat restored in the femur and tibia at a low dose of 5% dried plum powder ( w / w ) , but it took a 25% dose to improve lumbar vertebra . immobilization leads to bone loss and a dried plum diet performed as well as parathyroid hormone injections to restore bone during the reambulation period in bone mineral density ( bmd ) , bone strength and bone structure . a study in a rat male model of osteoporosis found that feeding 15% and 25% of the diet as dried plum powder ( w / w ) for 90 days postcastration prevented loss of bmd , whereas the 5% diet was effective only for the lumbar vertebra and femur . again , male rats allowed to lose bone after castration showed reversal of that loss with either 25% dried plum or parathyroid hormone . most interesting was a long - term study where adult ( 6 months ) and old ( 18 months ) mice were fed calorically adjusted diets of 15% and 25% dried plum ( w / w ) for 6 months . on the 25% diet , bone volume increased 50% in the adults and 40% in the old mice but the control groups lost 24% and 28% of their bone mass , respectively . the most recent study used lower doses ( 5% , 15% , and 25% w / w ) of dried plum in growing mice and found increased cancellous bone of 12% , 36% , and 64% , respectively.18 these investigators observed the dried plum appears to disrupt the balance between resorption and formation with a net effect of more bone and this might be due to a suppression of proinflammatory cytokines . almost all of them have used dried plum powder fed at 25% of the diet ( w / w ) , a much higher dose than would be reasonable for human beings . since these initial studies , efforts to find mechanism and the components of dried plum that are bioactive have led to understanding that dried plum phenolics are not the sole component19 and that dried plum has unique properties compared with other dried fruits.20 three human trials have been performed . both groups were supplemented with calcium and vitamin d.22 the third study assigned 48 osteopenic women to 6 months of either 50 , 100 , or 0 g of dried plums per day . all were supplemented with calcium and vitamin d. both doses of dried plum preserved bmd compared with the normal loss experienced by the control group . because we want to be sure of the dried plum effect in humans , we have decided to fund a larger clinical trial by a different set of investigators , using a standard serving size ( 45 dried plums ) and higher dose ( 1012 dried plums ) to characterize the effects on bone mass in this older population of women . bone loss caused by ionizing radiation is a health concern for radiotherapy patients , radiation workers , and astronauts . mice were divided into groups and given a selection of antioxidant and anti - inflammatory candidates ( antioxidant cocktail , dihydropipoic acid , ibuprofen , or dried plum 25% diet w / w ) . dried plum was the most effective in reducing expression of genes related to bone resorption and the prevention of cancellous bone loss . the authors concluded that dietary supplementation with dried plum may prevent the skeletal effects of radiation exposures either in space or on earth.24 the effect of whole dried plum on various dynamics of bone growth and resorption is certainly intriguing and worthy of further research and holds great promise as a dietary component that may promote bone health , not only in the elderly but throughout the life cycle . seeking to reposition the fruit away from the jokes and to younger consumers , the cdpb applied to the fda for a formal name change to dried plum in 2001 , which was approved . surprisingly little research has been published on the effect of dried plum or prune juice on gi function . there are 3 components of dried plum that could affect gi motility : sorbitol , dietary fiber , and phenolic compounds . the sorbitol content of dried plum ( 1012 g/100 g or 2 servings ) is uniquely high and is equivalent to consuming 12 sticks of sugarless gum or individual candies that can act as an osmotic laxative in high quantities.8 whether one is affected by consuming high levels of sorbitol is highly variable and seems to be related to the ability to absorb it . breath hydrogen studies indicated that 71% of healthy adults showed malabsorption after consuming 10 g of sorbitol and 20% reported symptoms of bloating , flatulence , and abdominal pain.9 however , a dose of pure sorbitol would enter the lower gi tract at a faster rate than found in an equivalent dose of whole dried plums. those with irritable bowel syndrome have a greater likelihood of having a lower absorption potential.8 phenolic compounds are also thought to have an effect on gi motility.6 high sorbitol content is likely reason for dried plum stool softening effect . with these data in hand , the cdpb applied for a health claim to the european safety authority.10 after weighing the evidence on these studies and plausible mechanisms , the european food safety authority stated on the basis of the data presented , the panel concludes that a cause and effect relationship has been established between consumption of dried plums of prune cultivars ( prunus domestica l. ) and maintenance of normal bowel function . subjects ( 41 men with mild hypercholesterolemia or 40 chronically constipated men and women ) consumed 12 dried plums a day for 8 and 3 weeks , respectively , in the 2 crossover studies . there was a notable lack of adverse gi effects and no increase in the number of bowel movements or decreased transit time . the number of complete spontaneous bowel movements per week and stool consistency scores improved significantly compared with psyllium.12 a 3-month study of 58 postmenopausal women ( 38 completers ) randomized to consume 100 g ( 1012 ) of dried plum or 75 g of dried apple ( 1.2 g sorbitol/100 g fresh apple ) not on hormone therapy ( bone health the major outcome ) was also submitted to the european food safety authority . both groups experienced the same mean number of spontaneous bowel movements with the sensation of complete evacuation.13 another study of 29 women consumed 84 g of dried plum or low - fat cookies for 2 weeks each in a crossover design . notable was that during the dried plum intervention , subjects reported significantly softer stools.14 a more extensive study of bowel health was subsequently funded by the cdpb in the united kingdom . a total of 120 healthy subjects were randomized to control or 80 or 120 g of dried plum per day ; 14 gi symptoms were tracked over the 4-week period . of the 104 subjects completing the study , for both doses , dried plum significantly increased stool weight and bowel movements but did not affect transit time . there was no reported diarrhea , but incidence of flatulence and/or reflux was higher in the dried plum groups compared with controls , but symptoms were rare and rated mild.15 we are beginning to explore the question , could dried plum consumption lower the risk of colon cancer by promoting a microbial population favorable to its prevention ? most of the following studies were funded by cdpb but not all of them . there was a notable lack of adverse gi effects and no increase in the number of bowel movements or decreased transit time . the number of complete spontaneous bowel movements per week and stool consistency scores improved significantly compared with psyllium.12 a 3-month study of 58 postmenopausal women ( 38 completers ) randomized to consume 100 g ( 1012 ) of dried plum or 75 g of dried apple ( 1.2 g sorbitol/100 g fresh apple ) not on hormone therapy ( bone health the major outcome ) was also submitted to the european food safety authority . both groups experienced the same mean number of spontaneous bowel movements with the sensation of complete evacuation.13 another study of 29 women consumed 84 g of dried plum or low - fat cookies for 2 weeks each in a crossover design . notable was that during the dried plum intervention , subjects reported significantly softer stools.14 a more extensive study of bowel health was subsequently funded by the cdpb in the united kingdom . a total of 120 healthy subjects were randomized to control or 80 or 120 g of dried plum per day ; 14 gi symptoms were tracked over the 4-week period . of the 104 subjects completing the study , for both doses , dried plum significantly increased stool weight and bowel movements but did not affect transit time . there was no reported diarrhea , but incidence of flatulence and/or reflux was higher in the dried plum groups compared with controls , but symptoms were rare and rated mild.15 we are beginning to explore the question , could dried plum consumption lower the risk of colon cancer by promoting a microbial population favorable to its prevention ? we also would like to determine whether 1 serving ( 5 prunes ) would also contribute to bowel health . exploring the role of dried plum on bone health seems to be an improbable leap . as i took on the role as nap member for the cdpb , i noticed that dried plum had an unusually high boron content . previous bone loss was somewhat restored in the femur and tibia at a low dose of 5% dried plum powder ( w / w ) , but it took a 25% dose to improve lumbar vertebra . immobilization leads to bone loss and a dried plum diet performed as well as parathyroid hormone injections to restore bone during the reambulation period in bone mineral density ( bmd ) , bone strength and bone structure . a study in a rat male model of osteoporosis found that feeding 15% and 25% of the diet as dried plum powder ( w / w ) for 90 days postcastration prevented loss of bmd , whereas the 5% diet was effective only for the lumbar vertebra and femur . again , male rats allowed to lose bone after castration showed reversal of that loss with either 25% dried plum or parathyroid hormone . most interesting was a long - term study where adult ( 6 months ) and old ( 18 months ) mice were fed calorically adjusted diets of 15% and 25% dried plum ( w / w ) for 6 months . the most recent study used lower doses ( 5% , 15% , and 25% w / w ) of dried plum in growing mice and found increased cancellous bone of 12% , 36% , and 64% , respectively.18 these investigators observed the dried plum appears to disrupt the balance between resorption and formation with a net effect of more bone and this might be due to a suppression of proinflammatory cytokines . almost all of them have used dried plum powder fed at 25% of the diet ( w / w ) , a much higher dose than would be reasonable for human beings . since these initial studies , efforts to find mechanism and the components of dried plum that are bioactive have led to understanding that dried plum phenolics are not the sole component19 and that dried plum has unique properties compared with other dried fruits.20 three human trials have been performed . three months of 100 g of dried plums a day ( 1012 dried plums ) improved the biomarkers of bone formation in postmenopausal women.21 a 12-month study showed that the same dose could prevent the normal loss of bmd in postmenopausal women . all were supplemented with calcium and vitamin d. both doses of dried plum preserved bmd compared with the normal loss experienced by the control group . because we want to be sure of the dried plum effect in humans , we have decided to fund a larger clinical trial by a different set of investigators , using a standard serving size ( 45 dried plums ) and higher dose ( 1012 dried plums ) to characterize the effects on bone mass in this older population of women . bone loss caused by ionizing radiation is a health concern for radiotherapy patients , radiation workers , and astronauts . animals exposed to such radiation experience skeletal damage and an imbalance between bone building and bone loss . dried plum was the most effective in reducing expression of genes related to bone resorption and the prevention of cancellous bone loss . the authors concluded that dietary supplementation with dried plum may prevent the skeletal effects of radiation exposures either in space or on earth.24 the effect of whole dried plum on various dynamics of bone growth and resorption is certainly intriguing and worthy of further research and holds great promise as a dietary component that may promote bone health , not only in the elderly but throughout the life cycle . the experience of scientists with the cdpb may be informative in how such commodity boards listen to scientific panels and determine how to fund and promote scientific studies . although there are grounds for concern when commodity boards and self - interested groups fund and then promote research that benefits the marketing of their products , great gains can be made with industry / academic partnerships that aid health and well - being . ( 4 ) the existence of independent scientific panels who direct the research programs of commodity boards is essential to guide commodity boards in the efficient and ethical use of funds set aside for research .

my role as a founding member of the nutrition advisory panel ( nap ) of cdpb may serve as an instructive example of responsible scientific partnership between academically based scientists ( public ) and commodity boards ( private ) . the nap was established in 1999 , in partial response to advice given to the cdpb who had joined the university of illinois functional foods for health program . * the cdpb was receiving proposals , in abstract form , from various university - based researchers to use prunes ( the fda approval to also use the term dried plums came in 2001 ) in their dietary fiber research . we suggested that they develop a panel of independent scientists that could evaluate research proposals , determine future direction for research efforts , and evaluate research results for efficacy and worthiness for public relations efforts . currently , the nap is composed of academic scientists with particular expertise in gastroenterology , oxidative stress , immunology , bone and energy metabolism , as well as health promotion to the public . our work consists of determining the most productive avenues of inquiry , issuing requests for proposals in particular areas of interest , evaluating research proposals , and suggesting further measurements to researchers with the goal of publication in respected scientific journals . the vision is to establish promising areas where dried plums ( prunes ) may improve human health and to draw additional researchers and funding sources to the effort . over the years , we have followed a number of leads : oxidative stress , cancer , heart disease , bone metabolism , and intestinal health . the nap advised the need for 2 extensive reviews of the literature ( 1 at the outset6 and 1 as an update7 ) of what was known of dried plum nutrient content and their health benefits . dried plum is made from a special plum ( prunus domestica l cv d'agen ) that can fully ripen on the tree without fermenting and has a sugar content that prevents it from molding during the drying process . the fresh prune plum or sugar plum is slightly smaller than the more common varieties of fresh plums and has a lovely purple skin . table 1 provides the highlights of dried plum nutrient content compared with their dietary reference intake ( dri ) , and table 2 lists notable phenolic compounds thought to provide antioxidant properties and may be bioactive in bone and intestinal health . major nutrients in dried plums and juice and percentage of dri / serving antioxidant compounds and major secondary metabolites in dried plums and prune juice dried plums have a high antioxidant capacity due to high phenolic content . a serving of dried plum ( ~5 plums/50 g ) offers a complex of nutrients and other compounds , many of which provide more than 10% of the dri . especially notable its complex sugar content with high amounts of sorbitol is probably the reason that its glycemic index ( 29 for a 60-g serving ) is so low for such a sweet food.7 dried plums have a high antioxidant capacity compared to other fruits , dried or not , depending upon the assay used . the low values of carotenoids , ascorbic acid , and vitamin e point to their high content of phenolic compounds as the contributing factor . yet the nap recommended to the cdpb that they focus on particular areas of research and seek to develop the science in those areas . gastrointestinal ( gi ) health , for obvious reasons , and bone health , which became an important area serendipitously . these studies are reviewed here with the objective that the reader sees the progression and type of studies funded , how other investigators not funded by cdpb become interested , and how paying close attention to the type of study needed as evidence leads to an official health claim . dried plums / prunes have been the subject of many a joke , especially in the united states . seeking to reposition the fruit away from the jokes and to younger consumers , the cdpb applied to the fda for a formal name change to dried plum in 2001 , which was approved . surprisingly little research has been published on the effect of dried plum or prune juice on gi function . the sorbitol content of dried plum ( 1012 g/100 g or 2 servings ) is uniquely high and is equivalent to consuming 12 sticks of sugarless gum or individual candies that can act as an osmotic laxative in high quantities.8 whether one is affected by consuming high levels of sorbitol is highly variable and seems to be related to the ability to absorb it . breath hydrogen studies indicated that 71% of healthy adults showed malabsorption after consuming 10 g of sorbitol and 20% reported symptoms of bloating , flatulence , and abdominal pain.9 however , a dose of pure sorbitol would enter the lower gi tract at a faster rate than found in an equivalent dose of whole dried plums. those with irritable bowel syndrome have a greater likelihood of having a lower absorption potential.8 phenolic compounds are also thought to have an effect on gi motility.6 high sorbitol content is likely reason for dried plum stool softening effect . with these data in hand , the cdpb applied for a health claim to the european safety authority.10 after weighing the evidence on these studies and plausible mechanisms , the european food safety authority stated on the basis of the data presented , the panel concludes that a cause and effect relationship has been established between consumption of dried plums of prune cultivars ( prunus domestica l. ) and maintenance of normal bowel function . subjects ( 41 men with mild hypercholesterolemia or 40 chronically constipated men and women ) consumed 12 dried plums a day for 8 and 3 weeks , respectively , in the 2 crossover studies . however , stools were softer and there was an increase in fecal weight.11,12 the study by attaluri et al was the most impressive ( not funded by cdpb ) . the number of complete spontaneous bowel movements per week and stool consistency scores improved significantly compared with psyllium.12 a 3-month study of 58 postmenopausal women ( 38 completers ) randomized to consume 100 g ( 1012 ) of dried plum or 75 g of dried apple ( 1.2 g sorbitol/100 g fresh apple ) not on hormone therapy ( bone health the major outcome ) was also submitted to the european food safety authority . both groups experienced the same mean number of spontaneous bowel movements with the sensation of complete evacuation.13 another study of 29 women consumed 84 g of dried plum or low - fat cookies for 2 weeks each in a crossover design . notable was that during the dried plum intervention , subjects reported significantly softer stools.14 a more extensive study of bowel health was subsequently funded by the cdpb in the united kingdom . of the 104 subjects completing the study , for both doses , dried plum significantly increased stool weight and bowel movements but did not affect transit time . there was no reported diarrhea , but incidence of flatulence and/or reflux was higher in the dried plum groups compared with controls , but symptoms were rare and rated mild.15 we are beginning to explore the question , could dried plum consumption lower the risk of colon cancer by promoting a microbial population favorable to its prevention ? exploring the role of dried plum on bone health seems to be an improbable leap . as i took on the role as nap member for the cdpb , i noticed that dried plum had an unusually high boron content . a young faculty member , bahram arjmandi , in my department at the university of illinois at chicago had a model for overiectomized animals to simulate osteoporosis , so i suggested that he put in a proposal to study dried plums with his animal model . the nap advised the cdpb that the proposal was low cost and scientifically worthwhile and cdpb funded the animal project although there was no previous evidence of a dried plum bone connection . to arjmandi 's surprise and ours , the dried plum treated ovariectomized rats ( a good model for postmenopausal osteoporosis ) experienced less bone loss than their counterparts.16 since that first experiment , a number of animal studies using various models of bone loss have been performed funded by the cdpb ( as reviewed17 ) . previous bone loss was somewhat restored in the femur and tibia at a low dose of 5% dried plum powder ( w / w ) , but it took a 25% dose to improve lumbar vertebra . immobilization leads to bone loss and a dried plum diet performed as well as parathyroid hormone injections to restore bone during the reambulation period in bone mineral density ( bmd ) , bone strength and bone structure . a study in a rat male model of osteoporosis found that feeding 15% and 25% of the diet as dried plum powder ( w / w ) for 90 days postcastration prevented loss of bmd , whereas the 5% diet was effective only for the lumbar vertebra and femur . again , male rats allowed to lose bone after castration showed reversal of that loss with either 25% dried plum or parathyroid hormone . most interesting was a long - term study where adult ( 6 months ) and old ( 18 months ) mice were fed calorically adjusted diets of 15% and 25% dried plum ( w / w ) for 6 months . on the 25% diet , bone volume increased 50% in the adults and 40% in the old mice but the control groups lost 24% and 28% of their bone mass , respectively . the most recent study used lower doses ( 5% , 15% , and 25% w / w ) of dried plum in growing mice and found increased cancellous bone of 12% , 36% , and 64% , respectively.18 these investigators observed the dried plum appears to disrupt the balance between resorption and formation with a net effect of more bone and this might be due to a suppression of proinflammatory cytokines . almost all of them have used dried plum powder fed at 25% of the diet ( w / w ) , a much higher dose than would be reasonable for human beings . since these initial studies , efforts to find mechanism and the components of dried plum that are bioactive have led to understanding that dried plum phenolics are not the sole component19 and that dried plum has unique properties compared with other dried fruits.20 three human trials have been performed . three months of 100 g of dried plums a day ( 1012 dried plums ) improved the biomarkers of bone formation in postmenopausal women.21 a 12-month study showed that the same dose could prevent the normal loss of bmd in postmenopausal women . both groups were supplemented with calcium and vitamin d.22 the third study assigned 48 osteopenic women to 6 months of either 50 , 100 , or 0 g of dried plums per day . all were supplemented with calcium and vitamin d. both doses of dried plum preserved bmd compared with the normal loss experienced by the control group . because we want to be sure of the dried plum effect in humans , we have decided to fund a larger clinical trial by a different set of investigators , using a standard serving size ( 45 dried plums ) and higher dose ( 1012 dried plums ) to characterize the effects on bone mass in this older population of women . mice were divided into groups and given a selection of antioxidant and anti - inflammatory candidates ( antioxidant cocktail , dihydropipoic acid , ibuprofen , or dried plum 25% diet w / w ) . dried plum was the most effective in reducing expression of genes related to bone resorption and the prevention of cancellous bone loss . the authors concluded that dietary supplementation with dried plum may prevent the skeletal effects of radiation exposures either in space or on earth.24 the effect of whole dried plum on various dynamics of bone growth and resorption is certainly intriguing and worthy of further research and holds great promise as a dietary component that may promote bone health , not only in the elderly but throughout the life cycle . dried plums / prunes have been the subject of many a joke , especially in the united states . seeking to reposition the fruit away from the jokes and to younger consumers , the cdpb applied to the fda for a formal name change to dried plum in 2001 , which was approved . the sorbitol content of dried plum ( 1012 g/100 g or 2 servings ) is uniquely high and is equivalent to consuming 12 sticks of sugarless gum or individual candies that can act as an osmotic laxative in high quantities.8 whether one is affected by consuming high levels of sorbitol is highly variable and seems to be related to the ability to absorb it . breath hydrogen studies indicated that 71% of healthy adults showed malabsorption after consuming 10 g of sorbitol and 20% reported symptoms of bloating , flatulence , and abdominal pain.9 however , a dose of pure sorbitol would enter the lower gi tract at a faster rate than found in an equivalent dose of whole dried plums. those with irritable bowel syndrome have a greater likelihood of having a lower absorption potential.8 phenolic compounds are also thought to have an effect on gi motility.6 high sorbitol content is likely reason for dried plum stool softening effect . with these data in hand , the cdpb applied for a health claim to the european safety authority.10 after weighing the evidence on these studies and plausible mechanisms , the european food safety authority stated on the basis of the data presented , the panel concludes that a cause and effect relationship has been established between consumption of dried plums of prune cultivars ( prunus domestica l. ) and maintenance of normal bowel function . the number of complete spontaneous bowel movements per week and stool consistency scores improved significantly compared with psyllium.12 a 3-month study of 58 postmenopausal women ( 38 completers ) randomized to consume 100 g ( 1012 ) of dried plum or 75 g of dried apple ( 1.2 g sorbitol/100 g fresh apple ) not on hormone therapy ( bone health the major outcome ) was also submitted to the european food safety authority . both groups experienced the same mean number of spontaneous bowel movements with the sensation of complete evacuation.13 another study of 29 women consumed 84 g of dried plum or low - fat cookies for 2 weeks each in a crossover design . notable was that during the dried plum intervention , subjects reported significantly softer stools.14 a more extensive study of bowel health was subsequently funded by the cdpb in the united kingdom . of the 104 subjects completing the study , for both doses , dried plum significantly increased stool weight and bowel movements but did not affect transit time . there was no reported diarrhea , but incidence of flatulence and/or reflux was higher in the dried plum groups compared with controls , but symptoms were rare and rated mild.15 we are beginning to explore the question , could dried plum consumption lower the risk of colon cancer by promoting a microbial population favorable to its prevention ? the number of complete spontaneous bowel movements per week and stool consistency scores improved significantly compared with psyllium.12 a 3-month study of 58 postmenopausal women ( 38 completers ) randomized to consume 100 g ( 1012 ) of dried plum or 75 g of dried apple ( 1.2 g sorbitol/100 g fresh apple ) not on hormone therapy ( bone health the major outcome ) was also submitted to the european food safety authority . both groups experienced the same mean number of spontaneous bowel movements with the sensation of complete evacuation.13 another study of 29 women consumed 84 g of dried plum or low - fat cookies for 2 weeks each in a crossover design . notable was that during the dried plum intervention , subjects reported significantly softer stools.14 a more extensive study of bowel health was subsequently funded by the cdpb in the united kingdom . of the 104 subjects completing the study , for both doses , dried plum significantly increased stool weight and bowel movements but did not affect transit time . there was no reported diarrhea , but incidence of flatulence and/or reflux was higher in the dried plum groups compared with controls , but symptoms were rare and rated mild.15 we are beginning to explore the question , could dried plum consumption lower the risk of colon cancer by promoting a microbial population favorable to its prevention ? as i took on the role as nap member for the cdpb , i noticed that dried plum had an unusually high boron content . the nap advised the cdpb that the proposal was low cost and scientifically worthwhile and cdpb funded the animal project although there was no previous evidence of a dried plum bone connection . treated ovariectomized rats ( a good model for postmenopausal osteoporosis ) experienced less bone loss than their counterparts.16 since that first experiment , a number of animal studies using various models of bone loss have been performed funded by the cdpb ( as reviewed17 ) . previous bone loss was somewhat restored in the femur and tibia at a low dose of 5% dried plum powder ( w / w ) , but it took a 25% dose to improve lumbar vertebra . immobilization leads to bone loss and a dried plum diet performed as well as parathyroid hormone injections to restore bone during the reambulation period in bone mineral density ( bmd ) , bone strength and bone structure . a study in a rat male model of osteoporosis found that feeding 15% and 25% of the diet as dried plum powder ( w / w ) for 90 days postcastration prevented loss of bmd , whereas the 5% diet was effective only for the lumbar vertebra and femur . most interesting was a long - term study where adult ( 6 months ) and old ( 18 months ) mice were fed calorically adjusted diets of 15% and 25% dried plum ( w / w ) for 6 months . on the 25% diet , bone volume increased 50% in the adults and 40% in the old mice but the control groups lost 24% and 28% of their bone mass , respectively . the most recent study used lower doses ( 5% , 15% , and 25% w / w ) of dried plum in growing mice and found increased cancellous bone of 12% , 36% , and 64% , respectively.18 these investigators observed the dried plum appears to disrupt the balance between resorption and formation with a net effect of more bone and this might be due to a suppression of proinflammatory cytokines . almost all of them have used dried plum powder fed at 25% of the diet ( w / w ) , a much higher dose than would be reasonable for human beings . since these initial studies , efforts to find mechanism and the components of dried plum that are bioactive have led to understanding that dried plum phenolics are not the sole component19 and that dried plum has unique properties compared with other dried fruits.20 three human trials have been performed . three months of 100 g of dried plums a day ( 1012 dried plums ) improved the biomarkers of bone formation in postmenopausal women.21 a 12-month study showed that the same dose could prevent the normal loss of bmd in postmenopausal women . both groups were supplemented with calcium and vitamin d.22 the third study assigned 48 osteopenic women to 6 months of either 50 , 100 , or 0 g of dried plums per day . because we want to be sure of the dried plum effect in humans , we have decided to fund a larger clinical trial by a different set of investigators , using a standard serving size ( 45 dried plums ) and higher dose ( 1012 dried plums ) to characterize the effects on bone mass in this older population of women . the authors concluded that dietary supplementation with dried plum may prevent the skeletal effects of radiation exposures either in space or on earth.24 the effect of whole dried plum on various dynamics of bone growth and resorption is certainly intriguing and worthy of further research and holds great promise as a dietary component that may promote bone health , not only in the elderly but throughout the life cycle .

one hundred years after carlos chagas identified and described the trypanosoma cruzi ( t. cruzi ) infection , there are still millions of infected people and thousands of newly diagnosed cases each year with chagas disease ( cd ) . the scientific community has intermittently increased the knowledge and understanding of how to manage patients with acute and chronic cd . nonetheless , much more research is still needed in order to improve care and answer many unknown questions regarding this debilitating and widespread disease , which has been estimated to affect about 8 million chronically infected people just in the americas . the goal of etiological treatment against chagas disease is to eliminate the parasite ( t. cruzi ) from the infected individual , to decrease the probability of developing clinical manifestations of the disease ( e.g. , cardiovascular or digestive diseases ) , and to break the chain of disease transmission . currently , there is a new scenario regarding the recommended etiological treatment against t. cruzi infection . it is based on several strong evidences supported by basic research , clinical trials , observational studies , and expert opinions . in this paper , we review the current evidence supporting etiological chagas disease therapy organized according to different levels of prevention . additionally , we discuss the tools available to demonstrate cure in these patients , and the need for further research required to improve care for t. cruzi infected people . cruzi pharmacotherapy ( etiological treatment ) in order to reduce or avoid the morbidity and mortality of chagas disease applied on different levels of prevention . in this regard , a medline search was conducted from january to july 2011 , using the term chagas disease with the subheadings diagnosis , prognosis , treatment , drug names ( nifurtimox , benznidazole , and other drugs ) , clinical trials , and observational studies . no restrictions regarding year recent guidelines as well as ongoing and unpublished studies were also identified by consulting researchers and experts in the field . evidence was organized according to the levels of prevention addressed by the retrieved guidelines or epidemiological researches . finally , we reviewed the strength of evidence for each indication in each level of prevention . leavell and clark 's have defined three different levels of prevention in human health ( primary , secondary , and tertiary ) in a classical textbook published in 1953 . each of them includes different means of intervention according to the natural history of the disease . primary preventionthese strategies intend to avoid the disease 's development , including the acquisition of new infection . most population - based health promotion activities are primary preventive measures . these strategies intend to avoid the disease 's development , including the acquisition of new infection . secondary preventionthese strategies attempt to diagnose and treat an existing disease in its early stages before it results in significant morbidity . these strategies attempt to diagnose and treat an existing disease in its early stages before it results in significant morbidity . tertiary preventionthese treatments aim to reduce the negative impact of established disease by restoring function and reducing disease - related complications . these treatments aim to reduce the negative impact of established disease by restoring function and reducing disease - related complications . in the last decades , jamoulle has proposed a fourth concept ( quaternary prevention ) , which was incorporated by the wonca international classification committee . in this regard , quaternary prevention describes the set of health activities aimed to mitigate or avoid the consequences of unnecessary or excessive interventions in the health system . strength of recommendations as well as the quality level of the evidence supporting these recommendations were addressed according to the quality standards subcommittee or the clinical affairs committee of the infectious diseases society of america ( idsa ) . both strong evidence for efficacy and substantial clinical benefit support recommendation for use . moderate evidence for efficacy or strong evidence for efficacy but only limited clinical benefit support recommendation for use . evidence for efficacy is insufficient to support a recommendation for or against use . or evidence for efficacy might not outweigh adverse consequences ( e.g. , drug toxicity , drug interactions ) or cost of the treatment under consideration : optional . moderate evidence for lack of efficacy or for adverse outcome supports a recommendation against use . good evidence for lack of efficacy or for adverse outcome supports a recommendation against use . evidence from at least one well - designed clinical trial without randomization , from cohort or case - controlled analytic studies ( preferably from more than one center ) , or from multiple time - series studies , or dramatic results from uncontrolled experiments . evidence from opinions of respected authorities based on clinical experience , descriptive studies , or reports of expert committees . several papers and guidelines have been published in the last years [ 740 ] , supporting with different levels of strength that etiological treatment is an effective intervention on both the individual and public health . these studies reached levels of evidence ranged from i to iii , providing strength of recommendations ( a ) , ( b ) , and ( c ) ( see table 1 ) . several studies have shown the benefit of treatment during acute phase with both benznidazole and nifurtimox with a level of evidence type i or ii [ 1015 ] . the assessment of failure and/or efficacy of treatment on patients treated during acute phase is demonstrable in short time because the parasitemia , whether direct or not ( parasitological test or molecular test ) , becomes negative a few days after the end of treatment . in addition , antibodies disappear completely ( seronegativization ) in at least 65% of cases , with some studies demonstrating seronegativity in 100% of cases up to 18 months of follow up after treatment . this effect is independent of the age of the patients , including newborns ( congenital transmission ) , children , and adults . the absence of parasitemia demonstrated by direct method such us strout or micromethod always precedes the reduction of antibodies [ 1015 ] . in general , treatment is well tolerated during the acute phase , and the risk of potential adverse events is counterbalanced by the reduction of clinical manifestations of the acute phase of chagas infection , and even the associated risk of death . there is wide consensus that all patients undergoing the acute phase of infection or reactivation of chronic infection must be treated ( strength of recommendation ( a ) ) . several studies ( evidence type i ) have provided support to the use of etiological treatment ( benznidazole ) during early stages of chronic infection in children [ 1622 ] . two studies have shown efficacy in this population through double blinded placebo - controlled trials of benznidazole for children aged 6 to 12 years old with asymptomatic t. cruzi infection demonstrated approximately 60% efficacy , as assessed by conversion from positive to negative serology results 3 to 4 years after treatment [ 16 , 17 ] . rates of seronegativization up to 70% were established with etiological treatment after long - term follow - up ( 15 years ) in south america , and up to 50% after short - term followup ( 3 years ) in central america . furthermore , additional studies ( evidence type ii ) have shown that seronegativization with etiological treatment is also possible in later stages of the chronic infections in adults [ 7 , 18 , 2331 ] . however , rate of seronegativization of antibodies ( serological test ) seems to be directly related to the age of patients . although a complete seronegativization can be obtained in more than 70% of the cases in children , the seronegativization rate have reached about 30% in adult patients after a long - term followup , around 20 years [ 7 , 1631 ] . fall in antibodies titers after treatment in children is faster than in adults , even if it does not cross the cutoff to become nonreactive . a statistically significant reduction is visible at 3 months with eia and ifa , and at 6 months with iha [ 16 , 17 ] . young children with longer - term followup have higher rates of seronegativization after treatment as compared to child patients with short - term followup , and equal phenomena occurs among adult patients with long - term followup in comparison with adult patients with short followup . demonstration of antiparasitic effects after treatment can be performed by the detection of antibodies , parasites , and/or parasite dna . the success of the treatment is determined by the disappearance of antibodies using serological tests , while therapeutic failure only can be demonstrated by showing the persistence of the parasite using parasitological methods . the assessment of failure of treatment on patients treated during the chronic phase could be demonstrable in short time because the parasitemia ( when it is present by parasitological test or molecular test ) disappears at the end of treatment if treatment is successful . when failure occurs , evidence of parasitemia remains positive after treatment ( not more than 5% in children or 10% in adults ) [ 9 , 10 , 17 , 18 , 20 , 41 , 42 ] . however , gallerano and sosa showed a higher rate of xeno positives including treatment with nifurtimox , benznidazole , and allopurinol . though , this last drug ( allopurinol ) did not show consistent results when they were tested in clinical trials [ 44 , 45 ] . other methodologies to evaluate the effectiveness of antiparasitic treatments have been tested , but have not reached consensus to change current testing strategy . methods to detect the parasite 's genomic fragments in tissues and body fluids using polymerase chain reaction ( pcr ) have proved to be promising tools for the assessment of therapy [ 13 , 36 , 4549 ] , and it was recently standardized for diagnosis . projects for standardization of pcr for assessing therapy which look for the presence of parasites in the blood are underway . there is agreement that , even with limitation , it will be a useful tool to improve the assessment of treatment failure . molecular methods are showing attributes for making a timely diagnosis at birth [ 11 , 13 , 51 ] . since tolerance to etiological treatment in children is better than that in adults ( see section 3.3 ) , there is a general agreement that children and adolescents undergoing chronic chagas phase have to be treated ( strength of recommendation ( a ) ) . on the other hand , the rate of seronegativization in adult patients ( about 30% ) based on evidence from observational studies linking the seronegativization to the prevention of clinical disease is currently under research [ 7 , 18 , 2331 ] . furthermore , higher rates of adverse events ( with a 17% of abandon rate ) are seen in adult patients in comparison with children , making this recommendation weaker in adults ( strength of recommendation ( b ) ) . in the case of adult patients undergoing the chronic phase of infection , treatment if accepted , therapy should be prescribed due to the strength of evidence available today . evidence type iii supports that health workers , researchers , and so forth who suffer accidents with infected blood have to be treated under specific protocols . regarding immunocompromised patients , available studies ( evidence type ii ) have shown that after etiological treatment , patients recover from severe manifestations of reactivation such as meningoencephalitis , myocarditis , and panniculitis [ 36 , 52 ] . however , in these cases the main objective is recovery from life - threatening acute events rather than seronegativization , due to the limited ability to interpret serological test results in states of immunosuppression . since the severity of reactivation and the risk of death are associated , there is a general agreement that these patients must be treated ( strength of recommendation ( a ) ) . on the other hand , no current evidence supports the use of etiological treatment as prophylaxis in immunocompromised patients with chronic chagasic infection without evidence of reactivation . although some studies have reported the etiologic treatment of pregnant women without adverse effects in the new born [ 32 , 33 ] , treatment using benznidazole or nifurtimox is currently not recommended for pregnant women ( absolute contraindication ) [ 7 , 9 ] . additional contraindications to use of etiological treatment include patients undergoing severe acute or chronic liver or kidney disease not related to t. cruzi infection ( relative contraindication ) , and lactation ( relative contraindication ) [ 7 , 9 ] . table 1 shows a summary of different scenarios for etiologic treatment against t. cruzi infection , and results of different ways for assessing therapeutic response . during treatment based on prior experiences , treatment tolerance is good and patients have not denoted serious side effects [ 8 , 19 , 27 , 41 , 53 , 54 ] . although cases with severe side effects have been reported , they have generally been associated with difficulties in seeking timely medical attention or receiving adequate care . side effects are more frequently observed in adolescents and adults than in children and babies . in neonates and in children up to 4 years old , tolerance is excellent . in all cases , types of side effects seen and their distribution during treatment are shown in figure 1 . other types of side effects include reversible clastogenesis and mutagenesis with benznidazole and nifurtimox without any associated manifestations [ 55 , 56 ] , toxicity against other tissues or increased risk of lymphomas in experimental animals have been described , but never demonstrated among a general population of infected patients undergoing treatment and never played a role in animal models . adequate management of side effects is necessary to carry out treatment as well as to diminish unfounded fears with the use of trypanocidal drugs [ 53 , 61 ] . the recommendations of etiological treatment allow for action on several levels of public health prevention . retrieved studies provide evidence for applying strategies of health care to control programs in several countries , whereby a greater portion of the population could get diagnosis , treatment , and cure , generating a new scenario for the reduction in disease burden in the future . if the goal is to avoid the acquisition of a new infection , etiological treatment could have an indirect effect when children and young people are treated . curing children and women in reproductive age would avoid future events of congenital t. cruzi transmission in newborns ( recommendation ( b ) , and evidence type iii ) . in addition , the availability of potential blood and organ donors will be increased by treating those infected . unfortunately , effectiveness of etiological treatment for these primary prevention indications remains unknown , although it can be assumed to be at least equal to seronegativization rates observed in available studies . another strategy would be the development of a treatment that can be administered to pregnant women , such as is used for hiv infection , to avoid congenital transmission during pregnancy . however , safety information on these drugs would be necessary for this strategy , and this is not currently available . etiological treatment in case of accidents with material contaminated with parasites or blood samples of patients infected with t. cruzi could also be considered as an indication for primary prevention . actually , the treatment is not strictly a prophylaxis because it is not possible to avoid the infections , but the infection can be aborted immediately after accidents with a timely treatment to get an appropriate concentration of specific drugs ( recommendation ( b ) , and evidence iii ) . if prevention activities can not avoid infection in children , the cure of infected children is still possible by prescribing etiological treatment [ 1022 ] . in this regard , etiological treatment is indicated when damages from cardiac or digestive disease are not strongly present in these children . this is the best opportunity to get seronegatization and avoid disease , thus preserving social , mental , and physical health into adulthood [ 63 , 64 ] . it has consisted of the screening of child populations as a regular strategy to offer opportunities for diagnosis and treatment ( recommendation ( a ) , and evidence type i ) , as well as timely diagnosis and treatment of children born with congenital infection ( recommendation ( a ) , and evidence type ii ) . the positive effect of curing children detected by serological screening must be assessed by taking into account patterns of disease transmission , evolution , and a calculation of the burden of disease attributable to chagas disease , in order to analyze the usefulness of serology as an indicator of the action against the vector . other indication for etiological treatment in secondary prevention is to avoid reactivation of a chronic infection . immunosuppression due to immunosuppressive therapies or hiv / aids increases the risk of reactivation in patients with chronic infection . although the effectiveness of etiological treatment for the clinical control of episodes of reactivation has been proven , it is necessary to gather evidence as to whether preventive treatment is effective in patients with no signs of clinical reactivation and with abnormal immunological parameters . in this regard , some protocols recommend the treatment of organ donors infected with t. cruzi in order to reduce risk of transmission by transplant . in this case , the treatment should be considered an act of primary prevention ( recommendation ( a ) , and evidence type ii ) . the use of etiological treatment against t. cruzi infection in order to reduce the negative impact of established disease is under evaluation through two randomized clinical trials , which assess the efficacy in patients with cardiac disease [ 6870 ] . several observational studies have been published showing effects of etiological treatment in patients infected with t. cruzi , on prevention of the progression of chronic chagasic cardiomyopathy [ 18 , 27 , 41 , 43 , 71 ] . these studies reached quality of evidence type ii , providing strength of recommendation ( b ) and ( c ) . the prognosis of patients with heart failure or advanced stages of chagas ' cardiomyopathy is poor , but similar to others that develop heart failure for other reasons . since the disease is chronic and heart damage develops over decades , it is very important to recognize factors that are determinant of disease progression in the early stages . etiological treatment should be considered as a protective factor in the model of physiopathology of chagas ' cardiomyopathy . as mentioned above , the effectiveness of etiological treatment for the control of episodes of reactivation has been proven , showing recovery of severe manifestations of reactivation such as meningoencephalitis , myocarditis , and panniculitis [ 36 , 52 ] . recommendations for appropriate care of patients are increasing , putting emphasis on the care of patients in the primary health care system , the use of other levels of care when necessary [ 7476 ] , and the incorporation of psychological aspects into care [ 63 , 64 ] . in this context , it is important to consider the available evidence about etiological treatment , and to maintain the perspective of etiological treatment as a public health tool in multiple levels of prevention , along with other interventions available for chagas disease control and treatment . in chagas disease , the best examples of primary prevention are vectorial control ( based on surveillance ) and control of blood and organ donors . however , etiological treatment has an important role in primary prevention and has to be considered a key element on among other strategies of the chagas disease control programs . the best example of secondary prevention in chagas disease is the control of congenital transmission , and the diagnosis of infection in children ( defined as chronic recent infection ) or young - adult patients in chronic phase without clinical manifestations ( sign and/or symptom ) . use of etiological treatment for tertiary prevention in chagas disease is currently supported by recommendation levels ( b ) and ( c ) , when given in addition to complementary therapies in patients with cardiac disease to reduce the clinical progression of the disease . for instance , cardiac transplant is a procedure that has been applied and has demonstrated clinical benefit in some patients with terminal heart failure . stem cell transplant is a new therapy applied to produce cardiac regeneration through distinction or increase heart myocytes or neovascular proliferation in patients in the final stage of congestive heart failure [ 7981 ] , but the results are still insufficient on chagas disease , and there is no consensus about its efficacy . regarding quaternary prevention , a national policy of etiological treatment of infected people should be considered as an activity . this approach has been utilized by several latin - american countries in the last decades . the assessment of effect of treatment against t. cruzi infection requires a clear understanding about the combination of variables to an appropriate interpretation of results to appraise . among others , the main variables are the tools used as indicators ( parasitological , molecular , and serological tests ) , the phase of infection ( acute or chronic ) that the patient is undergoing when he / she was treated , and the time elapsed between treatment and the application of the test to assess efficacy / failure . the ideal assessment of response to specific treatment is the detection of free parasites in the patient 's blood or tissues , which permits clear observation of failure of treatment . it is also necessary to validate new tools to confirm cure or failure in a timely manner after a full course of treatment has been given during the chronic phase , and studies are ongoing to validate pcr and standardized and validate qpcr . if persistence of the parasite is identified , after verifying if the drug was taken correctly , it is necessary to consider the possibility that the parasite has developed resistance [ 84 , 85 ] . possible regional differences ( host , t. cruzi strain , etc . ) , have also been described [ 10 , 22 ] , but more observation is needed to confirm this hypothesis . after etiologic treatment , even in cured patients , the antibodies may remain detectable in sera for a long term ( for years ) until they become negative . because of this phenomenon , it would be necessary to delve into the clinical history of patients with reactive serology , asking the question did he / she receive treatment in the past ? " when given an affirmative answer , the serological test has a limited value , because we must consider whether this reactivity is reflecting an active infection or if the patient was cured and he / she is becoming negative . current recommendations have put the bulk of the diagnostic and treatment responsibility on the primary health care system . yet the management of infected patients has some basic limitations , but several researches are looking for solutions . ( a ) current drugs are able to cure infection ( or prevent disease ) in adult patients during the chronic phase , which is when the first contact is made with most infected patients , and clinical trials are finishing or ongoing to demonstrate effects of conventional treatment on this population [ 69 , 70 ] . ( b ) new pediatric presentation of benznidazole is under evaluation to eliminate infection in newborns , and children with recent chronic infection . overall , the priorities in chagas disease research should be to produce new drugs providing a shorter treatment course with fewer side effects , and to devise pediatric formulas . some strategies , such as testing old drugs for extending current prescriptions , screening new compounds , testing drugs developed for other prescription such as pozanonzole , or developing new compounds are being used ( clinical trial for the treatment of chronic chagas disease with posaconazole and benznidazole ; nct01162967 ) [ 69 , 86 ] . associations of compounds with different mechanisms of action have been mentioned as another way to look for new treatment alternatives . based on current disease understanding during chronic phase of infection , there is consensus that every patient infected with t. cruzi must be ( children ) or should be treated ( adults ) . treatment can cure infection and reduce or prevent the progression to the chagas - related heart disease / cardiomyopathy . the current evidence of benefits and limitations of etiological treatment , based on clinical and implementation research , serve to prioritize strategies in primary health care , focusing on completing the scheme of treatment , rather than demonstrating serological negativization . to incorporate etiological treatment as a public health strategy which is useful at the primary , secondary , and tertiary prevention is essential to reduce burden of the disease and to eliminate chagas disease as a public health issue .

this paper reviews the evidence supporting the use of etiological treatment for chagas disease that has changed the standard of care for patients with trypanosoma cruzi infection in the last decades . implications of this evidence on different levels of prevention as well as gaps in current knowledge are also discussed . in this regard , etiological treatment has shown to be beneficial as an intervention for secondary prevention to successfully cure the infection or to delay , reduce , or prevent the progression to disease , and as primary disease prevention by breaking the chain of transmission . timely diagnosis during initial stages would allow for the prescription of appropriate therapies mainly in the primary health care system thus improving chances for a better quality of life . based on current evidence , etiological treatment has to be considered as an essential public health strategy useful to reduce disease burden and to eliminate chagas disease altogether .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

one hundred years after carlos chagas identified and described the trypanosoma cruzi ( t. cruzi ) infection , there are still millions of infected people and thousands of newly diagnosed cases each year with chagas disease ( cd ) . the scientific community has intermittently increased the knowledge and understanding of how to manage patients with acute and chronic cd . nonetheless , much more research is still needed in order to improve care and answer many unknown questions regarding this debilitating and widespread disease , which has been estimated to affect about 8 million chronically infected people just in the americas . the goal of etiological treatment against chagas disease is to eliminate the parasite ( t. cruzi ) from the infected individual , to decrease the probability of developing clinical manifestations of the disease ( e.g. , cardiovascular or digestive diseases ) , and to break the chain of disease transmission . currently , there is a new scenario regarding the recommended etiological treatment against t. cruzi infection . it is based on several strong evidences supported by basic research , clinical trials , observational studies , and expert opinions . in this paper , we review the current evidence supporting etiological chagas disease therapy organized according to different levels of prevention . additionally , we discuss the tools available to demonstrate cure in these patients , and the need for further research required to improve care for t. cruzi infected people . cruzi pharmacotherapy ( etiological treatment ) in order to reduce or avoid the morbidity and mortality of chagas disease applied on different levels of prevention . in this regard , a medline search was conducted from january to july 2011 , using the term chagas disease with the subheadings diagnosis , prognosis , treatment , drug names ( nifurtimox , benznidazole , and other drugs ) , clinical trials , and observational studies . no restrictions regarding year recent guidelines as well as ongoing and unpublished studies were also identified by consulting researchers and experts in the field . evidence was organized according to the levels of prevention addressed by the retrieved guidelines or epidemiological researches . finally , we reviewed the strength of evidence for each indication in each level of prevention . leavell and clark 's have defined three different levels of prevention in human health ( primary , secondary , and tertiary ) in a classical textbook published in 1953 . these strategies attempt to diagnose and treat an existing disease in its early stages before it results in significant morbidity . tertiary preventionthese treatments aim to reduce the negative impact of established disease by restoring function and reducing disease - related complications . these treatments aim to reduce the negative impact of established disease by restoring function and reducing disease - related complications . in the last decades , jamoulle has proposed a fourth concept ( quaternary prevention ) , which was incorporated by the wonca international classification committee . in this regard , quaternary prevention describes the set of health activities aimed to mitigate or avoid the consequences of unnecessary or excessive interventions in the health system . strength of recommendations as well as the quality level of the evidence supporting these recommendations were addressed according to the quality standards subcommittee or the clinical affairs committee of the infectious diseases society of america ( idsa ) . both strong evidence for efficacy and substantial clinical benefit support recommendation for use . moderate evidence for lack of efficacy or for adverse outcome supports a recommendation against use . evidence from at least one well - designed clinical trial without randomization , from cohort or case - controlled analytic studies ( preferably from more than one center ) , or from multiple time - series studies , or dramatic results from uncontrolled experiments . evidence from opinions of respected authorities based on clinical experience , descriptive studies , or reports of expert committees . several papers and guidelines have been published in the last years [ 740 ] , supporting with different levels of strength that etiological treatment is an effective intervention on both the individual and public health . these studies reached levels of evidence ranged from i to iii , providing strength of recommendations ( a ) , ( b ) , and ( c ) ( see table 1 ) . several studies have shown the benefit of treatment during acute phase with both benznidazole and nifurtimox with a level of evidence type i or ii [ 1015 ] . the assessment of failure and/or efficacy of treatment on patients treated during acute phase is demonstrable in short time because the parasitemia , whether direct or not ( parasitological test or molecular test ) , becomes negative a few days after the end of treatment . this effect is independent of the age of the patients , including newborns ( congenital transmission ) , children , and adults . the absence of parasitemia demonstrated by direct method such us strout or micromethod always precedes the reduction of antibodies [ 1015 ] . in general , treatment is well tolerated during the acute phase , and the risk of potential adverse events is counterbalanced by the reduction of clinical manifestations of the acute phase of chagas infection , and even the associated risk of death . there is wide consensus that all patients undergoing the acute phase of infection or reactivation of chronic infection must be treated ( strength of recommendation ( a ) ) . several studies ( evidence type i ) have provided support to the use of etiological treatment ( benznidazole ) during early stages of chronic infection in children [ 1622 ] . two studies have shown efficacy in this population through double blinded placebo - controlled trials of benznidazole for children aged 6 to 12 years old with asymptomatic t. cruzi infection demonstrated approximately 60% efficacy , as assessed by conversion from positive to negative serology results 3 to 4 years after treatment [ 16 , 17 ] . rates of seronegativization up to 70% were established with etiological treatment after long - term follow - up ( 15 years ) in south america , and up to 50% after short - term followup ( 3 years ) in central america . furthermore , additional studies ( evidence type ii ) have shown that seronegativization with etiological treatment is also possible in later stages of the chronic infections in adults [ 7 , 18 , 2331 ] . however , rate of seronegativization of antibodies ( serological test ) seems to be directly related to the age of patients . although a complete seronegativization can be obtained in more than 70% of the cases in children , the seronegativization rate have reached about 30% in adult patients after a long - term followup , around 20 years [ 7 , 1631 ] . a statistically significant reduction is visible at 3 months with eia and ifa , and at 6 months with iha [ 16 , 17 ] . young children with longer - term followup have higher rates of seronegativization after treatment as compared to child patients with short - term followup , and equal phenomena occurs among adult patients with long - term followup in comparison with adult patients with short followup . however , gallerano and sosa showed a higher rate of xeno positives including treatment with nifurtimox , benznidazole , and allopurinol . though , this last drug ( allopurinol ) did not show consistent results when they were tested in clinical trials [ 44 , 45 ] . methods to detect the parasite 's genomic fragments in tissues and body fluids using polymerase chain reaction ( pcr ) have proved to be promising tools for the assessment of therapy [ 13 , 36 , 4549 ] , and it was recently standardized for diagnosis . projects for standardization of pcr for assessing therapy which look for the presence of parasites in the blood are underway . there is agreement that , even with limitation , it will be a useful tool to improve the assessment of treatment failure . molecular methods are showing attributes for making a timely diagnosis at birth [ 11 , 13 , 51 ] . since tolerance to etiological treatment in children is better than that in adults ( see section 3.3 ) , there is a general agreement that children and adolescents undergoing chronic chagas phase have to be treated ( strength of recommendation ( a ) ) . on the other hand , the rate of seronegativization in adult patients ( about 30% ) based on evidence from observational studies linking the seronegativization to the prevention of clinical disease is currently under research [ 7 , 18 , 2331 ] . in the case of adult patients undergoing the chronic phase of infection , treatment if accepted , therapy should be prescribed due to the strength of evidence available today . evidence type iii supports that health workers , researchers , and so forth who suffer accidents with infected blood have to be treated under specific protocols . regarding immunocompromised patients , available studies ( evidence type ii ) have shown that after etiological treatment , patients recover from severe manifestations of reactivation such as meningoencephalitis , myocarditis , and panniculitis [ 36 , 52 ] . since the severity of reactivation and the risk of death are associated , there is a general agreement that these patients must be treated ( strength of recommendation ( a ) ) . on the other hand , no current evidence supports the use of etiological treatment as prophylaxis in immunocompromised patients with chronic chagasic infection without evidence of reactivation . although some studies have reported the etiologic treatment of pregnant women without adverse effects in the new born [ 32 , 33 ] , treatment using benznidazole or nifurtimox is currently not recommended for pregnant women ( absolute contraindication ) [ 7 , 9 ] . additional contraindications to use of etiological treatment include patients undergoing severe acute or chronic liver or kidney disease not related to t. cruzi infection ( relative contraindication ) , and lactation ( relative contraindication ) [ 7 , 9 ] . table 1 shows a summary of different scenarios for etiologic treatment against t. cruzi infection , and results of different ways for assessing therapeutic response . during treatment based on prior experiences , treatment tolerance is good and patients have not denoted serious side effects [ 8 , 19 , 27 , 41 , 53 , 54 ] . adequate management of side effects is necessary to carry out treatment as well as to diminish unfounded fears with the use of trypanocidal drugs [ 53 , 61 ] . the recommendations of etiological treatment allow for action on several levels of public health prevention . retrieved studies provide evidence for applying strategies of health care to control programs in several countries , whereby a greater portion of the population could get diagnosis , treatment , and cure , generating a new scenario for the reduction in disease burden in the future . if the goal is to avoid the acquisition of a new infection , etiological treatment could have an indirect effect when children and young people are treated . curing children and women in reproductive age would avoid future events of congenital t. cruzi transmission in newborns ( recommendation ( b ) , and evidence type iii ) . unfortunately , effectiveness of etiological treatment for these primary prevention indications remains unknown , although it can be assumed to be at least equal to seronegativization rates observed in available studies . however , safety information on these drugs would be necessary for this strategy , and this is not currently available . etiological treatment in case of accidents with material contaminated with parasites or blood samples of patients infected with t. cruzi could also be considered as an indication for primary prevention . actually , the treatment is not strictly a prophylaxis because it is not possible to avoid the infections , but the infection can be aborted immediately after accidents with a timely treatment to get an appropriate concentration of specific drugs ( recommendation ( b ) , and evidence iii ) . if prevention activities can not avoid infection in children , the cure of infected children is still possible by prescribing etiological treatment [ 1022 ] . in this regard , etiological treatment is indicated when damages from cardiac or digestive disease are not strongly present in these children . this is the best opportunity to get seronegatization and avoid disease , thus preserving social , mental , and physical health into adulthood [ 63 , 64 ] . it has consisted of the screening of child populations as a regular strategy to offer opportunities for diagnosis and treatment ( recommendation ( a ) , and evidence type i ) , as well as timely diagnosis and treatment of children born with congenital infection ( recommendation ( a ) , and evidence type ii ) . the positive effect of curing children detected by serological screening must be assessed by taking into account patterns of disease transmission , evolution , and a calculation of the burden of disease attributable to chagas disease , in order to analyze the usefulness of serology as an indicator of the action against the vector . other indication for etiological treatment in secondary prevention is to avoid reactivation of a chronic infection . immunosuppression due to immunosuppressive therapies or hiv / aids increases the risk of reactivation in patients with chronic infection . although the effectiveness of etiological treatment for the clinical control of episodes of reactivation has been proven , it is necessary to gather evidence as to whether preventive treatment is effective in patients with no signs of clinical reactivation and with abnormal immunological parameters . in this regard , some protocols recommend the treatment of organ donors infected with t. cruzi in order to reduce risk of transmission by transplant . in this case , the treatment should be considered an act of primary prevention ( recommendation ( a ) , and evidence type ii ) . the use of etiological treatment against t. cruzi infection in order to reduce the negative impact of established disease is under evaluation through two randomized clinical trials , which assess the efficacy in patients with cardiac disease [ 6870 ] . several observational studies have been published showing effects of etiological treatment in patients infected with t. cruzi , on prevention of the progression of chronic chagasic cardiomyopathy [ 18 , 27 , 41 , 43 , 71 ] . these studies reached quality of evidence type ii , providing strength of recommendation ( b ) and ( c ) . the prognosis of patients with heart failure or advanced stages of chagas ' cardiomyopathy is poor , but similar to others that develop heart failure for other reasons . since the disease is chronic and heart damage develops over decades , it is very important to recognize factors that are determinant of disease progression in the early stages . etiological treatment should be considered as a protective factor in the model of physiopathology of chagas ' cardiomyopathy . as mentioned above , the effectiveness of etiological treatment for the control of episodes of reactivation has been proven , showing recovery of severe manifestations of reactivation such as meningoencephalitis , myocarditis , and panniculitis [ 36 , 52 ] . recommendations for appropriate care of patients are increasing , putting emphasis on the care of patients in the primary health care system , the use of other levels of care when necessary [ 7476 ] , and the incorporation of psychological aspects into care [ 63 , 64 ] . in this context , it is important to consider the available evidence about etiological treatment , and to maintain the perspective of etiological treatment as a public health tool in multiple levels of prevention , along with other interventions available for chagas disease control and treatment . in chagas disease , the best examples of primary prevention are vectorial control ( based on surveillance ) and control of blood and organ donors . however , etiological treatment has an important role in primary prevention and has to be considered a key element on among other strategies of the chagas disease control programs . the best example of secondary prevention in chagas disease is the control of congenital transmission , and the diagnosis of infection in children ( defined as chronic recent infection ) or young - adult patients in chronic phase without clinical manifestations ( sign and/or symptom ) . use of etiological treatment for tertiary prevention in chagas disease is currently supported by recommendation levels ( b ) and ( c ) , when given in addition to complementary therapies in patients with cardiac disease to reduce the clinical progression of the disease . for instance , cardiac transplant is a procedure that has been applied and has demonstrated clinical benefit in some patients with terminal heart failure . stem cell transplant is a new therapy applied to produce cardiac regeneration through distinction or increase heart myocytes or neovascular proliferation in patients in the final stage of congestive heart failure [ 7981 ] , but the results are still insufficient on chagas disease , and there is no consensus about its efficacy . regarding quaternary prevention , a national policy of etiological treatment of infected people should be considered as an activity . this approach has been utilized by several latin - american countries in the last decades . the assessment of effect of treatment against t. cruzi infection requires a clear understanding about the combination of variables to an appropriate interpretation of results to appraise . among others , the main variables are the tools used as indicators ( parasitological , molecular , and serological tests ) , the phase of infection ( acute or chronic ) that the patient is undergoing when he / she was treated , and the time elapsed between treatment and the application of the test to assess efficacy / failure . the ideal assessment of response to specific treatment is the detection of free parasites in the patient 's blood or tissues , which permits clear observation of failure of treatment . it is also necessary to validate new tools to confirm cure or failure in a timely manner after a full course of treatment has been given during the chronic phase , and studies are ongoing to validate pcr and standardized and validate qpcr . after etiologic treatment , even in cured patients , the antibodies may remain detectable in sera for a long term ( for years ) until they become negative . because of this phenomenon , it would be necessary to delve into the clinical history of patients with reactive serology , asking the question did he / she receive treatment in the past ? " when given an affirmative answer , the serological test has a limited value , because we must consider whether this reactivity is reflecting an active infection or if the patient was cured and he / she is becoming negative . current recommendations have put the bulk of the diagnostic and treatment responsibility on the primary health care system . yet the management of infected patients has some basic limitations , but several researches are looking for solutions . ( a ) current drugs are able to cure infection ( or prevent disease ) in adult patients during the chronic phase , which is when the first contact is made with most infected patients , and clinical trials are finishing or ongoing to demonstrate effects of conventional treatment on this population [ 69 , 70 ] . ( b ) new pediatric presentation of benznidazole is under evaluation to eliminate infection in newborns , and children with recent chronic infection . overall , the priorities in chagas disease research should be to produce new drugs providing a shorter treatment course with fewer side effects , and to devise pediatric formulas . some strategies , such as testing old drugs for extending current prescriptions , screening new compounds , testing drugs developed for other prescription such as pozanonzole , or developing new compounds are being used ( clinical trial for the treatment of chronic chagas disease with posaconazole and benznidazole ; nct01162967 ) [ 69 , 86 ] . based on current disease understanding during chronic phase of infection , there is consensus that every patient infected with t. cruzi must be ( children ) or should be treated ( adults ) . treatment can cure infection and reduce or prevent the progression to the chagas - related heart disease / cardiomyopathy . the current evidence of benefits and limitations of etiological treatment , based on clinical and implementation research , serve to prioritize strategies in primary health care , focusing on completing the scheme of treatment , rather than demonstrating serological negativization . to incorporate etiological treatment as a public health strategy which is useful at the primary , secondary , and tertiary prevention is essential to reduce burden of the disease and to eliminate chagas disease as a public health issue .

one hundred years after carlos chagas identified and described the trypanosoma cruzi ( t. cruzi ) infection , there are still millions of infected people and thousands of newly diagnosed cases each year with chagas disease ( cd ) . the scientific community has intermittently increased the knowledge and understanding of how to manage patients with acute and chronic cd . nonetheless , much more research is still needed in order to improve care and answer many unknown questions regarding this debilitating and widespread disease , which has been estimated to affect about 8 million chronically infected people just in the americas . the goal of etiological treatment against chagas disease is to eliminate the parasite ( t. cruzi ) from the infected individual , to decrease the probability of developing clinical manifestations of the disease ( e.g. currently , there is a new scenario regarding the recommended etiological treatment against t. cruzi infection . in this paper , we review the current evidence supporting etiological chagas disease therapy organized according to different levels of prevention . additionally , we discuss the tools available to demonstrate cure in these patients , and the need for further research required to improve care for t. cruzi infected people . cruzi pharmacotherapy ( etiological treatment ) in order to reduce or avoid the morbidity and mortality of chagas disease applied on different levels of prevention . in this regard , a medline search was conducted from january to july 2011 , using the term chagas disease with the subheadings diagnosis , prognosis , treatment , drug names ( nifurtimox , benznidazole , and other drugs ) , clinical trials , and observational studies . no restrictions regarding year recent guidelines as well as ongoing and unpublished studies were also identified by consulting researchers and experts in the field . evidence was organized according to the levels of prevention addressed by the retrieved guidelines or epidemiological researches . finally , we reviewed the strength of evidence for each indication in each level of prevention . leavell and clark 's have defined three different levels of prevention in human health ( primary , secondary , and tertiary ) in a classical textbook published in 1953 . each of them includes different means of intervention according to the natural history of the disease . primary preventionthese strategies intend to avoid the disease 's development , including the acquisition of new infection . these strategies intend to avoid the disease 's development , including the acquisition of new infection . secondary preventionthese strategies attempt to diagnose and treat an existing disease in its early stages before it results in significant morbidity . these strategies attempt to diagnose and treat an existing disease in its early stages before it results in significant morbidity . tertiary preventionthese treatments aim to reduce the negative impact of established disease by restoring function and reducing disease - related complications . these treatments aim to reduce the negative impact of established disease by restoring function and reducing disease - related complications . in the last decades , jamoulle has proposed a fourth concept ( quaternary prevention ) , which was incorporated by the wonca international classification committee . in this regard , quaternary prevention describes the set of health activities aimed to mitigate or avoid the consequences of unnecessary or excessive interventions in the health system . strength of recommendations as well as the quality level of the evidence supporting these recommendations were addressed according to the quality standards subcommittee or the clinical affairs committee of the infectious diseases society of america ( idsa ) . moderate evidence for efficacy or strong evidence for efficacy but only limited clinical benefit support recommendation for use . , drug toxicity , drug interactions ) or cost of the treatment under consideration : optional . evidence from at least one well - designed clinical trial without randomization , from cohort or case - controlled analytic studies ( preferably from more than one center ) , or from multiple time - series studies , or dramatic results from uncontrolled experiments . evidence from opinions of respected authorities based on clinical experience , descriptive studies , or reports of expert committees . several papers and guidelines have been published in the last years [ 740 ] , supporting with different levels of strength that etiological treatment is an effective intervention on both the individual and public health . these studies reached levels of evidence ranged from i to iii , providing strength of recommendations ( a ) , ( b ) , and ( c ) ( see table 1 ) . several studies have shown the benefit of treatment during acute phase with both benznidazole and nifurtimox with a level of evidence type i or ii [ 1015 ] . the assessment of failure and/or efficacy of treatment on patients treated during acute phase is demonstrable in short time because the parasitemia , whether direct or not ( parasitological test or molecular test ) , becomes negative a few days after the end of treatment . in addition , antibodies disappear completely ( seronegativization ) in at least 65% of cases , with some studies demonstrating seronegativity in 100% of cases up to 18 months of follow up after treatment . this effect is independent of the age of the patients , including newborns ( congenital transmission ) , children , and adults . in general , treatment is well tolerated during the acute phase , and the risk of potential adverse events is counterbalanced by the reduction of clinical manifestations of the acute phase of chagas infection , and even the associated risk of death . there is wide consensus that all patients undergoing the acute phase of infection or reactivation of chronic infection must be treated ( strength of recommendation ( a ) ) . several studies ( evidence type i ) have provided support to the use of etiological treatment ( benznidazole ) during early stages of chronic infection in children [ 1622 ] . two studies have shown efficacy in this population through double blinded placebo - controlled trials of benznidazole for children aged 6 to 12 years old with asymptomatic t. cruzi infection demonstrated approximately 60% efficacy , as assessed by conversion from positive to negative serology results 3 to 4 years after treatment [ 16 , 17 ] . rates of seronegativization up to 70% were established with etiological treatment after long - term follow - up ( 15 years ) in south america , and up to 50% after short - term followup ( 3 years ) in central america . furthermore , additional studies ( evidence type ii ) have shown that seronegativization with etiological treatment is also possible in later stages of the chronic infections in adults [ 7 , 18 , 2331 ] . however , rate of seronegativization of antibodies ( serological test ) seems to be directly related to the age of patients . although a complete seronegativization can be obtained in more than 70% of the cases in children , the seronegativization rate have reached about 30% in adult patients after a long - term followup , around 20 years [ 7 , 1631 ] . fall in antibodies titers after treatment in children is faster than in adults , even if it does not cross the cutoff to become nonreactive . a statistically significant reduction is visible at 3 months with eia and ifa , and at 6 months with iha [ 16 , 17 ] . young children with longer - term followup have higher rates of seronegativization after treatment as compared to child patients with short - term followup , and equal phenomena occurs among adult patients with long - term followup in comparison with adult patients with short followup . demonstration of antiparasitic effects after treatment can be performed by the detection of antibodies , parasites , and/or parasite dna . the success of the treatment is determined by the disappearance of antibodies using serological tests , while therapeutic failure only can be demonstrated by showing the persistence of the parasite using parasitological methods . the assessment of failure of treatment on patients treated during the chronic phase could be demonstrable in short time because the parasitemia ( when it is present by parasitological test or molecular test ) disappears at the end of treatment if treatment is successful . when failure occurs , evidence of parasitemia remains positive after treatment ( not more than 5% in children or 10% in adults ) [ 9 , 10 , 17 , 18 , 20 , 41 , 42 ] . however , gallerano and sosa showed a higher rate of xeno positives including treatment with nifurtimox , benznidazole , and allopurinol . though , this last drug ( allopurinol ) did not show consistent results when they were tested in clinical trials [ 44 , 45 ] . other methodologies to evaluate the effectiveness of antiparasitic treatments have been tested , but have not reached consensus to change current testing strategy . methods to detect the parasite 's genomic fragments in tissues and body fluids using polymerase chain reaction ( pcr ) have proved to be promising tools for the assessment of therapy [ 13 , 36 , 4549 ] , and it was recently standardized for diagnosis . projects for standardization of pcr for assessing therapy which look for the presence of parasites in the blood are underway . there is agreement that , even with limitation , it will be a useful tool to improve the assessment of treatment failure . since tolerance to etiological treatment in children is better than that in adults ( see section 3.3 ) , there is a general agreement that children and adolescents undergoing chronic chagas phase have to be treated ( strength of recommendation ( a ) ) . on the other hand , the rate of seronegativization in adult patients ( about 30% ) based on evidence from observational studies linking the seronegativization to the prevention of clinical disease is currently under research [ 7 , 18 , 2331 ] . furthermore , higher rates of adverse events ( with a 17% of abandon rate ) are seen in adult patients in comparison with children , making this recommendation weaker in adults ( strength of recommendation ( b ) ) . in the case of adult patients undergoing the chronic phase of infection , treatment if accepted , therapy should be prescribed due to the strength of evidence available today . evidence type iii supports that health workers , researchers , and so forth who suffer accidents with infected blood have to be treated under specific protocols . regarding immunocompromised patients , available studies ( evidence type ii ) have shown that after etiological treatment , patients recover from severe manifestations of reactivation such as meningoencephalitis , myocarditis , and panniculitis [ 36 , 52 ] . however , in these cases the main objective is recovery from life - threatening acute events rather than seronegativization , due to the limited ability to interpret serological test results in states of immunosuppression . since the severity of reactivation and the risk of death are associated , there is a general agreement that these patients must be treated ( strength of recommendation ( a ) ) . on the other hand , no current evidence supports the use of etiological treatment as prophylaxis in immunocompromised patients with chronic chagasic infection without evidence of reactivation . although some studies have reported the etiologic treatment of pregnant women without adverse effects in the new born [ 32 , 33 ] , treatment using benznidazole or nifurtimox is currently not recommended for pregnant women ( absolute contraindication ) [ 7 , 9 ] . additional contraindications to use of etiological treatment include patients undergoing severe acute or chronic liver or kidney disease not related to t. cruzi infection ( relative contraindication ) , and lactation ( relative contraindication ) [ 7 , 9 ] . table 1 shows a summary of different scenarios for etiologic treatment against t. cruzi infection , and results of different ways for assessing therapeutic response . during treatment based on prior experiences , treatment tolerance is good and patients have not denoted serious side effects [ 8 , 19 , 27 , 41 , 53 , 54 ] . other types of side effects include reversible clastogenesis and mutagenesis with benznidazole and nifurtimox without any associated manifestations [ 55 , 56 ] , toxicity against other tissues or increased risk of lymphomas in experimental animals have been described , but never demonstrated among a general population of infected patients undergoing treatment and never played a role in animal models . adequate management of side effects is necessary to carry out treatment as well as to diminish unfounded fears with the use of trypanocidal drugs [ 53 , 61 ] . retrieved studies provide evidence for applying strategies of health care to control programs in several countries , whereby a greater portion of the population could get diagnosis , treatment , and cure , generating a new scenario for the reduction in disease burden in the future . if the goal is to avoid the acquisition of a new infection , etiological treatment could have an indirect effect when children and young people are treated . curing children and women in reproductive age would avoid future events of congenital t. cruzi transmission in newborns ( recommendation ( b ) , and evidence type iii ) . unfortunately , effectiveness of etiological treatment for these primary prevention indications remains unknown , although it can be assumed to be at least equal to seronegativization rates observed in available studies . another strategy would be the development of a treatment that can be administered to pregnant women , such as is used for hiv infection , to avoid congenital transmission during pregnancy . etiological treatment in case of accidents with material contaminated with parasites or blood samples of patients infected with t. cruzi could also be considered as an indication for primary prevention . actually , the treatment is not strictly a prophylaxis because it is not possible to avoid the infections , but the infection can be aborted immediately after accidents with a timely treatment to get an appropriate concentration of specific drugs ( recommendation ( b ) , and evidence iii ) . it has consisted of the screening of child populations as a regular strategy to offer opportunities for diagnosis and treatment ( recommendation ( a ) , and evidence type i ) , as well as timely diagnosis and treatment of children born with congenital infection ( recommendation ( a ) , and evidence type ii ) . the positive effect of curing children detected by serological screening must be assessed by taking into account patterns of disease transmission , evolution , and a calculation of the burden of disease attributable to chagas disease , in order to analyze the usefulness of serology as an indicator of the action against the vector . although the effectiveness of etiological treatment for the clinical control of episodes of reactivation has been proven , it is necessary to gather evidence as to whether preventive treatment is effective in patients with no signs of clinical reactivation and with abnormal immunological parameters . in this regard , some protocols recommend the treatment of organ donors infected with t. cruzi in order to reduce risk of transmission by transplant . in this case , the treatment should be considered an act of primary prevention ( recommendation ( a ) , and evidence type ii ) . the use of etiological treatment against t. cruzi infection in order to reduce the negative impact of established disease is under evaluation through two randomized clinical trials , which assess the efficacy in patients with cardiac disease [ 6870 ] . several observational studies have been published showing effects of etiological treatment in patients infected with t. cruzi , on prevention of the progression of chronic chagasic cardiomyopathy [ 18 , 27 , 41 , 43 , 71 ] . the prognosis of patients with heart failure or advanced stages of chagas ' cardiomyopathy is poor , but similar to others that develop heart failure for other reasons . since the disease is chronic and heart damage develops over decades , it is very important to recognize factors that are determinant of disease progression in the early stages . as mentioned above , the effectiveness of etiological treatment for the control of episodes of reactivation has been proven , showing recovery of severe manifestations of reactivation such as meningoencephalitis , myocarditis , and panniculitis [ 36 , 52 ] . recommendations for appropriate care of patients are increasing , putting emphasis on the care of patients in the primary health care system , the use of other levels of care when necessary [ 7476 ] , and the incorporation of psychological aspects into care [ 63 , 64 ] . in this context , it is important to consider the available evidence about etiological treatment , and to maintain the perspective of etiological treatment as a public health tool in multiple levels of prevention , along with other interventions available for chagas disease control and treatment . in chagas disease , the best examples of primary prevention are vectorial control ( based on surveillance ) and control of blood and organ donors . however , etiological treatment has an important role in primary prevention and has to be considered a key element on among other strategies of the chagas disease control programs . the best example of secondary prevention in chagas disease is the control of congenital transmission , and the diagnosis of infection in children ( defined as chronic recent infection ) or young - adult patients in chronic phase without clinical manifestations ( sign and/or symptom ) . use of etiological treatment for tertiary prevention in chagas disease is currently supported by recommendation levels ( b ) and ( c ) , when given in addition to complementary therapies in patients with cardiac disease to reduce the clinical progression of the disease . stem cell transplant is a new therapy applied to produce cardiac regeneration through distinction or increase heart myocytes or neovascular proliferation in patients in the final stage of congestive heart failure [ 7981 ] , but the results are still insufficient on chagas disease , and there is no consensus about its efficacy . the assessment of effect of treatment against t. cruzi infection requires a clear understanding about the combination of variables to an appropriate interpretation of results to appraise . among others , the main variables are the tools used as indicators ( parasitological , molecular , and serological tests ) , the phase of infection ( acute or chronic ) that the patient is undergoing when he / she was treated , and the time elapsed between treatment and the application of the test to assess efficacy / failure . the ideal assessment of response to specific treatment is the detection of free parasites in the patient 's blood or tissues , which permits clear observation of failure of treatment . it is also necessary to validate new tools to confirm cure or failure in a timely manner after a full course of treatment has been given during the chronic phase , and studies are ongoing to validate pcr and standardized and validate qpcr . if persistence of the parasite is identified , after verifying if the drug was taken correctly , it is necessary to consider the possibility that the parasite has developed resistance [ 84 , 85 ] . because of this phenomenon , it would be necessary to delve into the clinical history of patients with reactive serology , asking the question did he / she receive treatment in the past ? " current recommendations have put the bulk of the diagnostic and treatment responsibility on the primary health care system . ( a ) current drugs are able to cure infection ( or prevent disease ) in adult patients during the chronic phase , which is when the first contact is made with most infected patients , and clinical trials are finishing or ongoing to demonstrate effects of conventional treatment on this population [ 69 , 70 ] . some strategies , such as testing old drugs for extending current prescriptions , screening new compounds , testing drugs developed for other prescription such as pozanonzole , or developing new compounds are being used ( clinical trial for the treatment of chronic chagas disease with posaconazole and benznidazole ; nct01162967 ) [ 69 , 86 ] . based on current disease understanding during chronic phase of infection , there is consensus that every patient infected with t. cruzi must be ( children ) or should be treated ( adults ) . the current evidence of benefits and limitations of etiological treatment , based on clinical and implementation research , serve to prioritize strategies in primary health care , focusing on completing the scheme of treatment , rather than demonstrating serological negativization . to incorporate etiological treatment as a public health strategy which is useful at the primary , secondary , and tertiary prevention is essential to reduce burden of the disease and to eliminate chagas disease as a public health issue .

such techniques involve remote manipulations of tissues with thin instruments directed through small incisions made into an abdomen distended with gas . the surgeon interacts with images created by a camera lens assembly and displayed on a video monitor . surgeons accustomed to traditional techniques experience difficulty manipulating their operative instruments with reference to images on a video screen . because video - assisted surgery is significantly different from conventional surgery , physicians skilled in the conventional approach need to acquire skills and a capacity for visual - motor processing before becoming equally proficient in laparoscopic video surgery . the basic skill required for performing advanced laparoscopic surgery is an adequate visual - motor processing capacity for referencing the 2-dimensional image of an object projected on a video screen . acquiring such a capacity permits the surgeon to adapt to the loss of depth perception and lack of appreciation of the temporal proximity of structures displayed on a flat plane , and to command the orientation of instruments by motions projected in the operative field . more advanced skills call for precise control of instruments that pivot about access points remote from the surgical field ; the ability to adapt to limitation of access , range of operative motions , and tissue retraction ; and the capacity to execute complex manipulations requiring refined hand - eye coordination and manual dexterity . these skills may be acquired and perfected through instructions and repetitive practice on a laparoscopy training simulator . this report describes an improved training simulator designed to enhance laparoscopic proficiency and allow objective skill assessment and presents the results when this system was used to train 11 physicians who had various levels of prior experience . the laparoscopy training simulator 2000 ( lts 2000 ) was designed by the senior author . it is essentially a video training device that allows for simulated laparoscopic manipulations under realistic conditions . it is easily transportable and replicates the working dimensions of a dis - tended abdomen ; it is covered by a removable frame , or cassette , containing a resilient , multilayered membranous structure simulating the anterior abdominal wall . a flexible floor mat can be selectively adjusted to various heights at 3 axial positions forming various floor configurations . the floor mat is formed with velcro strips for attachment to corresponding strips mounted on exercise platforms . the laparoscopic skill exercise models are either integral with or can be replaced and attached to the platforms . a timer / stopwatch for measuring exercise time , and posts mounted on the outside of the simulator for knot - tying practice are provided . models that are integrated with dedicated platforms include : the peg board - and - posts coordination model that consists of a peg board and 2 thin metallic posts , 1 with a single curve , and the other with 2 curves , permanently attached to a platform.the 3-d ( dimensional)-to-2-d translation model is a platform constructed with 9 spring posts each with 3 rings , to create a virtual cube . the top 9 rings of the cube are red , the middle rings are yellow , and the bottom rings are blue.the oviduct cannulation model is composed of a malleable plastic tube , the distal end of which is shaped like the fimbriated end of an oviduct . the tube is suspended from a platform , which pivots to allow the fimbriated end to rise freely above the floor mat.the tube cannulation model is made up of a hard plastic tube joined along its entire length to a small platform . small beads , plastic rings , and pipe cleaners are also used for the exercises with these models . the peg board - and - posts coordination model that consists of a peg board and 2 thin metallic posts , 1 with a single curve , and the other with 2 curves , permanently attached to a platform . the 3-d ( dimensional)-to-2-d translation model is a platform constructed with 9 spring posts each with 3 rings , to create a virtual cube . the top 9 rings of the cube are red , the middle rings are yellow , and the bottom rings are blue . the oviduct cannulation model is composed of a malleable plastic tube , the distal end of which is shaped like the fimbriated end of an oviduct . the tube is suspended from a platform , which pivots to allow the fimbriated end to rise freely above the floor mat . the tube cannulation model is made up of a hard plastic tube joined along its entire length to a small platform . small beads , plastic rings , and pipe cleaners are also used for the exercises with these models . undedicated platforms are designed to fix interchangeable models to the floor mat : the small , single - clamp platform has a 3-cm central metallic post formed with an alligator - type clamp.the drill platform contains a centrally located 9-cm sharp drill capped with a winged nut.the 4-clamp platform has 4 metallic corner posts , each formed with an alligator - type clamp.the 4-nail platform is constructed with 4 upward pointing nails . the small , single - clamp platform has a 3-cm central metallic post formed with an alligator - type clamp . the drill platform contains a centrally located 9-cm sharp drill capped with a winged nut . the 4-clamp platform has 4 metallic corner posts , each formed with an alligator - type clamp . two preassembled models attach interchangeably to the 4-clamp or 4-nail platform : the sponge cloth model consists of a piece of cloth , with a centrally located incisional gap , which is peripherally fixed to a sponge.the blood vessel model is composed of a sponge with attached color - coded tubes bundled in close proximity to simulate artery , vein , and ureter . a thin rubber tube may be cut into short segments ; 1 segment may be fixed to the single clamp platform in a dangling position to constitute the appendix model . another segment may be suspended between 2 single clamp platforms or 2 of the 4 clamps of the 4-clamp platform to serve as the stapling model . a medium - sized balloon filled with water may be attached to the single clamp platform to serve as the ovarian cyst - gallbladder model . an orange may be turned into the drill platform to function as the myomectomy model . a piece of meat may be cooked medium rare and fixed to the drill platform to act as the morcellator model ( figures 1 and 2 ) . the sponge cloth model consists of a piece of cloth , with a centrally located incisional gap , which is peripherally fixed to a sponge . the blood vessel model is composed of a sponge with attached color - coded tubes bundled in close proximity to simulate artery , vein , and ureter . a thin rubber tube may be cut into short segments ; 1 segment may be fixed to the single clamp platform in a dangling position to constitute the appendix model . another segment may be suspended between 2 single clamp platforms or 2 of the 4 clamps of the 4-clamp platform to serve as the stapling model . a medium - sized balloon filled with water may be attached to the single clamp platform to serve as the ovarian cyst - gallbladder model . an orange may be turned into the drill platform to function as the myomectomy model . a piece of meat may be cooked medium rare and fixed to the drill platform to act as the morcellator model ( figures 1 and 2 ) . internal view of the laparoscopy training simulator 2000 ( lts 2000 ) showing the floor mat with velcro strips and attached exercise models . shown counter clockwise are the pegboard - and - post , straight tube , oviduct cannulation , blood vessel and virtual cube models . a small box with beads and rings is seen next to the pegboard - and - post model . internal view of the lts 2000 showing other exercises : balloon cyst inside a zip lock bag , sponge - cloth suturing model , meat morcellation and balloon cyst models . the simulator is used with a standard laparoscope attached to a video camera , light source , and video monitor placed at a comfortable height for viewing . alternatively , the camera lens assembly may be furnished by a camcorder mounted on a standard tripod to supply a stable and magnified view of any portion of the box 's interior when connected to a video monitor . the laparoscopic video camera or camcorder may be connected to a standard video recorder to permit subsequent review and analysis of the exercise manipulations ( figure 3 ) . ( a ) timer for measuring exercise time . ( b ) external post for practicing knot tying . ( c ) laparoscope with attached light source and camera ( not shown ) . ( d ) camcorder mounted on a standard tripod : an alternative camera - lens assembly . the simulator box is placed on a table with the top of the box at the level of the operator 's wrists . the 2 metal posts are inserted into corresponding openings on the exterior wall of the simulator . exercise models are attached to the floor mat , and the abdominal wall frame is returned to its original locked position . the first trocar is inserted through the rubbery abdominal wall in the midline , approximately 3 cm from 1 edge of the frame . once inserted , the cannulas remain stable on the abdominal wall and do not need to be changed or replaced . the laparoscope , with attached camera and light source , is introduced into the simulator through the primary port and exercise manipulations are initiated . practice with the laparoscope requires 2 individuals ; an assistant manipulates the scope , and the operator uses 2 hands to perform the exercise . all pre- and posttests were conducted with the laparoscope ; the camcorder was used for practice only . following conclusion of the pretest , the training exercises used for testing with the lts 2000 are summarized in table 1 . exercises not included in the testing program but used for practice purposes are summarized in table 2 . , the instructor ( nak ) recorded the number of successful exercise tasks completed in 1 minute . the instructor recorded the time taken to accomplish 1 successful task and then reset the timer for the following exercise . the name of the trainee , status ( resident or attending physician ) , and date of the test were entered on the pre- and posttest forms . a log was kept to detail the amount of time each trainee accumulated with the simulator . the mathematical basis of the scoring system was developed by one of the authors ( je ) . each exercise was given an equal rank and assigned a point value of 100 , giving the test a maximum score of 1100 points . exercises 1 through 6 were scored on the basis of the number of tasks completed in 1 minute , for instance , the number of beads placed on the peg board . a standard number of tasks were set prior to the training , based on the performance of two of the authors ( hmh , nak ) . a score of 100 was given to those trainees who equaled or exceeded the preestablished number of tasks . some of the trainees were able to complete a higher number of tasks after being trained . for example , making 3 roeder loop ties on the external post in 1 minute was preestablished by the authors as the desirable end point of the first exercise . subsequently , some of the trainees were able to set down 4 ties in 1 minute . therefore , instead of evaluating the other scores on the basis of the initial target of 3 , a new target value of 3.5 was set . the best score ( 100 ) was given to trainees who completed 3 or 4 tasks in 1 minute in exercise 1 and the worst score ( 0 ) was given to those who could not complete a single task . scores between the maximum and the minimum were set linearly as a function of those values . the equation for such a line was calculated on the basis of the upper limit ( 100 , target value ) and the bottom limit ( 0 , no tasks completed ) . for exercise 1 , for example , the upper limit in the calculation of the slope ( y2-y1)/(x2-x1 ) was 3.5 instead of 3 or 4 , ie , ( 100 - 0)/(3.5 - 0 ) . for exercises 7 to 11 , the best score ( 100 ) was established on the basis of the best performance ( shortest time to accomplish task ) . the worst score ( 0 ) was allocated to trainees who did not complete the task within a period of time 3 times greater than the best time . for example , in exercise 7 , trainees who cannulated the simulated oviduct using a pipe cleaner in the best time of 0.8 minutes were given a score of 100 . those who failed to cannulate the oviduct in 2.4 minutes were given a score of 0 . trainees who completed the exercise task in an intermediate period of time , between the best and the maximum allowed , received scores that were calculated linearly as a function of these 2 values ( table 3 ) . number of tasks completed in one minute number of minutes to complete one task . for methodological reasons , the authors modeled 2 other types of scoring systems , one based on a pure linear relationship between the best ( 100 ) and the worst score ( 0 ) , and the other based on a random distribution ( bell curve ) , where each trainee was graded as compared with his or her colleagues ' performance on each exercise . all 3 scoring systems led to very similar conclusions . between january 1999 and march 2000 , 11 physicians utilized the lts 2000 simulator , completed a pretest , received instructions to enhance their laparoscopic skills , and then completed a posttest after 2 to 23 hours of practice . all 11 physicians experienced significant improvement in the test scores in coordination , manipulation , suturing , and knot tying , as shown in tables 4 and 5 . fifteen other physicians were trained on the simulator but did not complete the posttest and were excluded from the study . e = exercise ; pre = pretest number of tasks completed in one minute for exercises 1 - 6 and number of minutes to complete one task for exercises 7 - 11 ; post = posttest values . effect of simulator training on skill scores . status ranked by level of pretest score . 1 - 4 yr . the average pretest score was 304.9 points ( sd 190.8 ) , with a range of 43.2 to 705.7 . the average posttest score was 834.2 points ( sd 141.2 ) , with a range of 547.3 to 1021.7 . when the pre- and posttraining scores were compared , the average increase in the posttest score was 529.3 points ( sd 200.2 ) , with a range of 280.3 to 934.7 points . the average number of hours spent in training was 5.9 with a range of 2 to 23 . this is not surprising because participating residents came from 3 programs in the city with different emphases on laparoscopic training , and one of the physicians had no previous experience with operative laparoscopy , but the other was an experienced laparoscopist . furthermore , individual variations in inherent abilities of the trainees played a role in explaining their performance on the pretest . however , after training with the lts 2000 , the variations on the posttest scores were not influenced much by the level of the pretest score . this finding indicates that the number of hours invested in the training was the primary reason for the noted improvement in the posttest score , as the inherent abilities of each trainee remains constant . improvement in the posttest scores correlated with the number of hours invested in training ( p = 0.055 ) . the greatest improvement was noted in physicians who had the lowest pretest score . when the benchmark for skill testing was set at the 70th percentile of the maximum achievable score , none of the physicians passed the test prior to training . eight of the 11 physicians scored higher than 73% after training and practice ( table 5 ) . the difference between pre- and posttest scores of individual exercises is shown in table 6 . the mean pretest scores for all exercises was 27.7 out of 100 and for posttest scores was 75.8 out of 100 . the need for frequent preparation and training is not unique to laparoscopic surgeons ; it is shared by all performers with highly developed skills including professional musicians and athletes , as previously noted by resnick . the technical expertise of a laparoscopic surgeon will ultimately depend on inherent genetic abilities , modulated by acquired knowledge and experience , and improved by practice . rosser et al pioneered the use of objective parameters to evaluate laparoscopic skills in 2 studies of a first - generation laparoscopy trainer . the dexterity exercises consisted of ( 1 ) the rope pass drill , grasping a rope with color coded bands and successively passing it from 1 hand to the other ; ( 2 ) the cup drop drill , dropping a pea into an aperture of an inverted cup using the nondominant hand ; ( 3 ) the triangle transfer drill , transferring a triangular wooden block from 1 area to another by engaging and disengaging a curved needle into and from a plastic loop at the apex of the triangle . the authors found that the time needed to perform the dexterity drills improved among participants with successive drills resulting in a significant difference ( p < .001 ) between the time required for the first and tenth drill . similarly , significant improvement ( p < .001 ) was noted in performing intracorporeal suturing with successive drills , after the participants received instruction . a significant correlation ( p < .001 ) was also observed between the performance efficiency of the dexterity and suturing drills , suggesting that dexterity drills help improve suturing . to evaluate the results , the authors correlated the mean time required to perform a drill with that of participants who completed all 10 drills and 10 suturing exercises . in the second study , these authors compared the performance of senior surgeons with that of residents after both groups had completed the training program the authors previously designed . the collective performance of the residents was similar to that of the trained surgeons in the rope pass drill and suturing exercises . however , the residents performed the triangle transfer drill faster and the cup drill more slowly than the trained surgeons . using objective parameters , derossis et al also used a first - generation laparoscopy trainer to train attending and resident surgeons with various degrees of experience . the simulated laparoscopic tasks consisted of ( 1 ) peg transfers , lifting a peg from 1 peg hole with 1 hand , transferring it to the other hand and placing it on another peg board ; ( 2 ) pattern cutting , cutting a circular pattern out of a larger piece of gauze ; ( 3 ) clip and divide , placing 2 hemostatic clips on a tubular foam structure and cutting in between ; ( 4 ) endo - looping , placing a pretied slip knot on a foam tubular appendage ; ( 5 ) mesh application , placing a mesh over a defect in a foam model and securing it to the foam with staples ; ( 6 ) intracorporeal knot , placing a suture into a penrose drain and tying it with an intracorporeal knot ; ( 7 ) extracorporeal knot , placing a suture into a penrose drain and tying it with an extracorporeal knot . all 7 tasks showed a significant improvement in performance with repetition ( p < 0.0001 ) . some of the tasks ( 1 , 2 , 6 ) showed a significant correlation between the degree of physician experience and score , but others did not . when the canadian group further studied the effect of practice on performance , they found that residents who had a pretest followed by 5 weekly practice sessions showed significant improvement in the posttest score for each task and for total score . residents who limited their practice to the pretest showed improvement in tasks 1 , 4 , 5 , and 7 , but not in 2 , 3 , and 6 in the posttest . the total score of the posttest was not statistically different from that of the pretest in this group . we used a new second - generation laparoscopy trainer , a complementary set of dexterity and suturing exercises , and a scoring system that is different from that described by the previous authors . the eekhout formula gives a precise score for any skill exercise based on the number of tasks completed in a unit of time or the number of minutes needed to complete a given task . a maximum score of 100 is given for achieving a target value or time ; a minimum score of 0 is given for not completing a single task in the unit of time or the assigned task in 3 times the target time . scores between 100 and 0 are set linearly as a function of those values . a user - friendly software computer program containing the algorithm has been developed for the benefit of future investigators . to calculate the skill scores of trainees for any given exercise with a known target value or time , the investigator simply needs to plug in the number of tasks the trainee completed per unit of time , or the number of minutes needed to complete a task . the program will then compute and tabulate the information presented in tables 36 , as required . physicians who are adept at performing conventional surgery possess the knowledge of surgical principles and the skills of operative manipulation . to become proficient in laparoscopic surgery , they need to master only the necessary adaptations in visual - motor processing through training and practice . physicians who do not perform advanced laparoscopic surgery on a regular basis have a particular need for ongoing simulator training to maintain a reasonable level of competence . the present study suggests that most physicians acquire competence in skill sets associated with advanced operative laparoscopy if they receive appropriate instructions and invest a reasonable amount of time practicing on a laparoscopy training simulator like the lts 2000 . expert laparoscopists can also benefit from utilizing the lts 2000 simulator for trying out new instruments and methods before using them in the operating room , and for practicing newly acquired skills . our group has been able to resolve many of the problematic subtleties associated with novel applications by testing them in the simulator before performing live surgery . the ultimate goal of any laparoscopy training system is to increase the comfort level and efficiency of laparoscopic surgeons in the operating room . the 11 physicians who participated in this training system experienced significant improvement in their laparoscopic skills regardless of prior level of expertise : all 11 scored below the 65th percentile of the maximum achievable score prior to training and 8 scored above the 73rd percentile after training and practice . the improvement in the posttest scores correlated with the number of hours invested in training ( p = 0.055 ) .

objective : to measure , using objective pre- and post - training assessments , the degree of improvement in laparoscopic skills following training with a new laparoscopy training simulator ( lts 2000).methods : this study utilized the lts 2000 training simulator . the simulator was used in conjunction with a laparoscope joined to a camera , light source , and monitor or with a camcorder . eleven exercises were planned with tasks designed to develop visual - motor - processing capabilities for referencing the 2-dimensional image of an object on a video screen , and to teach and allow practice of delicate manipulations , circular motions , oviductal cannulation , formation of roeder loops , and simple suturing and knot - tying . the skill level of individual trainees was assessed before and after training with objective means . each exercise was assigned a point value of 100 with a maximum score of 1100 . some exercises were scored in number of tasks per 1 minute , others in number of minutes per 1 task . a score of 100 was given for completing a target number of tasks in 1 minute and 0 for not completing any . a score of 100 was given for completing the assigned task in a target amount of time and 0 for not completing it in 3 times that amount . scores between 100 and 0 were set linearly as a function of those values.results:of the 11 participating physicians , none scored above the 65th percentile of the maximum achievable score before training ; 8 scored above the 73rd percentile after training . the average pretest score was 304.9 points ( sd 190.8 ) range 43.2 to 705.7 ; posttest score was 834.2 points ( sd 141.2 ) range 547.3 to 1021.7 . the average number of hours spent in practice was 5.9 ( range 2 to 23 ) . a positive correlation existed between hours of practice and posttest score improvement.conclusion:sustained training with the new simulator resulted in significant improvement in laparoscopic skills in all tested physicians , regardless of prior level of experience .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

the surgeon interacts with images created by a camera lens assembly and displayed on a video monitor . surgeons accustomed to traditional techniques experience difficulty manipulating their operative instruments with reference to images on a video screen . because video - assisted surgery is significantly different from conventional surgery , physicians skilled in the conventional approach need to acquire skills and a capacity for visual - motor processing before becoming equally proficient in laparoscopic video surgery . the basic skill required for performing advanced laparoscopic surgery is an adequate visual - motor processing capacity for referencing the 2-dimensional image of an object projected on a video screen . acquiring such a capacity permits the surgeon to adapt to the loss of depth perception and lack of appreciation of the temporal proximity of structures displayed on a flat plane , and to command the orientation of instruments by motions projected in the operative field . more advanced skills call for precise control of instruments that pivot about access points remote from the surgical field ; the ability to adapt to limitation of access , range of operative motions , and tissue retraction ; and the capacity to execute complex manipulations requiring refined hand - eye coordination and manual dexterity . these skills may be acquired and perfected through instructions and repetitive practice on a laparoscopy training simulator . this report describes an improved training simulator designed to enhance laparoscopic proficiency and allow objective skill assessment and presents the results when this system was used to train 11 physicians who had various levels of prior experience . the laparoscopy training simulator 2000 ( lts 2000 ) was designed by the senior author . it is essentially a video training device that allows for simulated laparoscopic manipulations under realistic conditions . the floor mat is formed with velcro strips for attachment to corresponding strips mounted on exercise platforms . the laparoscopic skill exercise models are either integral with or can be replaced and attached to the platforms . a timer / stopwatch for measuring exercise time , and posts mounted on the outside of the simulator for knot - tying practice are provided . models that are integrated with dedicated platforms include : the peg board - and - posts coordination model that consists of a peg board and 2 thin metallic posts , 1 with a single curve , and the other with 2 curves , permanently attached to a platform.the 3-d ( dimensional)-to-2-d translation model is a platform constructed with 9 spring posts each with 3 rings , to create a virtual cube . the top 9 rings of the cube are red , the middle rings are yellow , and the bottom rings are blue.the oviduct cannulation model is composed of a malleable plastic tube , the distal end of which is shaped like the fimbriated end of an oviduct . the tube is suspended from a platform , which pivots to allow the fimbriated end to rise freely above the floor mat.the tube cannulation model is made up of a hard plastic tube joined along its entire length to a small platform . small beads , plastic rings , and pipe cleaners are also used for the exercises with these models . the peg board - and - posts coordination model that consists of a peg board and 2 thin metallic posts , 1 with a single curve , and the other with 2 curves , permanently attached to a platform . the top 9 rings of the cube are red , the middle rings are yellow , and the bottom rings are blue . the oviduct cannulation model is composed of a malleable plastic tube , the distal end of which is shaped like the fimbriated end of an oviduct . the tube is suspended from a platform , which pivots to allow the fimbriated end to rise freely above the floor mat . the tube cannulation model is made up of a hard plastic tube joined along its entire length to a small platform . small beads , plastic rings , and pipe cleaners are also used for the exercises with these models . undedicated platforms are designed to fix interchangeable models to the floor mat : the small , single - clamp platform has a 3-cm central metallic post formed with an alligator - type clamp.the drill platform contains a centrally located 9-cm sharp drill capped with a winged nut.the 4-clamp platform has 4 metallic corner posts , each formed with an alligator - type clamp.the 4-nail platform is constructed with 4 upward pointing nails . the drill platform contains a centrally located 9-cm sharp drill capped with a winged nut . two preassembled models attach interchangeably to the 4-clamp or 4-nail platform : the sponge cloth model consists of a piece of cloth , with a centrally located incisional gap , which is peripherally fixed to a sponge.the blood vessel model is composed of a sponge with attached color - coded tubes bundled in close proximity to simulate artery , vein , and ureter . a thin rubber tube may be cut into short segments ; 1 segment may be fixed to the single clamp platform in a dangling position to constitute the appendix model . another segment may be suspended between 2 single clamp platforms or 2 of the 4 clamps of the 4-clamp platform to serve as the stapling model . a piece of meat may be cooked medium rare and fixed to the drill platform to act as the morcellator model ( figures 1 and 2 ) . the sponge cloth model consists of a piece of cloth , with a centrally located incisional gap , which is peripherally fixed to a sponge . the blood vessel model is composed of a sponge with attached color - coded tubes bundled in close proximity to simulate artery , vein , and ureter . a thin rubber tube may be cut into short segments ; 1 segment may be fixed to the single clamp platform in a dangling position to constitute the appendix model . another segment may be suspended between 2 single clamp platforms or 2 of the 4 clamps of the 4-clamp platform to serve as the stapling model . a piece of meat may be cooked medium rare and fixed to the drill platform to act as the morcellator model ( figures 1 and 2 ) . internal view of the laparoscopy training simulator 2000 ( lts 2000 ) showing the floor mat with velcro strips and attached exercise models . shown counter clockwise are the pegboard - and - post , straight tube , oviduct cannulation , blood vessel and virtual cube models . internal view of the lts 2000 showing other exercises : balloon cyst inside a zip lock bag , sponge - cloth suturing model , meat morcellation and balloon cyst models . the simulator is used with a standard laparoscope attached to a video camera , light source , and video monitor placed at a comfortable height for viewing . alternatively , the camera lens assembly may be furnished by a camcorder mounted on a standard tripod to supply a stable and magnified view of any portion of the box 's interior when connected to a video monitor . the laparoscopic video camera or camcorder may be connected to a standard video recorder to permit subsequent review and analysis of the exercise manipulations ( figure 3 ) . ( c ) laparoscope with attached light source and camera ( not shown ) . ( d ) camcorder mounted on a standard tripod : an alternative camera - lens assembly . the simulator box is placed on a table with the top of the box at the level of the operator 's wrists . the 2 metal posts are inserted into corresponding openings on the exterior wall of the simulator . exercise models are attached to the floor mat , and the abdominal wall frame is returned to its original locked position . the first trocar is inserted through the rubbery abdominal wall in the midline , approximately 3 cm from 1 edge of the frame . once inserted , the cannulas remain stable on the abdominal wall and do not need to be changed or replaced . the laparoscope , with attached camera and light source , is introduced into the simulator through the primary port and exercise manipulations are initiated . practice with the laparoscope requires 2 individuals ; an assistant manipulates the scope , and the operator uses 2 hands to perform the exercise . all pre- and posttests were conducted with the laparoscope ; the camcorder was used for practice only . following conclusion of the pretest , the training exercises used for testing with the lts 2000 are summarized in table 1 . , the instructor ( nak ) recorded the number of successful exercise tasks completed in 1 minute . the name of the trainee , status ( resident or attending physician ) , and date of the test were entered on the pre- and posttest forms . a log was kept to detail the amount of time each trainee accumulated with the simulator . the mathematical basis of the scoring system was developed by one of the authors ( je ) . each exercise was given an equal rank and assigned a point value of 100 , giving the test a maximum score of 1100 points . exercises 1 through 6 were scored on the basis of the number of tasks completed in 1 minute , for instance , the number of beads placed on the peg board . a standard number of tasks were set prior to the training , based on the performance of two of the authors ( hmh , nak ) . a score of 100 was given to those trainees who equaled or exceeded the preestablished number of tasks . some of the trainees were able to complete a higher number of tasks after being trained . for example , making 3 roeder loop ties on the external post in 1 minute was preestablished by the authors as the desirable end point of the first exercise . subsequently , some of the trainees were able to set down 4 ties in 1 minute . therefore , instead of evaluating the other scores on the basis of the initial target of 3 , a new target value of 3.5 was set . the best score ( 100 ) was given to trainees who completed 3 or 4 tasks in 1 minute in exercise 1 and the worst score ( 0 ) was given to those who could not complete a single task . scores between the maximum and the minimum were set linearly as a function of those values . the equation for such a line was calculated on the basis of the upper limit ( 100 , target value ) and the bottom limit ( 0 , no tasks completed ) . for exercise 1 , for example , the upper limit in the calculation of the slope ( y2-y1)/(x2-x1 ) was 3.5 instead of 3 or 4 , ie , ( 100 - 0)/(3.5 - 0 ) . for exercises 7 to 11 , the best score ( 100 ) was established on the basis of the best performance ( shortest time to accomplish task ) . the worst score ( 0 ) was allocated to trainees who did not complete the task within a period of time 3 times greater than the best time . for example , in exercise 7 , trainees who cannulated the simulated oviduct using a pipe cleaner in the best time of 0.8 minutes were given a score of 100 . those who failed to cannulate the oviduct in 2.4 minutes were given a score of 0 . trainees who completed the exercise task in an intermediate period of time , between the best and the maximum allowed , received scores that were calculated linearly as a function of these 2 values ( table 3 ) . number of tasks completed in one minute number of minutes to complete one task . for methodological reasons , the authors modeled 2 other types of scoring systems , one based on a pure linear relationship between the best ( 100 ) and the worst score ( 0 ) , and the other based on a random distribution ( bell curve ) , where each trainee was graded as compared with his or her colleagues ' performance on each exercise . between january 1999 and march 2000 , 11 physicians utilized the lts 2000 simulator , completed a pretest , received instructions to enhance their laparoscopic skills , and then completed a posttest after 2 to 23 hours of practice . all 11 physicians experienced significant improvement in the test scores in coordination , manipulation , suturing , and knot tying , as shown in tables 4 and 5 . fifteen other physicians were trained on the simulator but did not complete the posttest and were excluded from the study . e = exercise ; pre = pretest number of tasks completed in one minute for exercises 1 - 6 and number of minutes to complete one task for exercises 7 - 11 ; post = posttest values . status ranked by level of pretest score . the average pretest score was 304.9 points ( sd 190.8 ) , with a range of 43.2 to 705.7 . the average posttest score was 834.2 points ( sd 141.2 ) , with a range of 547.3 to 1021.7 . when the pre- and posttraining scores were compared , the average increase in the posttest score was 529.3 points ( sd 200.2 ) , with a range of 280.3 to 934.7 points . the average number of hours spent in training was 5.9 with a range of 2 to 23 . this is not surprising because participating residents came from 3 programs in the city with different emphases on laparoscopic training , and one of the physicians had no previous experience with operative laparoscopy , but the other was an experienced laparoscopist . furthermore , individual variations in inherent abilities of the trainees played a role in explaining their performance on the pretest . however , after training with the lts 2000 , the variations on the posttest scores were not influenced much by the level of the pretest score . this finding indicates that the number of hours invested in the training was the primary reason for the noted improvement in the posttest score , as the inherent abilities of each trainee remains constant . improvement in the posttest scores correlated with the number of hours invested in training ( p = 0.055 ) . the greatest improvement was noted in physicians who had the lowest pretest score . when the benchmark for skill testing was set at the 70th percentile of the maximum achievable score , none of the physicians passed the test prior to training . eight of the 11 physicians scored higher than 73% after training and practice ( table 5 ) . the difference between pre- and posttest scores of individual exercises is shown in table 6 . the mean pretest scores for all exercises was 27.7 out of 100 and for posttest scores was 75.8 out of 100 . the technical expertise of a laparoscopic surgeon will ultimately depend on inherent genetic abilities , modulated by acquired knowledge and experience , and improved by practice . rosser et al pioneered the use of objective parameters to evaluate laparoscopic skills in 2 studies of a first - generation laparoscopy trainer . the dexterity exercises consisted of ( 1 ) the rope pass drill , grasping a rope with color coded bands and successively passing it from 1 hand to the other ; ( 2 ) the cup drop drill , dropping a pea into an aperture of an inverted cup using the nondominant hand ; ( 3 ) the triangle transfer drill , transferring a triangular wooden block from 1 area to another by engaging and disengaging a curved needle into and from a plastic loop at the apex of the triangle . the authors found that the time needed to perform the dexterity drills improved among participants with successive drills resulting in a significant difference ( p < .001 ) between the time required for the first and tenth drill . similarly , significant improvement ( p < .001 ) was noted in performing intracorporeal suturing with successive drills , after the participants received instruction . a significant correlation ( p < .001 ) was also observed between the performance efficiency of the dexterity and suturing drills , suggesting that dexterity drills help improve suturing . to evaluate the results , the authors correlated the mean time required to perform a drill with that of participants who completed all 10 drills and 10 suturing exercises . the collective performance of the residents was similar to that of the trained surgeons in the rope pass drill and suturing exercises . however , the residents performed the triangle transfer drill faster and the cup drill more slowly than the trained surgeons . using objective parameters , derossis et al also used a first - generation laparoscopy trainer to train attending and resident surgeons with various degrees of experience . the simulated laparoscopic tasks consisted of ( 1 ) peg transfers , lifting a peg from 1 peg hole with 1 hand , transferring it to the other hand and placing it on another peg board ; ( 2 ) pattern cutting , cutting a circular pattern out of a larger piece of gauze ; ( 3 ) clip and divide , placing 2 hemostatic clips on a tubular foam structure and cutting in between ; ( 4 ) endo - looping , placing a pretied slip knot on a foam tubular appendage ; ( 5 ) mesh application , placing a mesh over a defect in a foam model and securing it to the foam with staples ; ( 6 ) intracorporeal knot , placing a suture into a penrose drain and tying it with an intracorporeal knot ; ( 7 ) extracorporeal knot , placing a suture into a penrose drain and tying it with an extracorporeal knot . all 7 tasks showed a significant improvement in performance with repetition ( p < 0.0001 ) . some of the tasks ( 1 , 2 , 6 ) showed a significant correlation between the degree of physician experience and score , but others did not . when the canadian group further studied the effect of practice on performance , they found that residents who had a pretest followed by 5 weekly practice sessions showed significant improvement in the posttest score for each task and for total score . residents who limited their practice to the pretest showed improvement in tasks 1 , 4 , 5 , and 7 , but not in 2 , 3 , and 6 in the posttest . the total score of the posttest was not statistically different from that of the pretest in this group . we used a new second - generation laparoscopy trainer , a complementary set of dexterity and suturing exercises , and a scoring system that is different from that described by the previous authors . the eekhout formula gives a precise score for any skill exercise based on the number of tasks completed in a unit of time or the number of minutes needed to complete a given task . a maximum score of 100 is given for achieving a target value or time ; a minimum score of 0 is given for not completing a single task in the unit of time or the assigned task in 3 times the target time . scores between 100 and 0 are set linearly as a function of those values . to calculate the skill scores of trainees for any given exercise with a known target value or time , the investigator simply needs to plug in the number of tasks the trainee completed per unit of time , or the number of minutes needed to complete a task . to become proficient in laparoscopic surgery , they need to master only the necessary adaptations in visual - motor processing through training and practice . physicians who do not perform advanced laparoscopic surgery on a regular basis have a particular need for ongoing simulator training to maintain a reasonable level of competence . the present study suggests that most physicians acquire competence in skill sets associated with advanced operative laparoscopy if they receive appropriate instructions and invest a reasonable amount of time practicing on a laparoscopy training simulator like the lts 2000 . expert laparoscopists can also benefit from utilizing the lts 2000 simulator for trying out new instruments and methods before using them in the operating room , and for practicing newly acquired skills . our group has been able to resolve many of the problematic subtleties associated with novel applications by testing them in the simulator before performing live surgery . the ultimate goal of any laparoscopy training system is to increase the comfort level and efficiency of laparoscopic surgeons in the operating room . the 11 physicians who participated in this training system experienced significant improvement in their laparoscopic skills regardless of prior level of expertise : all 11 scored below the 65th percentile of the maximum achievable score prior to training and 8 scored above the 73rd percentile after training and practice . the improvement in the posttest scores correlated with the number of hours invested in training ( p = 0.055 ) .

surgeons accustomed to traditional techniques experience difficulty manipulating their operative instruments with reference to images on a video screen . because video - assisted surgery is significantly different from conventional surgery , physicians skilled in the conventional approach need to acquire skills and a capacity for visual - motor processing before becoming equally proficient in laparoscopic video surgery . the basic skill required for performing advanced laparoscopic surgery is an adequate visual - motor processing capacity for referencing the 2-dimensional image of an object projected on a video screen . acquiring such a capacity permits the surgeon to adapt to the loss of depth perception and lack of appreciation of the temporal proximity of structures displayed on a flat plane , and to command the orientation of instruments by motions projected in the operative field . more advanced skills call for precise control of instruments that pivot about access points remote from the surgical field ; the ability to adapt to limitation of access , range of operative motions , and tissue retraction ; and the capacity to execute complex manipulations requiring refined hand - eye coordination and manual dexterity . this report describes an improved training simulator designed to enhance laparoscopic proficiency and allow objective skill assessment and presents the results when this system was used to train 11 physicians who had various levels of prior experience . the laparoscopy training simulator 2000 ( lts 2000 ) was designed by the senior author . it is essentially a video training device that allows for simulated laparoscopic manipulations under realistic conditions . it is easily transportable and replicates the working dimensions of a dis - tended abdomen ; it is covered by a removable frame , or cassette , containing a resilient , multilayered membranous structure simulating the anterior abdominal wall . a flexible floor mat can be selectively adjusted to various heights at 3 axial positions forming various floor configurations . the laparoscopic skill exercise models are either integral with or can be replaced and attached to the platforms . a timer / stopwatch for measuring exercise time , and posts mounted on the outside of the simulator for knot - tying practice are provided . models that are integrated with dedicated platforms include : the peg board - and - posts coordination model that consists of a peg board and 2 thin metallic posts , 1 with a single curve , and the other with 2 curves , permanently attached to a platform.the 3-d ( dimensional)-to-2-d translation model is a platform constructed with 9 spring posts each with 3 rings , to create a virtual cube . the top 9 rings of the cube are red , the middle rings are yellow , and the bottom rings are blue.the oviduct cannulation model is composed of a malleable plastic tube , the distal end of which is shaped like the fimbriated end of an oviduct . the tube is suspended from a platform , which pivots to allow the fimbriated end to rise freely above the floor mat.the tube cannulation model is made up of a hard plastic tube joined along its entire length to a small platform . small beads , plastic rings , and pipe cleaners are also used for the exercises with these models . the peg board - and - posts coordination model that consists of a peg board and 2 thin metallic posts , 1 with a single curve , and the other with 2 curves , permanently attached to a platform . the 3-d ( dimensional)-to-2-d translation model is a platform constructed with 9 spring posts each with 3 rings , to create a virtual cube . the top 9 rings of the cube are red , the middle rings are yellow , and the bottom rings are blue . the oviduct cannulation model is composed of a malleable plastic tube , the distal end of which is shaped like the fimbriated end of an oviduct . the tube is suspended from a platform , which pivots to allow the fimbriated end to rise freely above the floor mat . small beads , plastic rings , and pipe cleaners are also used for the exercises with these models . undedicated platforms are designed to fix interchangeable models to the floor mat : the small , single - clamp platform has a 3-cm central metallic post formed with an alligator - type clamp.the drill platform contains a centrally located 9-cm sharp drill capped with a winged nut.the 4-clamp platform has 4 metallic corner posts , each formed with an alligator - type clamp.the 4-nail platform is constructed with 4 upward pointing nails . the small , single - clamp platform has a 3-cm central metallic post formed with an alligator - type clamp . the 4-clamp platform has 4 metallic corner posts , each formed with an alligator - type clamp . two preassembled models attach interchangeably to the 4-clamp or 4-nail platform : the sponge cloth model consists of a piece of cloth , with a centrally located incisional gap , which is peripherally fixed to a sponge.the blood vessel model is composed of a sponge with attached color - coded tubes bundled in close proximity to simulate artery , vein , and ureter . a thin rubber tube may be cut into short segments ; 1 segment may be fixed to the single clamp platform in a dangling position to constitute the appendix model . another segment may be suspended between 2 single clamp platforms or 2 of the 4 clamps of the 4-clamp platform to serve as the stapling model . a medium - sized balloon filled with water may be attached to the single clamp platform to serve as the ovarian cyst - gallbladder model . an orange may be turned into the drill platform to function as the myomectomy model . a piece of meat may be cooked medium rare and fixed to the drill platform to act as the morcellator model ( figures 1 and 2 ) . the sponge cloth model consists of a piece of cloth , with a centrally located incisional gap , which is peripherally fixed to a sponge . the blood vessel model is composed of a sponge with attached color - coded tubes bundled in close proximity to simulate artery , vein , and ureter . a thin rubber tube may be cut into short segments ; 1 segment may be fixed to the single clamp platform in a dangling position to constitute the appendix model . another segment may be suspended between 2 single clamp platforms or 2 of the 4 clamps of the 4-clamp platform to serve as the stapling model . a medium - sized balloon filled with water may be attached to the single clamp platform to serve as the ovarian cyst - gallbladder model . an orange may be turned into the drill platform to function as the myomectomy model . a piece of meat may be cooked medium rare and fixed to the drill platform to act as the morcellator model ( figures 1 and 2 ) . internal view of the laparoscopy training simulator 2000 ( lts 2000 ) showing the floor mat with velcro strips and attached exercise models . shown counter clockwise are the pegboard - and - post , straight tube , oviduct cannulation , blood vessel and virtual cube models . a small box with beads and rings is seen next to the pegboard - and - post model . internal view of the lts 2000 showing other exercises : balloon cyst inside a zip lock bag , sponge - cloth suturing model , meat morcellation and balloon cyst models . the simulator is used with a standard laparoscope attached to a video camera , light source , and video monitor placed at a comfortable height for viewing . alternatively , the camera lens assembly may be furnished by a camcorder mounted on a standard tripod to supply a stable and magnified view of any portion of the box 's interior when connected to a video monitor . the laparoscopic video camera or camcorder may be connected to a standard video recorder to permit subsequent review and analysis of the exercise manipulations ( figure 3 ) . the simulator box is placed on a table with the top of the box at the level of the operator 's wrists . the 2 metal posts are inserted into corresponding openings on the exterior wall of the simulator . exercise models are attached to the floor mat , and the abdominal wall frame is returned to its original locked position . the first trocar is inserted through the rubbery abdominal wall in the midline , approximately 3 cm from 1 edge of the frame . once inserted , the cannulas remain stable on the abdominal wall and do not need to be changed or replaced . the laparoscope , with attached camera and light source , is introduced into the simulator through the primary port and exercise manipulations are initiated . practice with the laparoscope requires 2 individuals ; an assistant manipulates the scope , and the operator uses 2 hands to perform the exercise . all pre- and posttests were conducted with the laparoscope ; the camcorder was used for practice only . following conclusion of the pretest , the training exercises used for testing with the lts 2000 are summarized in table 1 . exercises not included in the testing program but used for practice purposes are summarized in table 2 . , the instructor ( nak ) recorded the number of successful exercise tasks completed in 1 minute . the instructor recorded the time taken to accomplish 1 successful task and then reset the timer for the following exercise . the name of the trainee , status ( resident or attending physician ) , and date of the test were entered on the pre- and posttest forms . the mathematical basis of the scoring system was developed by one of the authors ( je ) . exercises 1 through 6 were scored on the basis of the number of tasks completed in 1 minute , for instance , the number of beads placed on the peg board . a standard number of tasks were set prior to the training , based on the performance of two of the authors ( hmh , nak ) . a score of 100 was given to those trainees who equaled or exceeded the preestablished number of tasks . some of the trainees were able to complete a higher number of tasks after being trained . for example , making 3 roeder loop ties on the external post in 1 minute was preestablished by the authors as the desirable end point of the first exercise . subsequently , some of the trainees were able to set down 4 ties in 1 minute . therefore , instead of evaluating the other scores on the basis of the initial target of 3 , a new target value of 3.5 was set . the best score ( 100 ) was given to trainees who completed 3 or 4 tasks in 1 minute in exercise 1 and the worst score ( 0 ) was given to those who could not complete a single task . scores between the maximum and the minimum were set linearly as a function of those values . the equation for such a line was calculated on the basis of the upper limit ( 100 , target value ) and the bottom limit ( 0 , no tasks completed ) . for exercise 1 , for example , the upper limit in the calculation of the slope ( y2-y1)/(x2-x1 ) was 3.5 instead of 3 or 4 , ie , ( 100 - 0)/(3.5 - 0 ) . for exercises 7 to 11 , the best score ( 100 ) was established on the basis of the best performance ( shortest time to accomplish task ) . the worst score ( 0 ) was allocated to trainees who did not complete the task within a period of time 3 times greater than the best time . for example , in exercise 7 , trainees who cannulated the simulated oviduct using a pipe cleaner in the best time of 0.8 minutes were given a score of 100 . those who failed to cannulate the oviduct in 2.4 minutes were given a score of 0 . trainees who completed the exercise task in an intermediate period of time , between the best and the maximum allowed , received scores that were calculated linearly as a function of these 2 values ( table 3 ) . for methodological reasons , the authors modeled 2 other types of scoring systems , one based on a pure linear relationship between the best ( 100 ) and the worst score ( 0 ) , and the other based on a random distribution ( bell curve ) , where each trainee was graded as compared with his or her colleagues ' performance on each exercise . between january 1999 and march 2000 , 11 physicians utilized the lts 2000 simulator , completed a pretest , received instructions to enhance their laparoscopic skills , and then completed a posttest after 2 to 23 hours of practice . all 11 physicians experienced significant improvement in the test scores in coordination , manipulation , suturing , and knot tying , as shown in tables 4 and 5 . fifteen other physicians were trained on the simulator but did not complete the posttest and were excluded from the study . e = exercise ; pre = pretest number of tasks completed in one minute for exercises 1 - 6 and number of minutes to complete one task for exercises 7 - 11 ; post = posttest values . the average pretest score was 304.9 points ( sd 190.8 ) , with a range of 43.2 to 705.7 . the average posttest score was 834.2 points ( sd 141.2 ) , with a range of 547.3 to 1021.7 . when the pre- and posttraining scores were compared , the average increase in the posttest score was 529.3 points ( sd 200.2 ) , with a range of 280.3 to 934.7 points . the average number of hours spent in training was 5.9 with a range of 2 to 23 . this is not surprising because participating residents came from 3 programs in the city with different emphases on laparoscopic training , and one of the physicians had no previous experience with operative laparoscopy , but the other was an experienced laparoscopist . furthermore , individual variations in inherent abilities of the trainees played a role in explaining their performance on the pretest . however , after training with the lts 2000 , the variations on the posttest scores were not influenced much by the level of the pretest score . this finding indicates that the number of hours invested in the training was the primary reason for the noted improvement in the posttest score , as the inherent abilities of each trainee remains constant . improvement in the posttest scores correlated with the number of hours invested in training ( p = 0.055 ) . when the benchmark for skill testing was set at the 70th percentile of the maximum achievable score , none of the physicians passed the test prior to training . eight of the 11 physicians scored higher than 73% after training and practice ( table 5 ) . the mean pretest scores for all exercises was 27.7 out of 100 and for posttest scores was 75.8 out of 100 . the need for frequent preparation and training is not unique to laparoscopic surgeons ; it is shared by all performers with highly developed skills including professional musicians and athletes , as previously noted by resnick . the technical expertise of a laparoscopic surgeon will ultimately depend on inherent genetic abilities , modulated by acquired knowledge and experience , and improved by practice . rosser et al pioneered the use of objective parameters to evaluate laparoscopic skills in 2 studies of a first - generation laparoscopy trainer . the dexterity exercises consisted of ( 1 ) the rope pass drill , grasping a rope with color coded bands and successively passing it from 1 hand to the other ; ( 2 ) the cup drop drill , dropping a pea into an aperture of an inverted cup using the nondominant hand ; ( 3 ) the triangle transfer drill , transferring a triangular wooden block from 1 area to another by engaging and disengaging a curved needle into and from a plastic loop at the apex of the triangle . the authors found that the time needed to perform the dexterity drills improved among participants with successive drills resulting in a significant difference ( p < .001 ) between the time required for the first and tenth drill . similarly , significant improvement ( p < .001 ) was noted in performing intracorporeal suturing with successive drills , after the participants received instruction . a significant correlation ( p < .001 ) was also observed between the performance efficiency of the dexterity and suturing drills , suggesting that dexterity drills help improve suturing . to evaluate the results , the authors correlated the mean time required to perform a drill with that of participants who completed all 10 drills and 10 suturing exercises . in the second study , these authors compared the performance of senior surgeons with that of residents after both groups had completed the training program the authors previously designed . the collective performance of the residents was similar to that of the trained surgeons in the rope pass drill and suturing exercises . however , the residents performed the triangle transfer drill faster and the cup drill more slowly than the trained surgeons . the simulated laparoscopic tasks consisted of ( 1 ) peg transfers , lifting a peg from 1 peg hole with 1 hand , transferring it to the other hand and placing it on another peg board ; ( 2 ) pattern cutting , cutting a circular pattern out of a larger piece of gauze ; ( 3 ) clip and divide , placing 2 hemostatic clips on a tubular foam structure and cutting in between ; ( 4 ) endo - looping , placing a pretied slip knot on a foam tubular appendage ; ( 5 ) mesh application , placing a mesh over a defect in a foam model and securing it to the foam with staples ; ( 6 ) intracorporeal knot , placing a suture into a penrose drain and tying it with an intracorporeal knot ; ( 7 ) extracorporeal knot , placing a suture into a penrose drain and tying it with an extracorporeal knot . all 7 tasks showed a significant improvement in performance with repetition ( p < 0.0001 ) . some of the tasks ( 1 , 2 , 6 ) showed a significant correlation between the degree of physician experience and score , but others did not . when the canadian group further studied the effect of practice on performance , they found that residents who had a pretest followed by 5 weekly practice sessions showed significant improvement in the posttest score for each task and for total score . residents who limited their practice to the pretest showed improvement in tasks 1 , 4 , 5 , and 7 , but not in 2 , 3 , and 6 in the posttest . the total score of the posttest was not statistically different from that of the pretest in this group . we used a new second - generation laparoscopy trainer , a complementary set of dexterity and suturing exercises , and a scoring system that is different from that described by the previous authors . a maximum score of 100 is given for achieving a target value or time ; a minimum score of 0 is given for not completing a single task in the unit of time or the assigned task in 3 times the target time . to calculate the skill scores of trainees for any given exercise with a known target value or time , the investigator simply needs to plug in the number of tasks the trainee completed per unit of time , or the number of minutes needed to complete a task . physicians who are adept at performing conventional surgery possess the knowledge of surgical principles and the skills of operative manipulation . to become proficient in laparoscopic surgery , they need to master only the necessary adaptations in visual - motor processing through training and practice . the present study suggests that most physicians acquire competence in skill sets associated with advanced operative laparoscopy if they receive appropriate instructions and invest a reasonable amount of time practicing on a laparoscopy training simulator like the lts 2000 . our group has been able to resolve many of the problematic subtleties associated with novel applications by testing them in the simulator before performing live surgery . the ultimate goal of any laparoscopy training system is to increase the comfort level and efficiency of laparoscopic surgeons in the operating room . the 11 physicians who participated in this training system experienced significant improvement in their laparoscopic skills regardless of prior level of expertise : all 11 scored below the 65th percentile of the maximum achievable score prior to training and 8 scored above the 73rd percentile after training and practice . the improvement in the posttest scores correlated with the number of hours invested in training ( p = 0.055 ) .

one aim of nursing education is to motivate nurses to acquire skills for offering appropriate quality health care services to patients with multiple complex health problems . studies have shown that , in the clinical setting , the most important barrier to clinical education is students ' lack of interest and motivation . nursing students need long - term motivation to help others in the future . so paying due attention to the concept of motivation is of great importance in clinical education . clinical placements form a major part of nursing education and have an important role in students ' perceptions of nursing . nurses represent the largest category of health workers and provide 80% of direct patient care . they represent the professional image and the prospect of nursing career and are the main role model for nursing students . having a clear image of the career helps the students with choosing nursing as a career or retaining it [ 3 , 7 , 8 ] . conversely , a poor public image of nursing contributes to inadequate numbers of students entering the nursing education programmes and , for those that do enter nursing , it can influence the attrition rate . in contrast , miers et al . found that , although a service orientation remains a key factor in choosing nursing , students also look for a career that matches their interests and attributes , and offers professional values and rewards . dante et al . explored italian nursing students ' and leavers ' perspectives about leaving nursing education . personal reasons were the most common followed by difficult relationships with teaching staff , learning difficulties , and wrong career choice , as well as difficulties with practical training . nilsson and stomberg found that nursing students mainly grade their motivation positively distributed , and different throughout their entire education . nurses can be regarded as the source of learning at the clinic through social learning pattern . having substantial teaching background in clinical education , we found a theoretical sensitivity in this area . hence , considering the gap in the research literature , especially lack of enough studies in this field in iran , this study was conducted to achieve a deep and comprehensive view and to explore how the nursing students are motivated through clinical education , with an emphasize on the role of nurses in this regard . in iran , the nursing program offers a four - year baccalaureate in nursing accredited by the high council of medical education of the ministry of health and medical education . all schools are obliged to follow a basic curriculum established by the ministry , though some flexibility is allowed within the predetermined curriculum . the program consists of general education courses ( 20 credits ) , professional foundation courses ( 28 credits with a biological content ) ; and nursing courses ( 53 credits on theory with a biomedical nursing content and 33 credit on clinical preparation ) . a practicum is an integral part of professional nursing studies and includes a 40-week clinical placement , in which nursing students provide supervised care 7 hours/1day and 6days / week under the supervision of an academic nursing instructor . dissertation , titled the process of motivation formation in nursing students during clinical education , which was conducted using grounded theory method . grounded theory focuses on identification , description and explanation of interactional processes among individuals or groups within a given social context . the grounded theory method was selected in this work to explore the participants ' opinions on the conditions effective for clinical education . in the present research , 16 undergraduate nursing students at the second or higher semesters of study in the department of nursing and midwifery at the universities of tehran and zanjan were selected through purposive sampling . according to their viewpoints and suggestions , it was assumed that nursing instructors serve as suitable key informants regarding clinical education motivation . considering the wide range of experiences and perceptions among this group of participants , interviews were conducted theoretically according to the codes and categories as they emerged until data saturation . sixteen students and four instructors were invited to participate , and all of them attended an orientation to the study . after approval of the study by the ethical committee of the school of nursing ( tehran university of medical sciences ) and obtaining permission to proceed from the officials of the nursing schools of both tehran and zanjan universities of medical sciences , the first author began to collect the data . all participants provided informed consent and all interviews were conducted with prior appointment with the participants . in order to respect the participants ' privacy and confidentiality , the interviews were conducted only in the presence of the interviewer and the interviewee . also , for confidentiality of the information and keeping anonymity , all manuscripts and audio files were coded . all participants were informed that they could withdraw from the study at any time they wished and take their audio files and transcripts ; however , this did not happen during the study . the interviews took place at the first author 's room at the school of nursing & midwifery ( tehran or zanjan ) in a classroom , or in an instructor 's room . the interviews started with broad questions in order to encourage the participants to speak freely and express their personal experiences regarding motivation in clinical education . for example , can you talk about a typical day at your internship ? , what conditions influence students ' motivation during clinical education ? and how are nursing students motivated in clinical education ? can you talk about a typical day at your internship ? , what conditions influence students ' motivation during clinical education ? and how are nursing students motivated in clinical education ? as the interview progressed , the questions became more specific , allowing deeper investigation of the issues raised by the participants in the earlier interviews . the data required were collected and analyzed during a 18-month period from september 2010 until may 2011 . then they were managed and sort - coded using maxqda 2007 software ( verbi gmbh , berlin , germany ) . the process of conducting interviews , transcribing the recordings , and analyzing the data occurred simultaneously . the data collection and analysis continued until data saturation , that is , no additional data were found for development of the category properties . according to the constant comparative analysis , similar data were grouped and conceptually labeled during the so - called then the concepts were classified and the categories were linked and organized by relationships in a process , called axial coding . theory integration and refining were conducted by selective coding . in this step , the core variable monitoring and modeling through clinical education became a theoretical guide to the further collection and analysis of the data . therefore , the emerging categories and the core variable led the researcher toward interviewing several key informants who provided rich data about the nurses as role models . this is a repetitious concept and a referral category that can also be generalized to other categories . it further can create linkage among the categories . to maintain trustworthiness , several ways were used . maximum variant sampling and achievement of saturation were among other items that improved the credibility and the confidentiality of the data , in turn . the process of sampling and analysis of data took 18 months . this prolonged engagement with the participants and their interest helped the investigator to gain the participants ' trust and obtain more in - depth data . for member check , the codes extracted for accuracy of the meanings ' interpretation were returned back to all the participants and were approved by them . credibility and confirmability were established through prolonged engagement , allocation of adequate time , good communication , multiple methods ( such as data source triangulation ) , and maximum variant sampling , which provided plenty of opinions and points of view for the research . a second review of the transcripts , codes , and grouped codes and concepts by a number of colleagues ( as a peer check ) and participants ( as a member check ) was conducted . the majority of colleagues and participants agreed with the initial codes ; however , we modified a few codes . in addition , several nursing students who had not participated in the interviews reviewed the results and confirmed their suitability . ten out of the total of 16 student participants were females and 6 were males , with an average age of 2125 years . course in nursing . of the 4 participant instructors , 3 were females and one was male . in this work , concerns of becoming a nurse was emerged as one of the important patterns in the process of motivation formation in the nursing students during clinical education . this pattern consisted of three categories as follows : nurses clinical competency , nurses as full - scale mirror of the future , and monitoring and modeling through clinical education ( as the core variable ) , which will be described in detail in the coming paragraphs . in the view of all participants , nurses ' clinical competency has been effective on motivation of students in clinical education . this category comprises three subcategories : ( a ) caring about patient 's rights , ( b ) nurses clinical knowledge , and ( c ) submission to routines . ( a ) caring about patient 's rightsin the view of the participants , due attention of nurses to the spiritual and ideological beliefs of the patient and those accompanying him / her was among the items of caring about the patient 's rights by the nurse . this caring behavior would make the nursing students motivated through their clinical education . in the view of the participants , due attention of nurses to the spiritual and ideological beliefs of the patient and those accompanying him / her was among the items of caring about the patient 's rights by the nurse . yet , the participants frequently witnessed cases of not observing the patient 's rights by the nurses . the participants listed the aspects of disregard to the patient 's rights by the nurses as misbehavior with the patient , not paying attention to physical and psychological needs of the patient , not paying attention to the patient 's beliefs and/or his / her privacy , not preparing the patient , and not responding to the patient 's questions before conducting any procedure . one of the participants spoke about the disregard and inhumane behavior of the nurses toward the patients as follows:for example , even when the patient is screaming of the pain , it happens that the nurse says shut up to the patient . to give a analgesics for the patient is not that difficult but they refrain even from that they do n't consider the patient a human ( participant 7 , student ) . for example , even when the patient is screaming of the pain , it happens that the nurse says shut up to the patient . to give a analgesics for the patient is not that difficult but they refrain even from that they do n't consider the patient a human ( participant 7 , student ) . as a result , they became pessimistic about nursing , and this in turn decreases their motivation for clinical education . ( b ) nurses clinical knowledgemany participants mentioned that nurses ' high clinical knowledge leads to their high value , esteem toward them , and acceptance of their suggestions by the physician ; all of these improve the students ' clinical motivation . many participants mentioned that nurses ' high clinical knowledge leads to their high value , esteem toward them , and acceptance of their suggestions by the physician ; all of these improve the students ' clinical motivation . about the influence of the nurses ' high levels of clinical knowledge on the motivation of students for clinical education , a participant said:we observe some nurses who are really informed . for example , i know a nurse who is so educated that can easily comment on the patients ' treatment process for example , i know a nurse who is so educated that can easily comment on the patients ' treatment process another participant added:a nurse can increase his / her knowledge so that it is possible for him / her to speak with the doctors . it is his / her low level of knowledge that makes him / her stranger to the doctor ( participant 4 , student ) . a nurse can increase his / her knowledge so that it is possible for him / her to speak with the doctors . it is his / her low level of knowledge that makes him / her stranger to the doctor in contrast to the positive influence of high levels of clinical knowledge , nurses ' low clinical knowledge level is among the reasons for physicians , nursing students and patients considering nurses as noneducated . nurses ' low clinical knowledge leads to their inability to respond to the patient 's questions . as a result , the patient loses confidence on the nurse . another participant stated about the effect of nurses ' clinical knowledge on the motivation of student for clinical education : when my student observes an experienced , informed and skilled nurse , he / she would love to the same to receive the same respect . it is because they 're not educated when my student observes an experienced , informed and skilled nurse , he / she would love to the same to receive the same respect . a gap between nurses and other professional groups , especially the physicians , would make the participants unmotivated toward the clinical education . ( c ) submission to routinessubmission to routines made the students disinterested in the work in the clinic . submission to routines was stated as mechanical care , merely giving the drugs prescribed to the patient , taking vital signs , recording and reporting , lack of appropriate communication with the patient , and lack of teaching the patient . one of the participants spoke about submission to routines in nurses : you may see in the ward a nurse who has several years of experience but he / she knows nothing and acts just as a robot in giving the drugs , writing the report and handling it to the next shift ( participant 11 , student ) . submission to routines was stated as mechanical care , merely giving the drugs prescribed to the patient , taking vital signs , recording and reporting , lack of appropriate communication with the patient , and lack of teaching the patient . one of the participants spoke about submission to routines in nurses : you may see in the ward a nurse who has several years of experience but he / she knows nothing and acts just as a robot in giving the drugs , writing the report and handling it to the next shift ( participant 11 , student ) . you may see in the ward a nurse who has several years of experience but he / she knows nothing and acts just as a robot in giving the drugs , writing the report and handling it to the next shift ( participant 11 , student ) . one of the consequences of submission to routines in nurses is lack of appropriate care for the patients . a participant spoke about this fact:without seeing to the patient , the nurse sits in the station and writes the nursing report . student observing such kind of work would definitely wonder why then i should study that much when in the end i would be one like this . without seeing to the patient , the nurse sits in the station and writes the nursing report . student observing such kind of work would definitely wonder why then i should study that much when in the end i would be one like this . the participants believed that students would imagine their future career when observing the work conditions and the nurses ' clinical abilities . this category includes ( a ) extreme work conditions , and ( b ) nurses powerlessness . ( a ) extreme work conditions in the view of participants , qualified nurses are like a full - scale mirror of the future . therefore , the work conditions of nurses have a great effect on the students ' attitude and motivation . extreme work conditions is an item frequently referred to by the participants . by extreme work conditions , they refer to disproportionate ratio of nurses to patients , job stress and extreme work conditions versus low salary and benefits , lack of psychological security , carrying out the tasks of other professional groups by nurses , having night shifts , the hospital 's boring environment , permanent exposure of the nurses to the disease and death , and lack of an appropriate place for the nurses in the clinic and the community . a participant spoke about the extreme work conditions:when we go for internship , we ask the nurses about their work conditions when we see that they have to work so much for such a low salary . then we think why we should stand up that much and undergo such a hard work ( participant 9 , student ) . in the view of participants , qualified nurses are like a full - scale mirror of the future . therefore , the work conditions of nurses have a great effect on the students ' attitude and motivation . extreme work conditions is an item frequently referred to by the participants . by extreme work conditions , they refer to disproportionate ratio of nurses to patients , job stress and extreme work conditions versus low salary and benefits , lack of psychological security , carrying out the tasks of other professional groups by nurses , having night shifts , the hospital 's boring environment , permanent exposure of the nurses to the disease and death , and lack of an appropriate place for the nurses in the clinic and the community . a participant spoke about the extreme work conditions:when we go for internship , we ask the nurses about their work conditions when we see that they have to work so much for such a low salary . then we think why we should stand up that much and undergo such a hard work ( participant 9 , student ) . when we go for internship , we ask the nurses about their work conditions when we see that they have to work so much for such a low salary . then we think why we should stand up that much and undergo such a hard work ( participant 9 , student ) . in the view of participants , extreme work conditions made qualified nurses dissatisfied and unmotivated . consequently , they transferred this dissatisfaction and lack of motivation through their words to the students . course , or pursuing their study in nursing in order to work in educational environments and thus escaping from the clinical extreme work conditions could be the consequences of the nurses extreme work conditions . another student stated thatwhy do students think of perusing their studies ? they are trying to run away from clinical work . the participants stated that , in the clinical settings , they found no difference between the type of work done by the nurses holding b.s . or m.sc these conditions ultimately result in a decrease of the students ' motivation in making efforts to learn and to pursue their studies . in spite of extreme work conditions in nursing , the participants believed that existing appropriate job market as compared to other majors in iran will make students more optimistic to the job 's future , and to some extent , this is motivating for the students in the clinical education . a participant stated that:the fact that the graduates in other majors are jobless and that it is possible for the nursing students to get in the job market immediately , will be a motivating factor for them ( participant15 , instructor ) . the fact that the graduates in other majors are jobless and that it is possible for the nursing students to get in the job market immediately , will be a motivating factor for them ( participant15 , instructor ) . ( b ) nurses powerlessnessin the process of physician - nurse communication , the participants observed a boss - subordinate relation between them as well as the nurses ' submission to the physicians . she / he does nothing but to enter into the cardex the physician 's orders immediately . we as students will simply become ones like these after several years we see the future ! so no motivation will be left for the student ( participant 11 , student ) . in the process of physician - nurse communication , the participants observed a boss - subordinate relation between them as well as she / he does nothing but to enter into the cardex the physician 's orders immediately . we as students will simply become ones like these after several years we see the future ! so no motivation will be left for the student ( participant 11 , student ) . she / he does nothing but to enter into the cardex the physician 's orders immediately . we as students will simply become ones like these after several years we see the future ! so no motivation will be left for the student ( participant 11 , student ) . another student spoke about the fear of nurse in communicating with the physician:the relationship should be so appropriate that the nurse will not be afraid of the physician and ask about every problem he / she encounters . but now it 's turned to be so inappropriate that the head nurse even does not dare to ask the physician a single question she / he does not dare to say that a patient 's symptoms have changed the relationship should be so appropriate that the nurse will not be afraid of the physician and ask about every problem he / she encounters . but now it 's turned to be so inappropriate that the head nurse even does not dare to ask the physician a single question she / he does not dare to say that a patient 's symptoms have changed in contrast , a good cooperation atmosphere and nurses ' self - esteem were encouraging for the students to work in such an environment in the future . in the present study , the students monitored and judged on the performance of the nurses and moved on a spectrum of having or not having motivation accordingly . students frequently substituted nursing roles and imagined themselves in their future jobs . in this respect , a participant stated that:believe that from now on , we have the concerns of a nurse , and it is as if we are not students at all ( participant 5 , student ) . believe that from now on , we have the concerns of a nurse , and it is as if we are not students at all ( participant 5 , student ) . through this role replacement , students reach a stage of occupational understanding and cognition , by observing and role replacement of the nurses who have enough clinical knowledge , are not submitted to routines , observe patients ' rights , and are innovative in the decrease of clinical nursing problems , the students are motivated for clinical education and use different elements of clinical environment ( good communication environment and exploiting learning opportunities ) to learn more and better . in contrast , extreme work conditions and nurses ' clinical powerlessness can result in decreased motivation of the students in clinical education . the findings of this study may create a new insight into the relationship of nursing students ' challenges in facing the realities of the profession with clinical education motivation . concerns of becoming a nurse was recognized as one of the important patterns of this study . this pattern includes three categories of nurses clinical competency , nurses as full - scale mirror of the future , and monitoring and modeling through clinical education ( as the core variable ) . our findings showed that observing moral virtues in nurses as role models motivated the students . conversely , nonobservation of the patient 's rights had a negative effect on the students ' motivation . witnessing such behaviors , as nursing role models , plus receiving negative feedbacks from the patients about the nurses ' behaviors result in a decrease of clinical learning motivation in the students . recent reports have highlighted negative patient experiences , which reflect a clear lack of compassionate nursing care . in a study conducted by kyrkjeb and hage , the participating students experienced the fact that the patients were taken seriously by the staff . in this study , in the participants ' view , one of the criteria for clinical competency of nurses was their knowledge . the students , by witnessing the nurses ' high clinical knowledge that was materialized in their action , were motivated for continuing their study and clinical work and aspired to act as a knowledgeable nurse in the future . found that from the participants ' perspective , competence seemed to be related to instrumental - technical , intellectual - cognitive , organizational , social and communication skills , and attitudes . such a nurse can be an appropriate model for nursing students and motivate them for clinical learning . in contrast , the nurses with low clinical knowledge made an undesirable impression on the students and , in the opinion of the participants , it had a negative effect on the views of other professional groups in the clinic and , consequently , in the community . in the same context , the study made by mohammadi et al . showed that more than a half of the nurses in the ccu and icu units had a weak clinical knowledge . the students , when seeing the nurses ' submission to routines , refrained from any effort and lost their motivation in learning at the clinic . in submission to routines , a nurse performs his / her predefined duties without any thinking or questioning . in this type of performance , nurses do their duties without paying much attention to their job and , as a results , they have no decision - making power in different situations . hence , submission to routines takes their authority and power of making decision and adds to the nurses ' powerlessness and thus the students ' discouragement . it is widely accepted that nursing as a career is viewed favorably by the society in that it offers job security , mobility , and career variety . conversely , in this study , the majority of the students , following the observation of extreme nursing work conditions , sought a way to leave education . such a student has no motivation for clinical education . in agreement with the findings of this study , in the study of leong , concerns were raised about both salary and employee benefits , and one can conclude that policies in these areas are met with some dissatisfaction . therefore , imagination of imminent future demotivates nursing students . also , in jordan , the underpayment of nurses is one of the major reasons for nurses ' dissatisfaction and intention to leave hospitals . the lower social status of the nursing profession in terms of low salary drives students away from nursing . elsewhere , a study revealed that wage increases alone did not do much to attract or retain nursing staff : improvement in employment - based benefits was necessary . it was also revealed that the working environment , nurse - patient ratio , and health - care insurance were elements to be considered in the payment and welfare package . powerlessness of nurses in clinic forms a negative view in the mind of students towards the nursing career . also the study of brodie et al . showed that high clinical experience of the participant students in this study reinforced both their own and the society 's image of an underpaid and overworked profession that lacks respect and has low morale . low dignity of the nurses in the clinic decreases clinical learning motivation in the students and represents an unpleasant image from the nursing career to them , because students seek for their future dignity and respect by looking into the present nurses . health systems should take the promotion of the nurses ' dignity into account through providing a dignified work environment . according to the findings of the present research , students who model and substitute nursing roles get insight into their future profession . the findings also showed that nurses as role models had the most influence on the motivation or no motivation of students so that the performance , work conditions and power of nurses each somehow influenced the motivation of students during the clinical education . many of the strategies used by the students in escaping from clinical environment and future nursing reflect their undesirable evaluation of the nursing profession , which ends with lack of motivation toward clinical education . in return , the participants believed that in iran proper job market in nursing ( compared to the graduated students of other majors who do not find a job easily ) can absorb into the health system even those students who were unmotivated during their clinical education . this is in contrast to the research findings in other countries where unmotivated students refrain from entering the job market [ 25 , 27 , 29 ] . the entering of unmotivated students into nursing profession is reflected in the health system of iran society in facing unmotivated nurses , and each unmotivated student turns into an improper representative of nursing profession . therefore , it is of great importance to pay due attention to strategies for increasing motivation in nursing students . based on the findings of this study , educational and health care authorities can promote the quality of nurses ' work in offering better services to the patients through improving their work conditions . in this way , more appropriate nursing role models would be available for students , and thus their interest in the nursing career would increase . we recommend the conduct of action researches for improvement of the clinical setting in nursing education . although theoretical sampling was used in this work to collect the required data that encompassed multiple aspects of the experience in question , the small sample size were research limitations .

nurses are the first role models for students in clinical settings . they can have a significant role on students ' motivation . the purpose of this study was to explore the understanding of nursing students and instructors concerning the role of nurses in motivating nursing students through clinical education . the sampling was first started purposefully and continued with theoretical sampling . the study collected qualitative data through semistructured and interactive interviews with 16 nursing students and 4 nursing instructors . all interviews were recorded , transcribed , and analyzed using grounded theory approach . one important pattern emerged in this study was the concerns of becoming a nurse , which itself consisted of three categories : nurses clinical competency , nurses as full - scale mirror of the future , and monitoring and modeling through clinical education ( as the core variable ) . the findings showed that the nurses ' manners of performance as well as the profession 's prospect have a fundamental role in the process of formation of motivation through clinical education . students find an insight into the nursing profession by substituting themselves in the place of a nurse , and as result , are or are not motivated towards the clinical education .

1. Introduction 2. Materials and Methods 3. Discussion 4. Limits of the Present Research

studies have shown that , in the clinical setting , the most important barrier to clinical education is students ' lack of interest and motivation . nursing students need long - term motivation to help others in the future . so paying due attention to the concept of motivation is of great importance in clinical education . clinical placements form a major part of nursing education and have an important role in students ' perceptions of nursing . they represent the professional image and the prospect of nursing career and are the main role model for nursing students . conversely , a poor public image of nursing contributes to inadequate numbers of students entering the nursing education programmes and , for those that do enter nursing , it can influence the attrition rate . explored italian nursing students ' and leavers ' perspectives about leaving nursing education . personal reasons were the most common followed by difficult relationships with teaching staff , learning difficulties , and wrong career choice , as well as difficulties with practical training . nilsson and stomberg found that nursing students mainly grade their motivation positively distributed , and different throughout their entire education . having substantial teaching background in clinical education , we found a theoretical sensitivity in this area . hence , considering the gap in the research literature , especially lack of enough studies in this field in iran , this study was conducted to achieve a deep and comprehensive view and to explore how the nursing students are motivated through clinical education , with an emphasize on the role of nurses in this regard . in iran , the nursing program offers a four - year baccalaureate in nursing accredited by the high council of medical education of the ministry of health and medical education . dissertation , titled the process of motivation formation in nursing students during clinical education , which was conducted using grounded theory method . the grounded theory method was selected in this work to explore the participants ' opinions on the conditions effective for clinical education . in the present research , 16 undergraduate nursing students at the second or higher semesters of study in the department of nursing and midwifery at the universities of tehran and zanjan were selected through purposive sampling . according to their viewpoints and suggestions , it was assumed that nursing instructors serve as suitable key informants regarding clinical education motivation . sixteen students and four instructors were invited to participate , and all of them attended an orientation to the study . after approval of the study by the ethical committee of the school of nursing ( tehran university of medical sciences ) and obtaining permission to proceed from the officials of the nursing schools of both tehran and zanjan universities of medical sciences , the first author began to collect the data . all participants provided informed consent and all interviews were conducted with prior appointment with the participants . in order to respect the participants ' privacy and confidentiality , the interviews were conducted only in the presence of the interviewer and the interviewee . the interviews took place at the first author 's room at the school of nursing & midwifery ( tehran or zanjan ) in a classroom , or in an instructor 's room . the interviews started with broad questions in order to encourage the participants to speak freely and express their personal experiences regarding motivation in clinical education . , what conditions influence students ' motivation during clinical education ? and how are nursing students motivated in clinical education ? , what conditions influence students ' motivation during clinical education ? and how are nursing students motivated in clinical education ? as the interview progressed , the questions became more specific , allowing deeper investigation of the issues raised by the participants in the earlier interviews . the process of conducting interviews , transcribing the recordings , and analyzing the data occurred simultaneously . in this step , the core variable monitoring and modeling through clinical education became a theoretical guide to the further collection and analysis of the data . therefore , the emerging categories and the core variable led the researcher toward interviewing several key informants who provided rich data about the nurses as role models . maximum variant sampling and achievement of saturation were among other items that improved the credibility and the confidentiality of the data , in turn . the process of sampling and analysis of data took 18 months . credibility and confirmability were established through prolonged engagement , allocation of adequate time , good communication , multiple methods ( such as data source triangulation ) , and maximum variant sampling , which provided plenty of opinions and points of view for the research . a second review of the transcripts , codes , and grouped codes and concepts by a number of colleagues ( as a peer check ) and participants ( as a member check ) was conducted . in addition , several nursing students who had not participated in the interviews reviewed the results and confirmed their suitability . ten out of the total of 16 student participants were females and 6 were males , with an average age of 2125 years . in this work , concerns of becoming a nurse was emerged as one of the important patterns in the process of motivation formation in the nursing students during clinical education . this pattern consisted of three categories as follows : nurses clinical competency , nurses as full - scale mirror of the future , and monitoring and modeling through clinical education ( as the core variable ) , which will be described in detail in the coming paragraphs . in the view of all participants , nurses ' clinical competency has been effective on motivation of students in clinical education . this category comprises three subcategories : ( a ) caring about patient 's rights , ( b ) nurses clinical knowledge , and ( c ) submission to routines . ( a ) caring about patient 's rightsin the view of the participants , due attention of nurses to the spiritual and ideological beliefs of the patient and those accompanying him / her was among the items of caring about the patient 's rights by the nurse . this caring behavior would make the nursing students motivated through their clinical education . in the view of the participants , due attention of nurses to the spiritual and ideological beliefs of the patient and those accompanying him / her was among the items of caring about the patient 's rights by the nurse . the participants listed the aspects of disregard to the patient 's rights by the nurses as misbehavior with the patient , not paying attention to physical and psychological needs of the patient , not paying attention to the patient 's beliefs and/or his / her privacy , not preparing the patient , and not responding to the patient 's questions before conducting any procedure . one of the participants spoke about the disregard and inhumane behavior of the nurses toward the patients as follows:for example , even when the patient is screaming of the pain , it happens that the nurse says shut up to the patient . for example , even when the patient is screaming of the pain , it happens that the nurse says shut up to the patient . as a result , they became pessimistic about nursing , and this in turn decreases their motivation for clinical education . ( b ) nurses clinical knowledgemany participants mentioned that nurses ' high clinical knowledge leads to their high value , esteem toward them , and acceptance of their suggestions by the physician ; all of these improve the students ' clinical motivation . many participants mentioned that nurses ' high clinical knowledge leads to their high value , esteem toward them , and acceptance of their suggestions by the physician ; all of these improve the students ' clinical motivation . about the influence of the nurses ' high levels of clinical knowledge on the motivation of students for clinical education , a participant said:we observe some nurses who are really informed . it is his / her low level of knowledge that makes him / her stranger to the doctor in contrast to the positive influence of high levels of clinical knowledge , nurses ' low clinical knowledge level is among the reasons for physicians , nursing students and patients considering nurses as noneducated . another participant stated about the effect of nurses ' clinical knowledge on the motivation of student for clinical education : when my student observes an experienced , informed and skilled nurse , he / she would love to the same to receive the same respect . a gap between nurses and other professional groups , especially the physicians , would make the participants unmotivated toward the clinical education . submission to routines was stated as mechanical care , merely giving the drugs prescribed to the patient , taking vital signs , recording and reporting , lack of appropriate communication with the patient , and lack of teaching the patient . one of the participants spoke about submission to routines in nurses : you may see in the ward a nurse who has several years of experience but he / she knows nothing and acts just as a robot in giving the drugs , writing the report and handling it to the next shift ( participant 11 , student ) . submission to routines was stated as mechanical care , merely giving the drugs prescribed to the patient , taking vital signs , recording and reporting , lack of appropriate communication with the patient , and lack of teaching the patient . one of the participants spoke about submission to routines in nurses : you may see in the ward a nurse who has several years of experience but he / she knows nothing and acts just as a robot in giving the drugs , writing the report and handling it to the next shift ( participant 11 , student ) . you may see in the ward a nurse who has several years of experience but he / she knows nothing and acts just as a robot in giving the drugs , writing the report and handling it to the next shift ( participant 11 , student ) . one of the consequences of submission to routines in nurses is lack of appropriate care for the patients . a participant spoke about this fact:without seeing to the patient , the nurse sits in the station and writes the nursing report . without seeing to the patient , the nurse sits in the station and writes the nursing report . student observing such kind of work would definitely wonder why then i should study that much when in the end i would be one like this . the participants believed that students would imagine their future career when observing the work conditions and the nurses ' clinical abilities . ( a ) extreme work conditions in the view of participants , qualified nurses are like a full - scale mirror of the future . therefore , the work conditions of nurses have a great effect on the students ' attitude and motivation . by extreme work conditions , they refer to disproportionate ratio of nurses to patients , job stress and extreme work conditions versus low salary and benefits , lack of psychological security , carrying out the tasks of other professional groups by nurses , having night shifts , the hospital 's boring environment , permanent exposure of the nurses to the disease and death , and lack of an appropriate place for the nurses in the clinic and the community . in the view of participants , qualified nurses are like a full - scale mirror of the future . therefore , the work conditions of nurses have a great effect on the students ' attitude and motivation . by extreme work conditions , they refer to disproportionate ratio of nurses to patients , job stress and extreme work conditions versus low salary and benefits , lack of psychological security , carrying out the tasks of other professional groups by nurses , having night shifts , the hospital 's boring environment , permanent exposure of the nurses to the disease and death , and lack of an appropriate place for the nurses in the clinic and the community . when we go for internship , we ask the nurses about their work conditions when we see that they have to work so much for such a low salary . in the view of participants , extreme work conditions made qualified nurses dissatisfied and unmotivated . consequently , they transferred this dissatisfaction and lack of motivation through their words to the students . course , or pursuing their study in nursing in order to work in educational environments and thus escaping from the clinical extreme work conditions could be the consequences of the nurses extreme work conditions . the participants stated that , in the clinical settings , they found no difference between the type of work done by the nurses holding b.s . or m.sc these conditions ultimately result in a decrease of the students ' motivation in making efforts to learn and to pursue their studies . in spite of extreme work conditions in nursing , the participants believed that existing appropriate job market as compared to other majors in iran will make students more optimistic to the job 's future , and to some extent , this is motivating for the students in the clinical education . a participant stated that:the fact that the graduates in other majors are jobless and that it is possible for the nursing students to get in the job market immediately , will be a motivating factor for them ( participant15 , instructor ) . the fact that the graduates in other majors are jobless and that it is possible for the nursing students to get in the job market immediately , will be a motivating factor for them ( participant15 , instructor ) . ( b ) nurses powerlessnessin the process of physician - nurse communication , the participants observed a boss - subordinate relation between them as well as the nurses ' submission to the physicians . in the process of physician - nurse communication , the participants observed a boss - subordinate relation between them as well as she / he does nothing but to enter into the cardex the physician 's orders immediately . another student spoke about the fear of nurse in communicating with the physician:the relationship should be so appropriate that the nurse will not be afraid of the physician and ask about every problem he / she encounters . but now it 's turned to be so inappropriate that the head nurse even does not dare to ask the physician a single question she / he does not dare to say that a patient 's symptoms have changed the relationship should be so appropriate that the nurse will not be afraid of the physician and ask about every problem he / she encounters . but now it 's turned to be so inappropriate that the head nurse even does not dare to ask the physician a single question she / he does not dare to say that a patient 's symptoms have changed in contrast , a good cooperation atmosphere and nurses ' self - esteem were encouraging for the students to work in such an environment in the future . in the present study , the students monitored and judged on the performance of the nurses and moved on a spectrum of having or not having motivation accordingly . in this respect , a participant stated that:believe that from now on , we have the concerns of a nurse , and it is as if we are not students at all ( participant 5 , student ) . believe that from now on , we have the concerns of a nurse , and it is as if we are not students at all ( participant 5 , student ) . through this role replacement , students reach a stage of occupational understanding and cognition , by observing and role replacement of the nurses who have enough clinical knowledge , are not submitted to routines , observe patients ' rights , and are innovative in the decrease of clinical nursing problems , the students are motivated for clinical education and use different elements of clinical environment ( good communication environment and exploiting learning opportunities ) to learn more and better . in contrast , extreme work conditions and nurses ' clinical powerlessness can result in decreased motivation of the students in clinical education . the findings of this study may create a new insight into the relationship of nursing students ' challenges in facing the realities of the profession with clinical education motivation . concerns of becoming a nurse was recognized as one of the important patterns of this study . this pattern includes three categories of nurses clinical competency , nurses as full - scale mirror of the future , and monitoring and modeling through clinical education ( as the core variable ) . our findings showed that observing moral virtues in nurses as role models motivated the students . conversely , nonobservation of the patient 's rights had a negative effect on the students ' motivation . witnessing such behaviors , as nursing role models , plus receiving negative feedbacks from the patients about the nurses ' behaviors result in a decrease of clinical learning motivation in the students . in this study , in the participants ' view , one of the criteria for clinical competency of nurses was their knowledge . the students , by witnessing the nurses ' high clinical knowledge that was materialized in their action , were motivated for continuing their study and clinical work and aspired to act as a knowledgeable nurse in the future . found that from the participants ' perspective , competence seemed to be related to instrumental - technical , intellectual - cognitive , organizational , social and communication skills , and attitudes . such a nurse can be an appropriate model for nursing students and motivate them for clinical learning . in contrast , the nurses with low clinical knowledge made an undesirable impression on the students and , in the opinion of the participants , it had a negative effect on the views of other professional groups in the clinic and , consequently , in the community . in the same context , the study made by mohammadi et al . showed that more than a half of the nurses in the ccu and icu units had a weak clinical knowledge . the students , when seeing the nurses ' submission to routines , refrained from any effort and lost their motivation in learning at the clinic . in submission to routines , a nurse performs his / her predefined duties without any thinking or questioning . in this type of performance , nurses do their duties without paying much attention to their job and , as a results , they have no decision - making power in different situations . hence , submission to routines takes their authority and power of making decision and adds to the nurses ' powerlessness and thus the students ' discouragement . conversely , in this study , the majority of the students , following the observation of extreme nursing work conditions , sought a way to leave education . in agreement with the findings of this study , in the study of leong , concerns were raised about both salary and employee benefits , and one can conclude that policies in these areas are met with some dissatisfaction . also , in jordan , the underpayment of nurses is one of the major reasons for nurses ' dissatisfaction and intention to leave hospitals . the lower social status of the nursing profession in terms of low salary drives students away from nursing . it was also revealed that the working environment , nurse - patient ratio , and health - care insurance were elements to be considered in the payment and welfare package . powerlessness of nurses in clinic forms a negative view in the mind of students towards the nursing career . also the study of brodie et al . showed that high clinical experience of the participant students in this study reinforced both their own and the society 's image of an underpaid and overworked profession that lacks respect and has low morale . low dignity of the nurses in the clinic decreases clinical learning motivation in the students and represents an unpleasant image from the nursing career to them , because students seek for their future dignity and respect by looking into the present nurses . health systems should take the promotion of the nurses ' dignity into account through providing a dignified work environment . according to the findings of the present research , students who model and substitute nursing roles get insight into their future profession . the findings also showed that nurses as role models had the most influence on the motivation or no motivation of students so that the performance , work conditions and power of nurses each somehow influenced the motivation of students during the clinical education . many of the strategies used by the students in escaping from clinical environment and future nursing reflect their undesirable evaluation of the nursing profession , which ends with lack of motivation toward clinical education . in return , the participants believed that in iran proper job market in nursing ( compared to the graduated students of other majors who do not find a job easily ) can absorb into the health system even those students who were unmotivated during their clinical education . the entering of unmotivated students into nursing profession is reflected in the health system of iran society in facing unmotivated nurses , and each unmotivated student turns into an improper representative of nursing profession . based on the findings of this study , educational and health care authorities can promote the quality of nurses ' work in offering better services to the patients through improving their work conditions . in this way , more appropriate nursing role models would be available for students , and thus their interest in the nursing career would increase . we recommend the conduct of action researches for improvement of the clinical setting in nursing education . although theoretical sampling was used in this work to collect the required data that encompassed multiple aspects of the experience in question , the small sample size were research limitations .

one aim of nursing education is to motivate nurses to acquire skills for offering appropriate quality health care services to patients with multiple complex health problems . studies have shown that , in the clinical setting , the most important barrier to clinical education is students ' lack of interest and motivation . so paying due attention to the concept of motivation is of great importance in clinical education . clinical placements form a major part of nursing education and have an important role in students ' perceptions of nursing . nurses represent the largest category of health workers and provide 80% of direct patient care . they represent the professional image and the prospect of nursing career and are the main role model for nursing students . having a clear image of the career helps the students with choosing nursing as a career or retaining it [ 3 , 7 , 8 ] . conversely , a poor public image of nursing contributes to inadequate numbers of students entering the nursing education programmes and , for those that do enter nursing , it can influence the attrition rate . found that , although a service orientation remains a key factor in choosing nursing , students also look for a career that matches their interests and attributes , and offers professional values and rewards . personal reasons were the most common followed by difficult relationships with teaching staff , learning difficulties , and wrong career choice , as well as difficulties with practical training . nurses can be regarded as the source of learning at the clinic through social learning pattern . having substantial teaching background in clinical education , we found a theoretical sensitivity in this area . hence , considering the gap in the research literature , especially lack of enough studies in this field in iran , this study was conducted to achieve a deep and comprehensive view and to explore how the nursing students are motivated through clinical education , with an emphasize on the role of nurses in this regard . in iran , the nursing program offers a four - year baccalaureate in nursing accredited by the high council of medical education of the ministry of health and medical education . the program consists of general education courses ( 20 credits ) , professional foundation courses ( 28 credits with a biological content ) ; and nursing courses ( 53 credits on theory with a biomedical nursing content and 33 credit on clinical preparation ) . a practicum is an integral part of professional nursing studies and includes a 40-week clinical placement , in which nursing students provide supervised care 7 hours/1day and 6days / week under the supervision of an academic nursing instructor . dissertation , titled the process of motivation formation in nursing students during clinical education , which was conducted using grounded theory method . grounded theory focuses on identification , description and explanation of interactional processes among individuals or groups within a given social context . the grounded theory method was selected in this work to explore the participants ' opinions on the conditions effective for clinical education . in the present research , 16 undergraduate nursing students at the second or higher semesters of study in the department of nursing and midwifery at the universities of tehran and zanjan were selected through purposive sampling . according to their viewpoints and suggestions , it was assumed that nursing instructors serve as suitable key informants regarding clinical education motivation . considering the wide range of experiences and perceptions among this group of participants , interviews were conducted theoretically according to the codes and categories as they emerged until data saturation . sixteen students and four instructors were invited to participate , and all of them attended an orientation to the study . after approval of the study by the ethical committee of the school of nursing ( tehran university of medical sciences ) and obtaining permission to proceed from the officials of the nursing schools of both tehran and zanjan universities of medical sciences , the first author began to collect the data . in order to respect the participants ' privacy and confidentiality , the interviews were conducted only in the presence of the interviewer and the interviewee . the interviews took place at the first author 's room at the school of nursing & midwifery ( tehran or zanjan ) in a classroom , or in an instructor 's room . the interviews started with broad questions in order to encourage the participants to speak freely and express their personal experiences regarding motivation in clinical education . as the interview progressed , the questions became more specific , allowing deeper investigation of the issues raised by the participants in the earlier interviews . the process of conducting interviews , transcribing the recordings , and analyzing the data occurred simultaneously . the data collection and analysis continued until data saturation , that is , no additional data were found for development of the category properties . according to the constant comparative analysis , similar data were grouped and conceptually labeled during the so - called then the concepts were classified and the categories were linked and organized by relationships in a process , called axial coding . in this step , the core variable monitoring and modeling through clinical education became a theoretical guide to the further collection and analysis of the data . therefore , the emerging categories and the core variable led the researcher toward interviewing several key informants who provided rich data about the nurses as role models . maximum variant sampling and achievement of saturation were among other items that improved the credibility and the confidentiality of the data , in turn . this prolonged engagement with the participants and their interest helped the investigator to gain the participants ' trust and obtain more in - depth data . credibility and confirmability were established through prolonged engagement , allocation of adequate time , good communication , multiple methods ( such as data source triangulation ) , and maximum variant sampling , which provided plenty of opinions and points of view for the research . a second review of the transcripts , codes , and grouped codes and concepts by a number of colleagues ( as a peer check ) and participants ( as a member check ) was conducted . in addition , several nursing students who had not participated in the interviews reviewed the results and confirmed their suitability . ten out of the total of 16 student participants were females and 6 were males , with an average age of 2125 years . in this work , concerns of becoming a nurse was emerged as one of the important patterns in the process of motivation formation in the nursing students during clinical education . this pattern consisted of three categories as follows : nurses clinical competency , nurses as full - scale mirror of the future , and monitoring and modeling through clinical education ( as the core variable ) , which will be described in detail in the coming paragraphs . in the view of all participants , nurses ' clinical competency has been effective on motivation of students in clinical education . this category comprises three subcategories : ( a ) caring about patient 's rights , ( b ) nurses clinical knowledge , and ( c ) submission to routines . ( a ) caring about patient 's rightsin the view of the participants , due attention of nurses to the spiritual and ideological beliefs of the patient and those accompanying him / her was among the items of caring about the patient 's rights by the nurse . in the view of the participants , due attention of nurses to the spiritual and ideological beliefs of the patient and those accompanying him / her was among the items of caring about the patient 's rights by the nurse . yet , the participants frequently witnessed cases of not observing the patient 's rights by the nurses . the participants listed the aspects of disregard to the patient 's rights by the nurses as misbehavior with the patient , not paying attention to physical and psychological needs of the patient , not paying attention to the patient 's beliefs and/or his / her privacy , not preparing the patient , and not responding to the patient 's questions before conducting any procedure . one of the participants spoke about the disregard and inhumane behavior of the nurses toward the patients as follows:for example , even when the patient is screaming of the pain , it happens that the nurse says shut up to the patient . for example , even when the patient is screaming of the pain , it happens that the nurse says shut up to the patient . ( b ) nurses clinical knowledgemany participants mentioned that nurses ' high clinical knowledge leads to their high value , esteem toward them , and acceptance of their suggestions by the physician ; all of these improve the students ' clinical motivation . many participants mentioned that nurses ' high clinical knowledge leads to their high value , esteem toward them , and acceptance of their suggestions by the physician ; all of these improve the students ' clinical motivation . about the influence of the nurses ' high levels of clinical knowledge on the motivation of students for clinical education , a participant said:we observe some nurses who are really informed . for example , i know a nurse who is so educated that can easily comment on the patients ' treatment process for example , i know a nurse who is so educated that can easily comment on the patients ' treatment process another participant added:a nurse can increase his / her knowledge so that it is possible for him / her to speak with the doctors . it is his / her low level of knowledge that makes him / her stranger to the doctor in contrast to the positive influence of high levels of clinical knowledge , nurses ' low clinical knowledge level is among the reasons for physicians , nursing students and patients considering nurses as noneducated . another participant stated about the effect of nurses ' clinical knowledge on the motivation of student for clinical education : when my student observes an experienced , informed and skilled nurse , he / she would love to the same to receive the same respect . a gap between nurses and other professional groups , especially the physicians , would make the participants unmotivated toward the clinical education . submission to routines was stated as mechanical care , merely giving the drugs prescribed to the patient , taking vital signs , recording and reporting , lack of appropriate communication with the patient , and lack of teaching the patient . one of the participants spoke about submission to routines in nurses : you may see in the ward a nurse who has several years of experience but he / she knows nothing and acts just as a robot in giving the drugs , writing the report and handling it to the next shift ( participant 11 , student ) . submission to routines was stated as mechanical care , merely giving the drugs prescribed to the patient , taking vital signs , recording and reporting , lack of appropriate communication with the patient , and lack of teaching the patient . one of the participants spoke about submission to routines in nurses : you may see in the ward a nurse who has several years of experience but he / she knows nothing and acts just as a robot in giving the drugs , writing the report and handling it to the next shift ( participant 11 , student ) . you may see in the ward a nurse who has several years of experience but he / she knows nothing and acts just as a robot in giving the drugs , writing the report and handling it to the next shift ( participant 11 , student ) . one of the consequences of submission to routines in nurses is lack of appropriate care for the patients . a participant spoke about this fact:without seeing to the patient , the nurse sits in the station and writes the nursing report . without seeing to the patient , the nurse sits in the station and writes the nursing report . ( a ) extreme work conditions in the view of participants , qualified nurses are like a full - scale mirror of the future . therefore , the work conditions of nurses have a great effect on the students ' attitude and motivation . by extreme work conditions , they refer to disproportionate ratio of nurses to patients , job stress and extreme work conditions versus low salary and benefits , lack of psychological security , carrying out the tasks of other professional groups by nurses , having night shifts , the hospital 's boring environment , permanent exposure of the nurses to the disease and death , and lack of an appropriate place for the nurses in the clinic and the community . a participant spoke about the extreme work conditions:when we go for internship , we ask the nurses about their work conditions when we see that they have to work so much for such a low salary . therefore , the work conditions of nurses have a great effect on the students ' attitude and motivation . by extreme work conditions , they refer to disproportionate ratio of nurses to patients , job stress and extreme work conditions versus low salary and benefits , lack of psychological security , carrying out the tasks of other professional groups by nurses , having night shifts , the hospital 's boring environment , permanent exposure of the nurses to the disease and death , and lack of an appropriate place for the nurses in the clinic and the community . a participant spoke about the extreme work conditions:when we go for internship , we ask the nurses about their work conditions when we see that they have to work so much for such a low salary . when we go for internship , we ask the nurses about their work conditions when we see that they have to work so much for such a low salary . course , or pursuing their study in nursing in order to work in educational environments and thus escaping from the clinical extreme work conditions could be the consequences of the nurses extreme work conditions . the participants stated that , in the clinical settings , they found no difference between the type of work done by the nurses holding b.s . or m.sc these conditions ultimately result in a decrease of the students ' motivation in making efforts to learn and to pursue their studies . in spite of extreme work conditions in nursing , the participants believed that existing appropriate job market as compared to other majors in iran will make students more optimistic to the job 's future , and to some extent , this is motivating for the students in the clinical education . a participant stated that:the fact that the graduates in other majors are jobless and that it is possible for the nursing students to get in the job market immediately , will be a motivating factor for them ( participant15 , instructor ) . the fact that the graduates in other majors are jobless and that it is possible for the nursing students to get in the job market immediately , will be a motivating factor for them ( participant15 , instructor ) . ( b ) nurses powerlessnessin the process of physician - nurse communication , the participants observed a boss - subordinate relation between them as well as the nurses ' submission to the physicians . in the process of physician - nurse communication , the participants observed a boss - subordinate relation between them as well as she / he does nothing but to enter into the cardex the physician 's orders immediately . another student spoke about the fear of nurse in communicating with the physician:the relationship should be so appropriate that the nurse will not be afraid of the physician and ask about every problem he / she encounters . but now it 's turned to be so inappropriate that the head nurse even does not dare to ask the physician a single question she / he does not dare to say that a patient 's symptoms have changed the relationship should be so appropriate that the nurse will not be afraid of the physician and ask about every problem he / she encounters . but now it 's turned to be so inappropriate that the head nurse even does not dare to ask the physician a single question she / he does not dare to say that a patient 's symptoms have changed in contrast , a good cooperation atmosphere and nurses ' self - esteem were encouraging for the students to work in such an environment in the future . in the present study , the students monitored and judged on the performance of the nurses and moved on a spectrum of having or not having motivation accordingly . in this respect , a participant stated that:believe that from now on , we have the concerns of a nurse , and it is as if we are not students at all ( participant 5 , student ) . believe that from now on , we have the concerns of a nurse , and it is as if we are not students at all ( participant 5 , student ) . through this role replacement , students reach a stage of occupational understanding and cognition , by observing and role replacement of the nurses who have enough clinical knowledge , are not submitted to routines , observe patients ' rights , and are innovative in the decrease of clinical nursing problems , the students are motivated for clinical education and use different elements of clinical environment ( good communication environment and exploiting learning opportunities ) to learn more and better . in contrast , extreme work conditions and nurses ' clinical powerlessness can result in decreased motivation of the students in clinical education . the findings of this study may create a new insight into the relationship of nursing students ' challenges in facing the realities of the profession with clinical education motivation . this pattern includes three categories of nurses clinical competency , nurses as full - scale mirror of the future , and monitoring and modeling through clinical education ( as the core variable ) . witnessing such behaviors , as nursing role models , plus receiving negative feedbacks from the patients about the nurses ' behaviors result in a decrease of clinical learning motivation in the students . in a study conducted by kyrkjeb and hage , the participating students experienced the fact that the patients were taken seriously by the staff . in this study , in the participants ' view , one of the criteria for clinical competency of nurses was their knowledge . the students , by witnessing the nurses ' high clinical knowledge that was materialized in their action , were motivated for continuing their study and clinical work and aspired to act as a knowledgeable nurse in the future . found that from the participants ' perspective , competence seemed to be related to instrumental - technical , intellectual - cognitive , organizational , social and communication skills , and attitudes . in contrast , the nurses with low clinical knowledge made an undesirable impression on the students and , in the opinion of the participants , it had a negative effect on the views of other professional groups in the clinic and , consequently , in the community . showed that more than a half of the nurses in the ccu and icu units had a weak clinical knowledge . in this type of performance , nurses do their duties without paying much attention to their job and , as a results , they have no decision - making power in different situations . conversely , in this study , the majority of the students , following the observation of extreme nursing work conditions , sought a way to leave education . in agreement with the findings of this study , in the study of leong , concerns were raised about both salary and employee benefits , and one can conclude that policies in these areas are met with some dissatisfaction . also , in jordan , the underpayment of nurses is one of the major reasons for nurses ' dissatisfaction and intention to leave hospitals . the lower social status of the nursing profession in terms of low salary drives students away from nursing . it was also revealed that the working environment , nurse - patient ratio , and health - care insurance were elements to be considered in the payment and welfare package . showed that high clinical experience of the participant students in this study reinforced both their own and the society 's image of an underpaid and overworked profession that lacks respect and has low morale . low dignity of the nurses in the clinic decreases clinical learning motivation in the students and represents an unpleasant image from the nursing career to them , because students seek for their future dignity and respect by looking into the present nurses . according to the findings of the present research , students who model and substitute nursing roles get insight into their future profession . the findings also showed that nurses as role models had the most influence on the motivation or no motivation of students so that the performance , work conditions and power of nurses each somehow influenced the motivation of students during the clinical education . many of the strategies used by the students in escaping from clinical environment and future nursing reflect their undesirable evaluation of the nursing profession , which ends with lack of motivation toward clinical education . in return , the participants believed that in iran proper job market in nursing ( compared to the graduated students of other majors who do not find a job easily ) can absorb into the health system even those students who were unmotivated during their clinical education . based on the findings of this study , educational and health care authorities can promote the quality of nurses ' work in offering better services to the patients through improving their work conditions .

solar cells using hybrid perovskite semiconductors as the active material have recently seen a remarkable rise in performances with device efficiencies exceeding 20% . the recent renaissance of photovoltaic research has enabled hybrid mapbx3xyx ( ma = ch3nh3 ; x , y = i , br , cl ) perovskites , in their 3d structural form , to take center stage once more , leading to a plethora of intriguing results . tunable electroluminescence and amplified spontaneous emission have been demonstrated by adjusting the band gap of the hybrid semiconductor together with a proper optically pumped laser , both by constructing a simple vertical optical cavity in which the perovskite film was sandwiched and by simply pumping perovskite nanowires . while the majority of research has focused on thin films , colloidal synthesis routes to hybrid organic inorganic or to fully inorganic perovskite nanocrystals ( ncs ) have only been developed very recently . for instance , protesescu et al . reported a protocol for the synthesis of cspbx3 ncs ( x being either cl , br , i or a combination of both ) , with narrow size distributions as well as narrow emission line widths , and photoluminescence quantum yield ( plqy ) up to 90% . methylammonium based ncs ( mapbx3 , x = br , i , cl ) have also been recently reported with plqy up to 70% , narrow size distributions and wide color gamut , with each point in the chromaticity diagram corresponding to a different halide composition of the ncs . bandgap engineering of lead halide perovskites ( both in thin film or ncs form ) has been so far realized mainly at the synthesis step , for instance by substituting , partially or completely , the cations ( sn instead of pb or formamidium instead of ma ) , or by mixing different ratios of different halide salts in the reaction flask with the aim of preparing mixed - halide perovskites , or yet by tuning the average crystallite size . halide perovskites are additionally known to exhibit high ionic conductivities ( due to anion migration ) and cspbx3 perovskites have been identified as halide - ion conductors since the 80s . recently , high ion mobility has been recognized as one of the possible reasons for anomalous hystereses in the current voltage curves of perovskite based solar cells . the ability of halide ions to diffuse / migrate in the perovskite lattice explains their ease of replacement with other halide ions in hybrid halide perovskites thin films , using either ma - halides or halogen gases as sources of the entering halide ions . here , we propose a simple method to tune the optical properties of colloidal cspbx3 ( x = cl , br , i ) ncs via postsynthetic reactions with different compounds capable of delivering halide ions , and demonstrate fast halide exchange with preservation of shape and crystal structure of the initial ncs ( scheme 1 ) . in general , colloidal halide perovskite ncs ( for example both the ncs discussed here and the mapbbr3 ncs reported by schmidt et al . ) are unstable in polar solvents , which restricts the medium in which postsynthesis treatments can be carried out to nonpolar or moderately polar solvents ( toluene in the present case ) . in our experiments , the amount of added halide precursors regulated the pl of the resulting ncs : depending on the initial nc samples and on the type of exchanging halide ions , such pl could be tuned across the whole visible spectrum . except when lead halide salts were used as source of anions , the exchanged ncs had optical quality in terms of plqy and narrow emission linewidth in line with that of directly synthesized ncs . this suggests that the anion exchange process did not deteriorate the structure and the overall stability of the initial ncs . we additionally demonstrate that fast anion exchange can even take place between halide perovskite ncs bearing different halide ions . the inter - nc exchange of anions yielded ncs with homogeneous composition of halide ions in their respective lattices , with halide ratios dictated by the relative amounts of ions in the ncs that were initially mixed . lead(ii ) chloride ( pbcl2 , 99.999% trace metals basis ) , lead(ii ) bromide ( pbbr2 , 99.999% trace metals basis ) , lead(ii ) iodide ( pbi2 , 99.999% trace metals basis ) , cesium carbonate ( cs2co3 , reagentplus , 99% ) , tetrabutylammonium iodide ( tbai , reagentplus , 97.0% ) , tetrabutylammonium bromide ( tbabr , reagentplus , 99.0% ) , tetrabutylammonium chloride ( tbacl , 97.0% ) , iodine ( i2 , 99.99% ) , methylamine solution ( 33 wt % in absolute etoh ) , absolute ethanol ( etoh , acs reagent , 99.8% ) , octadecylamine ( oda , 97% ) , hydrochloric acid ( hcl , acs reagent , 37% ) , hydriodic acid ( hi , 57 wt % in h2o ) , hydrobromic acid ( hbr , 48 wt % in h2o , 99.99% ) , octadecene ( ode , technical grade , 90% ) , oleylamine ( olam , 70% ) , trichloroisocyanuric acid ( tcica , technical , 95% ) and oleic acid ( oa , 90% ) were purchased from sigma - aldrich . chloroform ( chcl3 , anhydrous , 99.95% ) and toluene ( tol , anhydrous , 99.8% ) were purchased from carlo erba reagents . all chemicals were used without any further purification , except for olam , oa , and ode , which were degassed at 100 c for 2 h under vacuum . cspbbr3 ( and cspbcl3/cspbi3 ) ncs were synthesized as described by protesescu et al . , with some minor adaptations . in a typical synthesis , 69 mg of pbbr2 ( 0.188 mmol ) ( or 52/87 mg of pbcl2/pbi2 , respectively ) , 5 ml of ode , 0.5 ml of oa and 0.5 ml of olam were loaded in a 25 ml 3-neck flask and dried under a vacuum for 1 h at 120 c . after degassing , the temperature was raised to 165 c and a mixture of 0.6 ml of ode with 0.4 ml of previously synthesized cs - oleate ( 0.4 g of cs2co3 degassed in 15 ml of ode and 1.75 ml of oa at 150 c ) was swiftly injected . immediately after the injection , the nc solution was quickly cooled down to room temperature with an ice bath , and the ncs were transferred to a glovebox . the ncs as delivered from the synthesis ( the crude ncs ) could be purified via high speed centrifugation ( at 12 000 rpm for 30 min ) , followed by redispersion in tol . methylammonium bromide ( ma - br ) used for anion exchange was synthesized following the protocol reported in the literature . 8.5 ml of hbr ( 48 wt % in h2o ) was slowly added to a 250 ml flask containing 24 ml of methylamine solution ( 33 wt % in absolute etoh ) and 100 ml of etoh . the solution was kept in ice bath under stirring for 2 h and then the solvent was removed by evaporation , yielding a white powder . the ma - br salt was then washed three times with diethyl ether and dried on a hot plate . octadecylammonium halides ( oda - x ) used for anion exchange were synthesized using the same procedure for ma - br : 0.08 ml of hcl ( or 0.5 ml of hi or 0.17 ml of hbr ) were slowly added in a flask containing 1 g of oda and 40 ml of etoh . the solution was stirred for 2 h and then the solvent was removed by evaporation . the olam - cl ( most likely a mixture of chloro - oleyalmine and oleylammonium chloride ) precursor was synthesized as follows : an excess of cl2 gas ( synthesized in situ by slowly adding 2 ml of 37% hcl to trichloroisocyanuric acid ) was bubbled into a flask containing 2 ml of olam , under nitrogen flow . from the flask , the n2/cl2 gaseous mixture was then bubbled in a naoh solution , to neutralize the unreacted cl2 gas . after all the trichloroisocyanuric acid had reacted , the oleylamine solution was diluted in 3 ml of tol . the corresponding olam - i precursor was prepared by reacting 0.5 mmol of i2 ( 1 mmol i ) with 0.250 ml of olam overnight . tba - x anion precursor solutions were prepared ( and stored in a glovebox ) by dissolving 1 mmol of halide salt ( 278 mg of tba - cl , 322 mg of tba - br or 370 mg of tba - i ) in 1 ml of chcl3 , diluted by 5 ml of tol . 0.25 ml of crude cspbx3 nc solution ( 0.0537 m in x ) were dispersed in 1 ml of tol , and different quantities ( normally ranging from 10 to 400 l ) of 0.17 m halide precursors solution were swiftly injected . the anion exchanges with solid lead halide salts were performed by addition of a 20-fold halide excess ( 0.14 mmol ) of the solid salt to a rapidly stirred nc solution . similarly , the exchange reaction with oda - x and ma - br was performed by adding a 20-fold halide excess ( 0.14 mmol ) of ma - br salt to 0.25 ml of cspbbr3 nc solution diluted in 0.75 ml of tol . conventional tem observations were carried out using a jeol jem 1011 microscope equipped with a thermionic gun operating at 100 kv of accelerating voltage . samples were prepared by dropping washed and diluted ( in tol ) nc solutions onto carbon - coated 200 mesh copper grids with subsequent solvent evaporation . a rigaku smartlab 9 kw diffractometer was used with a cu source operating at 40 kv and 150 ma . a gbel mirror was used to obtain a parallel beam and to suppress cu k radiation ( 1.392 ) . samples were prepared by dropcasting a concentrated and washed nc solution on a zero background silicon wafer . the spectra were recorded on a varian cary 5000 uv vis nir spectrophotometer . the nc solutions were prepared by diluting the crude nc solutions in tol ( 20 l in 1 ml ) , in 1 cm path length quartz cuvettes with airtight screw caps . thin films of cspbx3 ncs obtained via anion exchange were prepared as follows : 0.5 ml of crude cspbx3 nc solution was separated from the organic solvents by centrifugation at 10 000 rpm for 30 min . the ncs were redispersed in 20 l of tol and spin coated on a 1.5 by 1.5 cm quartz slide at 2000 rpm for 45 s. the crude nc solutions were diluted in tol ( 50 times ) , to reach an optical density of about 0.10.2 at the excitation wavelength . the steady - state pl spectra were collected with an edinburgh instruments fls920 spectrofluoremeter , by exciting the samples at 400 nm using a xe lamp coupled to a monochromator . for plqy measurements , the spectra were collected using a spectrofluorimeter ( horiba jobinyvon ) , by exciting the sample with a monochromated xenon lamp source . the central wavelength was set to exc= 400 nm for all the samples except for cspbcl3 , for which exc= 350 nm was used . the exciting light was coupled into an optical fiber connected to an integrating sphere where a quartz cuvette containing the sample was placed . the emitted light was then collected from the sphere with a second fiber coupled to the detection system , made of a spectrometer and a pmt detector . for each sample , four measurements were performed : ( i ) the sample emission ( sem ) that collects the photons emitted by the sample ; ( ii ) the blank emission ( bem ) , which is a measurement performed with the cuvette containing only the solvent ( blank ) in the same spectral range used for the sem measurement ; ( iii ) the sample excitation ( sex ) , which records the photons at the pumping wavelength that are not absorbed by the sample ; ( iv ) the blank excitation ( bex ) , which records the photons at the pumping wavelength going through the blank . the photoluminescence quantum yield plqy was then calculated asany reabsorption correction factor was neglected in our calculation of the plqy , since the solutions investigated were diluted to the point that reabsorption of the pl could be neglected . the halide precursors tested in the exchange reactions were : lead halide salts ( pbx2 ) , tetrabutylammonium halides ( tba - x ) , octadecylammonium halides ( oda - x ) and oleylammonium halides ( olam - x ) . the latter were prepared by reacting oleylamine with halogen molecules ( either cl2 or i2 in the present work , see the experimental section ) . it is known that primary and secondary amines react with halogen molecules to form haloamines ( at least for the cl2 case ) and ammonium halides . the rationale behind the use of olam - x is that the ncs synthesized by us were already coated with oleylamine molecules , therefore olam - x should be the reactant that would possibly entail the least interference with the nc ligand shell . in the tba - x case i br exchange routes , but not for the reverse cases ( cl br and br i ) . this is understandable in terms of hard / soft acid / base interactions : the lyophilic quaternary tba cation , with its four butyl chains and no possibility of forming hydrogen bonding , is a soft acid that prefers to bind to softer halide ions . therefore , cl ions in cspbcl3 ncs will not be exchanged with br ions coming from tba - br because the concurrent transformation from tba - br to tba - cl is not favored , as br ions are softer than cl ions and thus prefer to remain associated with tba . the same reasoning can explain the difficulty in exchanging br ions in cspbbr3 ncs with i ions coming from tba - i ( this time i ions are softer than br ions ) . interestingly , this reasoning does not apply to the much smaller methylammonium halide ( ma - x ) compounds , which however can be engaged in cation exchange reactions involving the ma cation , as discussed later . ( a ) pl of the exchanged ncs obtained from cspbbr3 by adding : ( from top to bottom ) olam - x , oda - x , tba - cl and pbx2 ( x = i , cl ) . ( b ) pl of the cspbbr3 ncs obtained starting from cspbcl3 and cspbi3 ncs using ( from top to bottom ) oda - br , tba - br and pbbr2 . all the anion exchange reactions discussed here led either to a blue shift ( for the br cl and i br routes ) or to a red shift ( for the br i and cl br routes ) of the optical features ( figure 1a and 1b ) , corroborating the incorporation of the new anions . highly soluble precursors such as olam - x and tba - x led to anion exchange within a few seconds ( see supporting movies ) at room temperature , and could be easily tracked by monitoring changes in the optical absorption and pl spectra of the ncs ( later discussed in figure s1 and figure 2a , respectively ) . the exchange with the less soluble oda - x and lead halides ( pbx2 ) salts was generally slower and required instead at least 1 day under stirring to record a full shift in the pl . furthermore , exchange was often incomplete in such cases ( broadened pl spectra were recorded ) . this could be easily explained by the low solubility of these compounds in the relatively nonpolar tol environment . attempts to improve their solubility , by working at 90 c , resulted in quick degradation of the ncs ( see also later ) . without loss of generality , we henceforth focus the discussion on the exchange routes starting from cspbbr3 ncs , due to their stability and strong fluorescence . the ncs , as synthesized , were characterized by a narrow emission band centered at 2.43 ev with a fwhm of 0.12 ev ( 26 nm ) and a plqy of 78% ( figure 2c , green star shaped marker ) . after one month from the synthesis , their plqy dropped only slightly ( to 70% ) . on the other hand , the cl- or i - based ncs prepared by direct synthesis exhibited a plqy of 1% and 30% , respectively ( figure 2c , violet and red star shaped markers , respectively ) . moreover the cspbi3 ncs were extremely sensitive to moisture , and were degraded in a couple of days if exposed to air . as for the halide precursors , we focus the discussion here on olam - i and tba - cl , although similar results were found when working with the other precursors , as reported in figure s1 . also , we focused on room temperature exchange , since the ncs were generally unstable at higher temperatures . for example , the pl intensity of samples of cspbbr3 ncs dispersed in tol decreased considerably upon prolonged exposure to heat ( 90 c for 2 h ) , as shown in figure s2 . ( a ) pl spectra of the cspb(br : x)3 ( x = cl , i ) ncs prepared by anion exchange from cspbbr3 ncs . ( b ) pl calibration curves : a targeted emission energy could be obtained by adding a precise amount of halide precursor to a crude solution of cspbbr3 ncs . the curves are reported as a function of the molar ratio between the added halide ( or exchange halide ) and the br amount in the starting ncs . ( c ) plqy recorded on the exchanged ncs ( dots ) as well as on the directly synthesized ncs ( stars ) . as shown in figure 2a , the pl of the pristine cspbbr3 ncs could be tuned within an energy interval comprised between 1.88 ev ( lowest value attainable by reaction with olam - i ) and 3.03 ev ( highest value attainable by reaction with tba - cl ) following the titration curves of figure 2b . similar tunabilities of the pl using olam - cl and tba - br are reported in figure s1 . the upper and lower pl positions of the corresponding samples prepared via anion exchange differed only slightly ( by less than 0.1 ev , see figure s4 ) from those of the cspbi3 and cspbcl3 ncs prepared by direct synthesis , signifying an almost full exchange . the pl emission lines for the cspbi3 and cspbcl3 ncs prepared by anion exchange remained narrow ( 0.11 and 0.10 ev , respectively ) . figure 2c shows the measured plqy , as a function of the energy of the emitted photons , for each of the studied samples . notably , upon a slight addition of tba - cl the plqy increased from 78% of the initial cspbbr3 ( green star shaped markers in figure 2c ) up to 95% . this is in line with the trend already reported by pellet et al . on ch3nh3pbbr3 thin films prepared via halide substitution from ch3nh3pbcl3 films ( apart from the above - mentioned exception , the plqy of the investigated samples generally dropped from the starting value of 78% when replacing br ions with either cl or i ions ( figure 2c ) . then , upon full halide exchange , the plqy settled to a value comparable to the one measured on the directly synthesized cspbi3 and cspbcl3 ncs . the lower plqys of cspbi3 and cspbcl3 with respect to cspbbr3 are most likely ascribable to intrinsic properties of halide perovskites . overall , our results indicate that the anion exchange process does not induce any remarkable formation of lattice / surface defects that would lead to deterioration of the optical properties of the ncs . for all nc samples ( regardless of whether the ncs were purified from the excess halide precursors or they were stored as crude solutions , see figure s5 ) , the pl spectral position , its narrow line width and the plqy remained stable over days . the exchanged ncs remained stable even if deposited as a thin film . these films exhibited plqys that were lower than the initial values of the ncs in solution ( figure s6 ) , but were comparable to those recorded from polycrystalline films grown by direct crystallization of chemical precursors on a substrate . also , it is worthy of note that the exchange worked only for the cl br and br i couples ( in both directions ) , but never from cspbcl3 directly to cspbi3 nor in the reverse direction . exchange attempts ended up in complete dissolution of the particles , with loss of pl from solution . one possible explanation here is that the direct exchange between cl and i anions would involve a structural stress on the perovskite lattice that can not be tolerated without serious degradation of the ncs . anion exchange did not alter the cubic shape of the initial cspbbr3 ncs ( figure 3d ) , although after exchange with cl their size decreased slightly , from ( 8.4 1.0 ) nm to ( 8.0 1.4 ) nm ( figure 3a ) , whereas the exchange with i led to a slight increase in size , to ( 9.1 1.3 ) nm ( figure 3c ; see also figure s7 and s8 ) . the xrd pattern collected on the pristine cspbbr3 ncs ( figure 3d ) could be indexed as cubic cspbbr3 ( a = 5.874 , space group pm3m , icsd 29073 ) as detailed in figure s9 , in agreement with what reported by protesescu et al . anion exchange reactions did not alter the crystal phase of the ncs ( see figures 3d , s4b and s9 ) and the patterns collected on the almost fully exchanged ncs were in good agreement with those recorded on directly synthesized cspbi3 ( a = 6.18 , space group pm3m , icsd 181288 ) and cspbcl3 ( a = 5.605 , space group pm3m , icsd 29072 ) ncs . the xrd patterns of partially exchanged ncs too could be ascribed to the same cubic phase : as expected , upon incorporation of cl , the cell shrunk and all the peaks shifted to higher angles , while the incorporation of i expanded the cell and the peaks shifted to lower angles ( figure 3d ) . table 1 lists the results of compositional analyses of the various samples on which xrd patterns were collected . these were performed via energy dispersive x - ray spectroscopy ( edx ) in the scanning electron microscope ( sem ) . the table also lists the molar ratios between the halide ions added to the nc crude solutions and the br ions in the starting ncs , and the corresponding ratios in the exchanged ncs . from the table , it can be seen that the yield of exchange differed in the two cases ( cl and i ) , the one with the larger i ions being less efficient than the one with the smaller cl ions ( see also the titration curves of figure 2b ) : while a 1.5 ratio of added cl ions to br ions initially present in the ncs yielded ncs with a cl : br ratio over 3 , the same reaction conditions , in the case of i ions , led to ncs with a i : br ratio of only 0.5 . ( a c ) tem images of pristine cspbbr3 ncs ( b ) and of fully exchanged cspbcl3 ( a ) and cspbi3 ncs ( c ) , indicating overall size and size preservation upon anion exchange . ( d ) zoom of the xrd patterns of pristine cspbbr3 ncs ( middle pattern in green ) , of the anion exchanged cspbcl3 ( top , violet ) , cspbi3 ( bottom , red ) and of two intermediate cl ( middle - top , light blue ) and i ( middle - bottom , yellow ) exchanged samples . the reagents listed above were obviously not the only ones capable of triggering anion exchange . however , the use of salts with small cations such as ma adds the further complication that the ma cation also may engage in exchange reactions , this time with the cs ions in the ncs . when for example cspbbr3 ncs were exposed to ma - br their photoluminescence ( pl ) peak shifted from 2.43 to 2.36 ev ( see figure s10 ) , and indeed the latter value is comparable to the one reported for mapbbr3 ncs . at the same time , a slight expansion of the lattice parameters could be inferred from the analysis of the xrd patterns ( figure s10 ) , further supporting the replacement of cs ions with the larger ma ions . similar results were found when cspbcl3 ncs were exposed to ma - br ( figure s11 ) . an extreme case of halide precursor is represented by the ncs themselves : anion exchange could be achieved even by mixing solutions containing perovskite ncs of different halide compositions and therefore emitting in different spectral ranges ( figure 4a ) . the resulting ncs emitted in a narrow spectral region that was intermediate between those of the parent particles . this suggests a fast partition dynamics of halide ions between the ncs and the solution phase , likely meditated by the residual excess organic stabilizers present in solution . pure cspbi3 ncs from a direct synthesis , mixed with cspbbr3 ncs , yielded different emitting samples , with pl energies depending on the ratios of the cspbx3 ncs that were mixed , and plqys in line with those observed when performing exchanges with the other halide precursors , as shown in table 2 . furthermore , interparticle exchange did not affect size and shape of the ncs : all nc samples , before mixing , had comparable average sizes and shapes . these remained practically the same after mixing ( see tem images of figure 4d , e ) . anion exchange between ncs took up to several minutes ( under stirring ) to reach steady state , most likely because the halides ions had to shuttle from nc to nc through the solution phase , a process that in these exploratory experiments was not optimized , for example by the addition of proper stabilizing agents . anion exchange was further supported by xrd patterns ( figure 4b ) of the cspb(br : cl)3 and cspb(br : i)3 samples prepared by mixing pure cspbbr3 and cspbi3 ncs solutions , which indicated that the halides ions redistributed homogeneously throughout the ncs : xrd patterns were indeed compatible with single phase ncs with average unit cells spacings that were intermediate between those of the parent particles . finally , in analogy with the failed cl i exchange attempts discusses earlier , mixing cspbcl3 and cspbi3 ncs resulted in a transparent clear sample and with a quenched pl ( see figure s13 ) , indicating dissolution of the ncs . ( b ) xrd patterns of the pristine cspbcl3 , cspbbr3 and cspbcl3 ncs and of the samples after mixing . ( c ) optical absorption and pl spectra of various cspb(br : cl)3 and cspb(br : i)3 ncs prepared via interparticle exchange . ( d ) and ( e ) tem images of cspbbr3:cspbcl3 1:1 ( 8.5 1.2 nm ) and cspbbr3:cspbcl3 1:1 ( 8.9 1.4 nm ) , respectively . , we have demonstrated a fast and simple postsynthesis anion exchange approach to access a wide variety of cspbx3 perovskite ncs , with preservation of shape and crystal structure of the starting cspbbr3 sample . the exchanged ncs span the whole visible region of the electromagnetic spectrum , with the pl easily controllable by the amount of added halide precursors , and in general with optical and structural properties in line with those of the same ncs prepared by direct synthesis . we have also shown that the ability of cspbx3 perovskite ncs to mutually exchange halide ions can be exploited as an additional tool to tune the optical and structural feature of the resulting materials , by simply mixing solutions of ncs having different halide compositions .

we demonstrate that , via controlled anion exchange reactions using a range of different halide precursors , we can finely tune the chemical composition and the optical properties of presynthesized colloidal cesium lead halide perovskite nanocrystals ( ncs ) , from green emitting cspbbr3 to bright emitters in any other region of the visible spectrum , and back , by displacement of cl or i ions and reinsertion of br ions . this approach gives access to perovskite semiconductor ncs with both structural and optical qualities comparable to those of directly synthesized ncs . we also show that anion exchange is a dynamic process that takes place in solution between ncs . therefore , by mixing solutions containing perovskite ncs emitting in different spectral ranges ( due to different halide compositions ) their mutual fast exchange dynamics leads to homogenization in their composition , resulting in ncs emitting in a narrow spectral region that is intermediate between those of the parent nanoparticles .

Introduction Experimental Section Results and Discussion

while the majority of research has focused on thin films , colloidal synthesis routes to hybrid organic inorganic or to fully inorganic perovskite nanocrystals ( ncs ) have only been developed very recently . reported a protocol for the synthesis of cspbx3 ncs ( x being either cl , br , i or a combination of both ) , with narrow size distributions as well as narrow emission line widths , and photoluminescence quantum yield ( plqy ) up to 90% . methylammonium based ncs ( mapbx3 , x = br , i , cl ) have also been recently reported with plqy up to 70% , narrow size distributions and wide color gamut , with each point in the chromaticity diagram corresponding to a different halide composition of the ncs . bandgap engineering of lead halide perovskites ( both in thin film or ncs form ) has been so far realized mainly at the synthesis step , for instance by substituting , partially or completely , the cations ( sn instead of pb or formamidium instead of ma ) , or by mixing different ratios of different halide salts in the reaction flask with the aim of preparing mixed - halide perovskites , or yet by tuning the average crystallite size . halide perovskites are additionally known to exhibit high ionic conductivities ( due to anion migration ) and cspbx3 perovskites have been identified as halide - ion conductors since the 80s . here , we propose a simple method to tune the optical properties of colloidal cspbx3 ( x = cl , br , i ) ncs via postsynthetic reactions with different compounds capable of delivering halide ions , and demonstrate fast halide exchange with preservation of shape and crystal structure of the initial ncs ( scheme 1 ) . in general , colloidal halide perovskite ncs ( for example both the ncs discussed here and the mapbbr3 ncs reported by schmidt et al . ) in our experiments , the amount of added halide precursors regulated the pl of the resulting ncs : depending on the initial nc samples and on the type of exchanging halide ions , such pl could be tuned across the whole visible spectrum . except when lead halide salts were used as source of anions , the exchanged ncs had optical quality in terms of plqy and narrow emission linewidth in line with that of directly synthesized ncs . this suggests that the anion exchange process did not deteriorate the structure and the overall stability of the initial ncs . we additionally demonstrate that fast anion exchange can even take place between halide perovskite ncs bearing different halide ions . the inter - nc exchange of anions yielded ncs with homogeneous composition of halide ions in their respective lattices , with halide ratios dictated by the relative amounts of ions in the ncs that were initially mixed . all chemicals were used without any further purification , except for olam , oa , and ode , which were degassed at 100 c for 2 h under vacuum . in a typical synthesis , 69 mg of pbbr2 ( 0.188 mmol ) ( or 52/87 mg of pbcl2/pbi2 , respectively ) , 5 ml of ode , 0.5 ml of oa and 0.5 ml of olam were loaded in a 25 ml 3-neck flask and dried under a vacuum for 1 h at 120 c . immediately after the injection , the nc solution was quickly cooled down to room temperature with an ice bath , and the ncs were transferred to a glovebox . the ncs as delivered from the synthesis ( the crude ncs ) could be purified via high speed centrifugation ( at 12 000 rpm for 30 min ) , followed by redispersion in tol . octadecylammonium halides ( oda - x ) used for anion exchange were synthesized using the same procedure for ma - br : 0.08 ml of hcl ( or 0.5 ml of hi or 0.17 ml of hbr ) were slowly added in a flask containing 1 g of oda and 40 ml of etoh . tba - x anion precursor solutions were prepared ( and stored in a glovebox ) by dissolving 1 mmol of halide salt ( 278 mg of tba - cl , 322 mg of tba - br or 370 mg of tba - i ) in 1 ml of chcl3 , diluted by 5 ml of tol . 0.25 ml of crude cspbx3 nc solution ( 0.0537 m in x ) were dispersed in 1 ml of tol , and different quantities ( normally ranging from 10 to 400 l ) of 0.17 m halide precursors solution were swiftly injected . the anion exchanges with solid lead halide salts were performed by addition of a 20-fold halide excess ( 0.14 mmol ) of the solid salt to a rapidly stirred nc solution . the steady - state pl spectra were collected with an edinburgh instruments fls920 spectrofluoremeter , by exciting the samples at 400 nm using a xe lamp coupled to a monochromator . for plqy measurements , the spectra were collected using a spectrofluorimeter ( horiba jobinyvon ) , by exciting the sample with a monochromated xenon lamp source . for each sample , four measurements were performed : ( i ) the sample emission ( sem ) that collects the photons emitted by the sample ; ( ii ) the blank emission ( bem ) , which is a measurement performed with the cuvette containing only the solvent ( blank ) in the same spectral range used for the sem measurement ; ( iii ) the sample excitation ( sex ) , which records the photons at the pumping wavelength that are not absorbed by the sample ; ( iv ) the blank excitation ( bex ) , which records the photons at the pumping wavelength going through the blank . the photoluminescence quantum yield plqy was then calculated asany reabsorption correction factor was neglected in our calculation of the plqy , since the solutions investigated were diluted to the point that reabsorption of the pl could be neglected . the halide precursors tested in the exchange reactions were : lead halide salts ( pbx2 ) , tetrabutylammonium halides ( tba - x ) , octadecylammonium halides ( oda - x ) and oleylammonium halides ( olam - x ) . this is understandable in terms of hard / soft acid / base interactions : the lyophilic quaternary tba cation , with its four butyl chains and no possibility of forming hydrogen bonding , is a soft acid that prefers to bind to softer halide ions . therefore , cl ions in cspbcl3 ncs will not be exchanged with br ions coming from tba - br because the concurrent transformation from tba - br to tba - cl is not favored , as br ions are softer than cl ions and thus prefer to remain associated with tba . the same reasoning can explain the difficulty in exchanging br ions in cspbbr3 ncs with i ions coming from tba - i ( this time i ions are softer than br ions ) . all the anion exchange reactions discussed here led either to a blue shift ( for the br cl and i br routes ) or to a red shift ( for the br i and cl br routes ) of the optical features ( figure 1a and 1b ) , corroborating the incorporation of the new anions . highly soluble precursors such as olam - x and tba - x led to anion exchange within a few seconds ( see supporting movies ) at room temperature , and could be easily tracked by monitoring changes in the optical absorption and pl spectra of the ncs ( later discussed in figure s1 and figure 2a , respectively ) . attempts to improve their solubility , by working at 90 c , resulted in quick degradation of the ncs ( see also later ) . without loss of generality , we henceforth focus the discussion on the exchange routes starting from cspbbr3 ncs , due to their stability and strong fluorescence . moreover the cspbi3 ncs were extremely sensitive to moisture , and were degraded in a couple of days if exposed to air . as for the halide precursors , we focus the discussion here on olam - i and tba - cl , although similar results were found when working with the other precursors , as reported in figure s1 . ( a ) pl spectra of the cspb(br : x)3 ( x = cl , i ) ncs prepared by anion exchange from cspbbr3 ncs . the curves are reported as a function of the molar ratio between the added halide ( or exchange halide ) and the br amount in the starting ncs . ( c ) plqy recorded on the exchanged ncs ( dots ) as well as on the directly synthesized ncs ( stars ) . as shown in figure 2a , the pl of the pristine cspbbr3 ncs could be tuned within an energy interval comprised between 1.88 ev ( lowest value attainable by reaction with olam - i ) and 3.03 ev ( highest value attainable by reaction with tba - cl ) following the titration curves of figure 2b . similar tunabilities of the pl using olam - cl and tba - br are reported in figure s1 . the upper and lower pl positions of the corresponding samples prepared via anion exchange differed only slightly ( by less than 0.1 ev , see figure s4 ) from those of the cspbi3 and cspbcl3 ncs prepared by direct synthesis , signifying an almost full exchange . on ch3nh3pbbr3 thin films prepared via halide substitution from ch3nh3pbcl3 films ( apart from the above - mentioned exception , the plqy of the investigated samples generally dropped from the starting value of 78% when replacing br ions with either cl or i ions ( figure 2c ) . then , upon full halide exchange , the plqy settled to a value comparable to the one measured on the directly synthesized cspbi3 and cspbcl3 ncs . overall , our results indicate that the anion exchange process does not induce any remarkable formation of lattice / surface defects that would lead to deterioration of the optical properties of the ncs . for all nc samples ( regardless of whether the ncs were purified from the excess halide precursors or they were stored as crude solutions , see figure s5 ) , the pl spectral position , its narrow line width and the plqy remained stable over days . these films exhibited plqys that were lower than the initial values of the ncs in solution ( figure s6 ) , but were comparable to those recorded from polycrystalline films grown by direct crystallization of chemical precursors on a substrate . anion exchange did not alter the cubic shape of the initial cspbbr3 ncs ( figure 3d ) , although after exchange with cl their size decreased slightly , from ( 8.4 1.0 ) nm to ( 8.0 1.4 ) nm ( figure 3a ) , whereas the exchange with i led to a slight increase in size , to ( 9.1 1.3 ) nm ( figure 3c ; see also figure s7 and s8 ) . anion exchange reactions did not alter the crystal phase of the ncs ( see figures 3d , s4b and s9 ) and the patterns collected on the almost fully exchanged ncs were in good agreement with those recorded on directly synthesized cspbi3 ( a = 6.18 , space group pm3m , icsd 181288 ) and cspbcl3 ( a = 5.605 , space group pm3m , icsd 29072 ) ncs . the xrd patterns of partially exchanged ncs too could be ascribed to the same cubic phase : as expected , upon incorporation of cl , the cell shrunk and all the peaks shifted to higher angles , while the incorporation of i expanded the cell and the peaks shifted to lower angles ( figure 3d ) . the table also lists the molar ratios between the halide ions added to the nc crude solutions and the br ions in the starting ncs , and the corresponding ratios in the exchanged ncs . from the table , it can be seen that the yield of exchange differed in the two cases ( cl and i ) , the one with the larger i ions being less efficient than the one with the smaller cl ions ( see also the titration curves of figure 2b ) : while a 1.5 ratio of added cl ions to br ions initially present in the ncs yielded ncs with a cl : br ratio over 3 , the same reaction conditions , in the case of i ions , led to ncs with a i : br ratio of only 0.5 . ( a c ) tem images of pristine cspbbr3 ncs ( b ) and of fully exchanged cspbcl3 ( a ) and cspbi3 ncs ( c ) , indicating overall size and size preservation upon anion exchange . ( d ) zoom of the xrd patterns of pristine cspbbr3 ncs ( middle pattern in green ) , of the anion exchanged cspbcl3 ( top , violet ) , cspbi3 ( bottom , red ) and of two intermediate cl ( middle - top , light blue ) and i ( middle - bottom , yellow ) exchanged samples . the reagents listed above were obviously not the only ones capable of triggering anion exchange . however , the use of salts with small cations such as ma adds the further complication that the ma cation also may engage in exchange reactions , this time with the cs ions in the ncs . when for example cspbbr3 ncs were exposed to ma - br their photoluminescence ( pl ) peak shifted from 2.43 to 2.36 ev ( see figure s10 ) , and indeed the latter value is comparable to the one reported for mapbbr3 ncs . at the same time , a slight expansion of the lattice parameters could be inferred from the analysis of the xrd patterns ( figure s10 ) , further supporting the replacement of cs ions with the larger ma ions . an extreme case of halide precursor is represented by the ncs themselves : anion exchange could be achieved even by mixing solutions containing perovskite ncs of different halide compositions and therefore emitting in different spectral ranges ( figure 4a ) . the resulting ncs emitted in a narrow spectral region that was intermediate between those of the parent particles . this suggests a fast partition dynamics of halide ions between the ncs and the solution phase , likely meditated by the residual excess organic stabilizers present in solution . pure cspbi3 ncs from a direct synthesis , mixed with cspbbr3 ncs , yielded different emitting samples , with pl energies depending on the ratios of the cspbx3 ncs that were mixed , and plqys in line with those observed when performing exchanges with the other halide precursors , as shown in table 2 . furthermore , interparticle exchange did not affect size and shape of the ncs : all nc samples , before mixing , had comparable average sizes and shapes . anion exchange between ncs took up to several minutes ( under stirring ) to reach steady state , most likely because the halides ions had to shuttle from nc to nc through the solution phase , a process that in these exploratory experiments was not optimized , for example by the addition of proper stabilizing agents . anion exchange was further supported by xrd patterns ( figure 4b ) of the cspb(br : cl)3 and cspb(br : i)3 samples prepared by mixing pure cspbbr3 and cspbi3 ncs solutions , which indicated that the halides ions redistributed homogeneously throughout the ncs : xrd patterns were indeed compatible with single phase ncs with average unit cells spacings that were intermediate between those of the parent particles . finally , in analogy with the failed cl i exchange attempts discusses earlier , mixing cspbcl3 and cspbi3 ncs resulted in a transparent clear sample and with a quenched pl ( see figure s13 ) , indicating dissolution of the ncs . ( d ) and ( e ) tem images of cspbbr3:cspbcl3 1:1 ( 8.5 1.2 nm ) and cspbbr3:cspbcl3 1:1 ( 8.9 1.4 nm ) , respectively . , we have demonstrated a fast and simple postsynthesis anion exchange approach to access a wide variety of cspbx3 perovskite ncs , with preservation of shape and crystal structure of the starting cspbbr3 sample . the exchanged ncs span the whole visible region of the electromagnetic spectrum , with the pl easily controllable by the amount of added halide precursors , and in general with optical and structural properties in line with those of the same ncs prepared by direct synthesis . we have also shown that the ability of cspbx3 perovskite ncs to mutually exchange halide ions can be exploited as an additional tool to tune the optical and structural feature of the resulting materials , by simply mixing solutions of ncs having different halide compositions .

tunable electroluminescence and amplified spontaneous emission have been demonstrated by adjusting the band gap of the hybrid semiconductor together with a proper optically pumped laser , both by constructing a simple vertical optical cavity in which the perovskite film was sandwiched and by simply pumping perovskite nanowires . reported a protocol for the synthesis of cspbx3 ncs ( x being either cl , br , i or a combination of both ) , with narrow size distributions as well as narrow emission line widths , and photoluminescence quantum yield ( plqy ) up to 90% . methylammonium based ncs ( mapbx3 , x = br , i , cl ) have also been recently reported with plqy up to 70% , narrow size distributions and wide color gamut , with each point in the chromaticity diagram corresponding to a different halide composition of the ncs . bandgap engineering of lead halide perovskites ( both in thin film or ncs form ) has been so far realized mainly at the synthesis step , for instance by substituting , partially or completely , the cations ( sn instead of pb or formamidium instead of ma ) , or by mixing different ratios of different halide salts in the reaction flask with the aim of preparing mixed - halide perovskites , or yet by tuning the average crystallite size . the ability of halide ions to diffuse / migrate in the perovskite lattice explains their ease of replacement with other halide ions in hybrid halide perovskites thin films , using either ma - halides or halogen gases as sources of the entering halide ions . here , we propose a simple method to tune the optical properties of colloidal cspbx3 ( x = cl , br , i ) ncs via postsynthetic reactions with different compounds capable of delivering halide ions , and demonstrate fast halide exchange with preservation of shape and crystal structure of the initial ncs ( scheme 1 ) . are unstable in polar solvents , which restricts the medium in which postsynthesis treatments can be carried out to nonpolar or moderately polar solvents ( toluene in the present case ) . in our experiments , the amount of added halide precursors regulated the pl of the resulting ncs : depending on the initial nc samples and on the type of exchanging halide ions , such pl could be tuned across the whole visible spectrum . the inter - nc exchange of anions yielded ncs with homogeneous composition of halide ions in their respective lattices , with halide ratios dictated by the relative amounts of ions in the ncs that were initially mixed . lead(ii ) chloride ( pbcl2 , 99.999% trace metals basis ) , lead(ii ) bromide ( pbbr2 , 99.999% trace metals basis ) , lead(ii ) iodide ( pbi2 , 99.999% trace metals basis ) , cesium carbonate ( cs2co3 , reagentplus , 99% ) , tetrabutylammonium iodide ( tbai , reagentplus , 97.0% ) , tetrabutylammonium bromide ( tbabr , reagentplus , 99.0% ) , tetrabutylammonium chloride ( tbacl , 97.0% ) , iodine ( i2 , 99.99% ) , methylamine solution ( 33 wt % in absolute etoh ) , absolute ethanol ( etoh , acs reagent , 99.8% ) , octadecylamine ( oda , 97% ) , hydrochloric acid ( hcl , acs reagent , 37% ) , hydriodic acid ( hi , 57 wt % in h2o ) , hydrobromic acid ( hbr , 48 wt % in h2o , 99.99% ) , octadecene ( ode , technical grade , 90% ) , oleylamine ( olam , 70% ) , trichloroisocyanuric acid ( tcica , technical , 95% ) and oleic acid ( oa , 90% ) were purchased from sigma - aldrich . in a typical synthesis , 69 mg of pbbr2 ( 0.188 mmol ) ( or 52/87 mg of pbcl2/pbi2 , respectively ) , 5 ml of ode , 0.5 ml of oa and 0.5 ml of olam were loaded in a 25 ml 3-neck flask and dried under a vacuum for 1 h at 120 c . after degassing , the temperature was raised to 165 c and a mixture of 0.6 ml of ode with 0.4 ml of previously synthesized cs - oleate ( 0.4 g of cs2co3 degassed in 15 ml of ode and 1.75 ml of oa at 150 c ) was swiftly injected . octadecylammonium halides ( oda - x ) used for anion exchange were synthesized using the same procedure for ma - br : 0.08 ml of hcl ( or 0.5 ml of hi or 0.17 ml of hbr ) were slowly added in a flask containing 1 g of oda and 40 ml of etoh . the olam - cl ( most likely a mixture of chloro - oleyalmine and oleylammonium chloride ) precursor was synthesized as follows : an excess of cl2 gas ( synthesized in situ by slowly adding 2 ml of 37% hcl to trichloroisocyanuric acid ) was bubbled into a flask containing 2 ml of olam , under nitrogen flow . tba - x anion precursor solutions were prepared ( and stored in a glovebox ) by dissolving 1 mmol of halide salt ( 278 mg of tba - cl , 322 mg of tba - br or 370 mg of tba - i ) in 1 ml of chcl3 , diluted by 5 ml of tol . 0.25 ml of crude cspbx3 nc solution ( 0.0537 m in x ) were dispersed in 1 ml of tol , and different quantities ( normally ranging from 10 to 400 l ) of 0.17 m halide precursors solution were swiftly injected . similarly , the exchange reaction with oda - x and ma - br was performed by adding a 20-fold halide excess ( 0.14 mmol ) of ma - br salt to 0.25 ml of cspbbr3 nc solution diluted in 0.75 ml of tol . for each sample , four measurements were performed : ( i ) the sample emission ( sem ) that collects the photons emitted by the sample ; ( ii ) the blank emission ( bem ) , which is a measurement performed with the cuvette containing only the solvent ( blank ) in the same spectral range used for the sem measurement ; ( iii ) the sample excitation ( sex ) , which records the photons at the pumping wavelength that are not absorbed by the sample ; ( iv ) the blank excitation ( bex ) , which records the photons at the pumping wavelength going through the blank . the halide precursors tested in the exchange reactions were : lead halide salts ( pbx2 ) , tetrabutylammonium halides ( tba - x ) , octadecylammonium halides ( oda - x ) and oleylammonium halides ( olam - x ) . the rationale behind the use of olam - x is that the ncs synthesized by us were already coated with oleylamine molecules , therefore olam - x should be the reactant that would possibly entail the least interference with the nc ligand shell . this is understandable in terms of hard / soft acid / base interactions : the lyophilic quaternary tba cation , with its four butyl chains and no possibility of forming hydrogen bonding , is a soft acid that prefers to bind to softer halide ions . therefore , cl ions in cspbcl3 ncs will not be exchanged with br ions coming from tba - br because the concurrent transformation from tba - br to tba - cl is not favored , as br ions are softer than cl ions and thus prefer to remain associated with tba . ( a ) pl of the exchanged ncs obtained from cspbbr3 by adding : ( from top to bottom ) olam - x , oda - x , tba - cl and pbx2 ( x = i , cl ) . ( b ) pl of the cspbbr3 ncs obtained starting from cspbcl3 and cspbi3 ncs using ( from top to bottom ) oda - br , tba - br and pbbr2 . all the anion exchange reactions discussed here led either to a blue shift ( for the br cl and i br routes ) or to a red shift ( for the br i and cl br routes ) of the optical features ( figure 1a and 1b ) , corroborating the incorporation of the new anions . highly soluble precursors such as olam - x and tba - x led to anion exchange within a few seconds ( see supporting movies ) at room temperature , and could be easily tracked by monitoring changes in the optical absorption and pl spectra of the ncs ( later discussed in figure s1 and figure 2a , respectively ) . without loss of generality , we henceforth focus the discussion on the exchange routes starting from cspbbr3 ncs , due to their stability and strong fluorescence . the ncs , as synthesized , were characterized by a narrow emission band centered at 2.43 ev with a fwhm of 0.12 ev ( 26 nm ) and a plqy of 78% ( figure 2c , green star shaped marker ) . on the other hand , the cl- or i - based ncs prepared by direct synthesis exhibited a plqy of 1% and 30% , respectively ( figure 2c , violet and red star shaped markers , respectively ) . as for the halide precursors , we focus the discussion here on olam - i and tba - cl , although similar results were found when working with the other precursors , as reported in figure s1 . as shown in figure 2a , the pl of the pristine cspbbr3 ncs could be tuned within an energy interval comprised between 1.88 ev ( lowest value attainable by reaction with olam - i ) and 3.03 ev ( highest value attainable by reaction with tba - cl ) following the titration curves of figure 2b . the upper and lower pl positions of the corresponding samples prepared via anion exchange differed only slightly ( by less than 0.1 ev , see figure s4 ) from those of the cspbi3 and cspbcl3 ncs prepared by direct synthesis , signifying an almost full exchange . notably , upon a slight addition of tba - cl the plqy increased from 78% of the initial cspbbr3 ( green star shaped markers in figure 2c ) up to 95% . on ch3nh3pbbr3 thin films prepared via halide substitution from ch3nh3pbcl3 films ( apart from the above - mentioned exception , the plqy of the investigated samples generally dropped from the starting value of 78% when replacing br ions with either cl or i ions ( figure 2c ) . overall , our results indicate that the anion exchange process does not induce any remarkable formation of lattice / surface defects that would lead to deterioration of the optical properties of the ncs . for all nc samples ( regardless of whether the ncs were purified from the excess halide precursors or they were stored as crude solutions , see figure s5 ) , the pl spectral position , its narrow line width and the plqy remained stable over days . these films exhibited plqys that were lower than the initial values of the ncs in solution ( figure s6 ) , but were comparable to those recorded from polycrystalline films grown by direct crystallization of chemical precursors on a substrate . also , it is worthy of note that the exchange worked only for the cl br and br i couples ( in both directions ) , but never from cspbcl3 directly to cspbi3 nor in the reverse direction . anion exchange did not alter the cubic shape of the initial cspbbr3 ncs ( figure 3d ) , although after exchange with cl their size decreased slightly , from ( 8.4 1.0 ) nm to ( 8.0 1.4 ) nm ( figure 3a ) , whereas the exchange with i led to a slight increase in size , to ( 9.1 1.3 ) nm ( figure 3c ; see also figure s7 and s8 ) . the xrd pattern collected on the pristine cspbbr3 ncs ( figure 3d ) could be indexed as cubic cspbbr3 ( a = 5.874 , space group pm3m , icsd 29073 ) as detailed in figure s9 , in agreement with what reported by protesescu et al . anion exchange reactions did not alter the crystal phase of the ncs ( see figures 3d , s4b and s9 ) and the patterns collected on the almost fully exchanged ncs were in good agreement with those recorded on directly synthesized cspbi3 ( a = 6.18 , space group pm3m , icsd 181288 ) and cspbcl3 ( a = 5.605 , space group pm3m , icsd 29072 ) ncs . the xrd patterns of partially exchanged ncs too could be ascribed to the same cubic phase : as expected , upon incorporation of cl , the cell shrunk and all the peaks shifted to higher angles , while the incorporation of i expanded the cell and the peaks shifted to lower angles ( figure 3d ) . the table also lists the molar ratios between the halide ions added to the nc crude solutions and the br ions in the starting ncs , and the corresponding ratios in the exchanged ncs . from the table , it can be seen that the yield of exchange differed in the two cases ( cl and i ) , the one with the larger i ions being less efficient than the one with the smaller cl ions ( see also the titration curves of figure 2b ) : while a 1.5 ratio of added cl ions to br ions initially present in the ncs yielded ncs with a cl : br ratio over 3 , the same reaction conditions , in the case of i ions , led to ncs with a i : br ratio of only 0.5 . ( a c ) tem images of pristine cspbbr3 ncs ( b ) and of fully exchanged cspbcl3 ( a ) and cspbi3 ncs ( c ) , indicating overall size and size preservation upon anion exchange . ( d ) zoom of the xrd patterns of pristine cspbbr3 ncs ( middle pattern in green ) , of the anion exchanged cspbcl3 ( top , violet ) , cspbi3 ( bottom , red ) and of two intermediate cl ( middle - top , light blue ) and i ( middle - bottom , yellow ) exchanged samples . at the same time , a slight expansion of the lattice parameters could be inferred from the analysis of the xrd patterns ( figure s10 ) , further supporting the replacement of cs ions with the larger ma ions . pure cspbi3 ncs from a direct synthesis , mixed with cspbbr3 ncs , yielded different emitting samples , with pl energies depending on the ratios of the cspbx3 ncs that were mixed , and plqys in line with those observed when performing exchanges with the other halide precursors , as shown in table 2 . anion exchange between ncs took up to several minutes ( under stirring ) to reach steady state , most likely because the halides ions had to shuttle from nc to nc through the solution phase , a process that in these exploratory experiments was not optimized , for example by the addition of proper stabilizing agents . anion exchange was further supported by xrd patterns ( figure 4b ) of the cspb(br : cl)3 and cspb(br : i)3 samples prepared by mixing pure cspbbr3 and cspbi3 ncs solutions , which indicated that the halides ions redistributed homogeneously throughout the ncs : xrd patterns were indeed compatible with single phase ncs with average unit cells spacings that were intermediate between those of the parent particles . finally , in analogy with the failed cl i exchange attempts discusses earlier , mixing cspbcl3 and cspbi3 ncs resulted in a transparent clear sample and with a quenched pl ( see figure s13 ) , indicating dissolution of the ncs . , we have demonstrated a fast and simple postsynthesis anion exchange approach to access a wide variety of cspbx3 perovskite ncs , with preservation of shape and crystal structure of the starting cspbbr3 sample . the exchanged ncs span the whole visible region of the electromagnetic spectrum , with the pl easily controllable by the amount of added halide precursors , and in general with optical and structural properties in line with those of the same ncs prepared by direct synthesis . we have also shown that the ability of cspbx3 perovskite ncs to mutually exchange halide ions can be exploited as an additional tool to tune the optical and structural feature of the resulting materials , by simply mixing solutions of ncs having different halide compositions .

polyynes , and materials based on chains of linear sp carbon atoms , have long been a focus of research , as models for carbyne , the elusive 1d allotrope of carbon . this field has been stimulated by the extraordinary mechanical , charge - transport , and nonlinear optical properties of these materials and by the use of polyynes as precursors to other carbon - rich nanostructures . recently , chalifoux and tykwinski developed efficient methods for preparing end - capped polyynes with up to 22 cc units ( the longest polyyne yet isolated , corresponding to a rod of carbyne of length 5.6 nm ) . the stabilities of polyynes generally decrease as the acetylenic chains become longer , suggesting that supramolecular encapsulation may be a valuable strategy for controlling reactivity . in this context , we , and others , reported the synthesis of polyyne rotaxanes , such as hm in which a phenanthroline - based macrocycle m(18 ) is threaded around a hexayne chain h ( chart 1 ) . these rotaxanes provide an opportunity to control the environment of the polyyne chain , for example , we showed that rhenium(i ) can be complexed to the threaded macrocycle to give hm(re ) , in which a re(co)3cl unit is held in contact with the hexayne core . most phenanthroline - re(co)3cl complexes exhibit bright luminescence , but during our initial study we found that the luminescence of m(re ) is totally quenched in hm(re ) . this result was surprising because the homo lumo gap of the hexayne ( absorption max 315 nm ) appears to be too large to accept excitation from the re(co)3cl unit ( emission max 605 nm ) , while the electron affinity of the polyyne is insufficient for it to quench the rhenium luminescence by photoinduced electron transfer . here we present a detailed investigation of the five compounds shown in chart 1 , using two ultrafast pump probe techniques : uv nir transient absorption ( ta ) and time - resolved infrared ( trir ) spectroscopy , both with pumping at uv wavelengths . the results provide many insights into the photophysics of polyyne chains and reveal that the luminescence quenching in hm(re ) originates from rapid triplet energy migration from the lowest metal - to - ligand charge transfer ( mlct ) excited state of m(re ) to the threaded hexayne ( time constant : = 1.5 ns ) . trir is an excellent technique for examining the kinetics of transient species , with a time - resolution of < 200 fs . when investigating the photophysics of polyynes , strong signals are observed for the cc triple - bond stretch ( 21002200 cm ) , providing information on ground - state depletion and recovery ( through the negative a signal ) and probing the evolution of singlet and triplet excited states , which can be distinguished by their different cc stretch frequencies ( reflecting their different extents of bond length alternation ) . despite these attractive features , there have been no previous ultrafast trir studies of polyynes , which is surprising because , despite extensive investigation , the excited states of polyynes remain poorly understood . theoretical and experimental studies agree that the first singlet excited states ( s1 ) are generally dark states , which means that s0s1 transitions are dipole forbidden . consequently , the absorption spectra are dominated by transitions to higher singlet excited states ( s0 sn ) , while the s0 s1 absorption band is weak or unobservable . internal conversion ( ic , sn s1 ) is very rapid , but fluorescence from s1 is normally too slow to compete with intersystem crossing ( isc , s1 t1 ) and nonradiative decay ( ic , s1 s0 ) . unsubstituted polyynes , h(cc)n h , exhibit an intense allowed absorption band to a high - energy singlet state ( g u ) as well as weak absorption bands at longer wavelengths , which are about 1000-times less intense and involve low - lying singlet states ( g u and g u ) . other polyynes , r(cc)n r , show similar behavior , although their electronic structures can be complicated by involvement of the end groups ( particularly if r = aryl ) , and it can be difficult to identify the dark states . knowledge of the energy of the dark s1 state is essential for estimating the optical homo computational studies have shown that the excited states of polyynes generally have reduced bond length alternation , i.e. , the cc triple bonds become longer and the c c single bonds become shorter . this prediction is supported by analysis of vibrational structure in electronic transitions and by time - resolved resonance raman studies on the t1 state of diphenyl butadiyne ( ph cc cc ph ) , and an intriguing metastable photoexcited polyyne has been identified at low temperatures which appears to have a cumulenic structure ( = c = c= rather than cc ) . here we show that optical excitation generates a short - lived higher singlet state ( sn ) with a lifetime of 0.9 ps in the free hexayne h and 1.8 ps in the rotaxane hm . trir also enables the s1 and t1 states of the polyyne to be directly observed ; comparison of the cc stretch frequencies ( s0 : 2191 , 2165 cm ; s1 : 2077 , 1737 cm ; t1 : 2047 , 1610 cm ) with results from dft calculations provides clear evidence for an increase in cumulenic character in the excited states . we also observe ultrafast singlet excitation energy transfer ( eet ) in hm , from the singlet excited state of the macrocycle to the dark s1 state of the polyyne ( eet , = 3.0 ps ) . the trir spectra show that this energy - transfer process directly populates the vibrationally equilibrated s1 state of the hexayne , whereas sn s1 internal conversion creates a hot vibrational state , which cools over about 5 ps . to gain more insights into this family of compounds , we have also compared the x - ray crystal structures of h , hm , hm(re ) , and m(re ) . the structure of the hm(re ) rotaxane complex shows that the re(co)3cl unit is pushed close to the c12 chain , with oc distances of less than the sum of the van der waals radii , with the polyyne bending round the metal center . the interactions between the re(co)3cl center and the hexayne chain appear to be weak in the ground state , while the excited - state trir spectra revealed mutual electronic perturbation of both components . the unthreaded hexayne dumbbell h , rotaxane hm , re(co)3clrotaxane hm(re ) , and re(co)3clmacrocycle m(re ) complexes and macrocycle m(18 ) were prepared as described previously . spectroscopic measurements were performed in air - saturated or deoxygenated dichloromethane solutions of spectroscopic quality ( aldrich ) . deoxygenation was achieved by three freeze pump thaw cycles , and sample preparation was carried out in glovebox under nitrogen atmosphere . crystals of re(co)3clmacrocycle m(re ) were grown by slow evaporation of a solution in toluene at 4 c ; single crystal x - ray diffraction data were collected at 150 k using an oxford diffraction ( agilent ) supernova a diffractometer . crystals of the re(co)3clrotaxane complex hm(re ) were grown from dichloromethane solutions by slow diffusion of methanol vapor at room temperature ; single crystal x - ray diffraction data were collected at 100 k using synchrotron radiation at the diamond light source , beamline i19 . in both cases , m(re ) was found to crystallize in p-1 with one molecule of m(re ) and two molecules of toluene in the asymmetric unit , while hm(re ) crystallized in p21/n with one molecule of hm(re ) in the asymmetric unit . the structures were solved using charge flipping with superflip and refined using least - squares within crystals . the difference map for hm(re ) indicated the presence of diffuse electron density believed to be disordered solvent . crystallographic data ( excluding structure factors ) have been deposited with the cambridge crystallographic data centre ( ccdc 10360721036073 ) , and copies of these data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif . the ultra instrument at the stfc rutherford appleton laboratory was used and is described in detail elsewhere . briefly , an amplified titanium sapphire laser ( thales optronique ) produces 50 fs pulses at a 10 khz repetition rate . the first generates uv pump pulses through an optical parametric amplifier ( topas opa ) at 310 or 350 nm . the second part of the fundamental is used to generate probe pulses : 400 cm broad mid - ir probe pulses through a second opa for trir experiments or a white light continuum ( 330680 nm ) through focusing of the fundamental into a 2 mm caf2 plate for ta experiments . typical pump and probe beam diameters in the sample were 130 and 80 m , respectively , the lower diameter probe being used to ensure only activated sample is interrogated . all experiments were carried out with the pump and probe set at the magic angle ( 54.7 ) . the probe pulses were split in two , one part subsequently focused into the sample overlapping with the pump beam and the other for use as a reference of laser spectral and intensity variations . the probe and reference pulses were dispersed onto array detectors to measure the spectrum of each laser shot ( using 128-element hgcdte detectors for trir experiments and 512-pixel silicon single - diode arrays for ta experiments ) . by modulating the pump pulses at 5 khz ( the probe measured at 10 khz ) normalized pump on pump off difference spectra can be collected in real time . the trir and ta spectra ( see below ) are presented as normalized difference spectra ( each spectrum averaged over a few seconds ) , thus , positive bands correspond to photogenerated ta , while negative bands indicate ground - state bleaching . the pump probe time delay was controlled by an optical delay line for femtosecond to nanosecond measurements , while the nanosecond to microsecond measurements were recorded using the same spectrometers but replacing the femtosecond pump laser with a 1 ns duration 5 khz ( for pump on pump off measurements as above ) laser operating at 266 nm . the sample absorbance was 0.20.3 at the pump wavelength ( 266 , 310 or 350 nm ) in a 0.1 mm path length flow cell with 2 mm caf2 windows . sample integrity was checked by ir and uv vis spectra measured before and after each experiment . all spectral fitting procedures and kinetic analysis were performed using originpro 8.5.1 software . the hexayne h was modeled as a methyl - capped hexayne , me c12me . the applicability of this model was assessed by comparison to bent structures and a tert - butyl capped model ( cf . ref ( 40 ) ) . the b3lyp functional was used in conjunction with the dunning cc - pvtz basis set . this level of theory has been shown to reproduce vibronic structures of polyyne absorption bands . similar results were obtained with b3lyp/6 - 31 g * * , pbe0/cc - pvtz , and pbe0/6 - 31 g * * ( see si ) . the first singlet excited state ( s1 ) was optimized in d3 symmetry using td - dft ( b3lyp ) . the first triplet excited state ( t1 ) was treated with ub3lyp , in c2 symmetry . vibrational frequencies were calculated from analytical force constants for s0 and t1 and from numerical force constants ( td - dft ) for s1 . the converged structures were confirmed to be minima by the absence of imaginary frequencies . a simple linear scaling factor ( 0.96 ) is applied to calculated vibrational frequencies presented in the main text ( unscaled frequencies are tabulated in the si ) . the structures of the hexayne dumbbell h , rotaxane hm , rhenium rotaxane hm(re ) , and rhenium - macrocycle complex m(re ) are compared in figure 1 . the two rotaxanes , hm and hm(re ) , show similar bond length alternation within experimental error : 0.143 0.008 in hm and 0.155 0.010 in hm(re ) , compared with 0.158 0.008 for h. the closest distance between carbonyl group oxygen and polyyne chain carbon in hm(re ) is 3.085(5 ) ( co c(5 ) ) , which is less than the sum of the van der waals radii ( 3.35 ) ; the three shortest cocc contacts are shown in figure 1e . the hexayne chain in hm(re ) is severely bent , with a single - arc conformation , unlike the double - arc of h. the angle between the two terminal c1 and c12 atoms and the centroid of the middle c6c7 bond of hm(re ) is 155.6(1) ( compared with 171.8(1) in hm and 180 in h ) ; the average value for this parameter for the 22 hexaynes in the cambridge structural database is 175.3 ( s.d . 7.1 ) , and only one other hexayne has been reported with a comparable angle ( 155.3(2) ) . the angles between the three carbonyl groups lie in the range 85.20(17)92.55(17) ( with an average of 88.7 ) in hm(re ) and 85.47(16)90.53(16) ( with an average of 87.9 ) in m(re ) , and the local cs symmetry of the rhenium center is preserved in both re(co)3cl complexes ( figure 1 ) . solid - state structures of ( a ) hexayne h , ( b ) rotaxane hm , ( c ) re(co)3clrotaxane hm(re ) , and ( d ) re(co)3clmacrocycle m(re ) . the uv absorption spectrum of hexayne h in dichloromethane ( figure 2a ) shows an intense band at 275325 nm , with a well - defined progression of 2060 cm , corresponding to the cc vibrational mode of the sn state . the vibrational progression matches well with the ground - state cc stretch frequency ( 21652191 cm ) seen in the ir spectrum ( figure 2b ) . hexayne h also displays a weak absorption band at 350450 nm , dominated by a vibrational progression of 20402169 cm ( inset in figure 2a ) . we assign this band to the forbidden s0 s1 transition , as reported in the absorption spectra of many other long polyynes , although we can not exclude the possibility that it is a transition to another low - lying singlet excited state , other than s1 . this s0 s1 transition at 350450 nm is about 500 times weaker than the s0 sn absorption at 275325 nm . for the hm rotaxane , the third vibrational band of the s0 s1 transition is obscured by the intense absorption of macrocycle at 390 nm , but the first two bands are clearly visible and do not change their position , while in the hm(re ) rotaxane , all the s0 s1 transitions are hidden by the intense m(re ) absorption . ( a ) uv vis absorption and ( b ) ir spectra of the hexayne h ( red ) , the rotaxane hm ( blue ) , hm(re ) ( green ) , m(re ) ( orange ) , and m ( brown ) , all recorded in ch2cl2 . concentrations of the compounds for ir measurements are 1.5 mm ; path length 0.5 mm . ( b ) the top picture depicts the three stretching modes of carbonyl groups . the same color code is used for spectra in ( a ) and ( b ) . the absorption spectrum of the macrocycle m ( figure 2a ) consists of a broad , lower energy * band , extending to 390 nm , and a n * band at around 275 nm . the absorption spectrum of the rotaxane hm is essentially the sum of its two components , thread and dumbbell , with a slight ( 4 nm ) bathochromic shift of the hexayne peaks ( figure 2a ) ; the presence of the threaded macrocycle slightly changes the environment of the polyyne chain . the m(re ) absorption spectrum consists of two transitions : a low - energy mlct , extending to about 490 nm , and a higher energy intraligand ( il ) * transition . the spectrum of the hm(re ) is the sum of its components with a slight ( 2 nm ) red - shift in the hexayne peaks ( figure 2a ) . the hexayne h has a broad collective band with two overlapping peaks at 2165 and 2191 cm , which are characteristic of long polyyne chains . the assignment of these bands is discussed below ( see section on calculated ir spectra ) . these cc bands are unshifted in the rotaxanes hm and hm(re ) , indicating that threading does not perturb the ground - state vibrational structure of hexayne . the three carbonyl groups of the re(co)3cl moiety give rise to three sharp , intense bands at 1894 ( a(2 ) ) , 1932 ( a ) , and 2025 ( a(1 ) ) cm for hm(re ) and 1885 ( a ) , 1924 ( a(2 ) ) , and 2022 ( a(1 ) ) cm for m(re ) , which are characteristic of fac - rhenium tricarbonyl polypyridyl complexes . the positions of the ir bands indicate the cs ( or pseudo c3 ) symmetry of the re center : two normal (co ) modes are symmetric ( a(1 ) and a(2 ) ) involving all three co ligands , while one mode ( a ) is antisymmetric involving only in the plane stretching of two co groups ( figure 2b ) . on threading , the a and a(2 ) bands of re(co)3clmacrocycle m(re ) shift to higher wavenumber , by 8 and 9 cm , respectively . we carried out a systematic study of the dynamic processes in the excited states of this family of compounds , using trir and ta spectroscopy . initially , we examined the excited - state behavior of the hexayne h and the macrocycle m separately , and then we moved to the topologically more complex rotaxane hm , rhenium tricarbonyl complex of macrocycle m(re ) , and rhenium tricarbonyl rotaxane complex hm(re ) . two regions of the trir spectra are particularly informative : the high - frequency region at 19002300 cm and the fingerprint region at 14001900 cm . the kinetic data for h , hm and hm(re ) are summarized in tables 14 . excitation of hexayne h at 310 nm results in an immediate bleach of the ground - state cc stretch at 21652191 cm ( within the time - resolution of our instrument , < 200 fs ) , and a new absorption band appears at 2063 cm ( figure 3a ) . this band , which corresponds to the cc stretch in a singlet excited state , shifts to 2077 cm ( = 0.8 0.2 ps , figures 3a and s3a ) in parallel with a 15% band narrowing ( measured at the half - maximum ) . these two spectral changes are indicative of rapid excited - state vibrational energy redistribution , convolved with solvent reorganization around the nascent excited state , and fast sn s1 internal conversion , as discussed below . previously , the formation of a vibrationally hot polyyne upon vertical excitation was postulated from the broadness of the ta spectra at short time delays . to our knowledge , this is the first time that the vibrational relaxation of a hot excited long polyyne has been characterized via vibrational spectroscopy . trir spectra and excited - state kinetics of hexayne dumbbell h excited at 310 nm ( a c ) , rotaxane hm excited at 310 nm ( d f ) and 350 nm ( g i ) . laser energy : 80100 nj , solvent : ch2cl2 . the s1 band at 2077 cm decays to a new band at 2047 cm ( figure 3b ) which we assign to the t1 triplet excited - state cc stretch ( based on the kinetics and on the match with calculated vibrational frequencies ; see below ) ; the rates of this decay ( 1 = 0.44 0.02 ns ) and of the growth of the band at 2047 cm ( 2 = 0.48 0.05 ns , figure s1c ) give the rate of isc , as summarized in table 1 . the intensity of the cc ground - state bleach band at 21752195 cm does not change during isc , indicating that the triplet yield is near unity . spectacular changes are also observed in the fingerprint region ( figure 3c ) , and these are useful for confirming the species assignments and associated kinetics . immediately after excitation , a band appears at 1740 cm , which decays gradually , giving a new band at 1610 cm . these two bands display similar kinetics ( 1740 cm decay time : 1 = 0.40 0.04 ns ; 1610 cm rise time : 2 = 0.38 0.02 ns , figure s1e ) to the bands at 20402080 cm and are attributed to the same excited species , s1 t1 . td dft calculations provide assignments for these low - energy excited - state ir bands , as discussed below ( see section on calculated ir spectra ) . the lifetime of the t1 state of h in deoxygenated dichloromethane is 16.0 3.6 s ( estimated from the 1610 cm band ; excitation with a 1 ns pulse at 266 nm ; figure s2 ) . at early times , the fingerprint band of the singlet excited state at 1741 cm shifts to 1737 cm , while decreasing in intensity , over 3 ps ( figure s3b ) . the direction of this spectral shift is opposite to that expected for intramolecular relaxation and solvation processes , and we tentatively assign it to the formation of s1 by internal conversion from sn , populated by a symmetry - allowed s0sn transition . ta spectra show a similar fast process : excitation at 310 nm generates a band at 421 nm , which grows with a rise time of 1 = 0.93 0.08 ps ( figure 4 ) . this rise is too slow for direct vertical excitation ( in trir spectra the bleach of ground - state cc stretching appears within the 200 fs instrument time - resolution ) . thus , the most reasonable explanation is the formation of the s1 state via the symmetry - allowed s0 sn transition . at longer times , ta confirms that the s1 state undergoes intersystem crossing , resulting in a band at 343 nm , corresponding to the t1 state ( figure 5a ) . the kinetics of t1 band growth at 343 nm ( 2 = 0.45 0.09 ns ) and s1 decay ( 1 = 0.44 0.02 ns ; figure s4 ) are similar to those extracted from trir spectra . growth of the s1 state of hexayne h following excitation at 310 nm in ch2cl2 : ( a ) ta spectra and ( b ) signal at 421 nm ( 1 = 0.93 0.08 ps ) . examination of the nir ta showed a short - lived broad band at 1185 nm which appears immediately following the excitation pulse ( figure s5 ) . the decay time of this band ( 1 = 0.82 0.13 ps ; figure s5b ) matches the growth of the s1 state in the visible region of ta spectrum ( 1 = 0.93 0.08 ps ) , and we therefore assign it to the sn state of the hexayne . to our knowledge , this is first time that a sn s1 transition of any polyynes has been characterized by trir and nir ta spectroscopy , although the s1 and sn states of the diphenylacetylene were studied by emission spectroscopy . -conjugated terminal aryl groups may play an important role in the excited - state dynamics of diphenylacetylene , while the supertrityl capped hexayne h presents a pure case of a polyyne chain , so its behavior can be regarded as intrinsic to the sp - hybridized carbyne chain . ta spectra of ( a ) hexayne h , ( b ) rotaxane hm , and ( c ) re(co)3clrotaxane hm(re ) excited at 310 nm . the excited states of 2,9-diaryl phenanthrolines have been studied before , and the * singlet - state lifetime is estimated to be a few ns in dichloromethane , while the triplet * state lives for about a second at 77 k. as expected , the trir spectrum of macrocycle m does not exhibit any features in the high - frequency region ( on excitation at 350 or 310 nm ) . in the fingerprint region , transient features appear at 1470 , 1510 , and 15201600 cm ( figure 6a ) . the transients have similar decay rates , indicating that they arise from the same excited species . the sharpest peak at 1504 cm was chosen for kinetic analysis and fitted to a single - exponential decay yielding a singlet lifetime of 2.1 0.2 ns ( figure s6 ) . the ta spectra show a very broad band between 350 and 700 nm ( figure s7 ) with distinguishable peaks at 365 and 652 nm . within a few ns , the peak at 365 nm decays and a new peak grows at 436 nm , but the total intensity of the broad envelope does not change dramatically over time . the decay constant of the 365 nm band is 1 = 1.8 0.2 ns , matching with the lifetime of the 1504 cm trir band . we ascribed the band at 365 nm to the singlet and the band at 436 nm to the triplet excited - state macrocycle . it is possible to excite the different components of the rotaxane separately : at 350 nm , where only the macrocycle absorbs , or at 310 nm , where absorption by the hexayne predominates ( both components absorb at 310 nm , but the molar absorption coefficient of the hexayne is higher by a factor of 12 ) . the trir spectra of hm , when excited at 310 nm , are almost identical to those of hexayne h ( compare figure 3d f with figure 3a c ) . s1 state undergoes rapid energy redistribution , and the observed frequency shift resembles that of the h ( figure 3d ) . the bleach of the cc stretch around 2190 cm does not change intensity during isc , indicating that the triplet yield is near unity , as in the hexayne h. the kinetics are summarized in table 2 . the data from the fingerprint region at late time delays ( > 15 ps ) match those from the high frequency region : the dumbbell s1 band at 1741 cm decays giving rise to a t1 band at 1610 cm ( figure 3f ) . excitation of the rotaxane hm at 350 nm , where only the macrocyclic component absorbs , was investigated to test for energy transfer between the two components . ( irradiation of free hexayne h at this wavelength does not produce detectable ta and trir signals , because h has negligible absorption at 350 nm . ) on excitation of hm at 350 nm , the ground - state cc stretch band gradually bleaches , at around 2190 cm , and the s1 polyyne band gradually grows at 2077 cm ( figure 3 g ) , indicating transfer of singlet excitation from the macrocycle to the dumbbell . after 20 ps , the trir spectra from excitation at 350 nm become identical to those for excitation at 310 nm ( compare figure 3g i with figure 3d f ) , and the decay kinetics are the same ( figure s1 m and table 3 ) . the spectral changes in the peak at 20602080 cm , which are observed on a time scale of 1 ps upon excitation of hm at 310 nm , do not occur when the system is excited at 350 nm ( compare figure 3 g with figure 3d ) . this difference indicates that the dominant eet process directly populates the s1 state ( without going via the sn state ) and that the early signal is not broadened or shifted by sn contributions , vibrational energy redistribution or solvent reorganization . examination of the fingerprint region of the trir spectra of hm after excitation at 350 nm showed a fast ( < 10 ps ) decay of a band at 15001600 cm corresponding to the singlet macrocycle and the parallel rise of the s1 excited hexayne ( 1741 cm ) ( figure 6b ) . this is direct evidence of singlet singlet energy transfer from the excited - state macrocycle to the ground - state hexayne . the decay of the macrocycle band at 1560 cm ( 1 = 3.2 0.2 ps ) and the growth of the singlet s1 hexayne at 1741 cm ( 1 = 2.7 0.2 ps ) are single - exponential ( figure 6c ) . the growth of the 2077 cm band of the singlet hexayne ( 1 = 2.6 0.1 ps ) and the bleach of the ground - state stretching mode at 2189 cm ( 1 = 2.9 0.3 ps ) are also single - exponential . the very weak s0 s1 oscillation strength of the polyyne implies that this fast eet occurs by a dexter mechanism . the triplet lifetime of the hexayne component in hm ( 1 = 14.0 2.2 s in deoxygenated solution , table 2 , figure s9 ) is similar to that of the hexayne h. trir spectra of ( a ) macrocycle m and ( b ) rotaxane hm excited at 350 nm . ( c ) kinetics of the singlet eet from m to the dumbbell estimated from the different spectral regions and bands , excited at 350 nm . ta spectra of rotaxane hm , with excitation at 350 nm , showed formation of a transient s1 absorption band at 421 nm ( figure 7 ) , similar to that of free dumbbell h. the rise - time of this band ( 1 = 3.2 0.4 ps , figure 7a ) , due to s1(macrocycle ) s1(hexayne ) , agrees with the trir data . on a longer time scale , this band decays ( 2 = 0.42 0.04 ns ) due to intersystem crossing . the growing t1 state was observed as a shoulder at 360 nm , but overlap with the excitation wavelength made it difficult to observe the triplet ( figure 7b ) . ( b ) decrease of singlet and increase of triplet ( as a shoulder ) ta bands . ta spectral changes of hm excited at 310 nm are similar to those in dumbbell h. the singlet s1 band at 422 nm grows at early time delays ( 1 = 1.8 0.2 ps , figure s10 ) due to sn s1 relaxation . later , this band decays , and the triplet band at 343 nm grows ( figures 5b and s4d and table 2 ) . the eventual formation of the polyyne triplet state was confirmed by carrying out ta experiments of the rotaxane hm in the presence of -carotene . the excited - state dynamics of 1,10-phenanthroline - based re(co)3cl complexes have been thoroughly described in the literature and will only be outlined briefly here for discussion of changes when part of the rotaxane . optical excitation creates both il and mlct states that undergo intersystem crossing to at least two thermally equilibrating , hot , triplet il and mlct states , over 150 fs . the il state arises from phenanthroline * transition , and the mlct state also involves the d orbitals of re atom . internal conversion between the il and mlct states takes place on a time scale of ps to ns , depending on the solvent and ligand structure . later , the mlct state relaxes to the vibrationally cooled lowest excited state , which decays on the ns time - scale via radiative and nonradiative pathways . on excitation of m(re ) at 350 nm , trir spectra show several positive bands from photogenerated species as well as negative bands originating from the depleted ground state ( figure 9a ) . indicate that the order of the bands in the excited state is different from that in the ground state ; bands were observed at 1944 , 1985 , and 2048 cm due to a , a(2 ) , and a(1 ) modes , respectively , and we adopt this assignment . the broad positive band at 19352005 cm represents the overlap of a and a(2 ) excited - state bands . the time - dependent shift of these two bands is complex , as it incorporates the instantaneous shift , as a result of franck condon excitation and formation of an excited state of mixed il and mlct character stemming from combined d * and * excitation . in addition , all of these processes are accompanied by solvent reorganization . over 10 ps , the low - energy shoulder of the excited - state a band decreases . the higher energy weak shoulder next to the excited - state a(2 ) band also decreases over 10 ps . the a band undergoes a 17 cm shift to higher frequency with a 26% intensity increase , while the ground - state a band bleach increases by 22% . the bleach and transient intensity changes can be attributed to changes in the relative intensity of overlapping ta bands , as the character of the il / mlct state evolves at early times , with the mlct becoming dominant . the a(1 ) excited band at 2054 cm shows a more pronounced intensity increase ( 62% ) , hypsochromic shift ( 12 cm ) , and band narrowing ( 64% ) ( figure 9a ) . whereas the shift to higher frequency is due to an increasing contribution of the mlct state , the band - narrowing apparently involves vibrational relaxation steps , stemming from local - solvent reorganization . these spectral changes resemble the excited - state dynamics of the re(co)3(4-et - pyridine ) 2,2-bipyridine complex . we ascribe the final trir spectral pattern , seen at longer time delays , to the relaxed lowest excited - state triplet ( see figure 8 for a summary of the excited states and kinetics ) . kinetic data were extracted from trir spectra , exciting m(re ) at 266 nm , so as to estimate the lifetime of the triplet excited state . the triplet lifetime is 93 ns under oxygen - free conditions ( figure s12 ) and 63 ns in the presence of air ( figure 9b , table s1 ) . energy diagram summarizing excited - state processes in ( a ) dumbbell h and rotaxane hm and ( b ) m(re ) and re(co)3clrotaxane complex hm(re ) . in the fingerprint region of the trir spectrum of m(re ) , two bleaches of ground - state bands at 1497 and 1608 cm appear immediately after excitation , and recover within 3040 ps , while the transient bands 1480 and 1595 cm grow slightly ( figure 9c ) . the positions of the bleach bands in m(re ) and m are similar , suggesting that they originate from the phenanthroline framework , and are not strongly perturbed by the presence of the rhenium . furthermore , in m(re ) , the recovery of these bleach bands is partial , due to overlapping positive bands , and shows single - exponential recovery kinetics ( 1497 cm : 1 = 6.8 0.6 ps ; 1608 cm : 1 = 14.0 1.2 ps ; figure s13d ) , on a time scale similar to the (co ) shifts observed in the transients in the carbonyl stretching region . thus , we tentatively assign the bleach recovery at 1497 and 1608 cm to the cooling and molecular reorganization processes of the initially formed unrelaxed states . the ground - state co and cc stretch vibrations of hm(re ) occur at different frequencies , allowing each component to be observed separately . trir spectra of hm(re ) following excitation of the macrocycle component at 350 nm show immediate bleaching of ground - state carbonyl stretching bands ( figure s14 ) . the excited - state co bands at 2051 and 1975 cm shift to high frequencies within 20 ps , due to the il mlct conversion and vibration relaxation , as in m(re ) . however , in contrast to m(re ) , the co bleaches of hm(re ) undergo rapid recovery ( figure 9d , e ) . the recovery of the co bleach is accompanied by bleaching of the cc band at 2190 cm , indicating energy transfer from re(co)3clmacrocycle to the hexayne chain . the rate of cc bleaching matches that of the recovery of all three co bands ( figure 9e ) . the trir spectra of hm(re ) in the high - frequency region do not reveal the nature of energy transfer , as the triplet and singlet - state bands of the hexayne at 20402080 cm overlap with excited - state a(1 ) band . the necessary information was obtained from the fingerprint region : the initial bleach of the excited re(co)3clmacrocycle at 1607 cm recovers , and then the band corresponding to the triplet - state hexayne at ca . 1610 cm grows over 2 ns ( figures 9f and s13 ) . at the same time the triplet re(co)3clmacrocycle band around these spectral changes clearly demonstrate triplet triplet energy transfer from the re(co)3clmacrocycle moiety to the threaded hexayne . the growth kinetics of the 1607 cm band ( figure s13h ) are biexponential ( 1 = 14.0 0.6 ps , a1 = 0.35 ; 2 = 1.13 0.05 ns , a2 = 0.65 ) with the fast component matching that of decay of the bleach at 1607 cm in re(co)3clmacrocycle m(re ) . this implies that in excited hm(re ) , il / mlct relaxation , vibrational cooling , and solvent reorganization take place in parallel with triplet energy transfer to the hexayne from a relaxed mlct state . indeed , the hexayne ground - state cc band at 2200 cm does not show any bleach over the first 2030 ps , while the il / mlct and other relaxations take place ( figures 9f and s14 ) . the trir spectra of hm(re ) using excitation at 350 nm did not show any singlet excited - state polyyne band , before or after energy transfer , excluding the possibility of singlet energy transfer from the re(co)3clmacrocycle . singlet energy transfer does not occur because intersystem crossing in the re(co)3clmacrocycle complex is ultrafast , and all spectral changes originate from triplet excited re(co)3clmacrocycle . thus , triplet sensitization of the hexayne in hm(re ) upon excitation of the molecule at 350 nm explains the quenching of emission of re(co)3clmacrocycle m(re ) at room temperature by the threaded dumbbell . the efficiency of eet was estimated by comparing the intensities of the cc band at 2200 cm and the a(1 ) c = o at 2054 cm in the ground - state ir spectra and in the trir spectra , at the time of maximum bleach ( figure s15 ) . trir spectra and excited - state kinetics of the m(re ) ( a c ) excited at 350 nm , hm(re ) excited at 350 nm ( d f ) , and 310 nm ( g i ) . . another noteworthy feature of the hm(re ) rotaxane is the perturbation of the ground - state hexayne by excited m(re ) ( after excitation , before energy transfer ) which appears as a slightly enhanced absorption at 2200 cm ( figures 9d and s16 ) . later , when m(re ) is in its relaxed state , after eet , the carbonyl bleaches do not decay to zero , although all positive transient bands disappear ( figure s16 ) as a result of electronic perturbation by the nearby triplet hexayne . in both cases , this effect serves to report the presence of a nearby excited - state constituent . the triplet - state polyyne lifetimes in deoxygenated and oxygen - containing solutions ( figure s17 ) are similar to those of the rotaxane hm ( table 2 ) . direct excitation of the hexayne component of hm(re ) at 310 nm generates the singlet - state hexayne via direct excitation as well as triplet macrocycle ( figure 2a ) followed by triplet energy transfer to the hexayne . in figure 9 g , for example , the band around 2070 cm , corresponding to singlet hexayne , decreases gradually ( 1 = 0.35 0.03 ns ) , while the cc bleach , which appeared immediately upon excitation , increases ( 2200 cm ) due to triplet energy transfer from macrocycle to hexayne . upon triplet eet the excited macrocycle bands also decrease and the (co ) bleach recovers ( figure s13 , table s1 ) . comparable changes are observed in the fingerprint region ( figure 9i ) where the singlet hexayne band at 1741 cm decreases and triplet band at 1610 cm increases . kinetic analysis of the fingerprint region shows that the singlet hexayne band at 1741 cm decays in a single - exponential fashion ( figure s13 , 1 = 0.35 0.01 ns ) ; the decay constant matches that of the singlet band at 2070 cm . ta spectra of hm(re ) excited at 310 nm show the formation of singlet and triplet excited states similar to rotaxane hm and hexayne h excited at 310 nm ( figure 5a , b ) . within 5 ps the band of the singlet hexayne at 422 nm grows over the broad transient envelope band of the macrocycle ( figure s18 ) . the singlet - state band at 422 nm decays single - exponentially with 1 = 0.34 0.01 ps . the broad band of the macrocycle also decreases , and the growth of hexayne triplet band at 343 nm is single exponential ( 2 = 0.49 0.02 ns , figure s4f ) . both 1 and 2 values we calculated the ir spectra of a model hexayne ( me c12me ) in its ground and excited states , to gain insights into the structural changes in the electronic excited states that are responsible for the observed trir bands of hexayne h. in particular , we sought to understand why the experimental s1 and t1 spectra each exhibit one vibration in the fingerprint region ( 15001900 cm ) and one vibration in the high - frequency region ( 20002250 cm ) . symmetric polyynes have been thoroughly studied by raman spectroscopy , but few investigations of their ir spectra have been reported . the combined computational and experimental results provide compelling evidence for cumulenic character in the s1 and t1 electronic states of h. the calculated bond lengths of the s0 , s1 , and t1 states ( figures 10 and s19 ) reveal a reduction in bond length alternation in s1 and t1 , compared with s0 , indicating that the excited states have significant cumulenic character , particularly near the center of the chain . these results agree well with previous work , and the calculated bond lengths in the s0 are close to those from the crystal structure of h. the presence of two cc excited - state bonding regions ( quasi - acetylenic at the ends and quasi - cumulenic in the middle ) is supported by the experimental and calculated vibrational spectra . within the spectral region of interest ( 15002250 cm ) , the calculated spectra show remarkable agreement with the experimental trir data ( figure 11 ) . the s0 vibrational spectrum shows two bands , 2 ( calculated : 2172 cm ; observed : 2165 cm ) and 1 ( calculated : 2207 cm ; observed : 2191 cm ) . for s1 and t1 , the calculated frequencies agree with experiment and predict a 1 band in the cc region ( s1 : 2104 cm ; t1 : 2089 cm ) , and a lower energy 2 band in the fingerprint region ( s1 : 1786 cm ; t1 : 1670 cm ) . on the basis of the match between the calculated and observed spectra , we assign the 1 and 2 bands to asymmetric stretch vibrations of the cc chain , as shown in the insets in figures 11 and s19 . the patterns of atomic displacement for both modes of s1 and t1 show close correspondence , as expected given the similar bonding in these two states ( figure 10 ) . bond length alternation in h ( from crystallographic data , black circles ; ref ( 9 ) ) and in calculated ( b3lyp / cc - pvtz ) s0 ( red circles ) , s1 ( yellow squares ) , and t1 ( purple stars ) states . calculated ir spectra for me - c12-me ( bars ) and experimental trir spectra for h ( lines ) ( a ) 20 ps after excitation ( s1 ) and ( b ) 3.4 ns after excitation ( t1 ) . red , yellow , and purple bars denote calculated ( b3lyp / cc - pvtz ) frequencies for s0 , s1 , and t1 states , respectively . inset plots show bond length displacements for each of the indicated ir modes of the c12 chain , the change in bond length being proportional to the direction and magnitude of the bar . the large change in frequency of the 2 mode ( in contrast to 1 ) in the excited states ( s1 and t1 ) , compared with the s0 ground state , for each electronic state , the higher energy 1 vibration involves greater displacement of the cc bonds at the ends of the hexayne , whereas the lower energy 2 vibration involves displacement of bonds near the center ( insets in figures 11 and s19 ) , making it much more sensitive to the excited - state cumulenic character around the center of the molecule . despite the alluring elemental simplicity of their molecular structures , polyyne chromophores exhibit remarkably rich and complex photophysical behavior . here we have shown that trir , coupled with dft calculations , is an excellent technique for probing their excited - state dynamics . we have also demonstrated that rotaxane synthesis is a powerful strategy for fixing a photoactive unit near the center of a polyyne chain , for studying singlet and triplet energy - transfer through - space , between nonbonded components . previously it was found that butadiyne bridges can mediate quantitative intramolecular triplet eet between covalently bonded chromophores . here we demonstrate the intermolecular triplet eet properties of polyynes in a covalently nonbonded rotaxane . the singlet and triplet excitation energy harvesting properties of polyynes resemble those of the carotenes , which harvest both triplet and singlet energy in photosynthetic systems . it is intriguing to consider why nature exclusively preferred conjugated double rather triple bonds for photosynthetic energy transfer . understanding the excited states of polyyne in supramolecular assemblies , such as the rotaxane studied here , is important for further application of materials based on chains of linear sp carbon atoms in molecular and optoelectronic devices . this study demonstrates how the photochemistry of a system can be controlled through nonbonded interactions , without altering the chromophore or relying on diffusion , through synthesis of rotaxane architectures .

we have used single - crystal x - ray diffraction and time - resolved uv nir ir absorption spectroscopy to gain insights into the structures and excited - state dynamics of a rotaxane consisting of a hexayne chain threaded through a phenanthroline macrocycle and a family of related compounds , including the rhenium(i ) chlorocarbonyl complex of this rotaxane . the hexayne unit in the rhenium - rotaxane is severely nonlinear ; it is bent into an arc with an angle of 155.6(1) between the terminal c1 and c12 atoms and the centroid of the central c c bond , with the most acute distortion at the point where the polyyne chain pushes against the re(co)3cl unit . there are strong through - space excited - state interactions between the components of the rotaxanes . in the metal - free rotaxane , there is rapid singlet excitation energy transfer ( eet ) from the macrocycle to the hexayne ( = 3.0 ps ) , whereas in the rhenium - rotaxane there is triplet eet , from the macrocycle complex 3mlct state to the hexayne ( = 1.5 ns ) . this study revealed detailed information on the short - lived higher excited state of the hexayne ( lifetime 1 ps ) and on structural reorganization and cooling of hot polyyne chains , following internal conversion ( over 5 ps ) . comparison of the observed ir bands of the excited states of the hexayne with results from time - dependent density functional calculations ( td dft ) shows that these excited states have high cumulenic character ( low bond length alternation ) around the central region of the chain . these findings shed light on the complex interactions between the components of this supramolecular rotaxane and are important for the development of materials for the emerging molecular and nanoscale electronics .

Introduction Experimental Section Results and Discussion Conclusion

these rotaxanes provide an opportunity to control the environment of the polyyne chain , for example , we showed that rhenium(i ) can be complexed to the threaded macrocycle to give hm(re ) , in which a re(co)3cl unit is held in contact with the hexayne core . this result was surprising because the homo lumo gap of the hexayne ( absorption max 315 nm ) appears to be too large to accept excitation from the re(co)3cl unit ( emission max 605 nm ) , while the electron affinity of the polyyne is insufficient for it to quench the rhenium luminescence by photoinduced electron transfer . here we present a detailed investigation of the five compounds shown in chart 1 , using two ultrafast pump probe techniques : uv nir transient absorption ( ta ) and time - resolved infrared ( trir ) spectroscopy , both with pumping at uv wavelengths . the results provide many insights into the photophysics of polyyne chains and reveal that the luminescence quenching in hm(re ) originates from rapid triplet energy migration from the lowest metal - to - ligand charge transfer ( mlct ) excited state of m(re ) to the threaded hexayne ( time constant : = 1.5 ns ) . when investigating the photophysics of polyynes , strong signals are observed for the cc triple - bond stretch ( 21002200 cm ) , providing information on ground - state depletion and recovery ( through the negative a signal ) and probing the evolution of singlet and triplet excited states , which can be distinguished by their different cc stretch frequencies ( reflecting their different extents of bond length alternation ) . knowledge of the energy of the dark s1 state is essential for estimating the optical homo computational studies have shown that the excited states of polyynes generally have reduced bond length alternation , i.e. this prediction is supported by analysis of vibrational structure in electronic transitions and by time - resolved resonance raman studies on the t1 state of diphenyl butadiyne ( ph cc cc ph ) , and an intriguing metastable photoexcited polyyne has been identified at low temperatures which appears to have a cumulenic structure ( = c = c= rather than cc ) . trir also enables the s1 and t1 states of the polyyne to be directly observed ; comparison of the cc stretch frequencies ( s0 : 2191 , 2165 cm ; s1 : 2077 , 1737 cm ; t1 : 2047 , 1610 cm ) with results from dft calculations provides clear evidence for an increase in cumulenic character in the excited states . we also observe ultrafast singlet excitation energy transfer ( eet ) in hm , from the singlet excited state of the macrocycle to the dark s1 state of the polyyne ( eet , = 3.0 ps ) . the trir spectra show that this energy - transfer process directly populates the vibrationally equilibrated s1 state of the hexayne , whereas sn s1 internal conversion creates a hot vibrational state , which cools over about 5 ps . to gain more insights into this family of compounds , we have also compared the x - ray crystal structures of h , hm , hm(re ) , and m(re ) . the structure of the hm(re ) rotaxane complex shows that the re(co)3cl unit is pushed close to the c12 chain , with oc distances of less than the sum of the van der waals radii , with the polyyne bending round the metal center . the interactions between the re(co)3cl center and the hexayne chain appear to be weak in the ground state , while the excited - state trir spectra revealed mutual electronic perturbation of both components . crystals of re(co)3clmacrocycle m(re ) were grown by slow evaporation of a solution in toluene at 4 c ; single crystal x - ray diffraction data were collected at 150 k using an oxford diffraction ( agilent ) supernova a diffractometer . crystals of the re(co)3clrotaxane complex hm(re ) were grown from dichloromethane solutions by slow diffusion of methanol vapor at room temperature ; single crystal x - ray diffraction data were collected at 100 k using synchrotron radiation at the diamond light source , beamline i19 . the structures of the hexayne dumbbell h , rotaxane hm , rhenium rotaxane hm(re ) , and rhenium - macrocycle complex m(re ) are compared in figure 1 . the two rotaxanes , hm and hm(re ) , show similar bond length alternation within experimental error : 0.143 0.008 in hm and 0.155 0.010 in hm(re ) , compared with 0.158 0.008 for h. the closest distance between carbonyl group oxygen and polyyne chain carbon in hm(re ) is 3.085(5 ) ( co c(5 ) ) , which is less than the sum of the van der waals radii ( 3.35 ) ; the three shortest cocc contacts are shown in figure 1e . the hexayne chain in hm(re ) is severely bent , with a single - arc conformation , unlike the double - arc of h. the angle between the two terminal c1 and c12 atoms and the centroid of the middle c6c7 bond of hm(re ) is 155.6(1) ( compared with 171.8(1) in hm and 180 in h ) ; the average value for this parameter for the 22 hexaynes in the cambridge structural database is 175.3 ( s.d . the angles between the three carbonyl groups lie in the range 85.20(17)92.55(17) ( with an average of 88.7 ) in hm(re ) and 85.47(16)90.53(16) ( with an average of 87.9 ) in m(re ) , and the local cs symmetry of the rhenium center is preserved in both re(co)3cl complexes ( figure 1 ) . for the hm rotaxane , the third vibrational band of the s0 s1 transition is obscured by the intense absorption of macrocycle at 390 nm , but the first two bands are clearly visible and do not change their position , while in the hm(re ) rotaxane , all the s0 s1 transitions are hidden by the intense m(re ) absorption . the absorption spectrum of the rotaxane hm is essentially the sum of its two components , thread and dumbbell , with a slight ( 4 nm ) bathochromic shift of the hexayne peaks ( figure 2a ) ; the presence of the threaded macrocycle slightly changes the environment of the polyyne chain . we carried out a systematic study of the dynamic processes in the excited states of this family of compounds , using trir and ta spectroscopy . initially , we examined the excited - state behavior of the hexayne h and the macrocycle m separately , and then we moved to the topologically more complex rotaxane hm , rhenium tricarbonyl complex of macrocycle m(re ) , and rhenium tricarbonyl rotaxane complex hm(re ) . excitation of hexayne h at 310 nm results in an immediate bleach of the ground - state cc stretch at 21652191 cm ( within the time - resolution of our instrument , < 200 fs ) , and a new absorption band appears at 2063 cm ( figure 3a ) . these two spectral changes are indicative of rapid excited - state vibrational energy redistribution , convolved with solvent reorganization around the nascent excited state , and fast sn s1 internal conversion , as discussed below . trir spectra and excited - state kinetics of hexayne dumbbell h excited at 310 nm ( a c ) , rotaxane hm excited at 310 nm ( d f ) and 350 nm ( g i ) . the s1 band at 2077 cm decays to a new band at 2047 cm ( figure 3b ) which we assign to the t1 triplet excited - state cc stretch ( based on the kinetics and on the match with calculated vibrational frequencies ; see below ) ; the rates of this decay ( 1 = 0.44 0.02 ns ) and of the growth of the band at 2047 cm ( 2 = 0.48 0.05 ns , figure s1c ) give the rate of isc , as summarized in table 1 . the decay time of this band ( 1 = 0.82 0.13 ps ; figure s5b ) matches the growth of the s1 state in the visible region of ta spectrum ( 1 = 0.93 0.08 ps ) , and we therefore assign it to the sn state of the hexayne . -conjugated terminal aryl groups may play an important role in the excited - state dynamics of diphenylacetylene , while the supertrityl capped hexayne h presents a pure case of a polyyne chain , so its behavior can be regarded as intrinsic to the sp - hybridized carbyne chain . the excited states of 2,9-diaryl phenanthrolines have been studied before , and the * singlet - state lifetime is estimated to be a few ns in dichloromethane , while the triplet * state lives for about a second at 77 k. as expected , the trir spectrum of macrocycle m does not exhibit any features in the high - frequency region ( on excitation at 350 or 310 nm ) . it is possible to excite the different components of the rotaxane separately : at 350 nm , where only the macrocycle absorbs , or at 310 nm , where absorption by the hexayne predominates ( both components absorb at 310 nm , but the molar absorption coefficient of the hexayne is higher by a factor of 12 ) . on excitation of hm at 350 nm , the ground - state cc stretch band gradually bleaches , at around 2190 cm , and the s1 polyyne band gradually grows at 2077 cm ( figure 3 g ) , indicating transfer of singlet excitation from the macrocycle to the dumbbell . examination of the fingerprint region of the trir spectra of hm after excitation at 350 nm showed a fast ( < 10 ps ) decay of a band at 15001600 cm corresponding to the singlet macrocycle and the parallel rise of the s1 excited hexayne ( 1741 cm ) ( figure 6b ) . this is direct evidence of singlet singlet energy transfer from the excited - state macrocycle to the ground - state hexayne . the decay of the macrocycle band at 1560 cm ( 1 = 3.2 0.2 ps ) and the growth of the singlet s1 hexayne at 1741 cm ( 1 = 2.7 0.2 ps ) are single - exponential ( figure 6c ) . the growth of the 2077 cm band of the singlet hexayne ( 1 = 2.6 0.1 ps ) and the bleach of the ground - state stretching mode at 2189 cm ( 1 = 2.9 0.3 ps ) are also single - exponential . ta spectra of rotaxane hm , with excitation at 350 nm , showed formation of a transient s1 absorption band at 421 nm ( figure 7 ) , similar to that of free dumbbell h. the rise - time of this band ( 1 = 3.2 0.4 ps , figure 7a ) , due to s1(macrocycle ) s1(hexayne ) , agrees with the trir data . the excited - state dynamics of 1,10-phenanthroline - based re(co)3cl complexes have been thoroughly described in the literature and will only be outlined briefly here for discussion of changes when part of the rotaxane . later , the mlct state relaxes to the vibrationally cooled lowest excited state , which decays on the ns time - scale via radiative and nonradiative pathways . indicate that the order of the bands in the excited state is different from that in the ground state ; bands were observed at 1944 , 1985 , and 2048 cm due to a , a(2 ) , and a(1 ) modes , respectively , and we adopt this assignment . these spectral changes resemble the excited - state dynamics of the re(co)3(4-et - pyridine ) 2,2-bipyridine complex . we ascribe the final trir spectral pattern , seen at longer time delays , to the relaxed lowest excited - state triplet ( see figure 8 for a summary of the excited states and kinetics ) . in the fingerprint region of the trir spectrum of m(re ) , two bleaches of ground - state bands at 1497 and 1608 cm appear immediately after excitation , and recover within 3040 ps , while the transient bands 1480 and 1595 cm grow slightly ( figure 9c ) . the positions of the bleach bands in m(re ) and m are similar , suggesting that they originate from the phenanthroline framework , and are not strongly perturbed by the presence of the rhenium . the recovery of the co bleach is accompanied by bleaching of the cc band at 2190 cm , indicating energy transfer from re(co)3clmacrocycle to the hexayne chain . the trir spectra of hm(re ) in the high - frequency region do not reveal the nature of energy transfer , as the triplet and singlet - state bands of the hexayne at 20402080 cm overlap with excited - state a(1 ) band . the necessary information was obtained from the fingerprint region : the initial bleach of the excited re(co)3clmacrocycle at 1607 cm recovers , and then the band corresponding to the triplet - state hexayne at ca . at the same time the triplet re(co)3clmacrocycle band around these spectral changes clearly demonstrate triplet triplet energy transfer from the re(co)3clmacrocycle moiety to the threaded hexayne . the efficiency of eet was estimated by comparing the intensities of the cc band at 2200 cm and the a(1 ) c = o at 2054 cm in the ground - state ir spectra and in the trir spectra , at the time of maximum bleach ( figure s15 ) . trir spectra and excited - state kinetics of the m(re ) ( a c ) excited at 350 nm , hm(re ) excited at 350 nm ( d f ) , and 310 nm ( g i ) . direct excitation of the hexayne component of hm(re ) at 310 nm generates the singlet - state hexayne via direct excitation as well as triplet macrocycle ( figure 2a ) followed by triplet energy transfer to the hexayne . both 1 and 2 values we calculated the ir spectra of a model hexayne ( me c12me ) in its ground and excited states , to gain insights into the structural changes in the electronic excited states that are responsible for the observed trir bands of hexayne h. in particular , we sought to understand why the experimental s1 and t1 spectra each exhibit one vibration in the fingerprint region ( 15001900 cm ) and one vibration in the high - frequency region ( 20002250 cm ) . the combined computational and experimental results provide compelling evidence for cumulenic character in the s1 and t1 electronic states of h. the calculated bond lengths of the s0 , s1 , and t1 states ( figures 10 and s19 ) reveal a reduction in bond length alternation in s1 and t1 , compared with s0 , indicating that the excited states have significant cumulenic character , particularly near the center of the chain . these results agree well with previous work , and the calculated bond lengths in the s0 are close to those from the crystal structure of h. the presence of two cc excited - state bonding regions ( quasi - acetylenic at the ends and quasi - cumulenic in the middle ) is supported by the experimental and calculated vibrational spectra . bond length alternation in h ( from crystallographic data , black circles ; ref ( 9 ) ) and in calculated ( b3lyp / cc - pvtz ) s0 ( red circles ) , s1 ( yellow squares ) , and t1 ( purple stars ) states . the large change in frequency of the 2 mode ( in contrast to 1 ) in the excited states ( s1 and t1 ) , compared with the s0 ground state , for each electronic state , the higher energy 1 vibration involves greater displacement of the cc bonds at the ends of the hexayne , whereas the lower energy 2 vibration involves displacement of bonds near the center ( insets in figures 11 and s19 ) , making it much more sensitive to the excited - state cumulenic character around the center of the molecule . here we have shown that trir , coupled with dft calculations , is an excellent technique for probing their excited - state dynamics . we have also demonstrated that rotaxane synthesis is a powerful strategy for fixing a photoactive unit near the center of a polyyne chain , for studying singlet and triplet energy - transfer through - space , between nonbonded components . understanding the excited states of polyyne in supramolecular assemblies , such as the rotaxane studied here , is important for further application of materials based on chains of linear sp carbon atoms in molecular and optoelectronic devices .

in this context , we , and others , reported the synthesis of polyyne rotaxanes , such as hm in which a phenanthroline - based macrocycle m(18 ) is threaded around a hexayne chain h ( chart 1 ) . these rotaxanes provide an opportunity to control the environment of the polyyne chain , for example , we showed that rhenium(i ) can be complexed to the threaded macrocycle to give hm(re ) , in which a re(co)3cl unit is held in contact with the hexayne core . this result was surprising because the homo lumo gap of the hexayne ( absorption max 315 nm ) appears to be too large to accept excitation from the re(co)3cl unit ( emission max 605 nm ) , while the electron affinity of the polyyne is insufficient for it to quench the rhenium luminescence by photoinduced electron transfer . here we present a detailed investigation of the five compounds shown in chart 1 , using two ultrafast pump probe techniques : uv nir transient absorption ( ta ) and time - resolved infrared ( trir ) spectroscopy , both with pumping at uv wavelengths . the results provide many insights into the photophysics of polyyne chains and reveal that the luminescence quenching in hm(re ) originates from rapid triplet energy migration from the lowest metal - to - ligand charge transfer ( mlct ) excited state of m(re ) to the threaded hexayne ( time constant : = 1.5 ns ) . when investigating the photophysics of polyynes , strong signals are observed for the cc triple - bond stretch ( 21002200 cm ) , providing information on ground - state depletion and recovery ( through the negative a signal ) and probing the evolution of singlet and triplet excited states , which can be distinguished by their different cc stretch frequencies ( reflecting their different extents of bond length alternation ) . knowledge of the energy of the dark s1 state is essential for estimating the optical homo computational studies have shown that the excited states of polyynes generally have reduced bond length alternation , i.e. this prediction is supported by analysis of vibrational structure in electronic transitions and by time - resolved resonance raman studies on the t1 state of diphenyl butadiyne ( ph cc cc ph ) , and an intriguing metastable photoexcited polyyne has been identified at low temperatures which appears to have a cumulenic structure ( = c = c= rather than cc ) . here we show that optical excitation generates a short - lived higher singlet state ( sn ) with a lifetime of 0.9 ps in the free hexayne h and 1.8 ps in the rotaxane hm . trir also enables the s1 and t1 states of the polyyne to be directly observed ; comparison of the cc stretch frequencies ( s0 : 2191 , 2165 cm ; s1 : 2077 , 1737 cm ; t1 : 2047 , 1610 cm ) with results from dft calculations provides clear evidence for an increase in cumulenic character in the excited states . we also observe ultrafast singlet excitation energy transfer ( eet ) in hm , from the singlet excited state of the macrocycle to the dark s1 state of the polyyne ( eet , = 3.0 ps ) . the structure of the hm(re ) rotaxane complex shows that the re(co)3cl unit is pushed close to the c12 chain , with oc distances of less than the sum of the van der waals radii , with the polyyne bending round the metal center . in both cases , m(re ) was found to crystallize in p-1 with one molecule of m(re ) and two molecules of toluene in the asymmetric unit , while hm(re ) crystallized in p21/n with one molecule of hm(re ) in the asymmetric unit . the two rotaxanes , hm and hm(re ) , show similar bond length alternation within experimental error : 0.143 0.008 in hm and 0.155 0.010 in hm(re ) , compared with 0.158 0.008 for h. the closest distance between carbonyl group oxygen and polyyne chain carbon in hm(re ) is 3.085(5 ) ( co c(5 ) ) , which is less than the sum of the van der waals radii ( 3.35 ) ; the three shortest cocc contacts are shown in figure 1e . the hexayne chain in hm(re ) is severely bent , with a single - arc conformation , unlike the double - arc of h. the angle between the two terminal c1 and c12 atoms and the centroid of the middle c6c7 bond of hm(re ) is 155.6(1) ( compared with 171.8(1) in hm and 180 in h ) ; the average value for this parameter for the 22 hexaynes in the cambridge structural database is 175.3 ( s.d . the angles between the three carbonyl groups lie in the range 85.20(17)92.55(17) ( with an average of 88.7 ) in hm(re ) and 85.47(16)90.53(16) ( with an average of 87.9 ) in m(re ) , and the local cs symmetry of the rhenium center is preserved in both re(co)3cl complexes ( figure 1 ) . for the hm rotaxane , the third vibrational band of the s0 s1 transition is obscured by the intense absorption of macrocycle at 390 nm , but the first two bands are clearly visible and do not change their position , while in the hm(re ) rotaxane , all the s0 s1 transitions are hidden by the intense m(re ) absorption . ( a ) uv vis absorption and ( b ) ir spectra of the hexayne h ( red ) , the rotaxane hm ( blue ) , hm(re ) ( green ) , m(re ) ( orange ) , and m ( brown ) , all recorded in ch2cl2 . the absorption spectrum of the rotaxane hm is essentially the sum of its two components , thread and dumbbell , with a slight ( 4 nm ) bathochromic shift of the hexayne peaks ( figure 2a ) ; the presence of the threaded macrocycle slightly changes the environment of the polyyne chain . the three carbonyl groups of the re(co)3cl moiety give rise to three sharp , intense bands at 1894 ( a(2 ) ) , 1932 ( a ) , and 2025 ( a(1 ) ) cm for hm(re ) and 1885 ( a ) , 1924 ( a(2 ) ) , and 2022 ( a(1 ) ) cm for m(re ) , which are characteristic of fac - rhenium tricarbonyl polypyridyl complexes . the positions of the ir bands indicate the cs ( or pseudo c3 ) symmetry of the re center : two normal (co ) modes are symmetric ( a(1 ) and a(2 ) ) involving all three co ligands , while one mode ( a ) is antisymmetric involving only in the plane stretching of two co groups ( figure 2b ) . initially , we examined the excited - state behavior of the hexayne h and the macrocycle m separately , and then we moved to the topologically more complex rotaxane hm , rhenium tricarbonyl complex of macrocycle m(re ) , and rhenium tricarbonyl rotaxane complex hm(re ) . excitation of hexayne h at 310 nm results in an immediate bleach of the ground - state cc stretch at 21652191 cm ( within the time - resolution of our instrument , < 200 fs ) , and a new absorption band appears at 2063 cm ( figure 3a ) . the s1 band at 2077 cm decays to a new band at 2047 cm ( figure 3b ) which we assign to the t1 triplet excited - state cc stretch ( based on the kinetics and on the match with calculated vibrational frequencies ; see below ) ; the rates of this decay ( 1 = 0.44 0.02 ns ) and of the growth of the band at 2047 cm ( 2 = 0.48 0.05 ns , figure s1c ) give the rate of isc , as summarized in table 1 . the direction of this spectral shift is opposite to that expected for intramolecular relaxation and solvation processes , and we tentatively assign it to the formation of s1 by internal conversion from sn , populated by a symmetry - allowed s0sn transition . the decay time of this band ( 1 = 0.82 0.13 ps ; figure s5b ) matches the growth of the s1 state in the visible region of ta spectrum ( 1 = 0.93 0.08 ps ) , and we therefore assign it to the sn state of the hexayne . -conjugated terminal aryl groups may play an important role in the excited - state dynamics of diphenylacetylene , while the supertrityl capped hexayne h presents a pure case of a polyyne chain , so its behavior can be regarded as intrinsic to the sp - hybridized carbyne chain . the excited states of 2,9-diaryl phenanthrolines have been studied before , and the * singlet - state lifetime is estimated to be a few ns in dichloromethane , while the triplet * state lives for about a second at 77 k. as expected , the trir spectrum of macrocycle m does not exhibit any features in the high - frequency region ( on excitation at 350 or 310 nm ) . it is possible to excite the different components of the rotaxane separately : at 350 nm , where only the macrocycle absorbs , or at 310 nm , where absorption by the hexayne predominates ( both components absorb at 310 nm , but the molar absorption coefficient of the hexayne is higher by a factor of 12 ) . on excitation of hm at 350 nm , the ground - state cc stretch band gradually bleaches , at around 2190 cm , and the s1 polyyne band gradually grows at 2077 cm ( figure 3 g ) , indicating transfer of singlet excitation from the macrocycle to the dumbbell . this difference indicates that the dominant eet process directly populates the s1 state ( without going via the sn state ) and that the early signal is not broadened or shifted by sn contributions , vibrational energy redistribution or solvent reorganization . examination of the fingerprint region of the trir spectra of hm after excitation at 350 nm showed a fast ( < 10 ps ) decay of a band at 15001600 cm corresponding to the singlet macrocycle and the parallel rise of the s1 excited hexayne ( 1741 cm ) ( figure 6b ) . the growth of the 2077 cm band of the singlet hexayne ( 1 = 2.6 0.1 ps ) and the bleach of the ground - state stretching mode at 2189 cm ( 1 = 2.9 0.3 ps ) are also single - exponential . the triplet lifetime of the hexayne component in hm ( 1 = 14.0 2.2 s in deoxygenated solution , table 2 , figure s9 ) is similar to that of the hexayne h. trir spectra of ( a ) macrocycle m and ( b ) rotaxane hm excited at 350 nm . ta spectra of rotaxane hm , with excitation at 350 nm , showed formation of a transient s1 absorption band at 421 nm ( figure 7 ) , similar to that of free dumbbell h. the rise - time of this band ( 1 = 3.2 0.4 ps , figure 7a ) , due to s1(macrocycle ) s1(hexayne ) , agrees with the trir data . the excited - state dynamics of 1,10-phenanthroline - based re(co)3cl complexes have been thoroughly described in the literature and will only be outlined briefly here for discussion of changes when part of the rotaxane . indicate that the order of the bands in the excited state is different from that in the ground state ; bands were observed at 1944 , 1985 , and 2048 cm due to a , a(2 ) , and a(1 ) modes , respectively , and we adopt this assignment . the time - dependent shift of these two bands is complex , as it incorporates the instantaneous shift , as a result of franck condon excitation and formation of an excited state of mixed il and mlct character stemming from combined d * and * excitation . in the fingerprint region of the trir spectrum of m(re ) , two bleaches of ground - state bands at 1497 and 1608 cm appear immediately after excitation , and recover within 3040 ps , while the transient bands 1480 and 1595 cm grow slightly ( figure 9c ) . furthermore , in m(re ) , the recovery of these bleach bands is partial , due to overlapping positive bands , and shows single - exponential recovery kinetics ( 1497 cm : 1 = 6.8 0.6 ps ; 1608 cm : 1 = 14.0 1.2 ps ; figure s13d ) , on a time scale similar to the (co ) shifts observed in the transients in the carbonyl stretching region . the trir spectra of hm(re ) in the high - frequency region do not reveal the nature of energy transfer , as the triplet and singlet - state bands of the hexayne at 20402080 cm overlap with excited - state a(1 ) band . thus , triplet sensitization of the hexayne in hm(re ) upon excitation of the molecule at 350 nm explains the quenching of emission of re(co)3clmacrocycle m(re ) at room temperature by the threaded dumbbell . the efficiency of eet was estimated by comparing the intensities of the cc band at 2200 cm and the a(1 ) c = o at 2054 cm in the ground - state ir spectra and in the trir spectra , at the time of maximum bleach ( figure s15 ) . another noteworthy feature of the hm(re ) rotaxane is the perturbation of the ground - state hexayne by excited m(re ) ( after excitation , before energy transfer ) which appears as a slightly enhanced absorption at 2200 cm ( figures 9d and s16 ) . kinetic analysis of the fingerprint region shows that the singlet hexayne band at 1741 cm decays in a single - exponential fashion ( figure s13 , 1 = 0.35 0.01 ns ) ; the decay constant matches that of the singlet band at 2070 cm . both 1 and 2 values we calculated the ir spectra of a model hexayne ( me c12me ) in its ground and excited states , to gain insights into the structural changes in the electronic excited states that are responsible for the observed trir bands of hexayne h. in particular , we sought to understand why the experimental s1 and t1 spectra each exhibit one vibration in the fingerprint region ( 15001900 cm ) and one vibration in the high - frequency region ( 20002250 cm ) . the combined computational and experimental results provide compelling evidence for cumulenic character in the s1 and t1 electronic states of h. the calculated bond lengths of the s0 , s1 , and t1 states ( figures 10 and s19 ) reveal a reduction in bond length alternation in s1 and t1 , compared with s0 , indicating that the excited states have significant cumulenic character , particularly near the center of the chain . these results agree well with previous work , and the calculated bond lengths in the s0 are close to those from the crystal structure of h. the presence of two cc excited - state bonding regions ( quasi - acetylenic at the ends and quasi - cumulenic in the middle ) is supported by the experimental and calculated vibrational spectra . for s1 and t1 , the calculated frequencies agree with experiment and predict a 1 band in the cc region ( s1 : 2104 cm ; t1 : 2089 cm ) , and a lower energy 2 band in the fingerprint region ( s1 : 1786 cm ; t1 : 1670 cm ) . the large change in frequency of the 2 mode ( in contrast to 1 ) in the excited states ( s1 and t1 ) , compared with the s0 ground state , for each electronic state , the higher energy 1 vibration involves greater displacement of the cc bonds at the ends of the hexayne , whereas the lower energy 2 vibration involves displacement of bonds near the center ( insets in figures 11 and s19 ) , making it much more sensitive to the excited - state cumulenic character around the center of the molecule . we have also demonstrated that rotaxane synthesis is a powerful strategy for fixing a photoactive unit near the center of a polyyne chain , for studying singlet and triplet energy - transfer through - space , between nonbonded components . understanding the excited states of polyyne in supramolecular assemblies , such as the rotaxane studied here , is important for further application of materials based on chains of linear sp carbon atoms in molecular and optoelectronic devices .

ischaemic heart disease is the leading cause of morbidity and mortality in the western world . it was recently estimated that 935 000 new cases of acute coronary occlusion are identified each year in the usa . several experimental studies have demonstrated that the region supplied by the occluded coronary artery , also known as myocardium at risk ( mar ) , is a major independent variable that determines the final infarct size , which in turn is closely related to the clinical outcome . the difference between mar and infarct size is used to calculate the myocardial salvage index , which is a measurement of the effectiveness of acute interventions aimed at reducing the extent of myocardial infarction . thus , a feasible and accurate method for determination of mar is of significant value in clinical studies evaluating the efficiency of cardioprotective therapies . cardiovascular magnetic resonance ( cmr ) imaging is currently considered the reference standard for in vivo assessment of myocardial infarction using late gadolinium - enhanced ( lge ) imaging . recently , cmr has also been introduced as a promising method for assessing mar using t2-weighted imaging up to 1 week after the acute event . thus , cmr can be used to calculate the myocardial salvage index by a single cmr imaging session several days after the acute event in a stable clinical setting . even though t2-weighted imaging is promising for the assessment of mar , this technique still has certain limitations such as difficulties in distinguishing blood pool from the myocardium , especially in the apical parts of the left ventricle ( lv ) where hypokinesia and trabeculation cause stagnant blood flow . more recently , a new cmr method for assessment of mar , referred to as contrast - enhanced steady - state free precession ( ce - ssfp ) , has been introduced . this technique is based on acquisition of time - resolved steady - state free precession images after injection of the gadolinium - based contrast agent and was recently validated against myocardial perfusion single - photon emission computed tomography ( spect ) . still , ce - ssfp has not been compared head - to - head with t2-weighted imaging . therefore , the aim of the present study was to explore the relationship between ce - ssfp and t2-weighted imaging with regard to mar in patients with first - time reperfused acute myocardial infarction . the protocol and procedures were approved by the regional research ethics committee and all patients gave their written consent . between april 2009 and october 2010 , 21 patients ( age 59 10 years , 17 males ) presenting with first - time acute st - elevation myocardial infarction ( stemi ) , due to an occluded coronary artery confirmed by angiography , were prospectively included in the study . all patients were treated with primary percutaneous coronary intervention with coronary stenting , resulting in timi grade iii flow in the culprit artery . one week after admission , cmr was performed using a 1.5 t philips intera cv ( philips , best , the netherlands ) . images were obtained using a five - element chest array surface coil with two anterior and three posterior elements . all subjects were placed in supine position and images were acquired at end - expiratory breath hold with vectrocardiographic gating . initial scout images were acquired to locate the heart , and a dark - blood t2-weighted triple inversion turbo spin - echo sequence ( t2-stir ) was employed to depict the mar . t2-weighted images were acquired in the short - axis view , covering the lv from the base to the apex . imaging parameters for the t2-weighted sequence were : echo time , 100 ms ; repetition time , 2 heart beats ; echo train length , 33 ; number of averages , 2 ; inversion time , 180 ms ; image resolution , 1.5 1.5 8 mm ; slice gap , 0 mm . no parallel imaging was performed , but surface coil intensity correction was performed to minimize the signal inhomogeneities due to differences in receiver coil sensitivity . acquisition of short - axis , retrospectively gated ssfp cine images , covering the lv from the base to the apex , were initiated within 8 min after the administration of 0.2 mmol / kg extracellular gadolinium - based contrast agent ( gadoteric acid , gd - dota ; guerbet , gothia medical ab , billdal , sweden ) . these images were referred to as ce - ssfp images ( see supplementary data online , movie s1 ) . typical image parameters were : echo time , 1.4 ms ; repetition time , 2.9 ms ; flip angle , 60 ; image resolution , 1.5 1.5 8 mm ; slice gap , 0 mm . no parallel imaging was performed to maximize the signal - to - noise ratio ( snr ) . long- and short - axis lge images covering the lv were then acquired 15 min after injection of gadolinium . the lge images were acquired with an inversion - recovery sequence with following image parameters : slice thickness , 8 mm ; field of view , 340 mm ; flip angle , 15 ; repetition time , 4.2 ms ; echo time , 1.3 ms ; image resolution , 1.4 1.4 8 mm ; slice gap , 0 mm ; parallel imaging with a sense factor of 2 . all cmr images were analysed using the freely available software segment v1.8 ( http://segment.heiberg.se ) . the mar derived from t2-weighted imaging was assessed according to a previously described methodology . in short , endocardial and epicardial borders of the lv were traced in all short - axis slices , followed by manual delineation of the hyperintense regions by two blinded observers . the mar was then defined as the total amount of hyperintense myocardium in all short - axis slices and expressed as percentage of lv . if present , hypo - intense myocardium within the area of increased signal intensity ( microvascular obstruction ) was included in the mar . the mar derived from ce - ssfp was also assessed according to previously described methodology . in short , endocardial and epicardial borders of the lv were traced in all short - axis slices in end - diastole and end - systole , followed by manual delineation of the hyperintense regions in both end - diastole and end - systole , by two observers blinded to both the t2-weighted and lge images . the values of mar in end - diastole and end - systole were averaged and expressed as a percentage of the lv . the contrast ratio ( cr ) for the ce - ssfp images was determined for each patient as the mean signal intensity in the mar divided by the mean signal intensity in remote myocardium . a slice - by - slice comparison of mar between t2-weighted imaging and ce - ssfp was also performed for corresponding slice positions . the infarcted myocardium was automatically quantified from the short - axis lge images according to a previously described method . in short , the lge myocardium was then defined using a computer algorithm that takes into consideration partial volume effects within the infarcted region . if present , a hypointense signal within the area of lge ( microvascular obstruction ) was included in the analysis as 100% infarction . finally , the myocardial infarct size was expressed as a percentage of the lv . the myocardial salvage index was defined as : 100*[(mar infarct size)/mar ] , where mar was assessed by both t2-weighted imaging and ce - ssfp . the snr ratio , contrast - to - noise ( cnr ) ratio and contrast ratio ( cr ) were determined for t2-weighted , ce - ssfp , and lge images , respectively . the snr was calculated as the mean signal intensity within the affected region ( mar or infarcted myocardium ) divided by the standard deviation of signal intensities within a background region of interest . the cnr was calculated as the snr in the affected region ( mar or infarcted myocardium ) snr within remote myocardium and cr was calculated as the mean signal intensity in the affected region ( mar or infarcted myocardium ) divided by the mean signal intensity in remote myocardium . pearson 's correlation was used to determine the relationship between t2-weighted imaging and ce - ssfp with regard to mar and myocardial salvage index . the agreement between t2-weigthed imaging and ce - ssfp was expressed as mean difference sd , and the limits of agreement were shown in a bland a paired t - test was used to compare the means of the contrast ratio between mar and remote myocardium for both t2-weighted imaging and ce - ssfp as well as difference in mar by t2-weighted imaging and ce - ssfp . spss version 17.0 software package ( chicago , il , usa ) was used for analysis . the protocol and procedures were approved by the regional research ethics committee and all patients gave their written consent . between april 2009 and october 2010 , 21 patients ( age 59 10 years , 17 males ) presenting with first - time acute st - elevation myocardial infarction ( stemi ) , due to an occluded coronary artery confirmed by angiography , were prospectively included in the study . all patients were treated with primary percutaneous coronary intervention with coronary stenting , resulting in timi grade iii flow in the culprit artery . one week after admission , cmr was performed using a 1.5 t philips intera cv ( philips , best , the netherlands ) . images were obtained using a five - element chest array surface coil with two anterior and three posterior elements . all subjects were placed in supine position and images were acquired at end - expiratory breath hold with vectrocardiographic gating . initial scout images were acquired to locate the heart , and a dark - blood t2-weighted triple inversion turbo spin - echo sequence ( t2-stir ) was employed to depict the mar . t2-weighted images were acquired in the short - axis view , covering the lv from the base to the apex . imaging parameters for the t2-weighted sequence were : echo time , 100 ms ; repetition time , 2 heart beats ; echo train length , 33 ; number of averages , 2 ; inversion time , 180 ms ; image resolution , 1.5 1.5 8 mm ; slice gap , 0 mm . no parallel imaging was performed , but surface coil intensity correction was performed to minimize the signal inhomogeneities due to differences in receiver coil sensitivity . acquisition of short - axis , retrospectively gated ssfp cine images , covering the lv from the base to the apex , were initiated within 8 min after the administration of 0.2 mmol / kg extracellular gadolinium - based contrast agent ( gadoteric acid , gd - dota ; guerbet , gothia medical ab , billdal , sweden ) . these images were referred to as ce - ssfp images ( see supplementary data online , movie s1 ) . typical image parameters were : echo time , 1.4 ms ; repetition time , 2.9 ms ; flip angle , 60 ; image resolution , 1.5 1.5 8 mm ; slice gap , 0 mm . no parallel imaging was performed to maximize the signal - to - noise ratio ( snr ) . long- and short - axis lge images covering the lv were then acquired 15 min after injection of gadolinium . the lge images were acquired with an inversion - recovery sequence with following image parameters : slice thickness , 8 mm ; field of view , 340 mm ; flip angle , 15 ; repetition time , 4.2 ms ; echo time , 1.3 ms ; image resolution , 1.4 1.4 8 mm ; slice gap , 0 mm ; parallel imaging with a sense factor of 2 . all cmr images were analysed using the freely available software segment v1.8 ( http://segment.heiberg.se ) . the mar derived from t2-weighted imaging was assessed according to a previously described methodology . in short , endocardial and epicardial borders of the lv were traced in all short - axis slices , followed by manual delineation of the hyperintense regions by two blinded observers . the mar was then defined as the total amount of hyperintense myocardium in all short - axis slices and expressed as percentage of lv . if present , hypo - intense myocardium within the area of increased signal intensity ( microvascular obstruction ) was included in the mar . the mar derived from ce - ssfp was also assessed according to previously described methodology . in short , endocardial and epicardial borders of the lv were traced in all short - axis slices in end - diastole and end - systole , followed by manual delineation of the hyperintense regions in both end - diastole and end - systole , by two observers blinded to both the t2-weighted and lge images . the values of mar in end - diastole and end - systole were averaged and expressed as a percentage of the lv . the contrast ratio ( cr ) for the ce - ssfp images was determined for each patient as the mean signal intensity in the mar divided by the mean signal intensity in remote myocardium . a slice - by - slice comparison of mar between t2-weighted imaging and ce - ssfp was also performed for corresponding slice positions . the infarcted myocardium was automatically quantified from the short - axis lge images according to a previously described method . in short , the lge myocardium was then defined using a computer algorithm that takes into consideration partial volume effects within the infarcted region . manual adjustments were made when image artefacts caused misinterpretation by the computer algorithm . if present , a hypointense signal within the area of lge ( microvascular obstruction ) was included in the analysis as 100% infarction . the myocardial salvage index was defined as : 100*[(mar infarct size)/mar ] , where mar was assessed by both t2-weighted imaging and ce - ssfp . the snr ratio , contrast - to - noise ( cnr ) ratio and contrast ratio ( cr ) were determined for t2-weighted , ce - ssfp , and lge images , respectively . the snr was calculated as the mean signal intensity within the affected region ( mar or infarcted myocardium ) divided by the standard deviation of signal intensities within a background region of interest . the cnr was calculated as the snr in the affected region ( mar or infarcted myocardium ) snr within remote myocardium and cr was calculated as the mean signal intensity in the affected region ( mar or infarcted myocardium ) divided by the mean signal intensity in remote myocardium . pearson 's correlation was used to determine the relationship between t2-weighted imaging and ce - ssfp with regard to mar and myocardial salvage index . the agreement between t2-weigthed imaging and ce - ssfp was expressed as mean difference sd , and the limits of agreement were shown in a bland a paired t - test was used to compare the means of the contrast ratio between mar and remote myocardium for both t2-weighted imaging and ce - ssfp as well as difference in mar by t2-weighted imaging and ce - ssfp . spss version 17.0 software package ( chicago , il , usa ) was used for analysis . in 59% ( 12 of 21 ) of the patients , the right coronary artery ( rca ) was the culprit vessel and in 29% ( 6 of 21 ) of the patients , the left anterior descending coronary artery ( lad ) was the culprit vessel . furthermore , 2 patients presented with an occlusion of the left circumflex coronary artery and 1 patient had a left main occlusion . in all patients , t2-weighted imaging and ce - ssfp identified the mar in the same perfusion territory and in concordance with angiography . figure 1a shows an example of multi - slice short - axis images from the base to the apex demonstrating mar by t2-weighted imaging and ce - ssfp as well as myocardial infarction by lge , in one patient with an occlusion in the left anterior descending coronary artery . for one patient , myocardial infarct size another patient had a clinical history of a prior infarction in a coronary territory different from that supplied by the current culprit vessel . this patient had no signs of infarction by lge imaging in the part of the myocardium previously reported to be infarcted . figure 1the myocardium at risk by t2-weighted imaging and ce - ssfp , and the infarct size by late gadolinium enhancement . ( a ) t2-weighted , ce - ssfp , and late gadolinium - enhanced short - axis images at the corresponding lv levels in a patient with a reperfused left anterior descending coronary artery occlusion . ( b ) single corresponding mid - ventricular short - axis images from a patient with an occlusion in the left anterior descending coronary artery ( lad ) , left circumflex coronary artery ( lcx ) , and the right coronary artery ( rca ) , respectively . the hyperenhanced regions constituting the myocardium at risk ( dashed arrows ) and the infarcted myocardium ( solid arrows ) are traced in white . note the similarity in location and the extent of the affected region between t2-weighted imaging and ce - ssfp . also note the significantly smaller infarction compared with the myocardium at risk indicating a significant myocardial salvage accomplished by the acute reperfusion therapy . the myocardium at risk by t2-weighted imaging and ce - ssfp , and the infarct size by late gadolinium enhancement . ( a ) t2-weighted , ce - ssfp , and late gadolinium - enhanced short - axis images at the corresponding lv levels in a patient with a reperfused left anterior descending coronary artery occlusion . ( b ) single corresponding mid - ventricular short - axis images from a patient with an occlusion in the left anterior descending coronary artery ( lad ) , left circumflex coronary artery ( lcx ) , and the right coronary artery ( rca ) , respectively . the hyperenhanced regions constituting the myocardium at risk ( dashed arrows ) and the infarcted myocardium ( solid arrows ) are traced in white . note the similarity in location and the extent of the affected region between t2-weighted imaging and ce - ssfp . also note the significantly smaller infarction compared with the myocardium at risk indicating a significant myocardial salvage accomplished by the acute reperfusion therapy . ce - ssfp , contrast - enhanced steady - state free precession . a region with increased signal intensity by t2-weighted imaging and ce - ssfp was observed in all patients , yielding a mean mar of 29 11% ( range 1265 ) and 32 12% ( range 870 ) of the lv , respectively ( table 1 ) . table 1myocardium at risk , infarct size and myocardial salvage index for each patientcase no.culprit vesselmyocardium at risk by t2w ( % ) myocardium at risk by ce - ssfp ( % ) infarct size by lge ( % ) myocardial salvage index ( % ) byobs 1obs 2meanobs 1obs 2meanobs 1obs 2meant2wce - ssfp1lm65606370666849434626322lad3327304334399101070763lad19202035283256672834rca39233124373123242424235rca22202119232166671716rca38383833443933323315167lad4342434246448rca27282832323216141544529rca221217212925111939510lad474446464144252224494611rca333132303030121011656312rca1412138109597472313lad383135444042212121384914lad362832383537333929315lcx171416171818878525716rca302729302829111212606017lcx321222311925444828418rca262526292527111312525519rca302025272928777737620rca373134363837191618495321rca2320222627271214133850ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging.infarct size could not be assessed due to poor image quality related to frequent arrhythmias . myocardium at risk , infarct size and myocardial salvage index for each patient ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging . infarct size could not be assessed due to poor image quality related to frequent arrhythmias . figure 1b shows three examples of the agreement of the mar by t2-weighted imaging and ce - ssfp . another example including the cine ce - ssfp can be seen in supplementary data online , movie s1 . figure 2a shows a scatter plot indicating the relationship between t2-weighted imaging and ce - ssfp ( mean of two observers ) . there was a strong correlation between the two methods ( r = 0.89 , p < 0.01 ) . figure 2b shows the limits of agreement between t2-weighted imaging and ce - ssfp , demonstrating a bias of 3.0 4.0% of the lv ( p < 0.01 ) . for the slice - by - slice comparison , r was 0.69 ( p < 0.01 ; y = 0.98x + 0.61 ) with a bias of 1.8 16% per slice . altman graph showing the difference between the mar quantified by t2w and ce - ssfp vs. the mean of the two methods . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; the solid line denotes mean difference ; dashed lines denote 2sd . the myocardium at risk by t2-weighted imaging and ce - ssfp . ( a ) mar by t2-weighted imaging ( t2w ) vs. ce - ssfp . altman graph showing the difference between the mar quantified by t2w and ce - ssfp vs. the mean of the two methods . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; the solid line denotes mean difference ; dashed lines denote 2sd . figure 3a shows the relationship between time after contrast agent administration at which the ce - ssfp image acquisition was commenced ( 28 min ) and the difference between t2-weighted imaging and ce - ssfp for the assessment of mar . the ce - ssfp imaging lasted for 24 min and the latest image was acquired 12 min after contrast agent administration . there was no change in the relationship between the two techniques as a function of time after contrast agent administration . this is illustrated in a patient with an lad occlusion in figure 3b . figure 3the mar by ce - ssfp as a function of time after gd administration . ( a ) the difference between mar by t2-weighted imaging and ce - ssfp as a function of time after contrast agent administration at which the ce - ssfp imaging was commenced . the relationship between mar by t2-weighted imaging and ce - ssfp was not affected by increased time after contrast injection at which the ce - ssfp was commenced . ( b ) it is shown that no hyperenhancement could be seen prior to gd administration ( a ) , whereas a hyperenhanced region was seen 2 , 8 , and 30 min after gd administration ( b d ) . note that the extent of hyperenhancement did not change during the first 30 min after gd injection ( arrows ) . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . ( a ) the difference between mar by t2-weighted imaging and ce - ssfp as a function of time after contrast agent administration at which the ce - ssfp imaging was commenced . the relationship between mar by t2-weighted imaging and ce - ssfp was not affected by increased time after contrast injection at which the ce - ssfp was commenced . ( b ) an example of a patient with an infarction within the lad territory . it is shown that no hyperenhancement could be seen prior to gd administration ( a ) , whereas a hyperenhanced region was seen 2 , 8 , and 30 min after gd administration ( b d ) . note that the extent of hyperenhancement did not change during the first 30 min after gd injection ( arrows ) . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . in six patients , cine ssfp short - axis imaging was also performed prior to contrast agent administration . in five of six patients , for one patient , a slight hyperenhancement with indistinct borders was found within the mar . snr within the mar was 156 7 and 132 10 for the t2-weighted imaging and ce - ssfp , respectively ( mean sem ) . the cnr was 58 3 and 27 6 for the t2-weighted imaging and ce - ssfp , respectively ( mean sem ) . the contrast ratio between mar and remote myocardium for t2-weighted imaging was 1.7 0.3 compared with 1.5 0.4 for ce - ssfp , which was not statistically significant different ( p > 0.05 ) . the interobserver variability was 5.0 5.4% of the lv for t2-weighted imaging and 0.1 6.2% of the lv for ce - ssfp . the mean infarct size by lge was 14 11% ( range 149 ) of the lv . the infarct size was smaller in all patients when compared with mar assessed by t2-weighted imaging ( p < 0.01 ) and ce - ssfp ( p < 0.01 ) . comparison of the infarct size by lge in relation to mar determined by t2-weighted imaging and ce - ssfp yielded a myocardial salvage index of 56 22% ( range : 1593 ) and 58 23% ( range : 1695 ) , respectively ( figure 4 , table 1 ) . there was a significant correlation ( r = 0.89 , p < 0.01 ) between the myocardial salvage index measured by the two methods ( figure 5 ) , with an insignificant bias of 2.3 7.4% of the lv ( p = 0.20 ) . mid - ventricular short - axis slices in a patient with a right coronary artery occlusion . the epicardium is traced in green , the endocardium is traced in red , and the affected region is traced in white . the infarcted myocardium on the late gadolinium - enhanced image is superimposed ( orange ) on the myocardium at risk within the t2-weighted image and the ce - ssfp image . the non - coloured part of the myocardium at risk defines the salvaged myocardium . for this patient , ( a ) the myocardial salvage index measured by t2-weighted imaging vs. myocardial salvage index measured by ce - ssfp ; the solid line denotes the line of identity . altman graph showing the difference between myocardial salvage index quantified by t2w and ce - ssfp vs. the mean of the two methods . the difference between t2w and ce - ssfp was 2.3 7.4% ; the solid line denotes the mean difference ; dashed lines represent 2sd . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging mid - ventricular short - axis slices in a patient with a right coronary artery occlusion . the epicardium is traced in green , the endocardium is traced in red , and the affected region is traced in white . the infarcted myocardium on the late gadolinium - enhanced image is superimposed ( orange ) on the myocardium at risk within the t2-weighted image and the ce - ssfp image . the non - coloured part of the myocardium at risk defines the salvaged myocardium . for this patient , ( a ) the myocardial salvage index measured by t2-weighted imaging vs. myocardial salvage index measured by ce - ssfp ; the solid line denotes the line of identity . altman graph showing the difference between myocardial salvage index quantified by t2w and ce - ssfp vs. the mean of the two methods . the difference between t2w and ce - ssfp was 2.3 7.4% ; the solid line denotes the mean difference ; dashed lines represent 2sd . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging . for the lge images , the snr within the infarction was 115 12 and the cnr was 73 8 . note that the lge images were acquired using parallel imaging increasing the noise levels in these images . the contrast ratio between remote and infarcted myocardium was 2.9 0.8 , which was significantly higher than for t2-weighted imaging and ce - ssfp ( p < 0.001 ) . a region with increased signal intensity by t2-weighted imaging and ce - ssfp was observed in all patients , yielding a mean mar of 29 11% ( range 1265 ) and 32 12% ( range 870 ) of the lv , respectively ( table 1 ) . table 1myocardium at risk , infarct size and myocardial salvage index for each patientcase no.culprit vesselmyocardium at risk by t2w ( % ) myocardium at risk by ce - ssfp ( % ) infarct size by lge ( % ) myocardial salvage index ( % ) byobs 1obs 2meanobs 1obs 2meanobs 1obs 2meant2wce - ssfp1lm65606370666849434626322lad3327304334399101070763lad19202035283256672834rca39233124373123242424235rca22202119232166671716rca38383833443933323315167lad4342434246448rca27282832323216141544529rca221217212925111939510lad474446464144252224494611rca333132303030121011656312rca1412138109597472313lad383135444042212121384914lad362832383537333929315lcx171416171818878525716rca302729302829111212606017lcx321222311925444828418rca262526292527111312525519rca302025272928777737620rca373134363837191618495321rca2320222627271214133850ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging.infarct size could not be assessed due to poor image quality related to frequent arrhythmias . myocardium at risk , infarct size and myocardial salvage index for each patient ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging . infarct size could not be assessed due to poor image quality related to frequent arrhythmias . figure 1b shows three examples of the agreement of the mar by t2-weighted imaging and ce - ssfp . another example including the cine ce - ssfp can be seen in supplementary data online , movie s1 . figure 2a shows a scatter plot indicating the relationship between t2-weighted imaging and ce - ssfp ( mean of two observers ) . there was a strong correlation between the two methods ( r = 0.89 , p < 0.01 ) . figure 2b shows the limits of agreement between t2-weighted imaging and ce - ssfp , demonstrating a bias of 3.0 4.0% of the lv ( p < 0.01 ) . for the slice - by - slice comparison , r was 0.69 ( p < 0.01 ; y = 0.98x + 0.61 ) with a bias of 1.8 16% per slice . figure 2the myocardium at risk by t2-weighted imaging and ce - ssfp . altman graph showing the difference between the mar quantified by t2w and ce - ssfp vs. the mean of the two methods . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; the solid line denotes mean difference ; dashed lines denote 2sd . altman graph showing the difference between the mar quantified by t2w and ce - ssfp vs. the mean of the two methods . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; the solid line denotes mean difference ; dashed lines denote 2sd . figure 3a shows the relationship between time after contrast agent administration at which the ce - ssfp image acquisition was commenced ( 28 min ) and the difference between t2-weighted imaging and ce - ssfp for the assessment of mar . the ce - ssfp imaging lasted for 24 min and the latest image was acquired 12 min after contrast agent administration . there was no change in the relationship between the two techniques as a function of time after contrast agent administration . this is illustrated in a patient with an lad occlusion in figure 3b . figure 3the mar by ce - ssfp as a function of time after gd administration . ( a ) the difference between mar by t2-weighted imaging and ce - ssfp as a function of time after contrast agent administration at which the ce - ssfp imaging was commenced . the relationship between mar by t2-weighted imaging and ce - ssfp was not affected by increased time after contrast injection at which the ce - ssfp was commenced . ( b ) an example of a patient with an infarction within the lad territory . it is shown that no hyperenhancement could be seen prior to gd administration ( a ) , whereas a hyperenhanced region was seen 2 , 8 , and 30 min after gd administration ( b d ) . note that the extent of hyperenhancement did not change during the first 30 min after gd injection ( arrows ) . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . ( a ) the difference between mar by t2-weighted imaging and ce - ssfp as a function of time after contrast agent administration at which the ce - ssfp imaging was commenced . the relationship between mar by t2-weighted imaging and ce - ssfp was not affected by increased time after contrast injection at which the ce - ssfp was commenced . ( b ) an example of a patient with an infarction within the lad territory . it is shown that no hyperenhancement could be seen prior to gd administration ( a ) , whereas a hyperenhanced region was seen 2 , 8 , and 30 min after gd administration ( b d ) . note that the extent of hyperenhancement did not change during the first 30 min after gd injection ( arrows ) . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . in six patients , cine ssfp short - axis imaging was also performed prior to contrast agent administration . in five of six patients , for one patient , a slight hyperenhancement with indistinct borders was found within the mar . snr within the mar was 156 7 and 132 10 for the t2-weighted imaging and ce - ssfp , respectively ( mean sem ) . the cnr was 58 3 and 27 6 for the t2-weighted imaging and ce - ssfp , respectively the contrast ratio between mar and remote myocardium for t2-weighted imaging was 1.7 0.3 compared with 1.5 0.4 for ce - ssfp , which was not statistically significant different ( p > 0.05 ) . the interobserver variability was 5.0 5.4% of the lv for t2-weighted imaging and 0.1 6.2% of the lv for ce - ssfp . the mean infarct size by lge was 14 11% ( range 149 ) of the lv . the infarct size was smaller in all patients when compared with mar assessed by t2-weighted imaging ( p < 0.01 ) and ce - ssfp ( p < 0.01 ) . comparison of the infarct size by lge in relation to mar determined by t2-weighted imaging and ce - ssfp yielded a myocardial salvage index of 56 22% ( range : 1593 ) and 58 23% ( range : 1695 ) , respectively ( figure 4 , table 1 ) . there was a significant correlation ( r = 0.89 , p < 0.01 ) between the myocardial salvage index measured by the two methods ( figure 5 ) , with an insignificant bias of 2.3 7.4% of the lv ( p = 0.20 ) . mid - ventricular short - axis slices in a patient with a right coronary artery occlusion . the epicardium is traced in green , the endocardium is traced in red , and the affected region is traced in white . the infarcted myocardium on the late gadolinium - enhanced image is superimposed ( orange ) on the myocardium at risk within the t2-weighted image and the ce - ssfp image . the non - coloured part of the myocardium at risk defines the salvaged myocardium . for this patient , t2-weighted imaging and ce - ssfp showed 52 and 55% myocardial salvage , respectively . ( a ) the myocardial salvage index measured by t2-weighted imaging vs. myocardial salvage index measured by ce - ssfp ; the solid line denotes the line of identity . altman graph showing the difference between myocardial salvage index quantified by t2w and ce - ssfp vs. the mean of the two methods . the difference between t2w and ce - ssfp was 2.3 7.4% ; the solid line denotes the mean difference ; dashed lines represent 2sd . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging . mid - ventricular short - axis slices in a patient with a right coronary artery occlusion . the epicardium is traced in green , the endocardium is traced in red , and the affected region is traced in white . the infarcted myocardium on the late gadolinium - enhanced image is superimposed ( orange ) on the myocardium at risk within the t2-weighted image and the ce - ssfp image . for this patient , t2-weighted imaging and ce - ssfp showed 52 and 55% myocardial salvage , respectively . ( a ) the myocardial salvage index measured by t2-weighted imaging vs. myocardial salvage index measured by ce - ssfp ; the solid line denotes the line of identity . altman graph showing the difference between myocardial salvage index quantified by t2w and ce - ssfp vs. the mean of the two methods . the difference between t2w and ce - ssfp was 2.3 7.4% ; the solid line denotes the mean difference ; dashed lines represent 2sd . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging . for the lge images , the snr within the infarction was 115 12 and the cnr was 73 8 . note that the lge images were acquired using parallel imaging increasing the noise levels in these images . the contrast ratio between remote and infarcted myocardium was 2.9 0.8 , which was significantly higher than for t2-weighted imaging and ce - ssfp ( p < 0.001 ) . this study explored the agreement between mar assessed by t2-weighted imaging and mar assessed by ce - ssfp . the agreement was shown to be good , both for the assessment of mar and myocardial salvage index . both t2-weighted imaging and ce - ssfp have previously been validated against mar assessed by myocardial perfusion spect , with a mean difference of 2.3 5.7 and 0.5 5.1% of the lv , respectively . in accordance with these results , the present study found a small bias of 3.0 4.0% when t2-weighted imaging was compared with ce - ssfp . this is , however , the first time these two techniques have been compared head - to - head which is important in order to ensure that both techniques can be used interchangeably in clinical cardioprotection trials using myocardial salvage as endpoint . the correlation between mar by t2-weighted imaging and ce - ssfp was not as strong for the slice - by - slice comparison as for the global measure of mar normalized to the entire lv . as the heart rate varies , the timing within the cardiac cycle at which the t2-weighted image is acquired varies resulting in difficulties with local registration of the myocardium when comparing the two techniques . even though the slice position was approximately the same for the two acquisitions , the part of the myocardium depicted may differ due to av - plane movement during the cardiac cycle . this is not a problem when comparing global measures of mar normalized to the entire lv . more recently , bright - blood t2-weighted sequences have been developed to increase the diagnostic performance of t2 cmr for oedema depiction . in a recent study by payne et al . , dark - blood t2-stir was shown to underestimate the mar with 9% units compared with the recently introduced bright - blood t2-weighted sequence ( acut2e ) . it was concluded that acut2e was more accurate for the determination of mar and myocardial salvage than was dark - blood t2-stir . thus , ce - ssfp might be more accurate than dark - blood t2 in the present study since ce - ssfp showed larger mar and , previously , showed no bias compared with myocardial perfusion spect . the pathophysiological basis for the enhanced myocardium observed using t2-weighted imaging is still not completely understood . following acute coronary occlusion , the ischaemic myocardium shifts from aerobic metabolism to anaerobic glycolysis and ceases to contract . this failure of the energy - regulated membrane channels results in swelling of the myocytes due to influx of water and sodium . furthermore , reperfusion leads to inflammatory - like response , increasing the amount of extracellular fluid . this increased water content in the affected myocardium is likely to explain the increased signal intensity compared with the non - affected myocardium as seen by t2-weighted imaging . the ischaemic episode causes post - ischaemic stunning , associated with a decreased contractility in the previously ischaemic myocardium . this decreased contractility is likely associated with a decreased lymphatic drainage from this part of the myocardium , which may also contribute to residual increased water content 1 week after the acute event . the mechanisms behind the enhanced myocardium observed in ce - ssfp are not completely known either . the contrast in ssfp images is dependent on the t2/t1 ratio . in the presence of paramagnetic gadolinium this is utilized for infarct visualization in t1-weighted inversion - recovery lge imaging , where the concentration of an extracellular gadolinium - based contrast agent is increased due to an increased distribution volume in ischaemically injured myocardium . it has been shown that even reversibly injured myocardium within the mar has an increased distribution volume in the acute phase after an ischaemic episode . hence , the t2/t1 ratio in the entire mar , including both reversible and irreversible injured myocardium , is affected by the presence of gadolinium . this might explain the increased signal intensity in the mar seen by ce - ssfp . furthermore , it has been shown that the relationship between the change in t1-relaxation rates before and after contrast agent administration ( r1 ) in different parts of the myocardium ( remote , salvaged , and infarcted ) in relation to r1 in blood ( r1 ratios ) remains constant from 4 to 29 min after contrast agent administration in acute myocardial infarction . these earlier findings indicate that the rate of exchange of contrast agent between the myocardium ( normal and injured ) and the blood pool is constant and much faster than the clearance rate in the kidneys during the first 30 min after contrast agent administration . . recently showed similar findings supporting this concept using t1 mapping before and after contrast administration in experimentally induced infarction . thus , this can explain why the relationship between mar assessed by t2-weighted imaging and ce - ssfp did not change with time after contrast agent administration in the present study ( figure 3 ) and why the timing of ce - ssfp after contrast agent administration is not critical . therefore , ce - ssfp could potentially be added to clinical protocols so that cine imaging is acquired after contrast agent administration for the assessment of lv function and mar . one advantage with ce - ssfp imaging is that this technique is based on a multi - phase acquisition throughout the cardiac cycle . this enables tracking of the mar and myocardial borders in multiple time frames ( see supplementary data online , movie s1 ) , making delineation of both mar and myocardial borders more robust . in the present protocol , mar in the ce - ssfp images were traced in both end - systole and end - diastole and averaged . another situation where ce - ssfp could be advantageous is when limited time for scanning is available , due to heavy clinical load or an unstable patient . in such a situation , gadolinium can be injected prior to the examination and the imaging protocol can be shortened since lv dimensions / function and mar can be assessed from the same set of images . on the other hand , t2-weighted imaging for the determination of mar can be performed in those patients where administration of a gadolinium - based contrast agent is contraindicated . thus , there are several advantages of having access to more than one method for determination of mar by cmr and we recommend both t2-weighted imaging and ce - ssfp to be implemented in the imaging protocol . owing to the relatively small differences in signal intensity between mar and remote myocardium , we recommend not to use parallel imaging when acquiring ce - ssfp images as this decreases the snr . no significant difference in the contrast ratio between t2-weighted imaging and ce - ssfp imaging was found in the present study where no parallel imaging was performed . the present study was performed on a limited number of stemi patients , all undergoing successful reperfusion . thus , how this would translate to a non - stemi population or to patients treated with thrombolytic therapy is not known . since only a few female patients where included , gender perspective can not be evaluated . no semi - quantitative method was used to determine the mar by both t2-weighted imaging and ce - ssfp , since the signal intensities of the mar and the remote myocardium varied between slices and between patients , making it difficult to choose a fixed standard deviation of signal intensities to differentiate mar from remote myocardium . there is a good agreement between mar assessed by t2-weighted imaging and mar assessed by ce - ssfp in patients with reperfused acute myocardial infarction 1 week after the acute event . thus , both methods can be used to determine mar and subsequently myocardial salvage at this point in time . this study was funded by the swedish research council ( 521 - 2008 - 2461 ) , the swedish heart and lung foundation , the medical faculty at lund university , sweden , european commission fp7 consortium cardiocell , region of scania , sweden , the stockholm county council and karolinska institutet / stockholm county council strategic cardiovascular program .

aimsto determine the myocardial salvage index , the extent of infarction needs to be related to the myocardium at risk ( mar ) . thus , the ability to assess both infarct size and mar is of central clinical and scientific importance . the aim of the present study was to explore the relationship between t2-weighted cardiac magnetic resonance ( cmr ) and contrast - enhanced steady - state free precession ( ce - ssfp ) cmr for the determination of mar in patients with acute myocardial infarction.methods and resultstwenty - one prospectively included patients with first - time st - elevation myocardial infarction underwent cmr 1 week after primary percutaneous coronary intervention . for the assessment of mar , t2-weighted images were acquired before and ce - ssfp images were acquired after the injection of a gadolinium - based contrast agent . for the assessment of infarct size , late gadolinium enhancement images were acquired . the mar by t2-weighted imaging and ce - ssfp was 29 11 and 32 12% of the left ventricle , respectively . thus , the mar with t2-weighted imaging was slightly smaller than that by ce - ssfp ( 3.0 4.0% ; p < 0.01 ) . there was a significant correlation between the two mar measures ( r2= 0.89 , p < 0.01 ) , independent of the time after contrast agent administration at which the ce - ssfp was commenced ( 28 min).conclusionthere is a good agreement between the mar assessed by t2-weighted imaging and that assessed by ce - ssfp in patients with reperfused acute myocardial infarction 1 week after the acute event . thus , both methods can be used to determine mar and myocardial salvage at this point in time .

Introduction Methods Study population Cardiovascular magnetic resonance Image analysis Statistics Results Myocardium at risk Myocardial salvage index Discussion Study limitations Conclusions Supplementary data Funding Supplementary Material

several experimental studies have demonstrated that the region supplied by the occluded coronary artery , also known as myocardium at risk ( mar ) , is a major independent variable that determines the final infarct size , which in turn is closely related to the clinical outcome . the difference between mar and infarct size is used to calculate the myocardial salvage index , which is a measurement of the effectiveness of acute interventions aimed at reducing the extent of myocardial infarction . thus , cmr can be used to calculate the myocardial salvage index by a single cmr imaging session several days after the acute event in a stable clinical setting . even though t2-weighted imaging is promising for the assessment of mar , this technique still has certain limitations such as difficulties in distinguishing blood pool from the myocardium , especially in the apical parts of the left ventricle ( lv ) where hypokinesia and trabeculation cause stagnant blood flow . more recently , a new cmr method for assessment of mar , referred to as contrast - enhanced steady - state free precession ( ce - ssfp ) , has been introduced . this technique is based on acquisition of time - resolved steady - state free precession images after injection of the gadolinium - based contrast agent and was recently validated against myocardial perfusion single - photon emission computed tomography ( spect ) . therefore , the aim of the present study was to explore the relationship between ce - ssfp and t2-weighted imaging with regard to mar in patients with first - time reperfused acute myocardial infarction . pearson 's correlation was used to determine the relationship between t2-weighted imaging and ce - ssfp with regard to mar and myocardial salvage index . the agreement between t2-weigthed imaging and ce - ssfp was expressed as mean difference sd , and the limits of agreement were shown in a bland a paired t - test was used to compare the means of the contrast ratio between mar and remote myocardium for both t2-weighted imaging and ce - ssfp as well as difference in mar by t2-weighted imaging and ce - ssfp . between april 2009 and october 2010 , 21 patients ( age 59 10 years , 17 males ) presenting with first - time acute st - elevation myocardial infarction ( stemi ) , due to an occluded coronary artery confirmed by angiography , were prospectively included in the study . pearson 's correlation was used to determine the relationship between t2-weighted imaging and ce - ssfp with regard to mar and myocardial salvage index . the agreement between t2-weigthed imaging and ce - ssfp was expressed as mean difference sd , and the limits of agreement were shown in a bland a paired t - test was used to compare the means of the contrast ratio between mar and remote myocardium for both t2-weighted imaging and ce - ssfp as well as difference in mar by t2-weighted imaging and ce - ssfp . figure 1a shows an example of multi - slice short - axis images from the base to the apex demonstrating mar by t2-weighted imaging and ce - ssfp as well as myocardial infarction by lge , in one patient with an occlusion in the left anterior descending coronary artery . figure 1the myocardium at risk by t2-weighted imaging and ce - ssfp , and the infarct size by late gadolinium enhancement . the myocardium at risk by t2-weighted imaging and ce - ssfp , and the infarct size by late gadolinium enhancement . a region with increased signal intensity by t2-weighted imaging and ce - ssfp was observed in all patients , yielding a mean mar of 29 11% ( range 1265 ) and 32 12% ( range 870 ) of the lv , respectively ( table 1 ) . table 1myocardium at risk , infarct size and myocardial salvage index for each patientcase no.culprit vesselmyocardium at risk by t2w ( % ) myocardium at risk by ce - ssfp ( % ) infarct size by lge ( % ) myocardial salvage index ( % ) byobs 1obs 2meanobs 1obs 2meanobs 1obs 2meant2wce - ssfp1lm65606370666849434626322lad3327304334399101070763lad19202035283256672834rca39233124373123242424235rca22202119232166671716rca38383833443933323315167lad4342434246448rca27282832323216141544529rca221217212925111939510lad474446464144252224494611rca333132303030121011656312rca1412138109597472313lad383135444042212121384914lad362832383537333929315lcx171416171818878525716rca302729302829111212606017lcx321222311925444828418rca262526292527111312525519rca302025272928777737620rca373134363837191618495321rca2320222627271214133850ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging.infarct size could not be assessed due to poor image quality related to frequent arrhythmias . myocardium at risk , infarct size and myocardial salvage index for each patient ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging . figure 2a shows a scatter plot indicating the relationship between t2-weighted imaging and ce - ssfp ( mean of two observers ) . there was a strong correlation between the two methods ( r = 0.89 , p < 0.01 ) . figure 2b shows the limits of agreement between t2-weighted imaging and ce - ssfp , demonstrating a bias of 3.0 4.0% of the lv ( p < 0.01 ) . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; the solid line denotes mean difference ; dashed lines denote 2sd . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; the solid line denotes mean difference ; dashed lines denote 2sd . figure 3a shows the relationship between time after contrast agent administration at which the ce - ssfp image acquisition was commenced ( 28 min ) and the difference between t2-weighted imaging and ce - ssfp for the assessment of mar . ( a ) the difference between mar by t2-weighted imaging and ce - ssfp as a function of time after contrast agent administration at which the ce - ssfp imaging was commenced . the relationship between mar by t2-weighted imaging and ce - ssfp was not affected by increased time after contrast injection at which the ce - ssfp was commenced . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . ( a ) the difference between mar by t2-weighted imaging and ce - ssfp as a function of time after contrast agent administration at which the ce - ssfp imaging was commenced . the relationship between mar by t2-weighted imaging and ce - ssfp was not affected by increased time after contrast injection at which the ce - ssfp was commenced . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . the infarct size was smaller in all patients when compared with mar assessed by t2-weighted imaging ( p < 0.01 ) and ce - ssfp ( p < 0.01 ) . comparison of the infarct size by lge in relation to mar determined by t2-weighted imaging and ce - ssfp yielded a myocardial salvage index of 56 22% ( range : 1593 ) and 58 23% ( range : 1695 ) , respectively ( figure 4 , table 1 ) . there was a significant correlation ( r = 0.89 , p < 0.01 ) between the myocardial salvage index measured by the two methods ( figure 5 ) , with an insignificant bias of 2.3 7.4% of the lv ( p = 0.20 ) . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging mid - ventricular short - axis slices in a patient with a right coronary artery occlusion . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging . a region with increased signal intensity by t2-weighted imaging and ce - ssfp was observed in all patients , yielding a mean mar of 29 11% ( range 1265 ) and 32 12% ( range 870 ) of the lv , respectively ( table 1 ) . table 1myocardium at risk , infarct size and myocardial salvage index for each patientcase no.culprit vesselmyocardium at risk by t2w ( % ) myocardium at risk by ce - ssfp ( % ) infarct size by lge ( % ) myocardial salvage index ( % ) byobs 1obs 2meanobs 1obs 2meanobs 1obs 2meant2wce - ssfp1lm65606370666849434626322lad3327304334399101070763lad19202035283256672834rca39233124373123242424235rca22202119232166671716rca38383833443933323315167lad4342434246448rca27282832323216141544529rca221217212925111939510lad474446464144252224494611rca333132303030121011656312rca1412138109597472313lad383135444042212121384914lad362832383537333929315lcx171416171818878525716rca302729302829111212606017lcx321222311925444828418rca262526292527111312525519rca302025272928777737620rca373134363837191618495321rca2320222627271214133850ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging.infarct size could not be assessed due to poor image quality related to frequent arrhythmias . myocardium at risk , infarct size and myocardial salvage index for each patient ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging . there was a strong correlation between the two methods ( r = 0.89 , p < 0.01 ) . figure 2b shows the limits of agreement between t2-weighted imaging and ce - ssfp , demonstrating a bias of 3.0 4.0% of the lv ( p < 0.01 ) . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; the solid line denotes mean difference ; dashed lines denote 2sd . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; the solid line denotes mean difference ; dashed lines denote 2sd . figure 3a shows the relationship between time after contrast agent administration at which the ce - ssfp image acquisition was commenced ( 28 min ) and the difference between t2-weighted imaging and ce - ssfp for the assessment of mar . ( a ) the difference between mar by t2-weighted imaging and ce - ssfp as a function of time after contrast agent administration at which the ce - ssfp imaging was commenced . the relationship between mar by t2-weighted imaging and ce - ssfp was not affected by increased time after contrast injection at which the ce - ssfp was commenced . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . ( a ) the difference between mar by t2-weighted imaging and ce - ssfp as a function of time after contrast agent administration at which the ce - ssfp imaging was commenced . the relationship between mar by t2-weighted imaging and ce - ssfp was not affected by increased time after contrast injection at which the ce - ssfp was commenced . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . the cnr was 58 3 and 27 6 for the t2-weighted imaging and ce - ssfp , respectively the contrast ratio between mar and remote myocardium for t2-weighted imaging was 1.7 0.3 compared with 1.5 0.4 for ce - ssfp , which was not statistically significant different ( p > 0.05 ) . the infarct size was smaller in all patients when compared with mar assessed by t2-weighted imaging ( p < 0.01 ) and ce - ssfp ( p < 0.01 ) . comparison of the infarct size by lge in relation to mar determined by t2-weighted imaging and ce - ssfp yielded a myocardial salvage index of 56 22% ( range : 1593 ) and 58 23% ( range : 1695 ) , respectively ( figure 4 , table 1 ) . there was a significant correlation ( r = 0.89 , p < 0.01 ) between the myocardial salvage index measured by the two methods ( figure 5 ) , with an insignificant bias of 2.3 7.4% of the lv ( p = 0.20 ) . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging . the infarcted myocardium on the late gadolinium - enhanced image is superimposed ( orange ) on the myocardium at risk within the t2-weighted image and the ce - ssfp image . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging . this study explored the agreement between mar assessed by t2-weighted imaging and mar assessed by ce - ssfp . the agreement was shown to be good , both for the assessment of mar and myocardial salvage index . in accordance with these results , the present study found a small bias of 3.0 4.0% when t2-weighted imaging was compared with ce - ssfp . the correlation between mar by t2-weighted imaging and ce - ssfp was not as strong for the slice - by - slice comparison as for the global measure of mar normalized to the entire lv . furthermore , it has been shown that the relationship between the change in t1-relaxation rates before and after contrast agent administration ( r1 ) in different parts of the myocardium ( remote , salvaged , and infarcted ) in relation to r1 in blood ( r1 ratios ) remains constant from 4 to 29 min after contrast agent administration in acute myocardial infarction . thus , this can explain why the relationship between mar assessed by t2-weighted imaging and ce - ssfp did not change with time after contrast agent administration in the present study ( figure 3 ) and why the timing of ce - ssfp after contrast agent administration is not critical . therefore , ce - ssfp could potentially be added to clinical protocols so that cine imaging is acquired after contrast agent administration for the assessment of lv function and mar . on the other hand , t2-weighted imaging for the determination of mar can be performed in those patients where administration of a gadolinium - based contrast agent is contraindicated . thus , there are several advantages of having access to more than one method for determination of mar by cmr and we recommend both t2-weighted imaging and ce - ssfp to be implemented in the imaging protocol . no significant difference in the contrast ratio between t2-weighted imaging and ce - ssfp imaging was found in the present study where no parallel imaging was performed . no semi - quantitative method was used to determine the mar by both t2-weighted imaging and ce - ssfp , since the signal intensities of the mar and the remote myocardium varied between slices and between patients , making it difficult to choose a fixed standard deviation of signal intensities to differentiate mar from remote myocardium . there is a good agreement between mar assessed by t2-weighted imaging and mar assessed by ce - ssfp in patients with reperfused acute myocardial infarction 1 week after the acute event . thus , both methods can be used to determine mar and subsequently myocardial salvage at this point in time .

several experimental studies have demonstrated that the region supplied by the occluded coronary artery , also known as myocardium at risk ( mar ) , is a major independent variable that determines the final infarct size , which in turn is closely related to the clinical outcome . the difference between mar and infarct size is used to calculate the myocardial salvage index , which is a measurement of the effectiveness of acute interventions aimed at reducing the extent of myocardial infarction . thus , a feasible and accurate method for determination of mar is of significant value in clinical studies evaluating the efficiency of cardioprotective therapies . even though t2-weighted imaging is promising for the assessment of mar , this technique still has certain limitations such as difficulties in distinguishing blood pool from the myocardium , especially in the apical parts of the left ventricle ( lv ) where hypokinesia and trabeculation cause stagnant blood flow . more recently , a new cmr method for assessment of mar , referred to as contrast - enhanced steady - state free precession ( ce - ssfp ) , has been introduced . this technique is based on acquisition of time - resolved steady - state free precession images after injection of the gadolinium - based contrast agent and was recently validated against myocardial perfusion single - photon emission computed tomography ( spect ) . therefore , the aim of the present study was to explore the relationship between ce - ssfp and t2-weighted imaging with regard to mar in patients with first - time reperfused acute myocardial infarction . between april 2009 and october 2010 , 21 patients ( age 59 10 years , 17 males ) presenting with first - time acute st - elevation myocardial infarction ( stemi ) , due to an occluded coronary artery confirmed by angiography , were prospectively included in the study . acquisition of short - axis , retrospectively gated ssfp cine images , covering the lv from the base to the apex , were initiated within 8 min after the administration of 0.2 mmol / kg extracellular gadolinium - based contrast agent ( gadoteric acid , gd - dota ; guerbet , gothia medical ab , billdal , sweden ) . in short , endocardial and epicardial borders of the lv were traced in all short - axis slices in end - diastole and end - systole , followed by manual delineation of the hyperintense regions in both end - diastole and end - systole , by two observers blinded to both the t2-weighted and lge images . the snr ratio , contrast - to - noise ( cnr ) ratio and contrast ratio ( cr ) were determined for t2-weighted , ce - ssfp , and lge images , respectively . the cnr was calculated as the snr in the affected region ( mar or infarcted myocardium ) snr within remote myocardium and cr was calculated as the mean signal intensity in the affected region ( mar or infarcted myocardium ) divided by the mean signal intensity in remote myocardium . the agreement between t2-weigthed imaging and ce - ssfp was expressed as mean difference sd , and the limits of agreement were shown in a bland a paired t - test was used to compare the means of the contrast ratio between mar and remote myocardium for both t2-weighted imaging and ce - ssfp as well as difference in mar by t2-weighted imaging and ce - ssfp . between april 2009 and october 2010 , 21 patients ( age 59 10 years , 17 males ) presenting with first - time acute st - elevation myocardial infarction ( stemi ) , due to an occluded coronary artery confirmed by angiography , were prospectively included in the study . acquisition of short - axis , retrospectively gated ssfp cine images , covering the lv from the base to the apex , were initiated within 8 min after the administration of 0.2 mmol / kg extracellular gadolinium - based contrast agent ( gadoteric acid , gd - dota ; guerbet , gothia medical ab , billdal , sweden ) . in short , endocardial and epicardial borders of the lv were traced in all short - axis slices in end - diastole and end - systole , followed by manual delineation of the hyperintense regions in both end - diastole and end - systole , by two observers blinded to both the t2-weighted and lge images . the snr ratio , contrast - to - noise ( cnr ) ratio and contrast ratio ( cr ) were determined for t2-weighted , ce - ssfp , and lge images , respectively . the cnr was calculated as the snr in the affected region ( mar or infarcted myocardium ) snr within remote myocardium and cr was calculated as the mean signal intensity in the affected region ( mar or infarcted myocardium ) divided by the mean signal intensity in remote myocardium . pearson 's correlation was used to determine the relationship between t2-weighted imaging and ce - ssfp with regard to mar and myocardial salvage index . the agreement between t2-weigthed imaging and ce - ssfp was expressed as mean difference sd , and the limits of agreement were shown in a bland a paired t - test was used to compare the means of the contrast ratio between mar and remote myocardium for both t2-weighted imaging and ce - ssfp as well as difference in mar by t2-weighted imaging and ce - ssfp . in 59% ( 12 of 21 ) of the patients , the right coronary artery ( rca ) was the culprit vessel and in 29% ( 6 of 21 ) of the patients , the left anterior descending coronary artery ( lad ) was the culprit vessel . figure 1a shows an example of multi - slice short - axis images from the base to the apex demonstrating mar by t2-weighted imaging and ce - ssfp as well as myocardial infarction by lge , in one patient with an occlusion in the left anterior descending coronary artery . ( b ) single corresponding mid - ventricular short - axis images from a patient with an occlusion in the left anterior descending coronary artery ( lad ) , left circumflex coronary artery ( lcx ) , and the right coronary artery ( rca ) , respectively . ( b ) single corresponding mid - ventricular short - axis images from a patient with an occlusion in the left anterior descending coronary artery ( lad ) , left circumflex coronary artery ( lcx ) , and the right coronary artery ( rca ) , respectively . a region with increased signal intensity by t2-weighted imaging and ce - ssfp was observed in all patients , yielding a mean mar of 29 11% ( range 1265 ) and 32 12% ( range 870 ) of the lv , respectively ( table 1 ) . table 1myocardium at risk , infarct size and myocardial salvage index for each patientcase no.culprit vesselmyocardium at risk by t2w ( % ) myocardium at risk by ce - ssfp ( % ) infarct size by lge ( % ) myocardial salvage index ( % ) byobs 1obs 2meanobs 1obs 2meanobs 1obs 2meant2wce - ssfp1lm65606370666849434626322lad3327304334399101070763lad19202035283256672834rca39233124373123242424235rca22202119232166671716rca38383833443933323315167lad4342434246448rca27282832323216141544529rca221217212925111939510lad474446464144252224494611rca333132303030121011656312rca1412138109597472313lad383135444042212121384914lad362832383537333929315lcx171416171818878525716rca302729302829111212606017lcx321222311925444828418rca262526292527111312525519rca302025272928777737620rca373134363837191618495321rca2320222627271214133850ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging.infarct size could not be assessed due to poor image quality related to frequent arrhythmias . myocardium at risk , infarct size and myocardial salvage index for each patient ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging . figure 3a shows the relationship between time after contrast agent administration at which the ce - ssfp image acquisition was commenced ( 28 min ) and the difference between t2-weighted imaging and ce - ssfp for the assessment of mar . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . comparison of the infarct size by lge in relation to mar determined by t2-weighted imaging and ce - ssfp yielded a myocardial salvage index of 56 22% ( range : 1593 ) and 58 23% ( range : 1695 ) , respectively ( figure 4 , table 1 ) . there was a significant correlation ( r = 0.89 , p < 0.01 ) between the myocardial salvage index measured by the two methods ( figure 5 ) , with an insignificant bias of 2.3 7.4% of the lv ( p = 0.20 ) . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging mid - ventricular short - axis slices in a patient with a right coronary artery occlusion . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging . a region with increased signal intensity by t2-weighted imaging and ce - ssfp was observed in all patients , yielding a mean mar of 29 11% ( range 1265 ) and 32 12% ( range 870 ) of the lv , respectively ( table 1 ) . table 1myocardium at risk , infarct size and myocardial salvage index for each patientcase no.culprit vesselmyocardium at risk by t2w ( % ) myocardium at risk by ce - ssfp ( % ) infarct size by lge ( % ) myocardial salvage index ( % ) byobs 1obs 2meanobs 1obs 2meanobs 1obs 2meant2wce - ssfp1lm65606370666849434626322lad3327304334399101070763lad19202035283256672834rca39233124373123242424235rca22202119232166671716rca38383833443933323315167lad4342434246448rca27282832323216141544529rca221217212925111939510lad474446464144252224494611rca333132303030121011656312rca1412138109597472313lad383135444042212121384914lad362832383537333929315lcx171416171818878525716rca302729302829111212606017lcx321222311925444828418rca262526292527111312525519rca302025272928777737620rca373134363837191618495321rca2320222627271214133850ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging.infarct size could not be assessed due to poor image quality related to frequent arrhythmias . myocardium at risk , infarct size and myocardial salvage index for each patient ce - ssfp , contrast - enhanced steady state free precession ; lad , left anterior descending ; lge , late gadolinium enhancement ; lm , left main ; lv , left ventricle ; obs , observer ; rca , right coronary artery ; t2w , t2-weighted imaging . figure 3a shows the relationship between time after contrast agent administration at which the ce - ssfp image acquisition was commenced ( 28 min ) and the difference between t2-weighted imaging and ce - ssfp for the assessment of mar . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . ce - ssfp , contrast - enhanced steady - state free precession ; gd - inj , gadolinium contrast agent injection ; t2w , t2-weighted imaging ; solid line , mean difference ; dashed lines = 2sd . the cnr was 58 3 and 27 6 for the t2-weighted imaging and ce - ssfp , respectively the contrast ratio between mar and remote myocardium for t2-weighted imaging was 1.7 0.3 compared with 1.5 0.4 for ce - ssfp , which was not statistically significant different ( p > 0.05 ) . comparison of the infarct size by lge in relation to mar determined by t2-weighted imaging and ce - ssfp yielded a myocardial salvage index of 56 22% ( range : 1593 ) and 58 23% ( range : 1695 ) , respectively ( figure 4 , table 1 ) . there was a significant correlation ( r = 0.89 , p < 0.01 ) between the myocardial salvage index measured by the two methods ( figure 5 ) , with an insignificant bias of 2.3 7.4% of the lv ( p = 0.20 ) . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging . ce - ssfp , contrast - enhanced steady - state free precession ; lv , left ventricle ; mar , myocardium at risk ; t2w , t2-weighted imaging . both t2-weighted imaging and ce - ssfp have previously been validated against mar assessed by myocardial perfusion spect , with a mean difference of 2.3 5.7 and 0.5 5.1% of the lv , respectively . this is , however , the first time these two techniques have been compared head - to - head which is important in order to ensure that both techniques can be used interchangeably in clinical cardioprotection trials using myocardial salvage as endpoint . thus , ce - ssfp might be more accurate than dark - blood t2 in the present study since ce - ssfp showed larger mar and , previously , showed no bias compared with myocardial perfusion spect . this increased water content in the affected myocardium is likely to explain the increased signal intensity compared with the non - affected myocardium as seen by t2-weighted imaging . in the presence of paramagnetic gadolinium this is utilized for infarct visualization in t1-weighted inversion - recovery lge imaging , where the concentration of an extracellular gadolinium - based contrast agent is increased due to an increased distribution volume in ischaemically injured myocardium . furthermore , it has been shown that the relationship between the change in t1-relaxation rates before and after contrast agent administration ( r1 ) in different parts of the myocardium ( remote , salvaged , and infarcted ) in relation to r1 in blood ( r1 ratios ) remains constant from 4 to 29 min after contrast agent administration in acute myocardial infarction . these earlier findings indicate that the rate of exchange of contrast agent between the myocardium ( normal and injured ) and the blood pool is constant and much faster than the clearance rate in the kidneys during the first 30 min after contrast agent administration . thus , this can explain why the relationship between mar assessed by t2-weighted imaging and ce - ssfp did not change with time after contrast agent administration in the present study ( figure 3 ) and why the timing of ce - ssfp after contrast agent administration is not critical . in the present protocol , mar in the ce - ssfp images were traced in both end - systole and end - diastole and averaged . thus , there are several advantages of having access to more than one method for determination of mar by cmr and we recommend both t2-weighted imaging and ce - ssfp to be implemented in the imaging protocol . no semi - quantitative method was used to determine the mar by both t2-weighted imaging and ce - ssfp , since the signal intensities of the mar and the remote myocardium varied between slices and between patients , making it difficult to choose a fixed standard deviation of signal intensities to differentiate mar from remote myocardium . this study was funded by the swedish research council ( 521 - 2008 - 2461 ) , the swedish heart and lung foundation , the medical faculty at lund university , sweden , european commission fp7 consortium cardiocell , region of scania , sweden , the stockholm county council and karolinska institutet / stockholm county council strategic cardiovascular program .

macroautophagy , which we will refer to as autophagy , is a highly conserved catabolic process in which cytoplasmic proteins and organelles are engulfed by double - membrane structures called autophagosomes , which are then transported to lysosomes for degradation . autophagy is a key regulator of many physiological and disease processes and mediates the removal of protein oligomers , mitochondria , and various pathogens . in organisms from yeast to man , autophagy is upregulated in response to nutrient deprivation to allow cells to generate energy - rich compounds from cytoplasmic macromolecules ( rubinsztein et al . , 2011 ) . autophagosomes are formed by the elongation and fusion of cup - shaped structures called phagophores . the biogenesis of mammalian ( and yeast ) autophagosomes involves two ubiquitin - like proteins , atg12 and lc3 ( atg8 ) ( ohsumi and mizushima , 2004 ) . the earlier of the ubiquitination - like events in autophagosome biogenesis involves the conjugation of atg12 to atg5 , after which atg12 - 5 can form a complex with atg16l1 ( mizushima et al . , 2003 ) . this atg12 - 5 - 16l1 complex decorates prephagophore structures and phagophores but dissociates from completed autophagosomes . the elongation of the edges of the phagophore involves a second ubiquitin - like protein , lc3 , an atg8 family member , which is cleaved by atg4 to form cytoplasmic lc3-i . cytoplasmic lc3-i is then covalently conjugated to phosphatidylethanolamine on the phagophore membrane ( where it is called lc3-ii ) . lc3-ii is specifically associated with phagophore and autophagosome membranes , and the intra - autophagosomal lc3-ii is degraded in the lysosome . thus , lc3-ii levels and the numbers of lc3 vesicles correlate with autophagosome numbers ( rubinsztein et al . , 2009 ) . the origin of the autophagosome membranes has been a major question in the field , and recent studies suggest that there may be contributions from multiple sources . under conditions in which autophagy is induced by various forms of starvation , autophagosomes appear to be formed at the endoplasmic reticulum ( er ) via structures called omegasomes ( axe et al . recently , we showed that plasma membrane contributes to nascent autophagosomes under both basal and autophagy induction conditions ( ravikumar et al . , 2010 ) . we found that atg16l1 associates with clathrin - coated pits , and after internalization and uncoating , the atg16l1-associated plasma membrane becomes associated with phagophore precursors , which mature into phagophores and then autophagosomes . inhibition of clathrin - mediated endocytosis causes defective autophagosome formation , which is associated with impaired uptake of plasma membrane into autophagic precursors and autophagosomes ( ravikumar et al . , 2010 ) . we recently showed that the small g protein arf6 also has a function in autophagy and that part of its effects on autophagy could be explained by the fact that it stimulates phosphatidylinositol 4,5-biphosphate ( pip2 ) formation at the plasma membrane . plasma membrane pip2 regulates endocytosis , and this is likely to explain part of its importance for autophagy ( moreau et al . , 2012 ) . the atg16l1-positive phagophore precursors undergo snare - mediated homotypic fusion events to give rise to tubulovesicular structures , and the increase in size resulting from these fusions enhances the capacity of these structures to acquire atg8/lc3 , which marks phagophores ( moreau et al . , 2011 ) . matg9 is the only known multipass transmembrane protein that is necessary for optimal autophagy ( orsi et al . , 2012 ; young et al . , 2006 ) . because some matg9 is seen in autophagosomes , its itinerary may provide important clues to additional membrane sources . in yeast and in mammalian cells , matg9 has been associated with the golgi apparatus , suggesting that this may provide yet additional membranes for autophagosomes ( van der vaart et al . however , these authors have argued on the basis of their recent data that this model may be overly simplistic because mammalian atg9 ( matg9 and atg9l1 ) was seen to associate with many other compartments , including recycling endosomes , early endosomes , and late endosomes ( orsi et al . furthermore , matg9 interactions with omegasomes and autophagosomes have been examined using live - cell imaging and fusion events were not observed , leading to the suggestion that matg9 undergoes dynamic interactions with phagophores and autophagosomes without being incorporated into their membranes and that matg9 traffics by tubulation from an existing reservoir compartment , analogous to what has been described in yeast ( mari et al . , 2010 ; it is therefore still unclear how matg9 traffics , how it ends up associated with some lc3-positive structures if no obvious fusion events appear to occur , and whether matg9 vesicles contribute membrane to mammalian autophagosomes . furthermore , although distinct sources of autophagosome membranes have been proposed , it is not clear to what extent they may be mutually exclusive ( possibly regulated via different autophagy signals ) or whether they may coalesce and cooperate . here , we find that matg9 vesicles are trafficked from the plasma membrane to the recycling endosome via clathrin - coated pits that are largely distinct from those that endocytose atg16l1 . we found that atg16l1 was also present in the recycling compartment and participates in vesicle fusions with matg9-postive vesicles in that site . the fusions between atg16l1 and matg9 are enhanced by reducing membrane egress from the recycling endosomes , a perturbation that also increases autophagosome formation . this process , which is dependent on the r - snare vamp3 , appears to be physiologically relevant , as starvation , an evolutionarily conserved autophagy stimulus , also reduces membrane exit from the recycling endosomes and results in increased associations of matg9 and atg16l1 . matg9 has been reported to be localized at the trans - golgi network ( tgn ) but also in recycling endosomes ( orsi et al . , 2012 ; the recycling endosome is so close to the tgn that it is sometimes difficult to distinguish between the two compartments at the em level ( puri et al . , 2010 ) . the most commonly used marker for recycling endosomes is rab11 , a small gtpase that regulates transferrin recycling through this compartment ( ren et al . , 1998 ; consistent with earlier data , we found good colocalization between matg9 and rab11 ( figures 1a and a ) . transferrin and its cognate receptor are internalized by clathrin - mediated endocytosis and recycled back to the plasma membrane through early endosomes or transferred to recycling endosomes for recycling back to the plasma membrane via a slower pathway ( maxfield and mcgraw , 2004 ) . at steady state , recycling endosomes are the predominant intracellular site at which one sees the transferrin receptor ( tfr ) , as well as its ligand transferrin . likewise , matg9 also appeared to colocalize with a tgn marker , tgn46 ( figure 1b ) . in order to examine how matg9 may arrive at recycling endosomes , we initially incubated cells at 18c because this inhibits membrane traffic between early and recycling endosomes ( ren et al . , 1998 ; as expected , the temperature block caused a peripherally biased redistribution of transferrin - positive vesicles , increased transferrin association with the early endosome marker eea1 , and decreased transferrin association with the recycling endosome marker rab11 ( figures s1a s1c and c ) . furthermore , the 18c incubation led to a peripherally biased dispersion of matg9 ( similar to what we observed with transferrin [ figure 1b ] ) , increased matg9 colocalization with the early endosome marker eea1 ( figures s1d and d ) , and reduced its colocalization with tgn46 ( a marker for tgn ) ( figure 1b ) . this suggests that the predominant effects of 18c on matg9 localization are not due to failed exit from the golgi - impaired golgi - recycling endosome trafficking , which has been described in polarized cells at 18c ( ang et al . , these results raised the possibility that most of the matg9 that appeared to be at the tgn in basal conditions may actually be in recycling endosomes nearby and that the tgn pool of matg9 may be quite small . furthermore , these data suggest that most of the matg9 in the recycling endosomes comes from the early endosomes / plasma membrane rather than from the tgn . ( the decreased transferrin - matg9 colocalization at 18c is very likely due to the known reduction of transferrin uptake that occurs at reduced temperature [ figure 1b ] . ) because transferrin enters the cell by clathrin - mediated endocytosis , we examined whether matg9 is trafficked to the plasma membrane and then internalized by clathrin - mediated endocytosis . first , we acutely inhibited endocytosis by treating cells with the dynamin inhibitor dynasore ( thompson and mcniven , 2006 ) . dynasore treatment caused a redistribution of matg9 from a perinuclear concentration to a more dispersed localization , and this was associated with decreased colocalization of matg9 with transferrin or a golgi marker ( figure 1c ) . total internal reflection fluorescence ( tirf ) microscopy , which enables high - resolution assessment of molecules close to the plasma membrane , suggested that matg9 redistributed close to the plasma membrane in dynasore - treated cells ( figure 1d ) . a similar redistribution of matg9 from its normal perinuclear localization close to the plasma membrane was seen in cells overexpressing a dominant - negative dynamin mutant or small interfering rna ( sirna ) to the clathrin adaptor ap2 ( to block clathrin - dependent endocytosis ) ( figures 1d and s1e s1 g ) . the presence of matg9 on the plasma membrane was confirmed by cell surface biotinylation , followed by immunoprecipitation ( figure 1e ) . moreover , electron microscopy ( em ) analysis showed matg9 on clathrin - coated structures on the plasma membrane ( figures 1fa1fc ) . as a control , we showed that almost no clathrin labeling was observed in cells transfected and labeled with gfp ( figure s1h ) . in addition to clathrin - coated structures , em also revealed matg9 associated with plasma membrane , endocytic structures , autophagic structures , and golgi ( figure s1i ) . because both atg16l1 ( ravikumar et al . , 2010 ) and matg9 are associated with clathrin - coated pits , we wondered whether they were in the same structures and followed the same internalization routes . to address this question , we transfected hela cells with gfp - atg16l1 , fixed , labeled for endogenous matg9 , and analyzed by tirf microscopy , which assesses structures within 200 nm of the plasma membrane ( figures 1 g and s1j ) . note that clathrin - coated structures on the plasma membrane are 100150 nm in diameter ( stoorvogel et al . , 1996 ) . in the same experiment , control cells were cotransfected with mstrawberry - atg16l1 and gfp - atg16l1 . the control experiment showed almost complete overlap of the mstrawberry - atg16l1 and gfp - atg16l1 ( figures 1 g and s1j ) . in order to analyze the clathrin - coated structures carrying these two proteins with greater resolution , we treated cells with dynasore to recruit as much as possible atg16l1 and matg9 to the plasma membrane and then performed immunogold labeling on cryosections to observe the two proteins . remarkably , at the em level , matg9 and atg16l1 appeared to associate with two different pools of clathrin - coated structures ( figures 1h1j ) . matg9 and atg16l1 appear to be internalized via different routes because matg9 was associated with eea1-positive early endosomes , whereas atg16l1 was not seen with eea1 ( consistent with what we have reported previously [ moreau et al . we confirmed this observation using the rab5 constitutive - active mutant q79l , which induces aberrant fusion of early endosomes ( stenmark et al . , 1994 ) and has been used widely to enable better resolution of proteins associated with early endosomes . this rab5 mutant colocalizes only with matg9 and not with atg16l1 ( figure s1l ) . because matg9 vesicles appear to arrive at recycling endosomes via endocytosis , we investigated whether atg16l1 vesicles also localized to that compartment . we performed em studies to identify possible intracellular sites associated with atg16l1 and observed atg16l1-positive tubulovesicular structures ( probably phagophore precursors ) localizing close to the golgi ( figure 2a ) . these atg16l1-positive structures are associated with endocytosed cholera toxin ( figure 2a ) , which binds to the plasma membrane and traffics to recycling endosomes and the golgi ( bonifacino and rojas , 2006 ; ravikumar et al . , 2010 ) . because the atg16l1 tubulovesicular structures appear to be distinct from the golgi , we tested whether atg16l1 trafficked through recycling endosomes . em analysis showed that atg16l1 and rab11 localized in the same tubulovesicular structures ( figure 2c ) . we have used very low levels of overexpressed atg16l1 to avoid artifacts ( ravikumar et al . , we have previously shown that low levels of exogenous atg16l1 decorate early autophagic structures and colocalize with atg5 and atg12 ( moreau et al . , 2011 ; ravikumar et al . , 2010 ) . homotypic fusion of atg16l1 vesicles is an important step that increases the size of these structures and facilitates their progression to the phagophore stage ( moreau et al . , 2011 ) . accordingly , we tested whether homotypic fusions of atg16l1 vesicles associated with the rab11 compartment . interestingly , live - imaging analysis of atg16l1 and rab11 confirmed that such homotypic fusions occurred , and there also appeared to be progressive recruitment of rab11 ( vesicles from recycling endosomes ) to the atg16l1 ( preautophagosome ) vesicles ( figure 2d and movie s1 ) , suggesting that the recycling compartment may provide membranes to preautophagosomal structures . because atg16l1 and matg9 are both localized to recycling endosomes , we tested and confirmed that they colocalize together with rab11 ( figure s2b ) . this was consistent with em data showing colocalization of atg16l1 and matg9 on structures that are associated with the transferrin receptor ( figure 2e ) . importantly , this colocalization is occurring prior to the phagophore stage because such colocalizations appear in some vesicles that are lc3 negative ( figure 2f ) . although previous studies have failed to see proper fusions between matg9-containing vesicles and autophagosome - related structures , these did not study atg16l1 phagophore precursors ( orsi et al . , 2012 ) . however , we observed authentic and long - lasting fusions between matg9 and atg16l1-positive structures , suggesting that matg9 vesicles and associated membranes mix with atg16l1-positive phagophore precursors and that matg9 becomes associated with autophagosome precursors at this early prephagophore stage ( figure 2 g and movie s2 ) . the colocalization of matg9 and atg16l1 in the recycling endosomes raised the possibility that these fusion events may be occurring in that compartment . our data suggest that the recycling endosome is the site of fusion between matg9 and atg16l1 vesicles and led us to hypothesize that membrane flow in and out of the recycling compartment may influence matg9-atg16l1 fusions and autophagy . accordingly , we initially studied the effect of reducing the temperature to 18c because this inhibits membrane traffic between early and recycling endosomes ( ren et al . , 1998 ; the most common method used to study autophagy is to assay lc3-ii levels , which correlates with autophagosome numbers . these possibilities can be deconvoluted by studying cells in the absence and presence of lysosomal inhibitors like bafilomycin a1 or nh4cl , which inhibit autophagosome degradation and hence allow one to assess autophagosome formation rates ( rubinsztein et al . , 2009 ) . the temperature block ( 18c ) that inhibits membrane traffic between early and recycling endosomes ( ren et al . ( figures s1a s1c ) and which causes endocytosed transferrin to accumulate mostly in early endosomes ( figures s1b and s1c ) also clearly impairs autophagosome formation as measured on lc3-ii blots ( figures 3a3c ) . cells incubated at 18c also had more numerous but smaller atg16l1 vesicles ( figures 3d and 3e)which is compatible with decreased efficiency and/or frequency of atg16l1 vesicle homotypic fusion events ( moreau et al . , 2011)and caused accumulation of atg16l1 in recycling endosomes but did not lead to a change in its negligible abundance in early endosomes ( note that the low level of colocalization we see may be background due to labeling of adjacent structures ) ( figures 3f and 3 g ) . in addition , the temperature block decreased the extent to which atg16l1 and matg9 colocalized and decreased the sizes of both matg9 and atg16l1 vesicles ( figures 3h and s3a for separate channels ) , suggesting that this treatment impaired homotypic and heterotypic fusion of atg16l1 and matg9 vesicles . because decreased temperature has diverse effects , one can not draw very strong conclusions that the 18c effects are specifically due to decreased membrane flow into the recycling compartment . accordingly , we aimed to test the converse situation by reducing membrane egress from the recycling compartment by overexpressing rab11 ( ren et al . , 1998 ) or by inhibiting myosin vb by overexpressing its tail domain ( lapierre et al . , 2001 ) . both rab11 and myosin vb tail overexpression enhanced autophagosome formation ( lc3-ii levels in the presence or absence of bafilomcyin a1 ) ( figure 3i ) . autophagy regulates the proportion of cells with mutant huntingtin q74 ( htt ) aggregates by regulating mutant htt clearance , and the percentage of cells with aggregates correlates inversely with autophagic activity ( luo and rubinsztein , 2010 ; ravikumar et al . , 2004 ) . the proportion of cells with mutant htt aggregates was reduced in cells transfected with rab11 or the myosin vb tail ( figure s3b ) . overexpression of rab11 and myosin vb tail also increased the size of atg16l1 vesicles ( figures 3j and s3c ) ( consistent with increased homotypic fusion of atg16l1 vesicles ) , increased the colocalization of atg16l1 with the transferrin ( figures 3k and s3d ) ( consistent with more atg16l1 in the recycling compartment ) , and increased the colocalization of atg16l1 and matg9 ( figures 3l and s3e ) ( consistent with enhanced heterotypic fusion of matg9 and atg16l vesicles ) . furthermore , homotypic atg16l1 interactions were also enhanced in real time when the cells overexpressed rab11 or the myosin vb tail ( figures s3f and s3 g ) . we examined the links between autophagy induction , recycling endosomes , homotypic atg16l1 fusion , and heterotypic atg16l1-matg9 fusion during starvation , an evolutionarily conserved autophagic stimulus . amino acid and serum starvation decreased transferrin recycling , similar to what is observed with rab11 or myosin vb tail overexpression ( lapierre et al . , 2001 ; ren et al . , 1998 ; wilcke et al . , 2000 ) again , similar to rab11 or myosin vb tail overexpression , starvation increased atg16l1 vesicle interactions in real time , consistent with what we reported previously ( moreau et al . , 2011 ) ( figure s3i and movies s3 and s4 ) . likewise , starvation increased the colocalization of atg16l1 with transferrin ( figure 3 m ) and increased the colocalization of atg16l1 with matg9 ( figure 3n ) . these data suggest that matg9 vesicles likely travel from early endosomes to recycling endosomes where they meet atg16l1-containing vesicles , which arrive at recycling endosomes via a route that does not appear to involve a significant residence time in early endosomes . the fusion of these vesicles appears to be rate limiting for autophagosome formation . in order to clarify the itineraries of these vesicles and their trafficking routes more robustly because intracellular vesicular fusions are generally snare dependent , we tested and confirmed that this was the case for atg16l1-matg9 fusions because n - ethylmaleimide ( nem ) , which abolishes snare fusion ( jahn and scheller , 2006 ; moreau et al . , 2011 ; galli et al . , 1994 ) , reduced the colocalization in cells between atg16l1 and matg9 ( figure 4a ) and impaired the fusion of these two vesicle populations in vitro using postnuclear supernatants isolated from either strawberry - atg16l1- or atg9l1-gfp - expressing cells ( figures 4b4d ) . typically , snare complexes comprise three molecules with highly conserved glutamine ( q ) residues in the snare motif and one with a highly conserved arginine ( r ) residue in this domain , namely qa- , qb- , qc- , and r - snares ( jahn and scheller , 2006 ) . in order to identify such snares pertinent to atg16l1 and matg9 vesicle fusion , we started screening for the levels of the autophagy substrate p62 ( renna et al . , 2011 ) in cells where different r - snares were knocked down with sirnas . vamp3 knockdown resulted in the strongest elevation of p62 levels ( figure 5a ) . we confirmed that vamp3 indeed regulated autophagy by showing that it decreased autophagosome numbers ( lc3-ii levels on western blots versus actin and a 60% reduction in the number of lc3-positive vesicles [ autophagosomes ] ) , which could be accounted for by impaired autophagosome biogenesis ( lc3-ii levels in the presence of bafilomycin a1 ) ( figures 5b , 5c , s4a , and s4b ) . vamp3 localizes in early endosomes ( eea1 positive ) and recycling endosomes ( rab11 positive ) ( figure 5d ) , as described previously ( yamazaki et al . , 2013 ; prekeris et al . , 1998 ) , suggesting that it was in appropriate compartments for regulating matg9-atg16l1 vesicle fusions . a temperature block at 18c , which impairs trafficking between early and recycling endosomes , did not affect the colocalization between matg9 and vamp3 , suggesting that the two proteins are cotrafficking ( figure 5e ) , but reduced the colocalization between atg16l1 and vamp3 ( figures 5e and s4c ) in a manner reminiscent to what we observed with matg9 and atg16l1 ( figure 3h ) . these data strengthen the likelihood of a connection between vamp3 and matg9-atg16l1 and support the hypothesis that vamp3 is trafficking with matg9 from early endosomes to recycling endosomes where atg16l1 localizes . vamp3 knockdown did not affect the morphology of the recycling endosomes ( marked by rab11 ) ( ren et al . , 1998 ) or the endocytosis of transferrin ( figures s4d and s4e ) , suggesting that it did not grossly affect endocytic trafficking . however , vamp3 knockdown caused the accumulation of matg9 in early endosomes ( ee ) and reduced its localization in recycling endosomes ( re ) ( figures 6a6c ) . in contrast to what we observed with matg9 , knockdown of vamp3 caused the accumulation of atg16l1 in recycling endosomes but did not lead to a change in its negligible abundance in early endosomes ( note that the low level of colocalization we see may be background due to labeling of adjacent structures ) ( figures 6d6f ) . vamp3 knockdown reduced the colocalization of matg9 and atg16l1 ( figure 7a ) , and live - cell imaging demonstrated that coalescence between atg16l1 and matg9 was abrogated by vamp3 knockdown as compared to control cells ( figures s5a and s5b ; see also movies s5 and s6 ) without affecting homotypic fusion of atg16l1 vesicles ( figure 7b ) . consistent with these data , vamp3 knockdown impaired in vitro fusion of atg16l1 and matg9 vesicles ( vesicles probably derived from recycling and early endosomes , respectively ) ( figure 7c ) . previous studies have shown that loss of matg9 compromises mammalian autophagy , suggesting that this protein is a critical part of the autophagy apparatus ( orsi et al . , 2012 ) . its role in autophagy has , however , been elusive . as the only known multipass transmembrane atg protein , it has been assumed that it may play a role in lipid delivery to nascent autophagosomes . however , despite having some colocalization with lc3-positive structures , matg9 was previously found to have less colocalization with autophagic markers than with other organelles ( longatti et al . , 2012 ; orsi et al . , 2012 ) . this observation could be interpreted as suggesting that matg9 has a complex itinerary and that it fuses with autophagosomes at some stage of their biogenesis . however , based on studies examining lc3- or dfcp1-positive structures , matg9 did not seem to fuse with these structures but instead circled around them , leaving the question open whether matg9 contributed membrane to autophagosomes or whether it had some other activities . our data suggest that matg9 does indeed follow a complex itinerary and also becomes associated with nascent autophagosomes . because matg9 is a transmembrane protein , it is likely that it is incorporated into autophagosomes precursors with some membrane from its originating compartment . previously , we showed that the plasma membrane is important for autophagosome biogenesis by contributing membrane to atg16l1-positive phagophore precursors ( moreau et al . interestingly , we found very low levels of colocalization between atg16l1 and eea1 , suggesting that the atg16l1 vesicles were routed away from early endosomes soon after clathrin uncoating ( moreau et al . , we have found that matg9 also traffics via the plasma membrane . in this case , matg9 appears to be localized to a largely distinct pool of clathrin pits compared to atg16l1 , and although both proteins end up in recycling endosomes , matg9 is trafficked to recycling endosomes via eea1-positive early endosomes , whereas atg16l1 has minimal residence in early endosomes ( supported by both temperature block and vamp3 knockdown experiments ) . the observation that matg9 and atg16l1 are trafficking via distinct routes is entirely consistent with the idea that they are internalized via largely distinct clathrin coats . there is a precedent for this type of process because it has been previously shown that different plasma membrane receptors are frequently endocytosed via different clathrin - coated structures ( tosoni et al . , 2005 ; warren et al . , 1997 , 1998 ; it is likely that some plasma membrane matg9 ends up being trafficked from early to recycling endosomes for fusion with atg16l1 . although we can not exclude other sources for early endosomal matg9 , our temperature block experiments suggest that matg9 in early endosomes is not predominantly derived from the golgi , as this protein accumulates in early endosomes rather than the golgi at 18c . our data suggest that the recycling endosome is where true fusions occur between distinct atg16l1 and matg9-positive structures . the fusion event occurs at the prephagophore stage , as it involves atg16l1-positive , lc3-negative vesicles . the coalescence of matg9 and atg16l1 depends on snare function , and we have identified vamp3 as one of the critical mediators . the matg9-atg16l1 fusions are distinct from the homotypic atg16l1 fusions , as the former , but not the latter , requires vamp3 . vamp3 appears to colocalize first with matg9 in early endosomes ( figure 5e ) and then likely fuses with partner snares on atg16l1 in recycling endosome - associated structures , resulting in colocalization with both proteins . this model is consistent with our observations with the 18c temperature block , which inhibits membrane trafficking between early and recycling endosomes ( ren et al . , as this treatment decreased the association of vamp3 with atg16l1 ( figures 5e and s4c ) , but not with matg9 ( figure 5e ) , and impaired the colocalization between atg16l1 and matg9 ( figure 3h ) . although vamp3 has previously been linked to autophagy , it is important to clarify that fader et al . ( 2009 ) suggested that vamp3 is involved in a much later stage of the pathway the fusion of already - completed autophagosomes with multivesicular bodies to form amphisomes . this is not the stage of autophagosome biogenesis we are concerned with we are describing the processes and structures preceding the formation of lc3-positive autophagosomes . ( 2009 ) do not formally demonstrate that vamp3 is the relevant snare , as they use tetanus toxin for their experiments , and this toxin inactivates many snares . our data reveal that membrane traffic from early to recycling endosomes is crucial for autophagosome formation and that the recycling compartment is not merely responsible for recycling of plasma membrane receptors but also serves as a station in the early stages of autophagosome biogenesis where matg9 and atg16l1 meet . the observation that vamp3 knockdown has no obvious effect on transferrin recycling ( compared to the dramatic consequences of the 18c temperature block ) suggests either that the fusion of the matg9 and atg16l1 compartments may have a higher dependency on this r - snare than conventional endocytic trafficking or that vamp3 may be mediating selective fusion of components of the early endosomal and recycling endosome compartments rather than bulk fusion . the relevant partner , q - snares , regulating this event will need to be elucidated in future studies ( and may be difficult to pinpoint because some snares participate in multiple distinct fusion events as a function of their partners [ jahn and scheller , 2006 ] ) however , the identification of vamp3 allowed us to propose that the coalescence between atg16l1 and matg9 in recycling endosomes mediated by vamp3 is likely a critical event required for autophagy because vamp3 knockdown also decreased autophagosome formation ( figure 7d ) . it is tempting to speculate that the vamp3-mediated fusion of the atg16l1 and matg9 compartments may also be important for exit of preautophagosomal structures containing these proteins from recycling endosomes , as atg16l1 accumulated in these structures when vamp3 was knocked down ( figures 6d and 6e ) . this model suggests that atg16l1-containing vesicles may require vamp3/matg9 fusion in order to be competent to exit the recycling endosomes and mature into phagophores ( figure 7d ) . our data do not exclude additional sources for autophagosome membrane , and our demonstration that matg9 and atg16l1 meet prior to acquisition of lc3 suggests the likelihood that multiple independent membrane sources and itineraries may contribute to autophagosomes . indeed , an important challenge for future work will be to define the distinct compartments that may contribute to autophagosome biogenesis and understand how their fusions are regulated . it is possible that such fusion events may occur in diverse cellular locations , depending on which components are examined . hela cells were cultured in dulbecco s modified eagle s medium ( dmem ) ( sigma d6548 ) supplemented with 2 mm l - glutamine , 100 u / ml penicillin / streptomycin , and 10% fetal bovine serum in 5% co2 at 37c . cells were treated with bafilomycin a1 ( 400 nm for 4 hr or 200 nm for 16 hr ) or with nh4cl ( 20 mm for 16 hr ) or not treated and then lysed in laemmli buffer . protein samples were boiled 57 min at 100c , separated by sds - page , transferred onto pvdf membranes , subjected to western blot analysis , and , finally , visualized using an ecl detection kit ( ge healthcare ) . hela cells were seeded on mattek petri dish ( mattek ) at a density of 1.5 10 cells per dish . cells were placed in hbss ( ca mg ) with hepes , after which they were imaged immediately at 37c . imaging was performed on a zeiss axiovert 200 m microscope with a lsm 710 confocal attachment using a 63 1.4 na plan apochromat oil - immersion lens . the assay was performed as described with some modifications ( moreau et al . , 2011 ) . briefly , two postnuclear supernatants ( pns ) from hela cells expressing either mstrawberry - atg16l1 or atg9l1-gfp were mixed for 60 min in the presence of an atp regenerative system at 37c ( a control sample was left on ice ) . after the reaction , the samples were fixed with 2% paraformaldehyde in pbs for 15 min , centrifuged to remove the fixative ( 13,000 rpm , 5 min ) , resuspended in distilled water , and mounted with mowiol 4 - 88 with dapi on glass coverslips for confocal observation . significance levels for comparisons between two groups were determined with two - tailed t test . p 0.05 ; p 0.01 ; p 0.001 . extended experimental proceduresantibodies and reagentsthe antibodies used include : rabbit anti - rab11 ( invitrogen-715300 ) , rabbit anti - atg16l1 ( cosmobio co. ltp- tmd - ph - atg16l ; cell signaling : 8089s ) , atg12 ( cell signaling : 2010s ) , mouse anti - tubulin ( sigma ) , rabbit anti - lc3 for western blotting ( novus biological- nb100 - 2220 ) , mouse anti - ha ( covance mms-10i ) , rabbit anti - atg9 ( abcam - ab108338 and sigma - a0732 ) , sheep anti - tgn46 ( abd serotec ah - p500 ) , mouse anti - human transferrin receptor ( atcc-5e9c11 ) , human transferrin alexa-555 , alexa-647 and alexa-488 ( invitrogen ) , mouse anti - eea1 ( abcam - ab70521 ) , rabbit anti - clathrin ( abcam- ab21679 ) , cholera toxin hrp ( invitrogen ) , mouse anti - ap2 2 ( bd ap-50 ) and anti - hrp ( sigma - p7899 ) , rabbit anti - vamp3 was a kind gift from a.a . protein - a gold is from cmc ( utrecht- nl).bafilomycin a1 and dynasore are from sigma.plasmidspmstrawberry-atg16l1 , pegfp - atg16l1 and httq74-ha have been described elsewhere ( cadwell et al . atg9l1-pegfp was a kind gift from dr y. takahashi , vamp3-ha was a kind gift from dr . peden and pegfp - lc3 was a kind gift from tamotsu yoshimori , pegfp - mvb tail was a kind gift from dr . the constructs were validated by sequencing.cell transfectionthe cells were seeded at 1 - 2 x10 per 6-well and transfections were performed using lipofectamine 2000 for sirna and myrus bio transit -2020 , according to the manufacturer s instructions , using 80 nm sirna ( ap2 2 sirna custom oligos that we used previously in ravikumar et al . ) and were compared with control sirna . atg16l1-pegfp and atg16l1-mstrawberry were transfected in a concentration of 0.3g per well , httq74-ha at 0.5 g and the other constructs at 1g.vamp3 was silenced using oligo a ( ggcaggcgcuucucaauuu ) or alternatively oligo b ( ggauuacuguuucugguuau ) and c ( gaguuaacguggacaaggu ) . all the smartpool oligos for the screening were from dharmacon.aggregate quantificationthe httq74-ha aggregation was detected by immunofluorescence ( primary ha antibody ) . statistics for aggregation assays were calculated as odds ratios ( the ratio of cells containing aggregates in each condition).immunofluorescence microscopyhela cells were grown on coverslips at confluency of 25% were fixed in 4% paraformaldehyde for 5 min and then permeabilized with 0.1% triton . imagej ( size and number of vesicles analysis ) and volocity software ( perkinelmer ) ( colocalization analysis using pearson s coefficient or manders coefficient ) were used for further analysis and processing of confocal images . the pearson s coefficient was used to measure the correlation between the signals from two different markers / proteins . the manders coefficient was used to quantify the localization of protein a in compartment b under different conditions . the background was fixed for all within - experiment analyses ( for manders).tirf and dynasore treatmenthela cells were treated with dynasore 100 m for 2 hr in serum - free medium , or transfected for 16h with dynamin k44a mutant or ap2 sirna kd ( 5 day of treatment ) fixed and labeled with anti - atg9 antibody.the samples were analyzed by total internal reflection fluorescence ( tirf ) microscopy ( tirf 3 , carl zeiss microimaging inc.).immunogold electron microscopyhela cells were transfected with gfp - atg16l1 for 24h . the cells were starved 1h in hbss , loaded with anti - tfr antibody ( 5e9d11 ) for 1h and then fixed with a mixture of 2% paraformaldehyde and 1% acrolein in phosphate buffer ( ph . cells were then prepared for ultrathin cryosectioning and immunogold - labeled , as previously described ( puri , 2009 ; slot and geuze , 2007 ) . briefly , fixed cells were washed once in pbs/0.02 m glycine , after which cells were scraped in 12% gelatin in pbs and embedded in the same solution . the cell - gelatin was cut into 1 mm blocks , infiltrated with 15% pvp in 1.7 m sucrose at 4c overnight , mounted on aluminum pins and frozen in liquid nitrogen . ultrathin cryosections were picked up in a mixture of 50% sucrose and 50% methylcellulose and incubated with primary antibodies ( atg16l1 , atg9 and rabbit anti - mouse to recognize tfr ) followed by protein a gold ( utrecht ) . double and triple labeling was performed as previously described ( puri et al . , 2005 ; slot and geuze , 2007).we optimized double and triple labeling as follows . briefly , we first performed the three separate single labelings to assess the specificity and the localization of the three different proteins ( clathrin , matg9 and atg16l1 ) . then we performed three sets of double labeling ( matg9/cla , atg16l1/cla , matg9/atg16l1 ) and the opposite sequential combinations ( cla / matg9 , cla / atg16l1 , atg16l1/matg9 ) . the protein with more abundant labeling is used first ( with gold 5 nm ) , which in our case is clathrin . we discovered from the double immunolabeling that matg9 is the weakest antibody and thus we labeled this last ( as the antibody and the gold tend to detach during the multiple immunolabeling procedures ) . thus , we decided on the order clathrin 5nm / atg16 10 nm/ atg9 15 nm . the protein a gold is always used from the smallest size to the largest . after each labeling step , the reaction was fixed 5 min with 1% glutaraldehyde in pbs to quench the free antibodies and stabilize the reaction . of course , with these multiple steps one acquires data with single as well as double labeling and one can monitor for any possible labeling abnormalities that may occur with the sequential process . as an example , we now show single , double and triple labeling in figures 1f , 1h , and 1i , respectively.morphometric analysis was performed directly on the pictures by counting the tubulo - vesicular structures carrying both markers ( atg16l1 and rab11 ) , compared with the total atg16l1-labeled structures ( figure 2c).facs - based endocytosis assaystransferrin - recycling assay was performed as previously described ( peden et al . , 2004 ) . briefly , the cells were incubated for 30 min at 37c in the continuous presence of tf alexa - fluor-647 . cells were then washed and incubated at 37c in media supplemented with 100 g / ml unlabeled transferrin for various times before fixation in 4% paraformaldehyde in pbs . alexa - fluor-647 was determined by facs analysis using bd facscalibur flow cytometer ( bd biosciences ) and flowjo software ( tree star inc.).biotinylation of cell surface proteinshela cells grown on 6-well plates were rinsed twice with ice - cold pbs ( containing 1 mm mgcl2 and 0.1 mm cacl2 ) . cells were subsequently incubated with freshly prepared ice - cold nhs - lc - biotin ( pierce ) solution ( 1 mg / ml in ice - cold pbs , 1 mm mgcl2 , 2 mm cacl2 , 150 mm nacl ) for 60 - 90 min at 4c with gentle agitation . unreacted nhs - lc - biotin was then quenched by washing the cells twice with ice - cold quenching buffer ( pbs , 1 mm mgcl2 , 0.1 mm cacl2 , 100 mm glycine ) for 20 min at 4c . after quenching , cells were rinsed twice with ice - cold pbs++ and were scraped in ripa buffer [ 50 mm tris - hcl , ph 7.4 , 150 mm nacl , 5 mm edta , 1% np-40 , 0.5% sodium deoxycholate , 0.1% sds and supplemented with phosphatase and protease inhibitor cocktails ] . after 30 min on ice and occasional brief vortexing , lysates were cleared by centrifugation at 16 000 g for 10 min at 4c . samples were then transferred to new tubes containing 40 l of streptavidin - agarose beads ( pierce ) . after 2 hr of incubation at 4c under agitation , the beads were washed three times with ripa buffer and one time with pbs . the final pellets were resuspended in 40 l of 2 laemmli buffer and denatured at 100c for 5 min . the beads were pelleted and the solubilized proteins were separated by sds - page , which was silver stained or transferred onto pvdf membranes and probed with antibodies against atg9 ( abcam ) . nonspecific binding of unlabeled proteins was assessed by including a control condition in which the biotinylation buffer did not contain biotin . the antibodies used include : rabbit anti - rab11 ( invitrogen-715300 ) , rabbit anti - atg16l1 ( cosmobio co. ltp- tmd - ph - atg16l ; cell signaling : 8089s ) , atg12 ( cell signaling : 2010s ) , mouse anti - tubulin ( sigma ) , rabbit anti - lc3 for western blotting ( novus biological- nb100 - 2220 ) , mouse anti - ha ( covance mms-10i ) , rabbit anti - atg9 ( abcam - ab108338 and sigma - a0732 ) , sheep anti - tgn46 ( abd serotec ah - p500 ) , mouse anti - human transferrin receptor ( atcc-5e9c11 ) , human transferrin alexa-555 , alexa-647 and alexa-488 ( invitrogen ) , mouse anti - eea1 ( abcam - ab70521 ) , rabbit anti - clathrin ( abcam- ab21679 ) , cholera toxin hrp ( invitrogen ) , mouse anti - ap2 2 ( bd ap-50 ) and anti - hrp ( sigma - p7899 ) , rabbit anti - vamp3 was a kind gift from a.a . protein - a gold is from cmc ( utrecht- nl ) . bafilomycin a1 and dynasore pmstrawberry - atg16l1 , pegfp - atg16l1 and httq74-ha have been described elsewhere ( cadwell et al . , 2008 ; ravikumar et al . , 2002 ) . atg9l1-pegfp was a kind gift from dr y. takahashi , vamp3-ha was a kind gift from dr . peden and pegfp - lc3 was a kind gift from tamotsu yoshimori , pegfp - mvb tail was a kind gift from dr . the cells were seeded at 1 - 2 x10 per 6-well and transfections were performed using lipofectamine 2000 for sirna and myrus bio transit -2020 , according to the manufacturer s instructions , using 80 nm sirna ( ap2 2 sirna custom oligos that we used previously in ravikumar et al . ) and were compared with control sirna . atg16l1-pegfp and atg16l1-mstrawberry were transfected in a concentration of 0.3g per well , httq74-ha at 0.5 g and the other constructs at 1g . vamp3 was silenced using oligo a ( ggcaggcgcuucucaauuu ) or alternatively oligo b ( ggauuacuguuucugguuau ) and c ( gaguuaacguggacaaggu ) . statistics for aggregation assays were calculated as odds ratios ( the ratio of cells containing aggregates in each condition ) . hela cells were grown on coverslips at confluency of 25% were fixed in 4% paraformaldehyde for 5 min and then permeabilized with 0.1% triton . imagej ( size and number of vesicles analysis ) and volocity software ( perkinelmer ) ( colocalization analysis using pearson s coefficient or manders coefficient ) were used for further analysis and processing of confocal images . the pearson s coefficient was used to measure the correlation between the signals from two different markers / proteins . the manders coefficient was used to quantify the localization of protein a in compartment b under different conditions . the background was fixed for all within - experiment analyses ( for mander s ) . hela cells were treated with dynasore 100 m for 2 hr in serum - free medium , or transfected for 16h with dynamin k44a mutant or ap2 sirna kd ( 5 day of treatment ) fixed and labeled with anti - atg9 antibody . the samples were analyzed by total internal reflection fluorescence ( tirf ) microscopy ( tirf 3 , carl zeiss microimaging inc . ) . the cells were starved 1h in hbss , loaded with anti - tfr antibody ( 5e9d11 ) for 1h and then fixed with a mixture of 2% paraformaldehyde and 1% acrolein in phosphate buffer ( ph . cells were then prepared for ultrathin cryosectioning and immunogold - labeled , as previously described ( puri , 2009 ; slot and geuze , 2007 ) . briefly , fixed cells were washed once in pbs/0.02 m glycine , after which cells were scraped in 12% gelatin in pbs and embedded in the same solution . the cell - gelatin was cut into 1 mm blocks , infiltrated with 15% pvp in 1.7 m sucrose at 4c overnight , mounted on aluminum pins and frozen in liquid nitrogen . ultrathin cryosections were picked up in a mixture of 50% sucrose and 50% methylcellulose and incubated with primary antibodies ( atg16l1 , atg9 and rabbit anti - mouse to recognize tfr ) followed by protein a gold ( utrecht ) . double and triple labeling was performed as previously described ( puri et al . , 2005 ; slot and geuze , 2007 ) . briefly , we first performed the three separate single labelings to assess the specificity and the localization of the three different proteins ( clathrin , matg9 and atg16l1 ) . then we performed three sets of double labeling ( matg9/cla , atg16l1/cla , matg9/atg16l1 ) and the opposite sequential combinations ( cla / matg9 , cla / atg16l1 , atg16l1/matg9 ) . the protein with more abundant labeling is used first ( with gold 5 nm ) , which in our case is clathrin . we discovered from the double immunolabeling that matg9 is the weakest antibody and thus we labeled this last ( as the antibody and the gold tend to detach during the multiple immunolabeling procedures ) . thus , we decided on the order clathrin 5nm / atg16 10 nm/ atg9 15 nm . the protein a gold is always used from the smallest size to the largest . after each labeling step , the reaction was fixed 5 min with 1% glutaraldehyde in pbs to quench the free antibodies and stabilize the reaction . of course , with these multiple steps one acquires data with single as well as double labeling and one can monitor for any possible labeling abnormalities that may occur with the sequential process . as an example , we now show single , double and triple labeling in figures 1f , 1h , and 1i , respectively . morphometric analysis was performed directly on the pictures by counting the tubulo - vesicular structures carrying both markers ( atg16l1 and rab11 ) , compared with the total atg16l1-labeled structures ( figure 2c ) . transferrin - recycling assay was performed as previously described ( peden et al . , 2004 ) . briefly , the cells were incubated for 30 min at 37c in the continuous presence of tf alexa - fluor-647 . cells were then washed and incubated at 37c in media supplemented with 100 g / ml unlabeled transferrin for various times before fixation in 4% paraformaldehyde in pbs . alexa - fluor-647 was determined by facs analysis using bd facscalibur flow cytometer ( bd biosciences ) and flowjo software ( tree star inc . ) . hela cells grown on 6-well plates were rinsed twice with ice - cold pbs ( containing 1 mm mgcl2 and 0.1 mm cacl2 ) . cells were subsequently incubated with freshly prepared ice - cold nhs - lc - biotin ( pierce ) solution ( 1 mg / ml in ice - cold pbs , 1 mm mgcl2 , 2 mm cacl2 , 150 mm nacl ) for 60 - 90 min at 4c with gentle agitation . unreacted nhs - lc - biotin was then quenched by washing the cells twice with ice - cold quenching buffer ( pbs , 1 mm mgcl2 , 0.1 mm cacl2 , 100 mm glycine ) for 20 min at 4c . after quenching , cells were rinsed twice with ice - cold pbs++ and were scraped in ripa buffer [ 50 mm tris - hcl , ph 7.4 , 150 mm nacl , 5 mm edta , 1% np-40 , 0.5% sodium deoxycholate , 0.1% sds and supplemented with phosphatase and protease inhibitor cocktails ] . after 30 min on ice and occasional brief vortexing , lysates were cleared by centrifugation at 16 000 g for 10 min at 4c . samples were then transferred to new tubes containing 40 l of streptavidin - agarose beads ( pierce ) . after 2 hr of incubation at 4c under agitation , the beads were washed three times with ripa buffer and one time with pbs . the final pellets were resuspended in 40 l of 2 laemmli buffer and denatured at 100c for 5 min . the beads were pelleted and the solubilized proteins were separated by sds - page , which was silver stained or transferred onto pvdf membranes and probed with antibodies against atg9 ( abcam ) . nonspecific binding of unlabeled proteins was assessed by including a control condition in which the biotinylation buffer did not contain biotin .

summaryautophagic protein degradation is mediated by autophagosomes that fuse with lysosomes , where their contents are degraded . the membrane origins of autophagosomes may involve multiple sources . however , it is unclear if and where distinct membrane sources fuse during autophagosome biogenesis . vesicles containing matg9 , the only transmembrane autophagy protein , are seen in many sites , and fusions with other autophagic compartments have not been visualized in mammalian cells . we observed that matg9 traffics from the plasma membrane to recycling endosomes in carriers that appear to be routed differently from atg16l1-containing vesicles , another source of autophagosome membrane . matg9- and atg16l1-containing vesicles traffic to recycling endosomes , where vamp3-dependent heterotypic fusions occur . these fusions correlate with autophagosome formation , and both processes are enhanced by perturbing membrane egress from recycling endosomes . starvation , a primordial autophagy activator , reduces membrane recycling from recycling endosomes and enhances matg9-atg16l1 vesicle fusion . thus , this mechanism may fine - tune physiological autophagic responses .

Introduction Results Discussion Experimental Procedures

thus , lc3-ii levels and the numbers of lc3 vesicles correlate with autophagosome numbers ( rubinsztein et al . the origin of the autophagosome membranes has been a major question in the field , and recent studies suggest that there may be contributions from multiple sources . under conditions in which autophagy is induced by various forms of starvation , autophagosomes appear to be formed at the endoplasmic reticulum ( er ) via structures called omegasomes ( axe et al . we found that atg16l1 associates with clathrin - coated pits , and after internalization and uncoating , the atg16l1-associated plasma membrane becomes associated with phagophore precursors , which mature into phagophores and then autophagosomes . inhibition of clathrin - mediated endocytosis causes defective autophagosome formation , which is associated with impaired uptake of plasma membrane into autophagic precursors and autophagosomes ( ravikumar et al . we recently showed that the small g protein arf6 also has a function in autophagy and that part of its effects on autophagy could be explained by the fact that it stimulates phosphatidylinositol 4,5-biphosphate ( pip2 ) formation at the plasma membrane . plasma membrane pip2 regulates endocytosis , and this is likely to explain part of its importance for autophagy ( moreau et al . the atg16l1-positive phagophore precursors undergo snare - mediated homotypic fusion events to give rise to tubulovesicular structures , and the increase in size resulting from these fusions enhances the capacity of these structures to acquire atg8/lc3 , which marks phagophores ( moreau et al . because some matg9 is seen in autophagosomes , its itinerary may provide important clues to additional membrane sources . in yeast and in mammalian cells , matg9 has been associated with the golgi apparatus , suggesting that this may provide yet additional membranes for autophagosomes ( van der vaart et al . however , these authors have argued on the basis of their recent data that this model may be overly simplistic because mammalian atg9 ( matg9 and atg9l1 ) was seen to associate with many other compartments , including recycling endosomes , early endosomes , and late endosomes ( orsi et al . furthermore , matg9 interactions with omegasomes and autophagosomes have been examined using live - cell imaging and fusion events were not observed , leading to the suggestion that matg9 undergoes dynamic interactions with phagophores and autophagosomes without being incorporated into their membranes and that matg9 traffics by tubulation from an existing reservoir compartment , analogous to what has been described in yeast ( mari et al . , 2010 ; it is therefore still unclear how matg9 traffics , how it ends up associated with some lc3-positive structures if no obvious fusion events appear to occur , and whether matg9 vesicles contribute membrane to mammalian autophagosomes . furthermore , although distinct sources of autophagosome membranes have been proposed , it is not clear to what extent they may be mutually exclusive ( possibly regulated via different autophagy signals ) or whether they may coalesce and cooperate . here , we find that matg9 vesicles are trafficked from the plasma membrane to the recycling endosome via clathrin - coated pits that are largely distinct from those that endocytose atg16l1 . the fusions between atg16l1 and matg9 are enhanced by reducing membrane egress from the recycling endosomes , a perturbation that also increases autophagosome formation . this process , which is dependent on the r - snare vamp3 , appears to be physiologically relevant , as starvation , an evolutionarily conserved autophagy stimulus , also reduces membrane exit from the recycling endosomes and results in increased associations of matg9 and atg16l1 . matg9 has been reported to be localized at the trans - golgi network ( tgn ) but also in recycling endosomes ( orsi et al . the most commonly used marker for recycling endosomes is rab11 , a small gtpase that regulates transferrin recycling through this compartment ( ren et al . transferrin and its cognate receptor are internalized by clathrin - mediated endocytosis and recycled back to the plasma membrane through early endosomes or transferred to recycling endosomes for recycling back to the plasma membrane via a slower pathway ( maxfield and mcgraw , 2004 ) . in order to examine how matg9 may arrive at recycling endosomes , we initially incubated cells at 18c because this inhibits membrane traffic between early and recycling endosomes ( ren et al . , 1998 ; as expected , the temperature block caused a peripherally biased redistribution of transferrin - positive vesicles , increased transferrin association with the early endosome marker eea1 , and decreased transferrin association with the recycling endosome marker rab11 ( figures s1a s1c and c ) . furthermore , the 18c incubation led to a peripherally biased dispersion of matg9 ( similar to what we observed with transferrin [ figure 1b ] ) , increased matg9 colocalization with the early endosome marker eea1 ( figures s1d and d ) , and reduced its colocalization with tgn46 ( a marker for tgn ) ( figure 1b ) . , these results raised the possibility that most of the matg9 that appeared to be at the tgn in basal conditions may actually be in recycling endosomes nearby and that the tgn pool of matg9 may be quite small . furthermore , these data suggest that most of the matg9 in the recycling endosomes comes from the early endosomes / plasma membrane rather than from the tgn . because transferrin enters the cell by clathrin - mediated endocytosis , we examined whether matg9 is trafficked to the plasma membrane and then internalized by clathrin - mediated endocytosis . dynasore treatment caused a redistribution of matg9 from a perinuclear concentration to a more dispersed localization , and this was associated with decreased colocalization of matg9 with transferrin or a golgi marker ( figure 1c ) . total internal reflection fluorescence ( tirf ) microscopy , which enables high - resolution assessment of molecules close to the plasma membrane , suggested that matg9 redistributed close to the plasma membrane in dynasore - treated cells ( figure 1d ) . a similar redistribution of matg9 from its normal perinuclear localization close to the plasma membrane was seen in cells overexpressing a dominant - negative dynamin mutant or small interfering rna ( sirna ) to the clathrin adaptor ap2 ( to block clathrin - dependent endocytosis ) ( figures 1d and s1e s1 g ) . the presence of matg9 on the plasma membrane was confirmed by cell surface biotinylation , followed by immunoprecipitation ( figure 1e ) . moreover , electron microscopy ( em ) analysis showed matg9 on clathrin - coated structures on the plasma membrane ( figures 1fa1fc ) . in addition to clathrin - coated structures , em also revealed matg9 associated with plasma membrane , endocytic structures , autophagic structures , and golgi ( figure s1i ) . to address this question , we transfected hela cells with gfp - atg16l1 , fixed , labeled for endogenous matg9 , and analyzed by tirf microscopy , which assesses structures within 200 nm of the plasma membrane ( figures 1 g and s1j ) . note that clathrin - coated structures on the plasma membrane are 100150 nm in diameter ( stoorvogel et al . in order to analyze the clathrin - coated structures carrying these two proteins with greater resolution , we treated cells with dynasore to recruit as much as possible atg16l1 and matg9 to the plasma membrane and then performed immunogold labeling on cryosections to observe the two proteins . matg9 and atg16l1 appear to be internalized via different routes because matg9 was associated with eea1-positive early endosomes , whereas atg16l1 was not seen with eea1 ( consistent with what we have reported previously [ moreau et al . because matg9 vesicles appear to arrive at recycling endosomes via endocytosis , we investigated whether atg16l1 vesicles also localized to that compartment . these atg16l1-positive structures are associated with endocytosed cholera toxin ( figure 2a ) , which binds to the plasma membrane and traffics to recycling endosomes and the golgi ( bonifacino and rojas , 2006 ; ravikumar et al . because the atg16l1 tubulovesicular structures appear to be distinct from the golgi , we tested whether atg16l1 trafficked through recycling endosomes . interestingly , live - imaging analysis of atg16l1 and rab11 confirmed that such homotypic fusions occurred , and there also appeared to be progressive recruitment of rab11 ( vesicles from recycling endosomes ) to the atg16l1 ( preautophagosome ) vesicles ( figure 2d and movie s1 ) , suggesting that the recycling compartment may provide membranes to preautophagosomal structures . because atg16l1 and matg9 are both localized to recycling endosomes , we tested and confirmed that they colocalize together with rab11 ( figure s2b ) . however , we observed authentic and long - lasting fusions between matg9 and atg16l1-positive structures , suggesting that matg9 vesicles and associated membranes mix with atg16l1-positive phagophore precursors and that matg9 becomes associated with autophagosome precursors at this early prephagophore stage ( figure 2 g and movie s2 ) . accordingly , we aimed to test the converse situation by reducing membrane egress from the recycling compartment by overexpressing rab11 ( ren et al . overexpression of rab11 and myosin vb tail also increased the size of atg16l1 vesicles ( figures 3j and s3c ) ( consistent with increased homotypic fusion of atg16l1 vesicles ) , increased the colocalization of atg16l1 with the transferrin ( figures 3k and s3d ) ( consistent with more atg16l1 in the recycling compartment ) , and increased the colocalization of atg16l1 and matg9 ( figures 3l and s3e ) ( consistent with enhanced heterotypic fusion of matg9 and atg16l vesicles ) . we examined the links between autophagy induction , recycling endosomes , homotypic atg16l1 fusion , and heterotypic atg16l1-matg9 fusion during starvation , an evolutionarily conserved autophagic stimulus . these data suggest that matg9 vesicles likely travel from early endosomes to recycling endosomes where they meet atg16l1-containing vesicles , which arrive at recycling endosomes via a route that does not appear to involve a significant residence time in early endosomes . the fusion of these vesicles appears to be rate limiting for autophagosome formation . we confirmed that vamp3 indeed regulated autophagy by showing that it decreased autophagosome numbers ( lc3-ii levels on western blots versus actin and a 60% reduction in the number of lc3-positive vesicles [ autophagosomes ] ) , which could be accounted for by impaired autophagosome biogenesis ( lc3-ii levels in the presence of bafilomycin a1 ) ( figures 5b , 5c , s4a , and s4b ) . a temperature block at 18c , which impairs trafficking between early and recycling endosomes , did not affect the colocalization between matg9 and vamp3 , suggesting that the two proteins are cotrafficking ( figure 5e ) , but reduced the colocalization between atg16l1 and vamp3 ( figures 5e and s4c ) in a manner reminiscent to what we observed with matg9 and atg16l1 ( figure 3h ) . these data strengthen the likelihood of a connection between vamp3 and matg9-atg16l1 and support the hypothesis that vamp3 is trafficking with matg9 from early endosomes to recycling endosomes where atg16l1 localizes . however , vamp3 knockdown caused the accumulation of matg9 in early endosomes ( ee ) and reduced its localization in recycling endosomes ( re ) ( figures 6a6c ) . in contrast to what we observed with matg9 , knockdown of vamp3 caused the accumulation of atg16l1 in recycling endosomes but did not lead to a change in its negligible abundance in early endosomes ( note that the low level of colocalization we see may be background due to labeling of adjacent structures ) ( figures 6d6f ) . consistent with these data , vamp3 knockdown impaired in vitro fusion of atg16l1 and matg9 vesicles ( vesicles probably derived from recycling and early endosomes , respectively ) ( figure 7c ) . as the only known multipass transmembrane atg protein , it has been assumed that it may play a role in lipid delivery to nascent autophagosomes . however , despite having some colocalization with lc3-positive structures , matg9 was previously found to have less colocalization with autophagic markers than with other organelles ( longatti et al . however , based on studies examining lc3- or dfcp1-positive structures , matg9 did not seem to fuse with these structures but instead circled around them , leaving the question open whether matg9 contributed membrane to autophagosomes or whether it had some other activities . because matg9 is a transmembrane protein , it is likely that it is incorporated into autophagosomes precursors with some membrane from its originating compartment . previously , we showed that the plasma membrane is important for autophagosome biogenesis by contributing membrane to atg16l1-positive phagophore precursors ( moreau et al . , we have found that matg9 also traffics via the plasma membrane . in this case , matg9 appears to be localized to a largely distinct pool of clathrin pits compared to atg16l1 , and although both proteins end up in recycling endosomes , matg9 is trafficked to recycling endosomes via eea1-positive early endosomes , whereas atg16l1 has minimal residence in early endosomes ( supported by both temperature block and vamp3 knockdown experiments ) . , 1997 , 1998 ; it is likely that some plasma membrane matg9 ends up being trafficked from early to recycling endosomes for fusion with atg16l1 . although we can not exclude other sources for early endosomal matg9 , our temperature block experiments suggest that matg9 in early endosomes is not predominantly derived from the golgi , as this protein accumulates in early endosomes rather than the golgi at 18c . although vamp3 has previously been linked to autophagy , it is important to clarify that fader et al . this is not the stage of autophagosome biogenesis we are concerned with we are describing the processes and structures preceding the formation of lc3-positive autophagosomes . our data reveal that membrane traffic from early to recycling endosomes is crucial for autophagosome formation and that the recycling compartment is not merely responsible for recycling of plasma membrane receptors but also serves as a station in the early stages of autophagosome biogenesis where matg9 and atg16l1 meet . the relevant partner , q - snares , regulating this event will need to be elucidated in future studies ( and may be difficult to pinpoint because some snares participate in multiple distinct fusion events as a function of their partners [ jahn and scheller , 2006 ] ) however , the identification of vamp3 allowed us to propose that the coalescence between atg16l1 and matg9 in recycling endosomes mediated by vamp3 is likely a critical event required for autophagy because vamp3 knockdown also decreased autophagosome formation ( figure 7d ) . it is tempting to speculate that the vamp3-mediated fusion of the atg16l1 and matg9 compartments may also be important for exit of preautophagosomal structures containing these proteins from recycling endosomes , as atg16l1 accumulated in these structures when vamp3 was knocked down ( figures 6d and 6e ) . this model suggests that atg16l1-containing vesicles may require vamp3/matg9 fusion in order to be competent to exit the recycling endosomes and mature into phagophores ( figure 7d ) . our data do not exclude additional sources for autophagosome membrane , and our demonstration that matg9 and atg16l1 meet prior to acquisition of lc3 suggests the likelihood that multiple independent membrane sources and itineraries may contribute to autophagosomes . after the reaction , the samples were fixed with 2% paraformaldehyde in pbs for 15 min , centrifuged to remove the fixative ( 13,000 rpm , 5 min ) , resuspended in distilled water , and mounted with mowiol 4 - 88 with dapi on glass coverslips for confocal observation . then we performed three sets of double labeling ( matg9/cla , atg16l1/cla , matg9/atg16l1 ) and the opposite sequential combinations ( cla / matg9 , cla / atg16l1 , atg16l1/matg9 ) . we discovered from the double immunolabeling that matg9 is the weakest antibody and thus we labeled this last ( as the antibody and the gold tend to detach during the multiple immunolabeling procedures ) . as an example , we now show single , double and triple labeling in figures 1f , 1h , and 1i , respectively.morphometric analysis was performed directly on the pictures by counting the tubulo - vesicular structures carrying both markers ( atg16l1 and rab11 ) , compared with the total atg16l1-labeled structures ( figure 2c).facs - based endocytosis assaystransferrin - recycling assay was performed as previously described ( peden et al . after 2 hr of incubation at 4c under agitation , the beads were washed three times with ripa buffer and one time with pbs . we discovered from the double immunolabeling that matg9 is the weakest antibody and thus we labeled this last ( as the antibody and the gold tend to detach during the multiple immunolabeling procedures ) .

macroautophagy , which we will refer to as autophagy , is a highly conserved catabolic process in which cytoplasmic proteins and organelles are engulfed by double - membrane structures called autophagosomes , which are then transported to lysosomes for degradation . the earlier of the ubiquitination - like events in autophagosome biogenesis involves the conjugation of atg12 to atg5 , after which atg12 - 5 can form a complex with atg16l1 ( mizushima et al . the elongation of the edges of the phagophore involves a second ubiquitin - like protein , lc3 , an atg8 family member , which is cleaved by atg4 to form cytoplasmic lc3-i . we found that atg16l1 associates with clathrin - coated pits , and after internalization and uncoating , the atg16l1-associated plasma membrane becomes associated with phagophore precursors , which mature into phagophores and then autophagosomes . inhibition of clathrin - mediated endocytosis causes defective autophagosome formation , which is associated with impaired uptake of plasma membrane into autophagic precursors and autophagosomes ( ravikumar et al . we recently showed that the small g protein arf6 also has a function in autophagy and that part of its effects on autophagy could be explained by the fact that it stimulates phosphatidylinositol 4,5-biphosphate ( pip2 ) formation at the plasma membrane . the atg16l1-positive phagophore precursors undergo snare - mediated homotypic fusion events to give rise to tubulovesicular structures , and the increase in size resulting from these fusions enhances the capacity of these structures to acquire atg8/lc3 , which marks phagophores ( moreau et al . however , these authors have argued on the basis of their recent data that this model may be overly simplistic because mammalian atg9 ( matg9 and atg9l1 ) was seen to associate with many other compartments , including recycling endosomes , early endosomes , and late endosomes ( orsi et al . furthermore , matg9 interactions with omegasomes and autophagosomes have been examined using live - cell imaging and fusion events were not observed , leading to the suggestion that matg9 undergoes dynamic interactions with phagophores and autophagosomes without being incorporated into their membranes and that matg9 traffics by tubulation from an existing reservoir compartment , analogous to what has been described in yeast ( mari et al . , 2010 ; it is therefore still unclear how matg9 traffics , how it ends up associated with some lc3-positive structures if no obvious fusion events appear to occur , and whether matg9 vesicles contribute membrane to mammalian autophagosomes . here , we find that matg9 vesicles are trafficked from the plasma membrane to the recycling endosome via clathrin - coated pits that are largely distinct from those that endocytose atg16l1 . this process , which is dependent on the r - snare vamp3 , appears to be physiologically relevant , as starvation , an evolutionarily conserved autophagy stimulus , also reduces membrane exit from the recycling endosomes and results in increased associations of matg9 and atg16l1 . , 1998 ; as expected , the temperature block caused a peripherally biased redistribution of transferrin - positive vesicles , increased transferrin association with the early endosome marker eea1 , and decreased transferrin association with the recycling endosome marker rab11 ( figures s1a s1c and c ) . furthermore , the 18c incubation led to a peripherally biased dispersion of matg9 ( similar to what we observed with transferrin [ figure 1b ] ) , increased matg9 colocalization with the early endosome marker eea1 ( figures s1d and d ) , and reduced its colocalization with tgn46 ( a marker for tgn ) ( figure 1b ) . this suggests that the predominant effects of 18c on matg9 localization are not due to failed exit from the golgi - impaired golgi - recycling endosome trafficking , which has been described in polarized cells at 18c ( ang et al . , these results raised the possibility that most of the matg9 that appeared to be at the tgn in basal conditions may actually be in recycling endosomes nearby and that the tgn pool of matg9 may be quite small . because transferrin enters the cell by clathrin - mediated endocytosis , we examined whether matg9 is trafficked to the plasma membrane and then internalized by clathrin - mediated endocytosis . dynasore treatment caused a redistribution of matg9 from a perinuclear concentration to a more dispersed localization , and this was associated with decreased colocalization of matg9 with transferrin or a golgi marker ( figure 1c ) . total internal reflection fluorescence ( tirf ) microscopy , which enables high - resolution assessment of molecules close to the plasma membrane , suggested that matg9 redistributed close to the plasma membrane in dynasore - treated cells ( figure 1d ) . a similar redistribution of matg9 from its normal perinuclear localization close to the plasma membrane was seen in cells overexpressing a dominant - negative dynamin mutant or small interfering rna ( sirna ) to the clathrin adaptor ap2 ( to block clathrin - dependent endocytosis ) ( figures 1d and s1e s1 g ) . in order to analyze the clathrin - coated structures carrying these two proteins with greater resolution , we treated cells with dynasore to recruit as much as possible atg16l1 and matg9 to the plasma membrane and then performed immunogold labeling on cryosections to observe the two proteins . interestingly , live - imaging analysis of atg16l1 and rab11 confirmed that such homotypic fusions occurred , and there also appeared to be progressive recruitment of rab11 ( vesicles from recycling endosomes ) to the atg16l1 ( preautophagosome ) vesicles ( figure 2d and movie s1 ) , suggesting that the recycling compartment may provide membranes to preautophagosomal structures . however , we observed authentic and long - lasting fusions between matg9 and atg16l1-positive structures , suggesting that matg9 vesicles and associated membranes mix with atg16l1-positive phagophore precursors and that matg9 becomes associated with autophagosome precursors at this early prephagophore stage ( figure 2 g and movie s2 ) . our data suggest that the recycling endosome is the site of fusion between matg9 and atg16l1 vesicles and led us to hypothesize that membrane flow in and out of the recycling compartment may influence matg9-atg16l1 fusions and autophagy . these possibilities can be deconvoluted by studying cells in the absence and presence of lysosomal inhibitors like bafilomycin a1 or nh4cl , which inhibit autophagosome degradation and hence allow one to assess autophagosome formation rates ( rubinsztein et al . , 2011)and caused accumulation of atg16l1 in recycling endosomes but did not lead to a change in its negligible abundance in early endosomes ( note that the low level of colocalization we see may be background due to labeling of adjacent structures ) ( figures 3f and 3 g ) . in addition , the temperature block decreased the extent to which atg16l1 and matg9 colocalized and decreased the sizes of both matg9 and atg16l1 vesicles ( figures 3h and s3a for separate channels ) , suggesting that this treatment impaired homotypic and heterotypic fusion of atg16l1 and matg9 vesicles . autophagy regulates the proportion of cells with mutant huntingtin q74 ( htt ) aggregates by regulating mutant htt clearance , and the percentage of cells with aggregates correlates inversely with autophagic activity ( luo and rubinsztein , 2010 ; ravikumar et al . overexpression of rab11 and myosin vb tail also increased the size of atg16l1 vesicles ( figures 3j and s3c ) ( consistent with increased homotypic fusion of atg16l1 vesicles ) , increased the colocalization of atg16l1 with the transferrin ( figures 3k and s3d ) ( consistent with more atg16l1 in the recycling compartment ) , and increased the colocalization of atg16l1 and matg9 ( figures 3l and s3e ) ( consistent with enhanced heterotypic fusion of matg9 and atg16l vesicles ) . we examined the links between autophagy induction , recycling endosomes , homotypic atg16l1 fusion , and heterotypic atg16l1-matg9 fusion during starvation , an evolutionarily conserved autophagic stimulus . in order to clarify the itineraries of these vesicles and their trafficking routes more robustly because intracellular vesicular fusions are generally snare dependent , we tested and confirmed that this was the case for atg16l1-matg9 fusions because n - ethylmaleimide ( nem ) , which abolishes snare fusion ( jahn and scheller , 2006 ; moreau et al . , 1994 ) , reduced the colocalization in cells between atg16l1 and matg9 ( figure 4a ) and impaired the fusion of these two vesicle populations in vitro using postnuclear supernatants isolated from either strawberry - atg16l1- or atg9l1-gfp - expressing cells ( figures 4b4d ) . typically , snare complexes comprise three molecules with highly conserved glutamine ( q ) residues in the snare motif and one with a highly conserved arginine ( r ) residue in this domain , namely qa- , qb- , qc- , and r - snares ( jahn and scheller , 2006 ) . we confirmed that vamp3 indeed regulated autophagy by showing that it decreased autophagosome numbers ( lc3-ii levels on western blots versus actin and a 60% reduction in the number of lc3-positive vesicles [ autophagosomes ] ) , which could be accounted for by impaired autophagosome biogenesis ( lc3-ii levels in the presence of bafilomycin a1 ) ( figures 5b , 5c , s4a , and s4b ) . a temperature block at 18c , which impairs trafficking between early and recycling endosomes , did not affect the colocalization between matg9 and vamp3 , suggesting that the two proteins are cotrafficking ( figure 5e ) , but reduced the colocalization between atg16l1 and vamp3 ( figures 5e and s4c ) in a manner reminiscent to what we observed with matg9 and atg16l1 ( figure 3h ) . in contrast to what we observed with matg9 , knockdown of vamp3 caused the accumulation of atg16l1 in recycling endosomes but did not lead to a change in its negligible abundance in early endosomes ( note that the low level of colocalization we see may be background due to labeling of adjacent structures ) ( figures 6d6f ) . vamp3 knockdown reduced the colocalization of matg9 and atg16l1 ( figure 7a ) , and live - cell imaging demonstrated that coalescence between atg16l1 and matg9 was abrogated by vamp3 knockdown as compared to control cells ( figures s5a and s5b ; see also movies s5 and s6 ) without affecting homotypic fusion of atg16l1 vesicles ( figure 7b ) . interestingly , we found very low levels of colocalization between atg16l1 and eea1 , suggesting that the atg16l1 vesicles were routed away from early endosomes soon after clathrin uncoating ( moreau et al . in this case , matg9 appears to be localized to a largely distinct pool of clathrin pits compared to atg16l1 , and although both proteins end up in recycling endosomes , matg9 is trafficked to recycling endosomes via eea1-positive early endosomes , whereas atg16l1 has minimal residence in early endosomes ( supported by both temperature block and vamp3 knockdown experiments ) . , as this treatment decreased the association of vamp3 with atg16l1 ( figures 5e and s4c ) , but not with matg9 ( figure 5e ) , and impaired the colocalization between atg16l1 and matg9 ( figure 3h ) . our data reveal that membrane traffic from early to recycling endosomes is crucial for autophagosome formation and that the recycling compartment is not merely responsible for recycling of plasma membrane receptors but also serves as a station in the early stages of autophagosome biogenesis where matg9 and atg16l1 meet . the observation that vamp3 knockdown has no obvious effect on transferrin recycling ( compared to the dramatic consequences of the 18c temperature block ) suggests either that the fusion of the matg9 and atg16l1 compartments may have a higher dependency on this r - snare than conventional endocytic trafficking or that vamp3 may be mediating selective fusion of components of the early endosomal and recycling endosome compartments rather than bulk fusion . the relevant partner , q - snares , regulating this event will need to be elucidated in future studies ( and may be difficult to pinpoint because some snares participate in multiple distinct fusion events as a function of their partners [ jahn and scheller , 2006 ] ) however , the identification of vamp3 allowed us to propose that the coalescence between atg16l1 and matg9 in recycling endosomes mediated by vamp3 is likely a critical event required for autophagy because vamp3 knockdown also decreased autophagosome formation ( figure 7d ) . it is tempting to speculate that the vamp3-mediated fusion of the atg16l1 and matg9 compartments may also be important for exit of preautophagosomal structures containing these proteins from recycling endosomes , as atg16l1 accumulated in these structures when vamp3 was knocked down ( figures 6d and 6e ) . extended experimental proceduresantibodies and reagentsthe antibodies used include : rabbit anti - rab11 ( invitrogen-715300 ) , rabbit anti - atg16l1 ( cosmobio co. ltp- tmd - ph - atg16l ; cell signaling : 8089s ) , atg12 ( cell signaling : 2010s ) , mouse anti - tubulin ( sigma ) , rabbit anti - lc3 for western blotting ( novus biological- nb100 - 2220 ) , mouse anti - ha ( covance mms-10i ) , rabbit anti - atg9 ( abcam - ab108338 and sigma - a0732 ) , sheep anti - tgn46 ( abd serotec ah - p500 ) , mouse anti - human transferrin receptor ( atcc-5e9c11 ) , human transferrin alexa-555 , alexa-647 and alexa-488 ( invitrogen ) , mouse anti - eea1 ( abcam - ab70521 ) , rabbit anti - clathrin ( abcam- ab21679 ) , cholera toxin hrp ( invitrogen ) , mouse anti - ap2 2 ( bd ap-50 ) and anti - hrp ( sigma - p7899 ) , rabbit anti - vamp3 was a kind gift from a.a . the background was fixed for all within - experiment analyses ( for manders).tirf and dynasore treatmenthela cells were treated with dynasore 100 m for 2 hr in serum - free medium , or transfected for 16h with dynamin k44a mutant or ap2 sirna kd ( 5 day of treatment ) fixed and labeled with anti - atg9 antibody.the samples were analyzed by total internal reflection fluorescence ( tirf ) microscopy ( tirf 3 , carl zeiss microimaging inc.).immunogold electron microscopyhela cells were transfected with gfp - atg16l1 for 24h . briefly , we first performed the three separate single labelings to assess the specificity and the localization of the three different proteins ( clathrin , matg9 and atg16l1 ) . as an example , we now show single , double and triple labeling in figures 1f , 1h , and 1i , respectively.morphometric analysis was performed directly on the pictures by counting the tubulo - vesicular structures carrying both markers ( atg16l1 and rab11 ) , compared with the total atg16l1-labeled structures ( figure 2c).facs - based endocytosis assaystransferrin - recycling assay was performed as previously described ( peden et al . the antibodies used include : rabbit anti - rab11 ( invitrogen-715300 ) , rabbit anti - atg16l1 ( cosmobio co. ltp- tmd - ph - atg16l ; cell signaling : 8089s ) , atg12 ( cell signaling : 2010s ) , mouse anti - tubulin ( sigma ) , rabbit anti - lc3 for western blotting ( novus biological- nb100 - 2220 ) , mouse anti - ha ( covance mms-10i ) , rabbit anti - atg9 ( abcam - ab108338 and sigma - a0732 ) , sheep anti - tgn46 ( abd serotec ah - p500 ) , mouse anti - human transferrin receptor ( atcc-5e9c11 ) , human transferrin alexa-555 , alexa-647 and alexa-488 ( invitrogen ) , mouse anti - eea1 ( abcam - ab70521 ) , rabbit anti - clathrin ( abcam- ab21679 ) , cholera toxin hrp ( invitrogen ) , mouse anti - ap2 2 ( bd ap-50 ) and anti - hrp ( sigma - p7899 ) , rabbit anti - vamp3 was a kind gift from a.a . briefly , we first performed the three separate single labelings to assess the specificity and the localization of the three different proteins ( clathrin , matg9 and atg16l1 ) .

low birth weight ( lbw ) is associated with an increased risk for a number of conditions arising in adulthood , such as type 2 diabetes , dyslipidemia , coronary artery disease , essential hypertension , and cerebrovascular accidents . insulin resistance ( i.e. , reduced insulin sensitivity ) is a well - established , early metabolic abnormality in the pathogenesis of these conditions . it has been proposed that a reduced insulin sensitivity in lbw subjects results from the adaptation to adverse in utero conditions during a critical period of development . however , it is well known that lbw newborns are also exposed to postnatal stress , which is reflected in higher neonatal morbidity and mortality . this has led to the hypothesis that postnatal stress may as well contribute to the metabolic modifications in lbw children , independently of the adequacy of their birth weight to gestational age . if early postnatal stress plays a role in long - term metabolic modifications , prematurity may be an important confounding factor . most previous studies linking lbw to the propensity toward disease in adulthood have focused on those who were small for gestational age ( sga ) and born at term . only few studies demonstrate the association of prematurity with a tendency to disease in letter adulthood but data 's are still conflicting , and further evidence is still required to establish a definitive role . moreover , it has been recognized that reduced insulin sensitivity , a hallmark in most lbw - related conditions may be present as early as the 1 year of life . hence , we planned a prospective cohort study to elaborate insulin sensitivity in infants who were lbw and born prematurely . in addition , it has been suggested that besides in utero stress and postnatal stress , other independent variables such as ponderal index ( pi ) , birth weight , catch - up growth for weight and length , and current weight and length may also contribute in insulin sensitivity . our second aim , therefore , was to determine the role of other variables to predict insulin sensitivity in infancy . the present study was a prospective ( cohort ) study , conducted in umaid hospital and dr . sample size was calculated at power 80% , confidence interval ( ci ) 95% , four independent variables and assuming expected multiple regression coefficient 0.4 as observed in a previous study titled as , determinants of insulin sensitivity and secretion in very - low - birth weight children bazaes et al . the resulting value of sample size came out to be 68 . in the beginning phase of the study , total 150 neonates born in this tertiary care center were enrolled to compensate loss during follow - up . those with a birth weight at par or above the 10 percentile were defined as being appropriate for gestational age ( aga ) , and those with a birth weight below 10 percentile as being sga . the study population was divided into four strata ( preterm aga , preterm sga , term aga , and term sga ) according to their gestational age and their birth weight for gestation thereafter sampling was done by stratified random sampling method . the study cohort was formed by 50 preterm aga and control was formed by 50 term aga neonates . another cohort of 50 sga neonates ( 25 preterm sga and 25 term sga ) was also included . this group was added for the sake of comparison and in order to determine other variables to predict insulin sensitivity in infancy . during follow - up ( till 9 months of the age ) numbers of participating infants were decreased , 15 were excluded ( 8 preterm aga , 1 term aga , 5 preterm sga , and 1 term sga ) and 35 did not come in follow - up ( 6 preterm aga , 13 term aga , 9 preterm sga , and 7 term sga ) . finally , 100 infants ( 36 preterm aga , 36 term aga , 11 preterm sga , and 17 term sga ) were gone through anthropometric and hormonal evaluation , with adequate sample size . exclusion criteria included neonates of diabetic mother and those having first degree relative with type-2 diabetes mellitus , chronic illness , or medical therapy known to influence insulin sensitivity , chromosomal syndromes , congenital anomalies , congenital infections and sick neonates ( intracranial hemorrhage , perinatal asphyxia , pyomeningitis , necrotizing enterocolitis ) , neonates requiring positive pressure ventilation , and vasopressors . the nature , purpose , and possible risks of the study were explained to the parents in detail before consent was obtained . complete history including family history and antenatal , natal , and postnatal history was taken in all subjects . general physical examination and systemic examination was done , and accurate assessment of gestational age was done by using the expanded new ballard score . detailed anthropometry ( length , weight , pi , head circumference , chest circumference , and weight - for - length ) was performed according to the world health organization ( who ) guidelines by a trained pediatrician . birth weight , length , and head circumference were converted into standard deviation scores ( sds ) ( z = [ observed value mean value]/sd ) according to the who reference to allow comparison of subjects with different gestational ages , chronological ages , and sexes . the weight - for - length index was used to provide an age - adjusted evaluation of relative obesity . routine blood investigation and fasting blood sugar level of all neonates ( after 24 h fasting ) were performed . however , the upper age cutoff for the enrolled subjects was limited to 9 months instead of 12 months in this study to avoid biases arising out of the transition to childhood from infancy during the last few months . the reason behind not using 6 months of age as cut off is because by that age , the term neonates may not be exposed to complementary feed , whereas preterm neonates would have received both breastfeeding and complementary feeding ( corrected age of 6 months for prematurity ) . both cohorts and controls were followed at corrected age of 3 months , 6 months , and 9 months . all infants were on exclusive breastfeed for 5.56 0.84 months ( preterm aga 5.47 0.78 , preterm sga 5.60 0.85 , term aga 5.60 0.85 , term sga 5.79 0.84 , p 0.05 , range 47 months ) and then started complementary feed according to the indian academy of pediatrics guideline . on each follow - up , anthropometry assessment was performed . at the age of 9 months ( 5 ml ) , blood sample was obtained for fasting plasma glucose and serum insulin ( si ) level ( after 24 h fasting ) . glucose was estimated by glucose oxidase - peroxidase aminophenazone - phenol method using the commercially available kit ( human mbh , weisbaden , germany ) . si was estimated by immunoradiometric assay using commercial kits ( dpc inc . , los angeles , usa ) . the intra- and inter - assay coefficients of variation for the insulin were 5.2 and 7.3% , respectively ; sensitivity was 1.2 u / ml and specificity was 80% . insulin resistance was determined using homeostasis model assessment version 2 ( homa-2 calculator available at http://www.dtu.ox.ac.uk/homa ) . for calculation of homa-2 , glucose values differences in demographic characteristics and clinical measures between control and premature groups were investigated by means of analysis of variance for continuous variables and the chi - square test for proportions . differences in neonatal characteristics were evaluated with the use of fisher 's exact test for proportions and the mann general linear model ( ancova ) was used to investigate differences in glucose - regulation variables among the four groups of subjects . the four groups , divided according to their gestational age ( preterm vs. term ) and weight for gestation ( aga vs. sga ) , were included as factor , whereas pi and weight - for - length at 9 months ( wfl ) were included as covariance in this model . the specific hypotheses tested were the difference in insulin sensitivity between appropriate- and small - for - gestational - age subjects within the premature groups and the difference in insulin sensitivity between the premature groups and the term group . further analysis was performed by multiple regression models using log insulin resistance index ( iri ) as depended variable and gestational age , pi , weight - for - length , and sga status as independent variables . the data on fasting si and homa iri were logarithmically transformed to meet the assumptions of normality . all analysis was carried out by using the spss version 20.0 statistical package ( spss , inc . , differences in demographic characteristics and clinical measures between control and premature groups were investigated by means of analysis of variance for continuous variables and the chi - square test for proportions . differences in neonatal characteristics were evaluated with the use of fisher 's exact test for proportions and the mann general linear model ( ancova ) was used to investigate differences in glucose - regulation variables among the four groups of subjects . the four groups , divided according to their gestational age ( preterm vs. term ) and weight for gestation ( aga vs. sga ) , were included as factor , whereas pi and weight - for - length at 9 months ( wfl ) were included as covariance in this model . the specific hypotheses tested were the difference in insulin sensitivity between appropriate- and small - for - gestational - age subjects within the premature groups and the difference in insulin sensitivity between the premature groups and the term group . further analysis was performed by multiple regression models using log insulin resistance index ( iri ) as depended variable and gestational age , pi , weight - for - length , and sga status as independent variables . the data on fasting si and homa iri were logarithmically transformed to meet the assumptions of normality . all analysis was carried out by using the spss version 20.0 statistical package ( spss , inc . , out of hundred subjects , 36 preterm aga , 11 preterm sga , and 17 term sga , as study cohorts and 36 term aga , as control cohort , were enrolled in this study . as it was expected , gestational age , weight ( sd score ) , length ( sd score ) , head circumference ( sd score ) , and weight - for - length index were significantly different among the groups . preterm sga babies were smaller ( length sds ) ( p = 0.013 ) and thinner than preterm aga ( p = 0.001 post hoc test ) . data about neonatal characteristics were summarized in table 2 , and no significant difference was observed in maternal characteristics and oxygen requirement , antibiotic days , and days until full oral feeding established . iri ( median 1.80 , interquartile range 3.90 [ 0.534.39 ] , skewness 1.60 [ rt skewed ] , kurtosis 2.42 , mean 2.95 , and sd 3.20 ) and si ( median 8.80 , interquartile range 17.55 [ 2.6220.16 ] , skewness 1.67 [ rt skewed ] , kurtosis 2.94 , mean 13.49 , sd 14.29 ) had abnormal distribution and converted to log 10 value to normalize the distribution [ figure 1 ] . generalized linear model ( ancova ) was constructed to adjust gestational age , weight for gestation , pi , and weight - for - length ( r = 0.76 , adjusted r = 0.75 ; means 75% variability explained by this model ) . change in r value for these factors show that gestational age , birth weight , and weight - for - length were good predictors for iri ( p = 0.0001 ) as compared to pi ( p = 0.002 ) . pairwise contrast shows that ( adjustment for multiple comparisons by bonferroni ) , both preterm aga ( mean difference 0.617 , 95% ci ; 0.430.80 , p = 0.0001 ) and preterm sga ( mean difference 0.764 , 95% ci ; 0.441.09 , p = 0.0001 ) similarly , infants who had been born term sga had significantly higher insulin resistance than term aga ( mean difference 0.725 , 95% ci ; 0.490.96 , p = 0.0001 ) . no significant difference was found in between preterm sga group and preterm aga group ( mean difference 0.147 95% ci ; 0.130.42 , p = 0.927 ) . it was noticed that when we combine both preterm cohort , insulin resistance gets increased by 5.2% but not become significantly higher ( mean difference 0.076 , 95% ci ; 0.260.11 , p = 0.95 ) than term sga . baseline characteristics of the study subjects * maternal and neonatal characteristics in two groups of premature neonates indicator of glucose homeostasis * right skewed distribution of insulin resistance index further analysis was done by using multiple regression models summarized in table 4 . iri at 9 months was found negatively correlated with gestational age ( = 0.42 , p = 0.001 ) , birth weight z - score ( = 0.35 , p = 0.001 ) , pi ( = 0.61 , p = 0.001 ) , length at 9 months ( z - score ) ( = 0.413 , p = 0.001 ) and catch - up growth for length ( = 0.311 , p = 0.002 ) and positively correlated with sga status ( = 0.511 , p = 0.001 ) , weight - for - length ( = 0.393 , p = 0.001 ) , and catch - up growth for weight ( = 0.198 , p = 0.048 ) . sequential multiple regression analysis for identifying influence of perinatal stress and anthropometric variables on insulin resistance index ( dependent variable ) multiple regression models were constructed using sequential method including gestational age , pi , weight - for - length as continuous variables , and type according to weight for gestation ( aga vs. sga ) as categorical variables . minimum variables which predict the largest amount of variability ( birth weight and length have collinearity with pi and weight and length at 9 months have collinearity with weight - for - length ) were included in this model to validate and stabilize the model . subsequently , pi , weight - for - length , and type according to weight for gestation were added in model 2 , 3 , and 4 , respectively . as a measure of goodness of fit , adjusted r was increased in each model from 0.188 in model 1 to 0.702 in model 4 , which indicates 70% of variability , now explained by model 4 ( significant r changes in each step p = 0.001 ) [ table 4 ] . substituting the variables and the unstandardized coefficients from the table 4 , the equation for model was as follows : log iri = 3.213 ( 0.173 gestational age ) ( 0.25 pi ) + ( 0.183 weight - for - length ) + ( 0.586 type ) because aga is coded zero , the final term in the equation was removed for aga . the term type indicates that after adjusting for gestational age , pi , and weight - for - length at 9 months , sga babies were 0.586 more iri than aga . in effect , this means that y - intercept was 3.799 for sga ( i.e. , 3.213 + 0.586 ) and 3.213 for aga . thus , the lines for aga and sga were parallel , but aga had a lower y - axis intercept [ figure 2 ] . scatter plot for insulin resistance index on gestational age with regression line ( appropriate for gestational age vs. small for gestational age ) standardized coefficients indicated the relative importance of each variable in comparable standardized units ( z - scores ) to predict iri . type according to weight for gestational age with a standardized coefficient of 0.505 was a more significant predictor of iri than gestational age ( = 0.481 ) , weight - for - length ( = 0.315 ) , and pi ( = 0.194 ) . as with an r - value , the negative sign was an indication of the direction of effect only [ table 4 ] . the partial correlation was the unique contribution of gestational age ( 0.596 ) , pi ( 0.258 ) , weight - for - length ( 0.504 ) , and type ( 0.579 ) to predicting iri after the effect of other three factor was removed and was an estimate of the relative importance of each predictive variable in isolation from other factors [ table 4 ] . model was validated for stability , precision , and reliability by testing for collinearity , interaction , and residuals . there was no collinearity ( significant relationship between explanatory variables ) in between variables ( correlation coefficient < 0.7 , variance inflation factor ( vif ) < 4 , and tolerance > 0.2 ) . interactions ( multiplicative rather than additive relationship between two explanatory variables ) were tested by including the interaction term ( gestational age type , pi type , and gestational age type pi ) in model , which shows no significant increase in adjusted r ( p > 0.05 ) . similarly , when iri plotted against gestational age , no significant difference between slope of regression line of aga and sga babies were present ( p > 0.05 ) [ figure 2 ] , which further favor in no interaction between gestational age and type according to weight for gestation . standard residuals ( distances between each data point and the value predicted by the regression equation ) showed normal distribution ( mean < 0.00001 , sd = 0.98 ) indicates equal spread of variance over the length of regression model , and the model was homoscedastic [ figure 3 ] . normal distribution of regression standardized residuals alternative regression model was constructed to determine role of catch - up growth in insulin resistance . in this model , gestational age , catch - up for weight , and type according to weight for gestation sequentially added ( adjusted r = 71.9% mean 71.9% variability explained by this model ) . catch - up growth ( = 0.714 ) is a significant predictor of insulin resistance independent of gestational age and sga status . weight - for - length and pi were not included in this model because of collinearity of catch - up growth with pi and weight - for - length at 9 months ( vif > 4 , tolerance < 0.2 , condition index in collinearity diagnostics > 30 ) . in collinearity , diagnostics condition index were 35.97 , 41.70 , and 57.73 for weight catch - up , weight - for - length at 9 months , and pi , respectively . substituting the variables and the unstandardized coefficients , the equation for this model was as follows : log iri = 5.621 ( 0.153 gestational age ) + ( 0.234 weight catch - up at 9 months ) + ( 0.320 type ) , the data are conflicting , with some studies demonstrating that fat mass is the major determinant of is with no effect of gestation whereas others identify a persisting effect of preterm birth . the present study demonstrates the association of prematurity with insulin resistance in infancy after adjusting for significant covariables . these findings provide additional evidence that preterm birth may be a risk factor for the future development of the insulin resistance and type-2 diabetes , and the insulin resistance may develop as early as infancy . we fill a gap in the knowledge , regarding insulin resistance in preterm born babies in 1 year of life which was unexplored till date . a particular strength of our study is that we also include term sga cohort in our study for comparison and explore relative importance of each variables to predict the insulin resistance in infants . the present study explores that sga status is the most significant predictor of insulin resistance followed by gestational age , weight - for - length , and pi in the descending order . most of the studies reveals that in preterm born babies , sga status exerts no effect on insulin sensitivity in childhood , adolescence , and adulthood . , respectively , found that sga preterm born babies have reduced insulin sensitivity in neonatal , childhood , and adolescence . gray used a milk tolerance test ( mtt ) and showed that sga preterm neonates had higher post - mtt insulin levels . reinehr study included both preterm and term sga born subjects thus insulin resistance might be due to confounding effect of term gestation . used both homa modeling and intravenous glucose tolerance test and found reduced insulin sensitivity in sga babies by using homa model but not after stimulated insulin release . however in term sga born babies ' insulin resistance in infancy , childhood , adolescent , and adulthood is well established . with concurrence to most of the studies , we also report that sga status exerts no impact on insulin sensitivity in preterm born babies , but its impact during infancy is a matter of special mention here . in multiple regression analysis , sga born infants have higher insulin resistance ( 0.586 more iri ) than aga born infants irrespective of their gestational age , pi , and current weight - for - length . this difference is insignificant in preterm babies and become significant with increasing gestational age in term babies [ figure 2 ] . furthermore , combination of both preterm cohorts does not result in significantly higher insulin resistance than term sga . these results indicate antenatal stress prior to the third trimester does not contribute in metabolic derangement , and critical window for the same is present in third trimester . similarly , hofman et al . also reported that third trimester is critical window period for metabolic imprinting , but there also remains a debate because studies in animals and humans have suggested several critical periods from the periconceptual period to later pregnancy . additional studies would require confirming the importance of this period and may provide further insight into the role of this critical period in the third trimester . our results also demonstrate that low pi ( thinness at birth ) is associated with higher insulin resistance with special concern to infancy . pi is a continuum with proportionality in sga fetuses and depends on the duration of intrauterine insult and the extent of its effects on weight and length before delivery . relatively brief duration intrauterine insult which affects more on weight than length results in lower pi ( asymmetrical intrauterine growth restriction [ iugr ] ) , and therefore higher insulin resistance . these results further favor that critical window period for metabolic derangement in lbw babies may present in third trimester in which maximum fetus weight growth occurs . insulin is an important growth factor during infancy , and insulin secretion could be relevant for fat deposition and weight gained shortly after birth . iugr followed by catch - up growth for weight in infancy and early childhood may be a sequence that led to high basal metabolic index ( bmi ) , obesity , and fat mass , and therefore insulin resistance in infancy , childhood , adolescent , and adulthood . similarly , prematurity followed by postnatal catch - up growth for weight in infancy and early childhood may be a sequence that led to high bmi , obesity , fat mass , and therefore insulin resistance in childhood , adolescent , and adulthood . in present study , we examine early changes in body composition by using weight - for - length , which provides a more accurate measure of adiposity ( bmi for > 2 years ) than weight alone . our study indicates that higher weight - for - length is associated with higher insulin resistance in infancy , and thus , it indicates an association of adiposity with insulin resistance as early as the 1 year of life . collinearity between catch - up growth for weight and weight - for - length indicate that excess adiposity is superimposed on normal growth related adiposity during catch - up growth . concomitant to this , converging evidence suggests that catch - up growth in preterm and small for gestation is intimately linked with a disproportionately faster rate to gain body fat rather than lean tissue , that is , to a preferential acceleration of fat recovery or catch - up fat . therefore , we create a hypothesis that catch - up growth for weight in infancy and early childhood and the higher bmi and obesity in letter childhood , adolescence , and adulthood are the same spectrum phenomenon in different window periods of life after exposure to environmental factors such as high energy intake or low physical activity and may be a result of metabolic imprinting after inutero stress or postnatal stress . first , it is one of the few to implement a prospective , longitudinal design to investigate the link between prematurity and insulin resistance as early as infancy . second , this study examines early changes in body composition using weight - for - length , which provides a more accurate measure of adiposity than weight alone . third , regression model is tested for stability , collinearity , and interaction between various variables and finally , this study also includes full - term sga infants , and thus can identify the relative importance of various variables to determine insulin sensitivity in infancy . our cohort did have better clinical outcomes than the total cohort of surviving children who had been born prematurely since we excluded subjects with major or moderate disability . on the basis of these neonatal characteristics and on the better developmental outcome of our cohort , we do not believe that a selection bias has occurred that influenced our findings . finally , we have drawn equation to estimate iri by using gestational age , pi , sga status , and current weight - for - length . by using weight - for - length which is age - independent variable , we can monitor insulin resistance during follow - up in both preterm and sga babies . identification of these infants may help to focus preventive measures aimed at controlling the current epidemic of obesity and its complications , but we should be cautious while making recommendations for nutrition till we have evidence available from randomized control trial

objective : this study was done to determine the role of prematurity and other variables to predict insulin sensitivity in infancy.subjects and methods : in this prospective study , 36 preterm appropriate for gestational age ( aga ) , 11 preterm small for gestational age ( sga ) , and 17 term sga included as study cohort and 36 term aga as control cohort . detailed anthropometry assessment was performed at birth , 3 , 6 , and 9 months and at 9 months , fasting plasma glucose and serum insulin was done . insulin resistance was determined by using homeostasis model assessment version 2.results:it is found that preterm aga ( mean difference 0.617 , 95% confidence interval [ ci ] ; 0.430.80 , p = 0.0001 ) , preterm sga ( mean difference 0.764 , 95% ci ; 0.441.09 , p = 0.0001 ) , and term aga ( mean difference 0.725 , 95% ci ; 0.490.96 , p = 0.0001 ) group had significantly higher insulin resistance than control . there was no significant difference in between preterm sga and preterm aga ( mean difference 0.147 95% ci ; 0.130.42 , p = 0.927 ) . in multiple regression models , sga status ( = 0.505 ) was more significant predictor of insulin resistance index than gestational age ( = 0.481 ) , weight - for - length ( = 0.315 ) , and ponderal index ( = 0.194).conclusion : preterm birth is a risk factor for the future development of insulin resistance which may develop as early as infancy .

I S Statistical methods R D Financial support and sponsorship Conflicts of interest

, reduced insulin sensitivity ) is a well - established , early metabolic abnormality in the pathogenesis of these conditions . most previous studies linking lbw to the propensity toward disease in adulthood have focused on those who were small for gestational age ( sga ) and born at term . only few studies demonstrate the association of prematurity with a tendency to disease in letter adulthood but data 's are still conflicting , and further evidence is still required to establish a definitive role . moreover , it has been recognized that reduced insulin sensitivity , a hallmark in most lbw - related conditions may be present as early as the 1 year of life . hence , we planned a prospective cohort study to elaborate insulin sensitivity in infants who were lbw and born prematurely . in addition , it has been suggested that besides in utero stress and postnatal stress , other independent variables such as ponderal index ( pi ) , birth weight , catch - up growth for weight and length , and current weight and length may also contribute in insulin sensitivity . our second aim , therefore , was to determine the role of other variables to predict insulin sensitivity in infancy . sample size was calculated at power 80% , confidence interval ( ci ) 95% , four independent variables and assuming expected multiple regression coefficient 0.4 as observed in a previous study titled as , determinants of insulin sensitivity and secretion in very - low - birth weight children bazaes et al . those with a birth weight at par or above the 10 percentile were defined as being appropriate for gestational age ( aga ) , and those with a birth weight below 10 percentile as being sga . the study population was divided into four strata ( preterm aga , preterm sga , term aga , and term sga ) according to their gestational age and their birth weight for gestation thereafter sampling was done by stratified random sampling method . the study cohort was formed by 50 preterm aga and control was formed by 50 term aga neonates . another cohort of 50 sga neonates ( 25 preterm sga and 25 term sga ) was also included . this group was added for the sake of comparison and in order to determine other variables to predict insulin sensitivity in infancy . during follow - up ( till 9 months of the age ) numbers of participating infants were decreased , 15 were excluded ( 8 preterm aga , 1 term aga , 5 preterm sga , and 1 term sga ) and 35 did not come in follow - up ( 6 preterm aga , 13 term aga , 9 preterm sga , and 7 term sga ) . finally , 100 infants ( 36 preterm aga , 36 term aga , 11 preterm sga , and 17 term sga ) were gone through anthropometric and hormonal evaluation , with adequate sample size . exclusion criteria included neonates of diabetic mother and those having first degree relative with type-2 diabetes mellitus , chronic illness , or medical therapy known to influence insulin sensitivity , chromosomal syndromes , congenital anomalies , congenital infections and sick neonates ( intracranial hemorrhage , perinatal asphyxia , pyomeningitis , necrotizing enterocolitis ) , neonates requiring positive pressure ventilation , and vasopressors . general physical examination and systemic examination was done , and accurate assessment of gestational age was done by using the expanded new ballard score . detailed anthropometry ( length , weight , pi , head circumference , chest circumference , and weight - for - length ) was performed according to the world health organization ( who ) guidelines by a trained pediatrician . the weight - for - length index was used to provide an age - adjusted evaluation of relative obesity . however , the upper age cutoff for the enrolled subjects was limited to 9 months instead of 12 months in this study to avoid biases arising out of the transition to childhood from infancy during the last few months . both cohorts and controls were followed at corrected age of 3 months , 6 months , and 9 months . all infants were on exclusive breastfeed for 5.56 0.84 months ( preterm aga 5.47 0.78 , preterm sga 5.60 0.85 , term aga 5.60 0.85 , term sga 5.79 0.84 , p 0.05 , range 47 months ) and then started complementary feed according to the indian academy of pediatrics guideline . on each follow - up , anthropometry assessment was performed . at the age of 9 months ( 5 ml ) , blood sample was obtained for fasting plasma glucose and serum insulin ( si ) level ( after 24 h fasting ) . insulin resistance was determined using homeostasis model assessment version 2 ( homa-2 calculator available at http://www.dtu.ox.ac.uk/homa ) . the four groups , divided according to their gestational age ( preterm vs. term ) and weight for gestation ( aga vs. sga ) , were included as factor , whereas pi and weight - for - length at 9 months ( wfl ) were included as covariance in this model . the specific hypotheses tested were the difference in insulin sensitivity between appropriate- and small - for - gestational - age subjects within the premature groups and the difference in insulin sensitivity between the premature groups and the term group . further analysis was performed by multiple regression models using log insulin resistance index ( iri ) as depended variable and gestational age , pi , weight - for - length , and sga status as independent variables . the four groups , divided according to their gestational age ( preterm vs. term ) and weight for gestation ( aga vs. sga ) , were included as factor , whereas pi and weight - for - length at 9 months ( wfl ) were included as covariance in this model . the specific hypotheses tested were the difference in insulin sensitivity between appropriate- and small - for - gestational - age subjects within the premature groups and the difference in insulin sensitivity between the premature groups and the term group . further analysis was performed by multiple regression models using log insulin resistance index ( iri ) as depended variable and gestational age , pi , weight - for - length , and sga status as independent variables . , out of hundred subjects , 36 preterm aga , 11 preterm sga , and 17 term sga , as study cohorts and 36 term aga , as control cohort , were enrolled in this study . as it was expected , gestational age , weight ( sd score ) , length ( sd score ) , head circumference ( sd score ) , and weight - for - length index were significantly different among the groups . preterm sga babies were smaller ( length sds ) ( p = 0.013 ) and thinner than preterm aga ( p = 0.001 post hoc test ) . data about neonatal characteristics were summarized in table 2 , and no significant difference was observed in maternal characteristics and oxygen requirement , antibiotic days , and days until full oral feeding established . iri ( median 1.80 , interquartile range 3.90 [ 0.534.39 ] , skewness 1.60 [ rt skewed ] , kurtosis 2.42 , mean 2.95 , and sd 3.20 ) and si ( median 8.80 , interquartile range 17.55 [ 2.6220.16 ] , skewness 1.67 [ rt skewed ] , kurtosis 2.94 , mean 13.49 , sd 14.29 ) had abnormal distribution and converted to log 10 value to normalize the distribution [ figure 1 ] . generalized linear model ( ancova ) was constructed to adjust gestational age , weight for gestation , pi , and weight - for - length ( r = 0.76 , adjusted r = 0.75 ; means 75% variability explained by this model ) . change in r value for these factors show that gestational age , birth weight , and weight - for - length were good predictors for iri ( p = 0.0001 ) as compared to pi ( p = 0.002 ) . pairwise contrast shows that ( adjustment for multiple comparisons by bonferroni ) , both preterm aga ( mean difference 0.617 , 95% ci ; 0.430.80 , p = 0.0001 ) and preterm sga ( mean difference 0.764 , 95% ci ; 0.441.09 , p = 0.0001 ) similarly , infants who had been born term sga had significantly higher insulin resistance than term aga ( mean difference 0.725 , 95% ci ; 0.490.96 , p = 0.0001 ) . no significant difference was found in between preterm sga group and preterm aga group ( mean difference 0.147 95% ci ; 0.130.42 , p = 0.927 ) . it was noticed that when we combine both preterm cohort , insulin resistance gets increased by 5.2% but not become significantly higher ( mean difference 0.076 , 95% ci ; 0.260.11 , p = 0.95 ) than term sga . baseline characteristics of the study subjects * maternal and neonatal characteristics in two groups of premature neonates indicator of glucose homeostasis * right skewed distribution of insulin resistance index further analysis was done by using multiple regression models summarized in table 4 . iri at 9 months was found negatively correlated with gestational age ( = 0.42 , p = 0.001 ) , birth weight z - score ( = 0.35 , p = 0.001 ) , pi ( = 0.61 , p = 0.001 ) , length at 9 months ( z - score ) ( = 0.413 , p = 0.001 ) and catch - up growth for length ( = 0.311 , p = 0.002 ) and positively correlated with sga status ( = 0.511 , p = 0.001 ) , weight - for - length ( = 0.393 , p = 0.001 ) , and catch - up growth for weight ( = 0.198 , p = 0.048 ) . sequential multiple regression analysis for identifying influence of perinatal stress and anthropometric variables on insulin resistance index ( dependent variable ) multiple regression models were constructed using sequential method including gestational age , pi , weight - for - length as continuous variables , and type according to weight for gestation ( aga vs. sga ) as categorical variables . minimum variables which predict the largest amount of variability ( birth weight and length have collinearity with pi and weight and length at 9 months have collinearity with weight - for - length ) were included in this model to validate and stabilize the model . subsequently , pi , weight - for - length , and type according to weight for gestation were added in model 2 , 3 , and 4 , respectively . as a measure of goodness of fit , adjusted r was increased in each model from 0.188 in model 1 to 0.702 in model 4 , which indicates 70% of variability , now explained by model 4 ( significant r changes in each step p = 0.001 ) [ table 4 ] . substituting the variables and the unstandardized coefficients from the table 4 , the equation for model was as follows : log iri = 3.213 ( 0.173 gestational age ) ( 0.25 pi ) + ( 0.183 weight - for - length ) + ( 0.586 type ) because aga is coded zero , the final term in the equation was removed for aga . the term type indicates that after adjusting for gestational age , pi , and weight - for - length at 9 months , sga babies were 0.586 more iri than aga . scatter plot for insulin resistance index on gestational age with regression line ( appropriate for gestational age vs. small for gestational age ) standardized coefficients indicated the relative importance of each variable in comparable standardized units ( z - scores ) to predict iri . type according to weight for gestational age with a standardized coefficient of 0.505 was a more significant predictor of iri than gestational age ( = 0.481 ) , weight - for - length ( = 0.315 ) , and pi ( = 0.194 ) . the partial correlation was the unique contribution of gestational age ( 0.596 ) , pi ( 0.258 ) , weight - for - length ( 0.504 ) , and type ( 0.579 ) to predicting iri after the effect of other three factor was removed and was an estimate of the relative importance of each predictive variable in isolation from other factors [ table 4 ] . there was no collinearity ( significant relationship between explanatory variables ) in between variables ( correlation coefficient < 0.7 , variance inflation factor ( vif ) < 4 , and tolerance > 0.2 ) . interactions ( multiplicative rather than additive relationship between two explanatory variables ) were tested by including the interaction term ( gestational age type , pi type , and gestational age type pi ) in model , which shows no significant increase in adjusted r ( p > 0.05 ) . similarly , when iri plotted against gestational age , no significant difference between slope of regression line of aga and sga babies were present ( p > 0.05 ) [ figure 2 ] , which further favor in no interaction between gestational age and type according to weight for gestation . standard residuals ( distances between each data point and the value predicted by the regression equation ) showed normal distribution ( mean < 0.00001 , sd = 0.98 ) indicates equal spread of variance over the length of regression model , and the model was homoscedastic [ figure 3 ] . normal distribution of regression standardized residuals alternative regression model was constructed to determine role of catch - up growth in insulin resistance . in this model , gestational age , catch - up for weight , and type according to weight for gestation sequentially added ( adjusted r = 71.9% mean 71.9% variability explained by this model ) . catch - up growth ( = 0.714 ) is a significant predictor of insulin resistance independent of gestational age and sga status . weight - for - length and pi were not included in this model because of collinearity of catch - up growth with pi and weight - for - length at 9 months ( vif > 4 , tolerance < 0.2 , condition index in collinearity diagnostics > 30 ) . in collinearity , diagnostics condition index were 35.97 , 41.70 , and 57.73 for weight catch - up , weight - for - length at 9 months , and pi , respectively . substituting the variables and the unstandardized coefficients , the equation for this model was as follows : log iri = 5.621 ( 0.153 gestational age ) + ( 0.234 weight catch - up at 9 months ) + ( 0.320 type ) , the data are conflicting , with some studies demonstrating that fat mass is the major determinant of is with no effect of gestation whereas others identify a persisting effect of preterm birth . the present study demonstrates the association of prematurity with insulin resistance in infancy after adjusting for significant covariables . these findings provide additional evidence that preterm birth may be a risk factor for the future development of the insulin resistance and type-2 diabetes , and the insulin resistance may develop as early as infancy . a particular strength of our study is that we also include term sga cohort in our study for comparison and explore relative importance of each variables to predict the insulin resistance in infants . the present study explores that sga status is the most significant predictor of insulin resistance followed by gestational age , weight - for - length , and pi in the descending order . most of the studies reveals that in preterm born babies , sga status exerts no effect on insulin sensitivity in childhood , adolescence , and adulthood . , respectively , found that sga preterm born babies have reduced insulin sensitivity in neonatal , childhood , and adolescence . reinehr study included both preterm and term sga born subjects thus insulin resistance might be due to confounding effect of term gestation . used both homa modeling and intravenous glucose tolerance test and found reduced insulin sensitivity in sga babies by using homa model but not after stimulated insulin release . however in term sga born babies ' insulin resistance in infancy , childhood , adolescent , and adulthood is well established . with concurrence to most of the studies , we also report that sga status exerts no impact on insulin sensitivity in preterm born babies , but its impact during infancy is a matter of special mention here . in multiple regression analysis , sga born infants have higher insulin resistance ( 0.586 more iri ) than aga born infants irrespective of their gestational age , pi , and current weight - for - length . furthermore , combination of both preterm cohorts does not result in significantly higher insulin resistance than term sga . additional studies would require confirming the importance of this period and may provide further insight into the role of this critical period in the third trimester . our results also demonstrate that low pi ( thinness at birth ) is associated with higher insulin resistance with special concern to infancy . relatively brief duration intrauterine insult which affects more on weight than length results in lower pi ( asymmetrical intrauterine growth restriction [ iugr ] ) , and therefore higher insulin resistance . iugr followed by catch - up growth for weight in infancy and early childhood may be a sequence that led to high basal metabolic index ( bmi ) , obesity , and fat mass , and therefore insulin resistance in infancy , childhood , adolescent , and adulthood . similarly , prematurity followed by postnatal catch - up growth for weight in infancy and early childhood may be a sequence that led to high bmi , obesity , fat mass , and therefore insulin resistance in childhood , adolescent , and adulthood . in present study , we examine early changes in body composition by using weight - for - length , which provides a more accurate measure of adiposity ( bmi for > 2 years ) than weight alone . our study indicates that higher weight - for - length is associated with higher insulin resistance in infancy , and thus , it indicates an association of adiposity with insulin resistance as early as the 1 year of life . collinearity between catch - up growth for weight and weight - for - length indicate that excess adiposity is superimposed on normal growth related adiposity during catch - up growth . therefore , we create a hypothesis that catch - up growth for weight in infancy and early childhood and the higher bmi and obesity in letter childhood , adolescence , and adulthood are the same spectrum phenomenon in different window periods of life after exposure to environmental factors such as high energy intake or low physical activity and may be a result of metabolic imprinting after inutero stress or postnatal stress . first , it is one of the few to implement a prospective , longitudinal design to investigate the link between prematurity and insulin resistance as early as infancy . second , this study examines early changes in body composition using weight - for - length , which provides a more accurate measure of adiposity than weight alone . third , regression model is tested for stability , collinearity , and interaction between various variables and finally , this study also includes full - term sga infants , and thus can identify the relative importance of various variables to determine insulin sensitivity in infancy . finally , we have drawn equation to estimate iri by using gestational age , pi , sga status , and current weight - for - length . by using weight - for - length which is age - independent variable , we can monitor insulin resistance during follow - up in both preterm and sga babies .

low birth weight ( lbw ) is associated with an increased risk for a number of conditions arising in adulthood , such as type 2 diabetes , dyslipidemia , coronary artery disease , essential hypertension , and cerebrovascular accidents . , reduced insulin sensitivity ) is a well - established , early metabolic abnormality in the pathogenesis of these conditions . it has been proposed that a reduced insulin sensitivity in lbw subjects results from the adaptation to adverse in utero conditions during a critical period of development . however , it is well known that lbw newborns are also exposed to postnatal stress , which is reflected in higher neonatal morbidity and mortality . this has led to the hypothesis that postnatal stress may as well contribute to the metabolic modifications in lbw children , independently of the adequacy of their birth weight to gestational age . if early postnatal stress plays a role in long - term metabolic modifications , prematurity may be an important confounding factor . most previous studies linking lbw to the propensity toward disease in adulthood have focused on those who were small for gestational age ( sga ) and born at term . only few studies demonstrate the association of prematurity with a tendency to disease in letter adulthood but data 's are still conflicting , and further evidence is still required to establish a definitive role . moreover , it has been recognized that reduced insulin sensitivity , a hallmark in most lbw - related conditions may be present as early as the 1 year of life . hence , we planned a prospective cohort study to elaborate insulin sensitivity in infants who were lbw and born prematurely . in addition , it has been suggested that besides in utero stress and postnatal stress , other independent variables such as ponderal index ( pi ) , birth weight , catch - up growth for weight and length , and current weight and length may also contribute in insulin sensitivity . our second aim , therefore , was to determine the role of other variables to predict insulin sensitivity in infancy . sample size was calculated at power 80% , confidence interval ( ci ) 95% , four independent variables and assuming expected multiple regression coefficient 0.4 as observed in a previous study titled as , determinants of insulin sensitivity and secretion in very - low - birth weight children bazaes et al . in the beginning phase of the study , total 150 neonates born in this tertiary care center were enrolled to compensate loss during follow - up . this group was added for the sake of comparison and in order to determine other variables to predict insulin sensitivity in infancy . during follow - up ( till 9 months of the age ) numbers of participating infants were decreased , 15 were excluded ( 8 preterm aga , 1 term aga , 5 preterm sga , and 1 term sga ) and 35 did not come in follow - up ( 6 preterm aga , 13 term aga , 9 preterm sga , and 7 term sga ) . exclusion criteria included neonates of diabetic mother and those having first degree relative with type-2 diabetes mellitus , chronic illness , or medical therapy known to influence insulin sensitivity , chromosomal syndromes , congenital anomalies , congenital infections and sick neonates ( intracranial hemorrhage , perinatal asphyxia , pyomeningitis , necrotizing enterocolitis ) , neonates requiring positive pressure ventilation , and vasopressors . the nature , purpose , and possible risks of the study were explained to the parents in detail before consent was obtained . detailed anthropometry ( length , weight , pi , head circumference , chest circumference , and weight - for - length ) was performed according to the world health organization ( who ) guidelines by a trained pediatrician . birth weight , length , and head circumference were converted into standard deviation scores ( sds ) ( z = [ observed value mean value]/sd ) according to the who reference to allow comparison of subjects with different gestational ages , chronological ages , and sexes . however , the upper age cutoff for the enrolled subjects was limited to 9 months instead of 12 months in this study to avoid biases arising out of the transition to childhood from infancy during the last few months . the reason behind not using 6 months of age as cut off is because by that age , the term neonates may not be exposed to complementary feed , whereas preterm neonates would have received both breastfeeding and complementary feeding ( corrected age of 6 months for prematurity ) . all infants were on exclusive breastfeed for 5.56 0.84 months ( preterm aga 5.47 0.78 , preterm sga 5.60 0.85 , term aga 5.60 0.85 , term sga 5.79 0.84 , p 0.05 , range 47 months ) and then started complementary feed according to the indian academy of pediatrics guideline . at the age of 9 months ( 5 ml ) , blood sample was obtained for fasting plasma glucose and serum insulin ( si ) level ( after 24 h fasting ) . for calculation of homa-2 , glucose values differences in demographic characteristics and clinical measures between control and premature groups were investigated by means of analysis of variance for continuous variables and the chi - square test for proportions . differences in neonatal characteristics were evaluated with the use of fisher 's exact test for proportions and the mann general linear model ( ancova ) was used to investigate differences in glucose - regulation variables among the four groups of subjects . the four groups , divided according to their gestational age ( preterm vs. term ) and weight for gestation ( aga vs. sga ) , were included as factor , whereas pi and weight - for - length at 9 months ( wfl ) were included as covariance in this model . the specific hypotheses tested were the difference in insulin sensitivity between appropriate- and small - for - gestational - age subjects within the premature groups and the difference in insulin sensitivity between the premature groups and the term group . further analysis was performed by multiple regression models using log insulin resistance index ( iri ) as depended variable and gestational age , pi , weight - for - length , and sga status as independent variables . , differences in demographic characteristics and clinical measures between control and premature groups were investigated by means of analysis of variance for continuous variables and the chi - square test for proportions . differences in neonatal characteristics were evaluated with the use of fisher 's exact test for proportions and the mann general linear model ( ancova ) was used to investigate differences in glucose - regulation variables among the four groups of subjects . the four groups , divided according to their gestational age ( preterm vs. term ) and weight for gestation ( aga vs. sga ) , were included as factor , whereas pi and weight - for - length at 9 months ( wfl ) were included as covariance in this model . the specific hypotheses tested were the difference in insulin sensitivity between appropriate- and small - for - gestational - age subjects within the premature groups and the difference in insulin sensitivity between the premature groups and the term group . further analysis was performed by multiple regression models using log insulin resistance index ( iri ) as depended variable and gestational age , pi , weight - for - length , and sga status as independent variables . , out of hundred subjects , 36 preterm aga , 11 preterm sga , and 17 term sga , as study cohorts and 36 term aga , as control cohort , were enrolled in this study . as it was expected , gestational age , weight ( sd score ) , length ( sd score ) , head circumference ( sd score ) , and weight - for - length index were significantly different among the groups . data about neonatal characteristics were summarized in table 2 , and no significant difference was observed in maternal characteristics and oxygen requirement , antibiotic days , and days until full oral feeding established . iri ( median 1.80 , interquartile range 3.90 [ 0.534.39 ] , skewness 1.60 [ rt skewed ] , kurtosis 2.42 , mean 2.95 , and sd 3.20 ) and si ( median 8.80 , interquartile range 17.55 [ 2.6220.16 ] , skewness 1.67 [ rt skewed ] , kurtosis 2.94 , mean 13.49 , sd 14.29 ) had abnormal distribution and converted to log 10 value to normalize the distribution [ figure 1 ] . change in r value for these factors show that gestational age , birth weight , and weight - for - length were good predictors for iri ( p = 0.0001 ) as compared to pi ( p = 0.002 ) . pairwise contrast shows that ( adjustment for multiple comparisons by bonferroni ) , both preterm aga ( mean difference 0.617 , 95% ci ; 0.430.80 , p = 0.0001 ) and preterm sga ( mean difference 0.764 , 95% ci ; 0.441.09 , p = 0.0001 ) similarly , infants who had been born term sga had significantly higher insulin resistance than term aga ( mean difference 0.725 , 95% ci ; 0.490.96 , p = 0.0001 ) . no significant difference was found in between preterm sga group and preterm aga group ( mean difference 0.147 95% ci ; 0.130.42 , p = 0.927 ) . it was noticed that when we combine both preterm cohort , insulin resistance gets increased by 5.2% but not become significantly higher ( mean difference 0.076 , 95% ci ; 0.260.11 , p = 0.95 ) than term sga . baseline characteristics of the study subjects * maternal and neonatal characteristics in two groups of premature neonates indicator of glucose homeostasis * right skewed distribution of insulin resistance index further analysis was done by using multiple regression models summarized in table 4 . iri at 9 months was found negatively correlated with gestational age ( = 0.42 , p = 0.001 ) , birth weight z - score ( = 0.35 , p = 0.001 ) , pi ( = 0.61 , p = 0.001 ) , length at 9 months ( z - score ) ( = 0.413 , p = 0.001 ) and catch - up growth for length ( = 0.311 , p = 0.002 ) and positively correlated with sga status ( = 0.511 , p = 0.001 ) , weight - for - length ( = 0.393 , p = 0.001 ) , and catch - up growth for weight ( = 0.198 , p = 0.048 ) . sequential multiple regression analysis for identifying influence of perinatal stress and anthropometric variables on insulin resistance index ( dependent variable ) multiple regression models were constructed using sequential method including gestational age , pi , weight - for - length as continuous variables , and type according to weight for gestation ( aga vs. sga ) as categorical variables . minimum variables which predict the largest amount of variability ( birth weight and length have collinearity with pi and weight and length at 9 months have collinearity with weight - for - length ) were included in this model to validate and stabilize the model . subsequently , pi , weight - for - length , and type according to weight for gestation were added in model 2 , 3 , and 4 , respectively . as a measure of goodness of fit , adjusted r was increased in each model from 0.188 in model 1 to 0.702 in model 4 , which indicates 70% of variability , now explained by model 4 ( significant r changes in each step p = 0.001 ) [ table 4 ] . substituting the variables and the unstandardized coefficients from the table 4 , the equation for model was as follows : log iri = 3.213 ( 0.173 gestational age ) ( 0.25 pi ) + ( 0.183 weight - for - length ) + ( 0.586 type ) because aga is coded zero , the final term in the equation was removed for aga . the term type indicates that after adjusting for gestational age , pi , and weight - for - length at 9 months , sga babies were 0.586 more iri than aga . scatter plot for insulin resistance index on gestational age with regression line ( appropriate for gestational age vs. small for gestational age ) standardized coefficients indicated the relative importance of each variable in comparable standardized units ( z - scores ) to predict iri . type according to weight for gestational age with a standardized coefficient of 0.505 was a more significant predictor of iri than gestational age ( = 0.481 ) , weight - for - length ( = 0.315 ) , and pi ( = 0.194 ) . the partial correlation was the unique contribution of gestational age ( 0.596 ) , pi ( 0.258 ) , weight - for - length ( 0.504 ) , and type ( 0.579 ) to predicting iri after the effect of other three factor was removed and was an estimate of the relative importance of each predictive variable in isolation from other factors [ table 4 ] . interactions ( multiplicative rather than additive relationship between two explanatory variables ) were tested by including the interaction term ( gestational age type , pi type , and gestational age type pi ) in model , which shows no significant increase in adjusted r ( p > 0.05 ) . similarly , when iri plotted against gestational age , no significant difference between slope of regression line of aga and sga babies were present ( p > 0.05 ) [ figure 2 ] , which further favor in no interaction between gestational age and type according to weight for gestation . standard residuals ( distances between each data point and the value predicted by the regression equation ) showed normal distribution ( mean < 0.00001 , sd = 0.98 ) indicates equal spread of variance over the length of regression model , and the model was homoscedastic [ figure 3 ] . normal distribution of regression standardized residuals alternative regression model was constructed to determine role of catch - up growth in insulin resistance . in this model , gestational age , catch - up for weight , and type according to weight for gestation sequentially added ( adjusted r = 71.9% mean 71.9% variability explained by this model ) . weight - for - length and pi were not included in this model because of collinearity of catch - up growth with pi and weight - for - length at 9 months ( vif > 4 , tolerance < 0.2 , condition index in collinearity diagnostics > 30 ) . in collinearity , diagnostics condition index were 35.97 , 41.70 , and 57.73 for weight catch - up , weight - for - length at 9 months , and pi , respectively . substituting the variables and the unstandardized coefficients , the equation for this model was as follows : log iri = 5.621 ( 0.153 gestational age ) + ( 0.234 weight catch - up at 9 months ) + ( 0.320 type ) , the data are conflicting , with some studies demonstrating that fat mass is the major determinant of is with no effect of gestation whereas others identify a persisting effect of preterm birth . the present study demonstrates the association of prematurity with insulin resistance in infancy after adjusting for significant covariables . these findings provide additional evidence that preterm birth may be a risk factor for the future development of the insulin resistance and type-2 diabetes , and the insulin resistance may develop as early as infancy . we fill a gap in the knowledge , regarding insulin resistance in preterm born babies in 1 year of life which was unexplored till date . a particular strength of our study is that we also include term sga cohort in our study for comparison and explore relative importance of each variables to predict the insulin resistance in infants . the present study explores that sga status is the most significant predictor of insulin resistance followed by gestational age , weight - for - length , and pi in the descending order . most of the studies reveals that in preterm born babies , sga status exerts no effect on insulin sensitivity in childhood , adolescence , and adulthood . with concurrence to most of the studies , we also report that sga status exerts no impact on insulin sensitivity in preterm born babies , but its impact during infancy is a matter of special mention here . in multiple regression analysis , sga born infants have higher insulin resistance ( 0.586 more iri ) than aga born infants irrespective of their gestational age , pi , and current weight - for - length . these results indicate antenatal stress prior to the third trimester does not contribute in metabolic derangement , and critical window for the same is present in third trimester . our results also demonstrate that low pi ( thinness at birth ) is associated with higher insulin resistance with special concern to infancy . iugr followed by catch - up growth for weight in infancy and early childhood may be a sequence that led to high basal metabolic index ( bmi ) , obesity , and fat mass , and therefore insulin resistance in infancy , childhood , adolescent , and adulthood . similarly , prematurity followed by postnatal catch - up growth for weight in infancy and early childhood may be a sequence that led to high bmi , obesity , fat mass , and therefore insulin resistance in childhood , adolescent , and adulthood . in present study , we examine early changes in body composition by using weight - for - length , which provides a more accurate measure of adiposity ( bmi for > 2 years ) than weight alone . our study indicates that higher weight - for - length is associated with higher insulin resistance in infancy , and thus , it indicates an association of adiposity with insulin resistance as early as the 1 year of life . collinearity between catch - up growth for weight and weight - for - length indicate that excess adiposity is superimposed on normal growth related adiposity during catch - up growth . concomitant to this , converging evidence suggests that catch - up growth in preterm and small for gestation is intimately linked with a disproportionately faster rate to gain body fat rather than lean tissue , that is , to a preferential acceleration of fat recovery or catch - up fat . therefore , we create a hypothesis that catch - up growth for weight in infancy and early childhood and the higher bmi and obesity in letter childhood , adolescence , and adulthood are the same spectrum phenomenon in different window periods of life after exposure to environmental factors such as high energy intake or low physical activity and may be a result of metabolic imprinting after inutero stress or postnatal stress . first , it is one of the few to implement a prospective , longitudinal design to investigate the link between prematurity and insulin resistance as early as infancy . second , this study examines early changes in body composition using weight - for - length , which provides a more accurate measure of adiposity than weight alone . third , regression model is tested for stability , collinearity , and interaction between various variables and finally , this study also includes full - term sga infants , and thus can identify the relative importance of various variables to determine insulin sensitivity in infancy . on the basis of these neonatal characteristics and on the better developmental outcome of our cohort , we do not believe that a selection bias has occurred that influenced our findings . by using weight - for - length which is age - independent variable , we can monitor insulin resistance during follow - up in both preterm and sga babies . identification of these infants may help to focus preventive measures aimed at controlling the current epidemic of obesity and its complications , but we should be cautious while making recommendations for nutrition till we have evidence available from randomized control trial

angiogenesis is one of the crucial steps in tumor progression and metastasis [ 1 , 2 ] . due to the lack of oxygen supply and the accumulation of toxic products , avascular tumors and tumor metastases can not grow beyond a critical size of 1 - 2 mm and thus will stay clinically occult [ 1 , 3 , 4 ] . tumor vessels can grow by sprouting of preexisting host vessels , intussusception or incorporation of bone marrow - derived endothelial progenitor cells ( epcs ) which are a subset of hematopoietic cells ( hcs ) . although epcs incorporate into tumors in only small numbers , targeting epcs has been shown to be effective in reducing tumor angiogenesis and tumor growth in experimental models . one of the most important factors mediating survival and proliferation of hcs is the stem cell factor ( scf ) which binds to the c - kit receptor on the surface of hcs . have shown that bone marrow suppression by anti - c - kit treatment induces a delay in tumor angiogenesis due to the inhibition of angiogenic sprouting in colon tumors and that c - kit blockade suppresses tumor growth of subcutaneously implanted prostate carcinomas ( pc3 ) by inhibition of tumor angiogenesis regulated by hcs . recruitment of hcs and epcs to avascular areas is orchestrated by different angiogenic growth factors and cytokines , predominantly by the cxc - chemokine stromal cell - derived factor ( sdf)-1 and its receptor cxcr4 [ 9 , 10 ] . hcs and epcs fluctuate to peripheral organs in antiphase with the expression of sdf-1 within the bone marrow microenvironment and migrate alongside a chemotactic gradient towards higher concentrations of sdf-1 , for example , sites of injury and tumor tissue [ 1214 ] . furthermore , it has been demonstrated that sdf-1-mediated recruitment of c - kit - positive cells to the periphery is dependent on cofactors , including tumor necrosis factor- and the endothelial nitric oxide synthase ( enos ) . however , the impact of circulating hcs and epcs on vasculogenesis and sprouting angiogenesis in tumor angio - development is still controversially discussed in the literature [ 1517 ] , and the role of the sdf-1/cxcr4 pathway is not yet fully understood . therefore , in the presented study we analyzed the influence of bone marrow suppression by anti - c - kit treatment combined with sdf-1 neutralization on tumor cell engraftment and neovascularization using a murine model of colorectal tumor metastasis . the ct26 cell line is an n - nitroso - n - methyl - urethane - induced undifferentiated adenocarcinoma of the colon , syngeneic with the balb / c mouse . for our studies , the ct26.wt cells ( atcc crl-2638 , lgc promochem gmbh , wesel , germany ) were transfected with the enhanced gfp expression vector pegfp - n1 ( clontech ) with the use of clonfectin ( clontech , palo alto , ca , usa ) according to the manufacturer 's instructions . for the individual experiments , ct26.wt-gfp cells were grown in cell culture as monolayers in rpmi-1640 medium with 2 mm l - glutamine ( sigma aldrich chemie gmbh , taufkirchen , germany ) supplemented with 10% fetal calf serum ( fcs gold , paa laboratories gmbh , clbe , germany ) , 100 u / ml penicillin , and 100 g / ml streptomycin ( paa laboratories gmbh ) . the cells were incubated at 37c in a humidified atmosphere containing 5% co2 , and only cells of the first three serial passages after cryostorage were used . at the day of implantation , tumor cells were harvested from subconfluent cultures ( 70 to 85% ) by trypsinization ( 0.05% trypsin and 0.02% edta , paa laboratories gmbh ) and washed twice in phosphate - buffered saline solution ( pbs ) . experiments were performed after approval by the local governmental ethic committee and conformed to the united kingdom coordinating committee on cancer research ( ukcccr ) guidelines for the welfare of animals in experimental neoplasia ( as described in 1998 in br j cancer 77 : 110 ) and the guide for care and use of laboratory animals ( institute of laboratory animal resources , national research council ; nih guide , vol . female balb / c mice ( charles river laboratories gmbh ; sulzfeld , germany ) with a body weight ( bw ) of 1820 g were used . the animals were housed in single cages at room temperature of 2224c and at a relative humidity of 6065% with a 12-hour light / dark cycle environment . the mice were allowed free access to drinking water and standard laboratory chow ( altromin ; lage , germany ) . to allow repetitive analyses of the microcirculation of the growing tumors , the dorsal skinfold chamber model was used for intravital microscopy as described previously in detail . for operative procedures , animals were anesthetized by intraperitoneal injection of 90 mg / kg bw ketamine ( ketavet , parke davis ; freiburg , germany ) and 20 mg / kg bw xylazine ( rompun , bayer ; leverkusen , germany ) . the chamber , consisting of two symmetrical titanium frames , was positioned to sandwich the extended double layer of the dorsal skin . one layer of skin and subcutis was completely removed in a circular area of 15 mm diameter . the remaining layers , consisting of the epidermis , subcutaneous tissue , and striated skin muscle , were covered with a glass coverslip incorporated into one of the titanium frames . the animals tolerated the chambers well and showed no signs of discomfort or changes in sleeping and feeding habits . for tumor cell implantation , the coverslip of the chamber was temporarily removed and 1 10 ct26.wt-gfp cells were implanted onto the surface of the striated muscle tissue within the chamber . immediately after cell implantation , the chamber tissue was covered again with the coverslip [ 2022 ] . animals were assigned to three different groups : the first group ( ckit - ab ; n = 8) received a pretreatment with a monoclonal anti - c - kit receptor antibody ( ack45 , bd biosciences , heidelberg , germany ) by daily intraperitoneal injections starting 4 days before tumor cell implantation . the ack45-antibody was given four times at a dose of 1 mg / kg bw daily as described by okamoto et al . . animals of the second group ( ckit / sdf1-ab ; n = 8) were also pretreated with ack45 as described above . additionally , these animals received 1 mg / kg bw of a monoclonal mouse anti - mouse sdf-1 antibody ( mab310 , r and d systems , wiesbaden , germany ) . mab310 application was performed intraperitoneally , starting at the day of tumor cell implantation ( d0 ) and repeated every second day thereafter until day 12 . the third group ( control ; n = 8) served as control and received pre- and posttreatment the same amount of the corresponding isotype - matched igg control antibodies ( a95 - 1 , bd biosciences , and mab002 r&d systems ) . all animals underwent repetitive intravital microscopic analyses directly ( d0 ) as well as 5 , 8 , 11 , and 14 days after tumor cell implantation . at the end of the experiment ( day 14 ) intravital fluorescence microscopy was performed in epi - illumination technique using a modified zeiss axio - tech microscope ( zeiss , oberkochen , germany ) with a 100-w hbo mercury lamp . microscopic images were monitored by a charge - coupled device video camera ( fk 6990 , cohu , prospective measurements inc . , san diego , ca , usa ) and were transferred to a video system ( vo-5800 ps , sony , mnchen , germany ) for subsequent off - line analysis . tumor size , growth kinetics , migration of tumor cell , and neovascularization were analyzed using blue light epi - illumination ( 450 to 490 nm excitation wavelength and > 520 nm emission wavelengths ) [ 2022 ] . microcirculatory parameters were assessed off line by frame - to - frame analysis of the videotaped images using a computer - assisted image analysis system ( capimage , zeintl software , heidelberg , germany ) . the fluorescent labeling of the tumor cells allowed precise delineation of the tumor from the surrounding host tissue . it also enabled for distinct identification of individual tumor cells to study tumor cell migration . at each observation time point , the surface of the fluorescently labeled tumor mass within the chamber was scanned for determination of the tumor size ( given as tumor area in mm ) . eight regions of interest ( rois ) were randomly chosen next to the tumor margin . the number of migrating cells was counted , and the distance to the tumor margin was measured ( given in m ) . first , eight representative rois within the tumor margin were chosen and analyzed for microcirculation parameters . in these rois , the onset of angiogenesis , that is , the existence of angiogenic buds , sprouts , and newly formed blood vessels , was documented and scored 0 to 8 , with 0 indicating existence of newly formed tumor microvessels in none of the rois and 8 indicating their existence in all of the rois . functional capillary density ( given in cm / cm ) of the tumor microvessels this parameter was defined as the length of red blood cell perfused microvessels per observation area and was analyzed within the eight rois of the tumor margin and within four additional rois of the tumor center . diameters of the newly formed tumor microvessels were measured perpendicularly to the vessel path ( given in m ) . to study vascular permeability of the newly formed tumor microvessels , petechial bleedings were documented in each of the rois , and given as percentage of all the rois analyzed [ 20 , 21 , 23 ] . at the end of the experiment ( day 14 ) , the tumor and the adjacent host tissue were harvested and immediately fixed in formalin . for light microscopy , formalin - fixed biopsies were embedded in paraffin . sections of 5 m were cut and stained with hematoxylin and eosin ( he ) according to standard procedures to analyze tumor growth characteristics . tumor cell invasion of the muscular layer on the surface of the dorsal skinfold chamber was quantified over the entire tumor basis and given as percentage of the length of the tumor basis . to study tumor cell proliferation and apoptotic cell death , proliferating cell nuclear antigen ( pcna ) and cleaved caspase-3 were stained using indirect immunoperoxidase techniques . therefore , deparaffinized sections were incubated with 3% h2o2 and 2% goat normal serum to block endogenous peroxidases and unspecific binding sites . a monoclonal mouse anti - pan pcna antibody ( pc10 , dakocytomation , hamburg , germany ) and a polyclonal rabbit anti - mouse cleaved caspase-3 antibody ( asp175 , cell signaling technology , frankfurt , germany ) were used as primary antibodies . goat anti - mouse and goat anti - rabbit pod - conjugated antibodies were used as secondary antibodies for streptavidin - biotin - complex peroxidase staining . sections were counterstained with hemalaun according to mayer and examined by light microscopy . to assess the expression of cd31 as a marker for endothelial cells , zinc fixative fixed paraffin sections of tumor tissue were used . after incubation with 3% h2o2 and 2% goat normal serum to block endogenous peroxidases and unspecific binding sites , deparaffinized sections were incubated with a rat anti - mouse cd31 antibody ( mec 13.3 , bd biosciences ) . a polyclonal goat anti - rat igg antibody ( bd biosciences ) facscan ( becton dickinson , mountain view , ca , usa ) analysis was performed to assess the expression of c - kit on the ct26.wt-gfp cells in triplicate . cells were fixed with 2% formalin , washed twice with pbs , resuspended in facs buffer and incubated with an fitc - conjugated rat anti - mouse igg2b c - kit antibody ( 1 : 50 ; 553354 , bd biosciences ) or an fitc - conjugated isotype - matched control antibody ( 553988 , bd biosciences ) . cells were washed again and then maintained in 2% paraformaldehyde in pbs . tumor cells were selectively analyzed for their fluorescence properties using the cellquest data handling program ( bd biosciences ) with assessment of 5000 events per sample . the flow cytometer was calibrated with fluorescent standard microbeads ( calibrite beads , bd biosciences ) . to study protein expression patterns , additional western blot analyses were performed on tumor specimen gained from the dorsal skinfold chamber . therefore , 12 additional animals received ct26.wt-gfp tumor cell implantation in the dorsal skinfold chamber and were assigned to the three groups as described above . eight animals were pretreated with the anti - c - kit antibody ( ack45 , bd biosciences ) . four of these pretreated animals received additional treatment with the neutralizing anti - sdf-1 antibody ( mab310 , r and d systems ) starting at the day of tumor cell implantation . four animals received the same amount of the corresponding isotype - matched control antibody ( a95 - 1 , bd biosciences ) . for whole protein extracts and western blot analysis of the expression of vascular endothelial growth factor ( vegf ) , the chemokine receptor cxcr4 and enos , ct26.wt-gfp-tumors were completely removed from the skinfold at day 5 . the tumor tissue samples were homogenized separately in lysis buffer and a protease inhibitor cocktail ( sigma , taufkirchen , germany ) , incubated on ice and centrifuged at 16.000 g . ten microgram protein per lane were separated discontinuously on sodium dodecyl sulfate polyacrylamide gels ( 10% sds - page ) and transferred to a polyvinyldifluoride membrane ( 0.2 m , biorad , mnchen , germany ) . after blockade of nonspecific binding sites , membranes were incubated with an anti - vegf antibody ( a-20 , santa cruz , heidelberg , germany ) , an anti - cxcr4 antibody ( ab2074 , abcam , heidelberg , germany ) , or an anti - enos antibody ( bd transduction lab . , heidelberg , germany ) followed by the corresponding secondary peroxidase - conjugated antibodies ( ge healthcare , freiburg , germany and santa cruz ) . protein expression was visualized by means of luminol enhanced chemiluminescence ( ecl , ge healthcare ) and exposure of the membranes to a blue - light - sensitive autoradiography film ( hyperfilm ecl , ge healthcare ) . signals were densitometrically assessed ( biorad , gel - dokumentationssystem ) and normalized to -actin signals ( mouse monoclonal anti--actin , sigma ) to correct for unequal loading . all values are expressed as means sem . after proving the assumption of normality and homogeneity of variance across groups , differences between groups were calculated by a one - way analysis of variance ( anova ) followed by the appropriate post hoc comparison , including correction of the alpha error according to bonferroni probabilities to compensate for multiple comparisons . overall statistical significance was set at p < 0.05 . statistical analysis was performed with the use of the software package sigmastat ( spss inc , chicago , ill ) . all animals had an uneventful postoperative recovery and tolerated well the dorsal skinfold chamber implantation and the repetitive intravital microscopic analyses . the general conditions of the mice were not affected and no changes in feeding or sleeping habits were observed . the take rate of the ct26.wt-gfp cells within the dorsal skinfold chamber was 100% , and progressive tumor growth was observed in all groups throughout the entire observation period ( figures 1(a)1(c ) ) . quantitative analysis of the increase of the tumor area revealed a significantly enhanced engraftment of tumor cells and a significant stimulation of tumor growth after pretreatment with the anti - c - kit antibody compared to controls ( p < 0.05 ) . interestingly , additional blockage of sdf-1 completely blunted this enhancement of tumor growth leading to similar tumor sizes as measured in controls ( p < 0.05 ; figure 1(d ) ) . in control animals , newly developed microvessels could be detected in 50% of the rois by day 5 after tumor cell implantation . at day 8 , angiogenesis was seen in almost 100% of the rois . pretreatment with the anti - c - kit antibody did not influence the onset of the angiogenic switch compared to controls . of interest , blockade of sdf-1 after pretreatment with anti - c - kit significantly delayed the angiogenic switch , with only 50% of the rois showing newly formed microvessels by day 8 and 70% by day 11 ( p < 0.05 ) . in this group , angiogenesis was observed in all rois only at the end of the observation period ( figure 2 ) . the differential effects of c - kit blockade with or without additional sdf-1 blockade were also reflected by quantitative analysis of the microvascular densities of the newly formed tumor vessels . as characteristic for tumor vessels , the vascular network of the tumors consisted of irregularly shaped and chaotically arranged microvessels ( figures 3(a)3(c ) ) . whereas no differences of microvascular density within the tumor vasculature could be observed between controls and anti - c - kit pretreated animals , additional treatment with the anti - sdf-1 antibody significantly decreased the microvascular density within the tumor center compared to the other groups from day 8 until the end of the observation period ( figure 3(d ) ) . quantitative analysis of the functional microvascular density within the tumor margin showed similar results ( data not shown ) . the functional microvascular density within the tumor margin and the tumor center was not significantly different within the individual treatment groups . in contrast to the highly vascularized ct26.wt-gfp tumors within the intravital fluorescence microscopy , immunohistological staining for cd31 as a marker for endothelial cells displayed positive staining of only a few cells within tumor microvessels without significant differences between the three groups ( data not shown ) . as neovascularization is usually associated with vasodilation due to the action of vegf , microvessel diameters within the tumor were analyzed . however , no overall differences between the diameters of the microvessels within the tumor margin and the tumor center could be observed ( table 1 ) . during the 14 days observation period , diameters slightly increased in all groups until day 11 , particularly in the control and anti - c - kit - pretreated groups . of interest , at day 14 , microvessel diameters within tumors of mice treated with anti - c - kit and anti - sdf-1 were markedly smaller compared to the other two groups ( table 1 ) . petechial bleedings within the areas of angiogenesis of growing tumors are a characteristic indicator of the vegf action on vascular permeability . to study this vegf - induced increase of vascular permeability , we analyzed the petechial bleedings within the rois by intravital fluorescence microscopy . microbleedings were observed in all tumors . at day 5 , signs of petechial bleedings were observed in 6% of the tumor area of control tumors , whereas petechial bleedings occurred in up to 30% of the tumor area in animals pretreated with anti - c - kit at that time . in all groups , petechial bleedings were most pronounced 8 days after tumor cell implantation ( control : 1520% , anti - c - kit : 30% , anti - c - kit / anti - sdf-1 : 3540% ) . comparing the three groups , no statistical significance could be found due to the high standard deviation within the individual groups ( data not shown ) . as sdf-1 has been demonstrated to exert chemotactic effects on ct26.wt-gfp tumor cells , we additionally focused on migrating tumor cells next to the tumor margin . the migrating tumor cells were easily detectable due to their gfp labeling ( table 2 ) . starting at day 5 after tumor cell implantation , tumor cell migration was detectable in all tumors until the end of the observation period with slightly increasing distances from the tumor margin . the number of migrating tumor cells ranged between 20 and 25 per representative field during the whole observation period . treatment with anti - c - kit alone or additional blockade of sdf-1 did not impair tumor cell migration compared to controls ( table 2 ) . histological examinations on hematoxylin - eosin stained sections revealed solid tumor growth within the dorsal skinfold chamber . signs of malignant tumor growth such as invasion of the adjacent host tissue by the tumor cells were detectable in each group ( figure 4 ) . quantitative analysis of tumor cell invasion showed a significant increase of tumor cell infiltration through the muscular layer of the dorsal skinfold chamber in tumors of animals which were pretreated with anti - c - kit compared to controls ( p < 0.05 ) . of interest , tumors of animals additionally treated with anti - sdf-1 showed a significant reduction of muscular infiltration compared to anti - c - kit pretreated tumors ( p < 0.05 ) , resulting in invasive growth characteristics which were comparable to controls ( figure 4(c ) ) . pcna as an indicator of cell proliferation displayed positive staining in 46.9 3.1% of the tumor cells in control animals ( figure 5 ) . after pretreatment with anti - c - kit , the number of pcna - positive tumor cells was significantly higher compared to controls ( p < 0.05 , figures 5(a ) and 5(c ) ) . in contrast , additional blockade of sdf-1 resulted in a significantly lower number of pcna - positive tumor cells compared to controls and to anti - c - kit pretreated animals ( p < 0.05 , figures 5(b ) and 5(c ) ) . to study apoptotic cell death , 14 days after tumor cell implantation , only a minor fraction of the total number of 351.3 5.2 tumor cells within the high power fields ( hpf ) showed positive staining for caspase-3 . whereas 0.73 0.13 tumor cells / hpf were positive for caspase-3 in controls , pretreatment with anti - c - kit reduced apoptotic cell death within the tumors ( 0.26 0.06 tumor cells / hpf ) . in tumors of animals additionally treated with anti - sdf-1 antibodies , this effect was even more pronounced ( 0.15 0.03 tumor cells / hpf , p < 0.05 ) . facscan analysis demonstrated that 8.2 0.8% of the ct26-gfp cells transfected with the enhanced gfp expression vector pegfp - n1 was c - kit receptor positive . at day 5 after tumor cell implantation , the tumors of animals pretreated with anti - c - kit antibodies showed a significantly higher expression of vegf and cxcr4 compared to controls ( p < 0.05 ; figures 6(a ) and 6(b ) ) . furthermore , enos expression within the tumors was decreased by anti - c - kit pretreatment with and without additional anti - sdf-1 treatment compared to controls ( data not shown ) . the major finding of the present study is that bone marrow suppression by anti - c - kit treatment significantly enhances tumor cell engraftment of colorectal tumors due to an increase of tumor cell proliferation and invasion . additional anti - sdf-1 treatment neutralizes this increased tumor outgrowth by inhibition of tumor cell proliferation and tumor neovascularization . these findings suggest that the enhanced tumor growth under the conditions of bone marrow suppression induced by anti - c - kit treatment is related to the sdf-1/cxcr4 pathway . bone - marrow - derived hematopoetic cells ( hcs ) contribute to physiological and pathological vessel formation . during tumor growth , the release of cytokines and chemokines mediates the recruitment of epcs and hcs contributing to the early initiation and stabilization of newly formed blood vessels , so - called vasculogenesis [ 2426 ] . whereas many studies indicate that bone - marrow - derived epcs incorporate into tumor neovessels [ 25 , 27 ] , hcs may promote vasculogenesis via paracrine release of angiogenic factors enhancing the recruitment and incorporation of epcs into neovessels . in the early phases of tumor growth , 5090% of the neovessels within the tumor mass are derived from the bone marrow dependent on the tumor type . however , recent reports have already doubted the impact of vasculogenesis from bone - marrow - derived cells for tumor neovascularization and claim an exclusive role for sprouting angiogenesis in tumor blood vessel development [ 15 , 16 ] . in a model of syngeneic bone marrow transplantation , patil et al . demonstrated that gpf - expressing c - kit bone - marrow - derived progenitor cells are recruited to subcutaneously implanted lewis lung carcinoma but do not directly contribute to microvascular structure . they , therefore , concluded that even if circulating hcs and epcs home to sites of tumor growth , they do not contribute to tumor angiogenesis . in a model of subcutaneously implanted prostate carcinoma , okamoto et al . observed an accumulation of hcs around newly formed blood vessels , but did not find these cells to be part of the tumor microvessels themselves . because of these findings , the authors postulated a stabilizing and supportive role of hcs for the developing vascular network . in the present study , intravital fluorescence microscopy showed highly vascularized ct26.wt-gfp tumors 14 days after tumor cell implantation . however , immunohistological staining using the endothelial cell marker cd31 displayed positive staining of only a few endothelial cells within these tumors . this finding reflects the diverging structure between normal microvessels and tumor neovessels which consist of only cancer cells or a mosaic of cancer and endothelial cells . previous experimental studies in mice have shown that a depletion of the myeloid and erythroid cell lineages including epcs and hcs from the bone marrow could be performed by daily injection of 1 mg / kg ack2 , an antagonistic anti - c - kit antibody , which blocks the function of c - kit without cytotoxic side effects on c - kit cells [ 30 , 31 ] . of interest , whereas no polymorphonuclear cells or erythroblasts were present in the bone marrow of these ack2-treated animals , b lineage cells continued to grow to fill the space from which myeloid and erythroid progenitor cells were purged [ 30 , 31 ] . moreover , the reduction rate of the colony forming cells within the bone marrow was dependent on the amount of anti - c - kit antibodies . in the present study , we , therefore , used the anti - c - kit antibody in a dosage of 1 mg / kg bw for the induction of bone marrow suppression over a time period of 4 days to avoid surgical complications and death of the animals resulting from the immune incompetence by bone marrow depletion . as shown by okamoto et al . , bone marrow suppression by anti - c - kit pretreatment over a time period of 4 days before subcutaneous implantation of colon tumor cells induced leucopenia which was still detectable 10 days after the last injection . although tumor growth and sprouting of tumor vessels were slightly reduced in these studies of okamoto et al . during the first 57 days , tumor growth was rapidly reinitiated as the number of circulating hcs in the peripheral blood increased again . therefore , the authors concluded that hcs migrating into the tumor mass promote the initiation of tumor neovascularization . in our present study , although neoangiogenesis was not influenced after administration of anti - c - kit , tumor growth was stimulated as a result of an increased tumor cell proliferation and invasion . this observation might be the result of immunological mechanisms that suppress tumor growth in animals with an intact immune system . c - kit blockade for the induction of bone marrow suppression concurrently stimulates b lymphopoiesis [ 30 , 31 ] . as the number of b cell precursors normally decreases in tumor bearing mice [ 32 , 33 ] , it must be speculated that in our experiment of bone marrow suppression , c - kit blockade stimulates b cell genesis and thus increases tumor cell engraftment . furthermore , b lymphopoiesis is associated with increased angiogenesis and cellular proliferation [ 3436 ] . in our study , neoangiogenesis within the colorectal tumors of animals pretreated with anti - c - kit was comparable to controls despite the lack of functional hcs which are necessary for angiogenesis and vasculogenesis . thus , we hypothesize that the lack of hcs was compensated by local angiogenic factors or b lymphopoiesis - associated angiogenesis , resulting in a tumor neovascularization comparable to that observed in controls . the scf receptor c - kit has been shown to be essential for the development of blood cells , melanocytes , germ cells , interstitial cells of cajal in the gastrointestinal tract , and mast cells . furthermore , the majority of tumor cells , especially those of the neural axis , breast , lung , prostate , and colon show an aberrant c - kit expression . especially gastrointestinal stromal tumors ( gists ) express c - kit on the cell - surface , and mutations of kit in these tumors results in an activation of kit signaling , which leads to uncontrolled cell proliferation and resistance to apoptosis [ 39 , 40 ] . today , patients with gist are treated with imatinib , an inhibitor of certain protein tyrosine kinases including kit , depending on the mitotic index of the gist . imatinib induces an arrest of tumor cell proliferation and causes apoptotic cell death . in established mca26 tumors , pan et al showed that injection of anti - c - kit antibodies markedly reduces tumor - induced immune tolerance exhibited by myeloid - derived suppressors in mice . furthermore , their experiments demonstrate that anti - c - kit treatment can prevent tumor - specific t - cell anergy and development of t regulatory cells ( treg ) . in combination with an immune modulatory therapy of il-12 plus 4 - 1bb activation , treatment with anti - c - kit antibodies significantly improved the long - term survival of mca26 tumor bearing mice . in our study , only 8% of the ct26.wt-gfp cells were c - kit receptor positive . therefore , we do not expect a direct inhibitory effect of anti - c - kit treatment on tumor cell proliferation . recruitment of hcs and epc is predominantly mediated by sdf-1 and its receptor cxcr4 [ 9 , 10 ] because hcs and epcs migrate along a chemotactic gradient towards higher concentrations of sdf-1 [ 1214 ] . have shown that almost 100% of bone marrow and peripheral blood c - kit cells are positive for cxcr4 . in the present study , we could demonstrate that neutralization of sdf-1 after anti - c - kit pretreatment significantly reduces neovascularization most probably by the inhibition of hc and epc recruitment within the tumors . additionally , in combination with anti - c - kit pretreatment , sdf-1 neutralization is capable of decreasing tumor cell proliferation and invasion . taken together , neutralization of sdf-1 counteracts the stimulating effects of bone marrow suppression by anti - c - kit treatment on tumor cell engraftment and inhibits compensatory local and b lymphopoiesis - associated angiogenesis . as described by kaminski et al . , the presence of sdf-1 chemoattractant activity and inflammatory endothelial activation by tnf- is required for c - kit cells to form functionally relevant interactions with the endothelium in postcapillary venules . blockade of icam-1 and cxcr4 abolishes adhesion of c - kit cells to the vascular endothelium despite application of sdf-1 and tnf-. moreover , in their cremaster muscle microcirculation model , stem cell adhesion was significantly reduced when enos was not present or systemic nos inhibited . as sdf-1 and tnf- stimulation activates the endothelium by an increase of the cxcr4 expression leading to relevant stem cell attraction , our results indicate that the increase of cxcr4 and vegf expression within the tumors represents a compensatory pathway after anti - c - kit pretreatment . neutralization of sdf-1 inhibits interactions of c - kit positive cells with tumor vessels and as a consequence , leads to inhibition of tumor neovascularization . in conclusion , bone marrow suppression by anti - c - kit pretreatment significantly enhances tumor cell engraftment of colorectal tumors . as anti - sdf-1 treatment counteracts this increased tumor outgrowth by inhibition of neovascularization , the sdf-1/cxcr4 pathway seems to be crucial for tumor angiogenesis mediated by hcs and epcs .

background . mobilization of c - kit+ hematopoietic cells ( hcs ) contributes to tumor vascularization . whereas survival and proliferation of hcs are regulated by binding of the stem cell factor to its receptor c - kit , migration of hcs is directed by stromal cell - derived factor ( sdf)-1 . therefore , targeting migration of hcs provides a promising new strategy of anti - tumor therapy . methods . balb / c mice ( n = 16 ) were pretreated with an anti - c - kit antibody followed by implantation of ct26.wt-gfp colorectal cancer cells into dorsal skinfold chambers . animals ( n = 8) additionally received a neutralizing anti - sdf-1 antibody . animals ( n = 8) treated with a control antibody served as controls . investigations were performed using intravital fluorescence microscopy , immunohistochemistry , flow cytometry and western blot analysis . results . blockade of c - kit significantly enhanced tumor cell engraftment compared to controls due to stimulation of tumor cell proliferation and invasion without markedly affecting tumor vascularization . c - kit blockade significantly increased vegf and cxcr4 expression within the growing tumors . neutralization of sdf-1 completely antagonized this anti - c - kit - associated tumor growth by suppression of tumor neovascularization , inhibition of tumor cell proliferation and reduction of muscular infiltration . conclusion . our study indicates that bone marrow suppression via anti - c - kit pretreatment enhances tumor cell engraftment of colorectal metastases due to interaction with the sdf-1/cxcr4 pathway which is involved in hc - mediated tumor angiogenesis .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

tumor vessels can grow by sprouting of preexisting host vessels , intussusception or incorporation of bone marrow - derived endothelial progenitor cells ( epcs ) which are a subset of hematopoietic cells ( hcs ) . one of the most important factors mediating survival and proliferation of hcs is the stem cell factor ( scf ) which binds to the c - kit receptor on the surface of hcs . have shown that bone marrow suppression by anti - c - kit treatment induces a delay in tumor angiogenesis due to the inhibition of angiogenic sprouting in colon tumors and that c - kit blockade suppresses tumor growth of subcutaneously implanted prostate carcinomas ( pc3 ) by inhibition of tumor angiogenesis regulated by hcs . recruitment of hcs and epcs to avascular areas is orchestrated by different angiogenic growth factors and cytokines , predominantly by the cxc - chemokine stromal cell - derived factor ( sdf)-1 and its receptor cxcr4 [ 9 , 10 ] . hcs and epcs fluctuate to peripheral organs in antiphase with the expression of sdf-1 within the bone marrow microenvironment and migrate alongside a chemotactic gradient towards higher concentrations of sdf-1 , for example , sites of injury and tumor tissue [ 1214 ] . furthermore , it has been demonstrated that sdf-1-mediated recruitment of c - kit - positive cells to the periphery is dependent on cofactors , including tumor necrosis factor- and the endothelial nitric oxide synthase ( enos ) . however , the impact of circulating hcs and epcs on vasculogenesis and sprouting angiogenesis in tumor angio - development is still controversially discussed in the literature [ 1517 ] , and the role of the sdf-1/cxcr4 pathway is not yet fully understood . therefore , in the presented study we analyzed the influence of bone marrow suppression by anti - c - kit treatment combined with sdf-1 neutralization on tumor cell engraftment and neovascularization using a murine model of colorectal tumor metastasis . the ct26 cell line is an n - nitroso - n - methyl - urethane - induced undifferentiated adenocarcinoma of the colon , syngeneic with the balb / c mouse . female balb / c mice ( charles river laboratories gmbh ; sulzfeld , germany ) with a body weight ( bw ) of 1820 g were used . to allow repetitive analyses of the microcirculation of the growing tumors , the dorsal skinfold chamber model was used for intravital microscopy as described previously in detail . for tumor cell implantation , the coverslip of the chamber was temporarily removed and 1 10 ct26.wt-gfp cells were implanted onto the surface of the striated muscle tissue within the chamber . animals were assigned to three different groups : the first group ( ckit - ab ; n = 8) received a pretreatment with a monoclonal anti - c - kit receptor antibody ( ack45 , bd biosciences , heidelberg , germany ) by daily intraperitoneal injections starting 4 days before tumor cell implantation . animals of the second group ( ckit / sdf1-ab ; n = 8) were also pretreated with ack45 as described above . the third group ( control ; n = 8) served as control and received pre- and posttreatment the same amount of the corresponding isotype - matched igg control antibodies ( a95 - 1 , bd biosciences , and mab002 r&d systems ) . at the end of the experiment ( day 14 ) intravital fluorescence microscopy was performed in epi - illumination technique using a modified zeiss axio - tech microscope ( zeiss , oberkochen , germany ) with a 100-w hbo mercury lamp . tumor size , growth kinetics , migration of tumor cell , and neovascularization were analyzed using blue light epi - illumination ( 450 to 490 nm excitation wavelength and > 520 nm emission wavelengths ) [ 2022 ] . tumor cell invasion of the muscular layer on the surface of the dorsal skinfold chamber was quantified over the entire tumor basis and given as percentage of the length of the tumor basis . to study tumor cell proliferation and apoptotic cell death , proliferating cell nuclear antigen ( pcna ) and cleaved caspase-3 were stained using indirect immunoperoxidase techniques . a polyclonal goat anti - rat igg antibody ( bd biosciences ) facscan ( becton dickinson , mountain view , ca , usa ) analysis was performed to assess the expression of c - kit on the ct26.wt-gfp cells in triplicate . cells were fixed with 2% formalin , washed twice with pbs , resuspended in facs buffer and incubated with an fitc - conjugated rat anti - mouse igg2b c - kit antibody ( 1 : 50 ; 553354 , bd biosciences ) or an fitc - conjugated isotype - matched control antibody ( 553988 , bd biosciences ) . therefore , 12 additional animals received ct26.wt-gfp tumor cell implantation in the dorsal skinfold chamber and were assigned to the three groups as described above . eight animals were pretreated with the anti - c - kit antibody ( ack45 , bd biosciences ) . four of these pretreated animals received additional treatment with the neutralizing anti - sdf-1 antibody ( mab310 , r and d systems ) starting at the day of tumor cell implantation . for whole protein extracts and western blot analysis of the expression of vascular endothelial growth factor ( vegf ) , the chemokine receptor cxcr4 and enos , ct26.wt-gfp-tumors were completely removed from the skinfold at day 5 . after blockade of nonspecific binding sites , membranes were incubated with an anti - vegf antibody ( a-20 , santa cruz , heidelberg , germany ) , an anti - cxcr4 antibody ( ab2074 , abcam , heidelberg , germany ) , or an anti - enos antibody ( bd transduction lab . the take rate of the ct26.wt-gfp cells within the dorsal skinfold chamber was 100% , and progressive tumor growth was observed in all groups throughout the entire observation period ( figures 1(a)1(c ) ) . quantitative analysis of the increase of the tumor area revealed a significantly enhanced engraftment of tumor cells and a significant stimulation of tumor growth after pretreatment with the anti - c - kit antibody compared to controls ( p < 0.05 ) . interestingly , additional blockage of sdf-1 completely blunted this enhancement of tumor growth leading to similar tumor sizes as measured in controls ( p < 0.05 ; figure 1(d ) ) . pretreatment with the anti - c - kit antibody did not influence the onset of the angiogenic switch compared to controls . of interest , blockade of sdf-1 after pretreatment with anti - c - kit significantly delayed the angiogenic switch , with only 50% of the rois showing newly formed microvessels by day 8 and 70% by day 11 ( p < 0.05 ) . the differential effects of c - kit blockade with or without additional sdf-1 blockade were also reflected by quantitative analysis of the microvascular densities of the newly formed tumor vessels . whereas no differences of microvascular density within the tumor vasculature could be observed between controls and anti - c - kit pretreated animals , additional treatment with the anti - sdf-1 antibody significantly decreased the microvascular density within the tumor center compared to the other groups from day 8 until the end of the observation period ( figure 3(d ) ) . in contrast to the highly vascularized ct26.wt-gfp tumors within the intravital fluorescence microscopy , immunohistological staining for cd31 as a marker for endothelial cells displayed positive staining of only a few cells within tumor microvessels without significant differences between the three groups ( data not shown ) . during the 14 days observation period , diameters slightly increased in all groups until day 11 , particularly in the control and anti - c - kit - pretreated groups . of interest , at day 14 , microvessel diameters within tumors of mice treated with anti - c - kit and anti - sdf-1 were markedly smaller compared to the other two groups ( table 1 ) . petechial bleedings within the areas of angiogenesis of growing tumors are a characteristic indicator of the vegf action on vascular permeability . to study this vegf - induced increase of vascular permeability , we analyzed the petechial bleedings within the rois by intravital fluorescence microscopy . at day 5 , signs of petechial bleedings were observed in 6% of the tumor area of control tumors , whereas petechial bleedings occurred in up to 30% of the tumor area in animals pretreated with anti - c - kit at that time . in all groups , petechial bleedings were most pronounced 8 days after tumor cell implantation ( control : 1520% , anti - c - kit : 30% , anti - c - kit / anti - sdf-1 : 3540% ) . treatment with anti - c - kit alone or additional blockade of sdf-1 did not impair tumor cell migration compared to controls ( table 2 ) . quantitative analysis of tumor cell invasion showed a significant increase of tumor cell infiltration through the muscular layer of the dorsal skinfold chamber in tumors of animals which were pretreated with anti - c - kit compared to controls ( p < 0.05 ) . of interest , tumors of animals additionally treated with anti - sdf-1 showed a significant reduction of muscular infiltration compared to anti - c - kit pretreated tumors ( p < 0.05 ) , resulting in invasive growth characteristics which were comparable to controls ( figure 4(c ) ) . pcna as an indicator of cell proliferation displayed positive staining in 46.9 3.1% of the tumor cells in control animals ( figure 5 ) . after pretreatment with anti - c - kit , the number of pcna - positive tumor cells was significantly higher compared to controls ( p < 0.05 , figures 5(a ) and 5(c ) ) . in contrast , additional blockade of sdf-1 resulted in a significantly lower number of pcna - positive tumor cells compared to controls and to anti - c - kit pretreated animals ( p < 0.05 , figures 5(b ) and 5(c ) ) . to study apoptotic cell death , 14 days after tumor cell implantation , only a minor fraction of the total number of 351.3 5.2 tumor cells within the high power fields ( hpf ) showed positive staining for caspase-3 . whereas 0.73 0.13 tumor cells / hpf were positive for caspase-3 in controls , pretreatment with anti - c - kit reduced apoptotic cell death within the tumors ( 0.26 0.06 tumor cells / hpf ) . in tumors of animals additionally treated with anti - sdf-1 antibodies , this effect was even more pronounced ( 0.15 0.03 tumor cells / hpf , p < 0.05 ) . facscan analysis demonstrated that 8.2 0.8% of the ct26-gfp cells transfected with the enhanced gfp expression vector pegfp - n1 was c - kit receptor positive . at day 5 after tumor cell implantation , the tumors of animals pretreated with anti - c - kit antibodies showed a significantly higher expression of vegf and cxcr4 compared to controls ( p < 0.05 ; figures 6(a ) and 6(b ) ) . furthermore , enos expression within the tumors was decreased by anti - c - kit pretreatment with and without additional anti - sdf-1 treatment compared to controls ( data not shown ) . the major finding of the present study is that bone marrow suppression by anti - c - kit treatment significantly enhances tumor cell engraftment of colorectal tumors due to an increase of tumor cell proliferation and invasion . additional anti - sdf-1 treatment neutralizes this increased tumor outgrowth by inhibition of tumor cell proliferation and tumor neovascularization . these findings suggest that the enhanced tumor growth under the conditions of bone marrow suppression induced by anti - c - kit treatment is related to the sdf-1/cxcr4 pathway . bone - marrow - derived hematopoetic cells ( hcs ) contribute to physiological and pathological vessel formation . in the early phases of tumor growth , 5090% of the neovessels within the tumor mass are derived from the bone marrow dependent on the tumor type . demonstrated that gpf - expressing c - kit bone - marrow - derived progenitor cells are recruited to subcutaneously implanted lewis lung carcinoma but do not directly contribute to microvascular structure . they , therefore , concluded that even if circulating hcs and epcs home to sites of tumor growth , they do not contribute to tumor angiogenesis . in the present study , intravital fluorescence microscopy showed highly vascularized ct26.wt-gfp tumors 14 days after tumor cell implantation . previous experimental studies in mice have shown that a depletion of the myeloid and erythroid cell lineages including epcs and hcs from the bone marrow could be performed by daily injection of 1 mg / kg ack2 , an antagonistic anti - c - kit antibody , which blocks the function of c - kit without cytotoxic side effects on c - kit cells [ 30 , 31 ] . moreover , the reduction rate of the colony forming cells within the bone marrow was dependent on the amount of anti - c - kit antibodies . in the present study , we , therefore , used the anti - c - kit antibody in a dosage of 1 mg / kg bw for the induction of bone marrow suppression over a time period of 4 days to avoid surgical complications and death of the animals resulting from the immune incompetence by bone marrow depletion . , bone marrow suppression by anti - c - kit pretreatment over a time period of 4 days before subcutaneous implantation of colon tumor cells induced leucopenia which was still detectable 10 days after the last injection . therefore , the authors concluded that hcs migrating into the tumor mass promote the initiation of tumor neovascularization . in our present study , although neoangiogenesis was not influenced after administration of anti - c - kit , tumor growth was stimulated as a result of an increased tumor cell proliferation and invasion . c - kit blockade for the induction of bone marrow suppression concurrently stimulates b lymphopoiesis [ 30 , 31 ] . as the number of b cell precursors normally decreases in tumor bearing mice [ 32 , 33 ] , it must be speculated that in our experiment of bone marrow suppression , c - kit blockade stimulates b cell genesis and thus increases tumor cell engraftment . in our study , neoangiogenesis within the colorectal tumors of animals pretreated with anti - c - kit was comparable to controls despite the lack of functional hcs which are necessary for angiogenesis and vasculogenesis . thus , we hypothesize that the lack of hcs was compensated by local angiogenic factors or b lymphopoiesis - associated angiogenesis , resulting in a tumor neovascularization comparable to that observed in controls . the scf receptor c - kit has been shown to be essential for the development of blood cells , melanocytes , germ cells , interstitial cells of cajal in the gastrointestinal tract , and mast cells . furthermore , the majority of tumor cells , especially those of the neural axis , breast , lung , prostate , and colon show an aberrant c - kit expression . especially gastrointestinal stromal tumors ( gists ) express c - kit on the cell - surface , and mutations of kit in these tumors results in an activation of kit signaling , which leads to uncontrolled cell proliferation and resistance to apoptosis [ 39 , 40 ] . today , patients with gist are treated with imatinib , an inhibitor of certain protein tyrosine kinases including kit , depending on the mitotic index of the gist . imatinib induces an arrest of tumor cell proliferation and causes apoptotic cell death . in established mca26 tumors , pan et al showed that injection of anti - c - kit antibodies markedly reduces tumor - induced immune tolerance exhibited by myeloid - derived suppressors in mice . furthermore , their experiments demonstrate that anti - c - kit treatment can prevent tumor - specific t - cell anergy and development of t regulatory cells ( treg ) . in combination with an immune modulatory therapy of il-12 plus 4 - 1bb activation , treatment with anti - c - kit antibodies significantly improved the long - term survival of mca26 tumor bearing mice . in our study , only 8% of the ct26.wt-gfp cells were c - kit receptor positive . therefore , we do not expect a direct inhibitory effect of anti - c - kit treatment on tumor cell proliferation . recruitment of hcs and epc is predominantly mediated by sdf-1 and its receptor cxcr4 [ 9 , 10 ] because hcs and epcs migrate along a chemotactic gradient towards higher concentrations of sdf-1 [ 1214 ] . have shown that almost 100% of bone marrow and peripheral blood c - kit cells are positive for cxcr4 . in the present study , we could demonstrate that neutralization of sdf-1 after anti - c - kit pretreatment significantly reduces neovascularization most probably by the inhibition of hc and epc recruitment within the tumors . additionally , in combination with anti - c - kit pretreatment , sdf-1 neutralization is capable of decreasing tumor cell proliferation and invasion . taken together , neutralization of sdf-1 counteracts the stimulating effects of bone marrow suppression by anti - c - kit treatment on tumor cell engraftment and inhibits compensatory local and b lymphopoiesis - associated angiogenesis . , the presence of sdf-1 chemoattractant activity and inflammatory endothelial activation by tnf- is required for c - kit cells to form functionally relevant interactions with the endothelium in postcapillary venules . blockade of icam-1 and cxcr4 abolishes adhesion of c - kit cells to the vascular endothelium despite application of sdf-1 and tnf-. as sdf-1 and tnf- stimulation activates the endothelium by an increase of the cxcr4 expression leading to relevant stem cell attraction , our results indicate that the increase of cxcr4 and vegf expression within the tumors represents a compensatory pathway after anti - c - kit pretreatment . neutralization of sdf-1 inhibits interactions of c - kit positive cells with tumor vessels and as a consequence , leads to inhibition of tumor neovascularization . in conclusion , bone marrow suppression by anti - c - kit pretreatment significantly enhances tumor cell engraftment of colorectal tumors . as anti - sdf-1 treatment counteracts this increased tumor outgrowth by inhibition of neovascularization , the sdf-1/cxcr4 pathway seems to be crucial for tumor angiogenesis mediated by hcs and epcs .

due to the lack of oxygen supply and the accumulation of toxic products , avascular tumors and tumor metastases can not grow beyond a critical size of 1 - 2 mm and thus will stay clinically occult [ 1 , 3 , 4 ] . tumor vessels can grow by sprouting of preexisting host vessels , intussusception or incorporation of bone marrow - derived endothelial progenitor cells ( epcs ) which are a subset of hematopoietic cells ( hcs ) . one of the most important factors mediating survival and proliferation of hcs is the stem cell factor ( scf ) which binds to the c - kit receptor on the surface of hcs . have shown that bone marrow suppression by anti - c - kit treatment induces a delay in tumor angiogenesis due to the inhibition of angiogenic sprouting in colon tumors and that c - kit blockade suppresses tumor growth of subcutaneously implanted prostate carcinomas ( pc3 ) by inhibition of tumor angiogenesis regulated by hcs . recruitment of hcs and epcs to avascular areas is orchestrated by different angiogenic growth factors and cytokines , predominantly by the cxc - chemokine stromal cell - derived factor ( sdf)-1 and its receptor cxcr4 [ 9 , 10 ] . hcs and epcs fluctuate to peripheral organs in antiphase with the expression of sdf-1 within the bone marrow microenvironment and migrate alongside a chemotactic gradient towards higher concentrations of sdf-1 , for example , sites of injury and tumor tissue [ 1214 ] . furthermore , it has been demonstrated that sdf-1-mediated recruitment of c - kit - positive cells to the periphery is dependent on cofactors , including tumor necrosis factor- and the endothelial nitric oxide synthase ( enos ) . however , the impact of circulating hcs and epcs on vasculogenesis and sprouting angiogenesis in tumor angio - development is still controversially discussed in the literature [ 1517 ] , and the role of the sdf-1/cxcr4 pathway is not yet fully understood . therefore , in the presented study we analyzed the influence of bone marrow suppression by anti - c - kit treatment combined with sdf-1 neutralization on tumor cell engraftment and neovascularization using a murine model of colorectal tumor metastasis . the ct26 cell line is an n - nitroso - n - methyl - urethane - induced undifferentiated adenocarcinoma of the colon , syngeneic with the balb / c mouse . for our studies , the ct26.wt cells ( atcc crl-2638 , lgc promochem gmbh , wesel , germany ) were transfected with the enhanced gfp expression vector pegfp - n1 ( clontech ) with the use of clonfectin ( clontech , palo alto , ca , usa ) according to the manufacturer 's instructions . for the individual experiments , ct26.wt-gfp cells were grown in cell culture as monolayers in rpmi-1640 medium with 2 mm l - glutamine ( sigma aldrich chemie gmbh , taufkirchen , germany ) supplemented with 10% fetal calf serum ( fcs gold , paa laboratories gmbh , clbe , germany ) , 100 u / ml penicillin , and 100 g / ml streptomycin ( paa laboratories gmbh ) . at the day of implantation , tumor cells were harvested from subconfluent cultures ( 70 to 85% ) by trypsinization ( 0.05% trypsin and 0.02% edta , paa laboratories gmbh ) and washed twice in phosphate - buffered saline solution ( pbs ) . experiments were performed after approval by the local governmental ethic committee and conformed to the united kingdom coordinating committee on cancer research ( ukcccr ) guidelines for the welfare of animals in experimental neoplasia ( as described in 1998 in br j cancer 77 : 110 ) and the guide for care and use of laboratory animals ( institute of laboratory animal resources , national research council ; nih guide , vol . to allow repetitive analyses of the microcirculation of the growing tumors , the dorsal skinfold chamber model was used for intravital microscopy as described previously in detail . for operative procedures , animals were anesthetized by intraperitoneal injection of 90 mg / kg bw ketamine ( ketavet , parke davis ; freiburg , germany ) and 20 mg / kg bw xylazine ( rompun , bayer ; leverkusen , germany ) . the remaining layers , consisting of the epidermis , subcutaneous tissue , and striated skin muscle , were covered with a glass coverslip incorporated into one of the titanium frames . for tumor cell implantation , the coverslip of the chamber was temporarily removed and 1 10 ct26.wt-gfp cells were implanted onto the surface of the striated muscle tissue within the chamber . animals were assigned to three different groups : the first group ( ckit - ab ; n = 8) received a pretreatment with a monoclonal anti - c - kit receptor antibody ( ack45 , bd biosciences , heidelberg , germany ) by daily intraperitoneal injections starting 4 days before tumor cell implantation . the third group ( control ; n = 8) served as control and received pre- and posttreatment the same amount of the corresponding isotype - matched igg control antibodies ( a95 - 1 , bd biosciences , and mab002 r&d systems ) . at the end of the experiment ( day 14 ) intravital fluorescence microscopy was performed in epi - illumination technique using a modified zeiss axio - tech microscope ( zeiss , oberkochen , germany ) with a 100-w hbo mercury lamp . tumor size , growth kinetics , migration of tumor cell , and neovascularization were analyzed using blue light epi - illumination ( 450 to 490 nm excitation wavelength and > 520 nm emission wavelengths ) [ 2022 ] . at each observation time point , the surface of the fluorescently labeled tumor mass within the chamber was scanned for determination of the tumor size ( given as tumor area in mm ) . in these rois , the onset of angiogenesis , that is , the existence of angiogenic buds , sprouts , and newly formed blood vessels , was documented and scored 0 to 8 , with 0 indicating existence of newly formed tumor microvessels in none of the rois and 8 indicating their existence in all of the rois . functional capillary density ( given in cm / cm ) of the tumor microvessels this parameter was defined as the length of red blood cell perfused microvessels per observation area and was analyzed within the eight rois of the tumor margin and within four additional rois of the tumor center . to study vascular permeability of the newly formed tumor microvessels , petechial bleedings were documented in each of the rois , and given as percentage of all the rois analyzed [ 20 , 21 , 23 ] . at the end of the experiment ( day 14 ) , the tumor and the adjacent host tissue were harvested and immediately fixed in formalin . tumor cell invasion of the muscular layer on the surface of the dorsal skinfold chamber was quantified over the entire tumor basis and given as percentage of the length of the tumor basis . a polyclonal goat anti - rat igg antibody ( bd biosciences ) facscan ( becton dickinson , mountain view , ca , usa ) analysis was performed to assess the expression of c - kit on the ct26.wt-gfp cells in triplicate . therefore , 12 additional animals received ct26.wt-gfp tumor cell implantation in the dorsal skinfold chamber and were assigned to the three groups as described above . four of these pretreated animals received additional treatment with the neutralizing anti - sdf-1 antibody ( mab310 , r and d systems ) starting at the day of tumor cell implantation . for whole protein extracts and western blot analysis of the expression of vascular endothelial growth factor ( vegf ) , the chemokine receptor cxcr4 and enos , ct26.wt-gfp-tumors were completely removed from the skinfold at day 5 . protein expression was visualized by means of luminol enhanced chemiluminescence ( ecl , ge healthcare ) and exposure of the membranes to a blue - light - sensitive autoradiography film ( hyperfilm ecl , ge healthcare ) . the take rate of the ct26.wt-gfp cells within the dorsal skinfold chamber was 100% , and progressive tumor growth was observed in all groups throughout the entire observation period ( figures 1(a)1(c ) ) . quantitative analysis of the increase of the tumor area revealed a significantly enhanced engraftment of tumor cells and a significant stimulation of tumor growth after pretreatment with the anti - c - kit antibody compared to controls ( p < 0.05 ) . interestingly , additional blockage of sdf-1 completely blunted this enhancement of tumor growth leading to similar tumor sizes as measured in controls ( p < 0.05 ; figure 1(d ) ) . pretreatment with the anti - c - kit antibody did not influence the onset of the angiogenic switch compared to controls . of interest , blockade of sdf-1 after pretreatment with anti - c - kit significantly delayed the angiogenic switch , with only 50% of the rois showing newly formed microvessels by day 8 and 70% by day 11 ( p < 0.05 ) . in this group , angiogenesis was observed in all rois only at the end of the observation period ( figure 2 ) . the differential effects of c - kit blockade with or without additional sdf-1 blockade were also reflected by quantitative analysis of the microvascular densities of the newly formed tumor vessels . as characteristic for tumor vessels , the vascular network of the tumors consisted of irregularly shaped and chaotically arranged microvessels ( figures 3(a)3(c ) ) . whereas no differences of microvascular density within the tumor vasculature could be observed between controls and anti - c - kit pretreated animals , additional treatment with the anti - sdf-1 antibody significantly decreased the microvascular density within the tumor center compared to the other groups from day 8 until the end of the observation period ( figure 3(d ) ) . quantitative analysis of the functional microvascular density within the tumor margin showed similar results ( data not shown ) . in contrast to the highly vascularized ct26.wt-gfp tumors within the intravital fluorescence microscopy , immunohistological staining for cd31 as a marker for endothelial cells displayed positive staining of only a few cells within tumor microvessels without significant differences between the three groups ( data not shown ) . however , no overall differences between the diameters of the microvessels within the tumor margin and the tumor center could be observed ( table 1 ) . of interest , at day 14 , microvessel diameters within tumors of mice treated with anti - c - kit and anti - sdf-1 were markedly smaller compared to the other two groups ( table 1 ) . to study this vegf - induced increase of vascular permeability , we analyzed the petechial bleedings within the rois by intravital fluorescence microscopy . at day 5 , signs of petechial bleedings were observed in 6% of the tumor area of control tumors , whereas petechial bleedings occurred in up to 30% of the tumor area in animals pretreated with anti - c - kit at that time . in all groups , petechial bleedings were most pronounced 8 days after tumor cell implantation ( control : 1520% , anti - c - kit : 30% , anti - c - kit / anti - sdf-1 : 3540% ) . starting at day 5 after tumor cell implantation , tumor cell migration was detectable in all tumors until the end of the observation period with slightly increasing distances from the tumor margin . quantitative analysis of tumor cell invasion showed a significant increase of tumor cell infiltration through the muscular layer of the dorsal skinfold chamber in tumors of animals which were pretreated with anti - c - kit compared to controls ( p < 0.05 ) . of interest , tumors of animals additionally treated with anti - sdf-1 showed a significant reduction of muscular infiltration compared to anti - c - kit pretreated tumors ( p < 0.05 ) , resulting in invasive growth characteristics which were comparable to controls ( figure 4(c ) ) . after pretreatment with anti - c - kit , the number of pcna - positive tumor cells was significantly higher compared to controls ( p < 0.05 , figures 5(a ) and 5(c ) ) . in contrast , additional blockade of sdf-1 resulted in a significantly lower number of pcna - positive tumor cells compared to controls and to anti - c - kit pretreated animals ( p < 0.05 , figures 5(b ) and 5(c ) ) . to study apoptotic cell death , 14 days after tumor cell implantation , only a minor fraction of the total number of 351.3 5.2 tumor cells within the high power fields ( hpf ) showed positive staining for caspase-3 . whereas 0.73 0.13 tumor cells / hpf were positive for caspase-3 in controls , pretreatment with anti - c - kit reduced apoptotic cell death within the tumors ( 0.26 0.06 tumor cells / hpf ) . at day 5 after tumor cell implantation , the tumors of animals pretreated with anti - c - kit antibodies showed a significantly higher expression of vegf and cxcr4 compared to controls ( p < 0.05 ; figures 6(a ) and 6(b ) ) . furthermore , enos expression within the tumors was decreased by anti - c - kit pretreatment with and without additional anti - sdf-1 treatment compared to controls ( data not shown ) . the major finding of the present study is that bone marrow suppression by anti - c - kit treatment significantly enhances tumor cell engraftment of colorectal tumors due to an increase of tumor cell proliferation and invasion . these findings suggest that the enhanced tumor growth under the conditions of bone marrow suppression induced by anti - c - kit treatment is related to the sdf-1/cxcr4 pathway . during tumor growth , the release of cytokines and chemokines mediates the recruitment of epcs and hcs contributing to the early initiation and stabilization of newly formed blood vessels , so - called vasculogenesis [ 2426 ] . in the early phases of tumor growth , 5090% of the neovessels within the tumor mass are derived from the bone marrow dependent on the tumor type . however , recent reports have already doubted the impact of vasculogenesis from bone - marrow - derived cells for tumor neovascularization and claim an exclusive role for sprouting angiogenesis in tumor blood vessel development [ 15 , 16 ] . in the present study , intravital fluorescence microscopy showed highly vascularized ct26.wt-gfp tumors 14 days after tumor cell implantation . previous experimental studies in mice have shown that a depletion of the myeloid and erythroid cell lineages including epcs and hcs from the bone marrow could be performed by daily injection of 1 mg / kg ack2 , an antagonistic anti - c - kit antibody , which blocks the function of c - kit without cytotoxic side effects on c - kit cells [ 30 , 31 ] . of interest , whereas no polymorphonuclear cells or erythroblasts were present in the bone marrow of these ack2-treated animals , b lineage cells continued to grow to fill the space from which myeloid and erythroid progenitor cells were purged [ 30 , 31 ] . moreover , the reduction rate of the colony forming cells within the bone marrow was dependent on the amount of anti - c - kit antibodies . in the present study , we , therefore , used the anti - c - kit antibody in a dosage of 1 mg / kg bw for the induction of bone marrow suppression over a time period of 4 days to avoid surgical complications and death of the animals resulting from the immune incompetence by bone marrow depletion . , bone marrow suppression by anti - c - kit pretreatment over a time period of 4 days before subcutaneous implantation of colon tumor cells induced leucopenia which was still detectable 10 days after the last injection . as the number of b cell precursors normally decreases in tumor bearing mice [ 32 , 33 ] , it must be speculated that in our experiment of bone marrow suppression , c - kit blockade stimulates b cell genesis and thus increases tumor cell engraftment . in our study , neoangiogenesis within the colorectal tumors of animals pretreated with anti - c - kit was comparable to controls despite the lack of functional hcs which are necessary for angiogenesis and vasculogenesis . thus , we hypothesize that the lack of hcs was compensated by local angiogenic factors or b lymphopoiesis - associated angiogenesis , resulting in a tumor neovascularization comparable to that observed in controls . the scf receptor c - kit has been shown to be essential for the development of blood cells , melanocytes , germ cells , interstitial cells of cajal in the gastrointestinal tract , and mast cells . furthermore , the majority of tumor cells , especially those of the neural axis , breast , lung , prostate , and colon show an aberrant c - kit expression . especially gastrointestinal stromal tumors ( gists ) express c - kit on the cell - surface , and mutations of kit in these tumors results in an activation of kit signaling , which leads to uncontrolled cell proliferation and resistance to apoptosis [ 39 , 40 ] . in combination with an immune modulatory therapy of il-12 plus 4 - 1bb activation , treatment with anti - c - kit antibodies significantly improved the long - term survival of mca26 tumor bearing mice . in the present study , we could demonstrate that neutralization of sdf-1 after anti - c - kit pretreatment significantly reduces neovascularization most probably by the inhibition of hc and epc recruitment within the tumors . taken together , neutralization of sdf-1 counteracts the stimulating effects of bone marrow suppression by anti - c - kit treatment on tumor cell engraftment and inhibits compensatory local and b lymphopoiesis - associated angiogenesis . , the presence of sdf-1 chemoattractant activity and inflammatory endothelial activation by tnf- is required for c - kit cells to form functionally relevant interactions with the endothelium in postcapillary venules . as sdf-1 and tnf- stimulation activates the endothelium by an increase of the cxcr4 expression leading to relevant stem cell attraction , our results indicate that the increase of cxcr4 and vegf expression within the tumors represents a compensatory pathway after anti - c - kit pretreatment . as anti - sdf-1 treatment counteracts this increased tumor outgrowth by inhibition of neovascularization , the sdf-1/cxcr4 pathway seems to be crucial for tumor angiogenesis mediated by hcs and epcs .

however , the incidence of gbc is relatively high in korea , where it accounts for 6.3 per 100,000 individuals and is the sixth leading cause of cancer - related death . the prognosis of gbc is poor , with a 5-year overall survival ( os ) rate of 17% to 45% , even in patients treated with curative intent surgery [ 3 - 5 ] . advanced stage , high - grade histological differentiation , and a positive resection margin are known to be poor prognostic factors in gbc patients who have undergone surgical resection . given the high relapse rate and poor prognosis , adjuvant therapy would appear to be a rational therapeutic strategy in gbc patients following surgical resection . nevertheless , there is controversy surrounding the role of adjuvant therapy in gbc . current national comprehensive cancer network guidelines include consideration of fluoropyrimidine chemoradiotherapy , fluoropyrimidine or gemcitabine chemotherapy , and observation as postoperative options , with no preference given to any particular option . in addition , unlike cholangiocarcinoma , due to the limited clinical data available , the guidelines for gbc do not provide information on treatment strategy according to known prognostic factors , such as resection margin status . several retrospective studies reported that patients with node - positive disease had a survival benefit from adjuvant chemoradiotherapy or radiotherapy . a recent study showed that gemcitabine - based adjuvant chemotherapy improved the survival of gbc patients after r1 or r2 resections . reported that gbc patients who have undergone non - curative resections may derive a slight benefit from systemic chemotherapy , but this was not observed in patients who underwent curative surgery in a subgroup analysis of the study , which combined patients with pancreatic , bile duct , and gallbladder cancers . however , the question of whether patients who have undergone r0 resection benefit from adjuvant therapy is controversial . a few studies reported that , compared with surgery alone , adjuvant chemoradiotherapy resulted in improved survival in gbc patients following r0 resection . in contrast , other studies did not show any survival benefit of adjuvant therapy in gbc patients who underwent r0 resection . importantly , many previous studies included a small number of patients ; heterogeneous populations with respect to tumor location , tnm stage , nodal status , and resection margin ; or unbalanced comparative groups . these limitations make it difficult for physicians to identify patients who will truly benefit from adjuvant therapy . a meta - analysis of 20 studies involving 6,712 patients reported that gbc patients who had undergone r1 resection , but not r0 resection , benefited from adjuvant therapy . another recent meta - analysis also reported similar results showing that gbc patients who underwent r0 resection did not benefit from adjuvant therapy compared with surgery alone . however , due to the potential selection bias caused by unbalanced clinical characteristics , which is an inherent drawback of a retrospective study , these two meta - analyses can not confirm the ineffectiveness of adjuvant therapy in gbc patients who have undergone r0 resection . therefore , we attempted to determine whether adjuvant therapy is necessary for non - metastatic gbc patients who have undergone r0 resection through a multicenter , case - control study with propensity score matching ( psm ) to reduce the selection bias . information on patients who consecutively underwent curative r0 resection for gbc from march 1999 to february 2014 at 6 tertiary medical centers was collected retrospectively . of the 441 patients who were initially screened , those in the following categories 1 ) : ( 1 ) stage iv disease ; ( 2 ) unknown histological subtype ; ( 3 ) minor histological subtype such as adenosquamous , mucinous , mucosal , or neuroendocrine carcinoma ; ( 4 ) follow - up duration less than 3 months ; ( 5 ) relapse within 1 month of surgical resection ; ( 6 ) adjuvant therapy with radiotherapy alone ; or ( 7 ) unknown preoperative serum carbohydrate antigen 19 - 9 ( ca 19 - 9 ) level . curative r0 resection was performed in all patients and confirmed by the operation record and the pathology report . then , postoperative management was planned according to each center s protocol . in patients treated with adjuvant chemotherapy alone , fluoropyrimidine - based chemotherapy , including 5-fluorouracil / leucovorin , doxifluridine , tegafur / uracil , or s-1 , was performed . adjuvant concurrent chemoradiotherapy ( ccrt ) was also performed using fluoropyrimidine - based agents including 5-fluorouracil and capecitabine . patients managed with surveillance - only received follow - up care after surgical resection without adjuvant chemotherapy or ccrt . regular monitoring was performed in all patients via comprehensive physical examinations , serum tumor markers , and computed tomography for detection of relapse according to each center s protocol . clinical data , including demographics , histological type , tnm stage based on the seventh edition of the american joint committee on cancer , preoperative serum level of ca 19 - 9 , treatment modality , and clinical outcomes ( e.g. , relapse and survival ) , were collected by medical chart review . the cut - off level of preoperative serum ca 19 - 9 was defined as 37 u / ml ( the upper limit of the normal range ) . relapse - free survival ( rfs ) was defined as the time from the date of surgery to the date of either the first relapse at any site or death . os was defined as the time from the date of surgery to the date of death . we hypothesized that patients with advanced disease may be more likely to be treated with adjuvant therapy at the discretion of the clinician . we therefore performed psm to reduce the selection bias between patients treated with adjuvant therapy and those managed with surveillance only using the matchit package in r statistical software ver . the propensity score was calculated from a logistic regression model that included age ( years ) , sex , histological type , tnm stage , and preoperative serum ca 19 - 9 level ( normal or elevated ) . with the propensity score estimated , 84 pairs of patients in the adjuvant therapy group and the surveillance - only group were matched using a 1:1 nearest neighbor matching algorithm . the standardized mean difference ( smd ) in each covariate was calculated to determine whether the psm was adequately conducted . an smd less than 0.1 was considered a negligible difference in the mean or prevalence of a covariate between two treatment groups . statistical comparisons were made using the chi - square test or fisher exact test for categorical variables . rfs and os were estimated using the kaplan - meier method , and comparisons between groups were performed using the log - rank test . multivariate cox regression analysis using the enter selection method was performed to identify independent predictors of mortality . all variables with a p - value less than 0.1 in the univariate analysis were entered into the multivariate analysis . the results were reported as hazard ratios ( hrs ) and 95% confidence intervals ( cis ) . spss ver . 21.0 ( ibm co. , armonk , ny ) was used for statistical analyses . information on patients who consecutively underwent curative r0 resection for gbc from march 1999 to february 2014 at 6 tertiary medical centers was collected retrospectively . of the 441 patients who were initially screened , those in the following categories 1 ) : ( 1 ) stage iv disease ; ( 2 ) unknown histological subtype ; ( 3 ) minor histological subtype such as adenosquamous , mucinous , mucosal , or neuroendocrine carcinoma ; ( 4 ) follow - up duration less than 3 months ; ( 5 ) relapse within 1 month of surgical resection ; ( 6 ) adjuvant therapy with radiotherapy alone ; or ( 7 ) unknown preoperative serum carbohydrate antigen 19 - 9 ( ca 19 - 9 ) level . curative r0 resection was performed in all patients and confirmed by the operation record and the pathology report . then , postoperative management was planned according to each center s protocol . in patients treated with adjuvant chemotherapy alone , fluoropyrimidine - based chemotherapy , including 5-fluorouracil / leucovorin , doxifluridine , tegafur / uracil , or s-1 , was performed . adjuvant concurrent chemoradiotherapy ( ccrt ) was also performed using fluoropyrimidine - based agents including 5-fluorouracil and capecitabine . patients managed with surveillance - only received follow - up care after surgical resection without adjuvant chemotherapy or ccrt . regular monitoring was performed in all patients via comprehensive physical examinations , serum tumor markers , and computed tomography for detection of relapse according to each center s protocol . clinical data , including demographics , histological type , tnm stage based on the seventh edition of the american joint committee on cancer , preoperative serum level of ca 19 - 9 , treatment modality , and clinical outcomes ( e.g. , relapse and survival ) , were collected by medical chart review . the cut - off level of preoperative serum ca 19 - 9 was defined as 37 u / ml ( the upper limit of the normal range ) . relapse - free survival ( rfs ) was defined as the time from the date of surgery to the date of either the first relapse at any site or death . os was defined as the time from the date of surgery to the date of death . we hypothesized that patients with advanced disease may be more likely to be treated with adjuvant therapy at the discretion of the clinician . we therefore performed psm to reduce the selection bias between patients treated with adjuvant therapy and those managed with surveillance only using the matchit package in r statistical software ver . the propensity score was calculated from a logistic regression model that included age ( years ) , sex , histological type , tnm stage , and preoperative serum ca 19 - 9 level ( normal or elevated ) . with the propensity score estimated , 84 pairs of patients in the adjuvant therapy group and the surveillance - only group were matched using a 1:1 nearest neighbor matching algorithm . the standardized mean difference ( smd ) in each covariate was calculated to determine whether the psm was adequately conducted . an smd less than 0.1 was considered a negligible difference in the mean or prevalence of a covariate between two treatment groups . statistical comparisons were made using the chi - square test or fisher exact test for categorical variables . rfs and os were estimated using the kaplan - meier method , and comparisons between groups were performed using the log - rank test . multivariate cox regression analysis using the enter selection method was performed to identify independent predictors of mortality . all variables with a p - value less than 0.1 in the univariate analysis were entered into the multivariate analysis . the results were reported as hazard ratios ( hrs ) and 95% confidence intervals ( cis ) . spss ver . 21.0 ( ibm co. , armonk , ny ) was used for statistical analyses . of the 363 patients included in the study , 84 patients were treated with adjuvant therapy ( chemotherapy , n=39 ; ccrt , n=45 ) and 279 were managed with surveillance only after surgical resection . the baseline characteristics before and after psm are shown in table 1 . in the unmatched cohort , the distributions of age , sex , and histological type were not significantly different between the two groups . however , the proportions of patients with stage iii disease ( 51.2% vs. 14.7% , p < 0.001 ) , node - positive disease ( 46.4% vs. 12.2% , p < 0.001 ) , and elevated ca 19 - 9 levels ( 29.8% vs. 18.6% , p=0.029 ) were higher in the adjuvant therapy group than in the surveillance - only group . after psm , the smds were reduced in all variables except age , and none of the variables showed an smd of more than 0.1 . there were no significant differences in the baseline characteristics between the two treatment groups in the matched cohort ( table 1 ) . in the unmatched cohort , the median follow - up durations were 60.4 and 53.4 months in all patients and in surviving patients at the time of analysis , respectively ( range , 5.3 to 177.6 months in both groups ) . thirty - seven ( 44.0% ) and 143 ( 51.3% ) patients were followed up for less than 5 years in the adjuvant therapy and surveillance only groups , respectively . follow - up was lost in 17 patients ( 20.2% ) in the adjuvant therapy group and in 46 patients ( 16.5% ) in the surveillance only group . of the 363 total patients , the 5-year rfs and os rates were 68.9% and 76.3% , respectively . in comparison of treatment outcomes between the two treatment groups , the adjuvant therapy group showed a shorter rfs compared with the surveillance - only group ( 3- and 5-year rfs rates , adjuvant therapy 58.0% and 50.8% vs. surveillance only 78.0% and 74.8% ; p < 0.001 ) ( fig . os also tended to be worse in the adjuvant therapy group than in the surveillance - only group ( 3- and 5-year os rates , adjuvant therapy 79.9% and 66.2% vs. surveillance only 85.5% and 79.5% ; p=0.089 ) ( fig . the median follow - up durations were 62.1 and 53.4 months in all patients and in surviving patients at the time of analysis , respectively ( range , 4.9 to 177.1 months in both groups ) . thirty - seven ( 44.0% ) and 44 ( 52.4% ) patients were followed up for less than 5 years in the adjuvant therapy and surveillance only groups , respectively . follow - up was lost in 17 patients ( 20.2% ) in the adjuvant therapy group and 15 patients ( 17.9% ) in the surveillance only group . among a total of 168 patients , 55 patients relapsed and 42 patients died . in this cohort , rfs was not significantly different between the two treatment groups ( 3- and 5-year rfs rates , adjuvant therapy 58.0% and 50.8% vs. surveillance only 67.1% and 64.8% ; p=0.319 ) ( fig . there was also no significant difference in os between the two treatment groups ( 3- and 5-year os rates , adjuvant therapy 79.9% and 66.2% vs. surveillance only 74.8% and 70.4% ; p=0.703 ) ( fig . none of the variables , including age , sex , histological type , tnm stage , nodal status , and preoperative serum ca 19 - 9 level , were associated with the efficacy of adjuvant therapy ( fig . although there was a tendency in favor of adjuvant therapy in elderly patients , this result was biased by a higher proportion of stage iii disease in the surveillance - only group compared with the adjuvant therapy group ( 54.0% vs. 36.8% , p=0.110 ) among elderly patients . in the matched cohort , advanced tnm stage , higher preoperative serum ca 19 - 9 level , and histological type other than well - differentiated grade disease were potential poor prognostic factors for os in the univariate analysis . in the multivariate analysis , advanced tnm stage was the only poor prognostic factor for os . adjuvant therapy did not affect the prognosis of this cohort in the multivariate analysis ( table 2 ) . next , we evaluated the prognosis according to treatment modality among 84 patients treated with adjuvant therapy . patients treated with chemotherapy had better rfs than those treated with ccrt ( 3- and 5-year rfs rates , chemotherapy 70.3% and 57.0% vs. ccrt 47.3% and 47.3% ; p=0.027 ) ( fig . 4a ) . however , os was not significantly different between patients treated with chemotherapy and those treated with ccrt ( 3- and 5-year os rates , chemotherapy 87.7% and 71.0% vs. ccrt 72.4% and 62.0% ; p=0.203 ) ( fig . 4b ) . in addition , patients treated with ccrt were more likely to have poor prognostic characteristics compared to those treated with chemotherapy , as follows : histological type other than well - differentiated grade ( 82.2% vs. 41.0% , p < 0.001 ) , stage iii disease ( 80.0% vs. 17.9% , p < 0.001 ) , and a higher preoperative serum ca 19 - 9 level ( 40.0% vs. 17.9% , p=0.027 ) ( table 3 ) . in a multivariate analysis adjusted for these poor prognostic markers , ccrt was not associated with worse rfs ( hr , 1.501 ; 95% ci , 0.685 to 3.286 ; p=0.310 ) as well as os ( hr , 1.062 ; 95% ci , 0.367 to 3.075 ; p=0.912 ) compared with chemotherapy ( table 4 ) . of the 363 patients included in the study , 84 patients were treated with adjuvant therapy ( chemotherapy , n=39 ; ccrt , n=45 ) and 279 were managed with surveillance only after surgical resection . the baseline characteristics before and after psm are shown in table 1 . in the unmatched cohort , the distributions of age , sex , and histological type were not significantly different between the two groups . however , the proportions of patients with stage iii disease ( 51.2% vs. 14.7% , p < 0.001 ) , node - positive disease ( 46.4% vs. 12.2% , p < 0.001 ) , and elevated ca 19 - 9 levels ( 29.8% vs. 18.6% , p=0.029 ) were higher in the adjuvant therapy group than in the surveillance - only group . after psm , the smds were reduced in all variables except age , and none of the variables showed an smd of more than 0.1 . there were no significant differences in the baseline characteristics between the two treatment groups in the matched cohort ( table 1 ) . in the unmatched cohort , the median follow - up durations were 60.4 and 53.4 months in all patients and in surviving patients at the time of analysis , respectively ( range , 5.3 to 177.6 months in both groups ) . thirty - seven ( 44.0% ) and 143 ( 51.3% ) patients were followed up for less than 5 years in the adjuvant therapy and surveillance only groups , respectively . follow - up was lost in 17 patients ( 20.2% ) in the adjuvant therapy group and in 46 patients ( 16.5% ) in the surveillance only group . of the 363 total patients , the 5-year rfs and os rates were 68.9% and 76.3% , respectively . in comparison of treatment outcomes between the two treatment groups , the adjuvant therapy group showed a shorter rfs compared with the surveillance - only group ( 3- and 5-year rfs rates , adjuvant therapy 58.0% and 50.8% vs. surveillance only 78.0% and 74.8% ; p < 0.001 ) ( fig . os also tended to be worse in the adjuvant therapy group than in the surveillance - only group ( 3- and 5-year os rates , adjuvant therapy 79.9% and 66.2% vs. surveillance only 85.5% and 79.5% ; p=0.089 ) ( fig . the median follow - up durations were 62.1 and 53.4 months in all patients and in surviving patients at the time of analysis , respectively ( range , 4.9 to 177.1 months in both groups ) . thirty - seven ( 44.0% ) and 44 ( 52.4% ) patients were followed up for less than 5 years in the adjuvant therapy and surveillance only groups , respectively . follow - up was lost in 17 patients ( 20.2% ) in the adjuvant therapy group and 15 patients ( 17.9% ) in the surveillance only group . among a total of 168 patients , 55 patients relapsed and 42 patients died . in this cohort , rfs was not significantly different between the two treatment groups ( 3- and 5-year rfs rates , adjuvant therapy 58.0% and 50.8% vs. surveillance only 67.1% and 64.8% ; p=0.319 ) ( fig . there was also no significant difference in os between the two treatment groups ( 3- and 5-year os rates , adjuvant therapy 79.9% and 66.2% vs. surveillance only 74.8% and 70.4% ; p=0.703 ) ( fig . none of the variables , including age , sex , histological type , tnm stage , nodal status , and preoperative serum ca 19 - 9 level , were associated with the efficacy of adjuvant therapy ( fig . although there was a tendency in favor of adjuvant therapy in elderly patients , this result was biased by a higher proportion of stage iii disease in the surveillance - only group compared with the adjuvant therapy group ( 54.0% vs. 36.8% , p=0.110 ) among elderly patients . in the matched cohort , advanced tnm stage , higher preoperative serum ca 19 - 9 level , and histological type other than well - differentiated grade disease were potential poor prognostic factors for os in the univariate analysis . in the multivariate analysis , advanced tnm stage was the only poor prognostic factor for os . adjuvant therapy did not affect the prognosis of this cohort in the multivariate analysis ( table 2 ) . next , we evaluated the prognosis according to treatment modality among 84 patients treated with adjuvant therapy . patients treated with chemotherapy had better rfs than those treated with ccrt ( 3- and 5-year rfs rates , chemotherapy 70.3% and 57.0% vs. ccrt 47.3% and 47.3% ; p=0.027 ) ( fig . 4a ) . however , os was not significantly different between patients treated with chemotherapy and those treated with ccrt ( 3- and 5-year os rates , chemotherapy 87.7% and 71.0% vs. ccrt 72.4% and 62.0% ; p=0.203 ) ( fig . 4b ) . in addition , patients treated with ccrt were more likely to have poor prognostic characteristics compared to those treated with chemotherapy , as follows : histological type other than well - differentiated grade ( 82.2% vs. 41.0% , p < 0.001 ) , stage iii disease ( 80.0% vs. 17.9% , p < 0.001 ) , and a higher preoperative serum ca 19 - 9 level ( 40.0% vs. 17.9% , p=0.027 ) ( table 3 ) . in a multivariate analysis adjusted for these poor prognostic markers , ccrt was not associated with worse rfs ( hr , 1.501 ; 95% ci , 0.685 to 3.286 ; p=0.310 ) as well as os ( hr , 1.062 ; 95% ci , 0.367 to 3.075 ; p=0.912 ) compared with chemotherapy ( table 4 ) . our data suggests that there is no role for adjuvant therapy after r0 resection in gbc patients . upon initiating this study , we assessed which patients were likely to be treated with adjuvant therapy after r0 resection for gbc in clinical practice . patients with characteristics indicating high tumor burden , such as more advanced tnm stage , node - positive disease , and a higher preoperative serum ca 19 - 9 level , were more frequently treated with adjuvant therapy compared to those with a low tumor burden . however , adjuvant therapy failed to improve the prognosis of patients with a high tumor burden , resulting in shorter rfs and os rates compared with the prognosis of patients managed with surveillance only . this paradoxical trend is similar to the results of a large , non - randomized japanese study in which , after surgical resection with or without a negative surgical margin , the prognosis of patients who received adjuvant chemotherapy was worse than that of patients who did not receive adjuvant chemotherapy . to minimize the selection bias , psm was performed in this study . however , even after the characteristics of patients were balanced via psm , there was no difference in survival , regardless of the postoperative treatment strategy . previous studies on adjuvant therapy in gbc patients who have undergone r0 resection are summarized in table 5 . in accordance with the results of our study , a few studies have suggested that adjuvant therapy is ineffective in gbc patients following r0 resection . a study of 279 gbc patients who had undergone surgery ( r0 resection in 164 patients ) found that neither adjuvant chemotherapy nor radiotherapy improved the os of patients following r0 resection . a subgroup analysis of 51 gbc patients who underwent curative resections in a phase iii trial showed that they did not benefit from adjuvant chemotherapy . another recent study of 61 patients with stage iii gbc also did not support the benefit of adjuvant therapy . however , these previous studies included a small number of patients or enrolled patients who had undergone r1 resection or had stage iv disease , therefore , the results are difficult to interpret . the strengths of our study compared with previous studies are that the role of adjuvant therapy was assessed in selected patients ( r0 resection , no stage iv disease ) and the inevitable selection bias caused by the retrospective design was minimized by psm . in addition , to the best of our knowledge , our study included the largest number of cases among case - control studies of adjuvant therapy in gbc patients following r0 resection . two previous retrospective studies in gbc patients who had undergone r0 resection yielded opposite results from those obtained in our study . reported that adjuvant chemoradiotherapy reduced the risk of death by 70% compared with surgery alone in stage i - ii gbc patients who had undergone r0 resection . however , the study had a small sample size ( n=73 ) , and the prognosis of the surgery - only group was relatively worse compared with those reported in other studies . for example , in a large japanese study of 4,770 gbc patients , the 5-year survival rates among the patients in the surgery - only group with stage i and ii disease were 78% and 65% , respectively . similarly , in our study , the 5-year survival rate was 79.5% among the patients in the surgery - only group , 85.3% of whom had stage i or ii disease before psm . , the 5-year survival rate in the surgery - only group was approximately 40% , even though 80% of the patients had stage i disease . this result indicates that the survival difference between patients who underwent surgery alone and those who received adjuvant therapy observed in the study by gold et al . may be attributed to the poorer prognosis of the surgery - only group compared with those in both the japanese study as well as our own study . furthermore , in a previous western study of over 10,000 cases of gbc based on data from the national cancer database ( ncdb ) , the 5-year survival rates were only 50% and 29% among patients with stage i and ii disease , respectively , and these trends were identical in the subset of 7,462 patients who underwent potentially curative surgical resection . cho et al . also reported that adjuvant chemoradiotherapy after surgical resection ( r0 and r1 resection in 38 and 2 patients , respectively ) is beneficial for node - positive t2/t3 gbc patients . however , the data are difficult to interpret because the sample size was too small and there were few end - point events . based on both the lack of strong evidence supporting adjuvant therapy and on our findings , we recommend against the routine use of adjuvant therapy in stage i - iii gbc patients who have undergone r0 resection , particularly in asian patients . therefore , the clinical data may not be as accurate as those collected prospectively , and there may be potential selection bias . we tried to control selection bias by using psm , and patient characteristics were balanced between the groups after psm . second , because the data were collected from six institutions , there was heterogeneity in treatment strategies . in this study , however , there was no difference in survival between patients treated with chemotherapy and those treated with ccrt . in addition , all patients enrolled in the study were treated with a fluoropyrimidine - based regimen , mainly due to the national insurance coverage policy in korea , although the specific dosage and the schedule of chemotherapy and radiotherapy varied . third , the efficacy of adjuvant radiotherapy alone was not assessed in this study due to the small sample size . however , based on the two previous meta - analyses showing that patients receiving adjuvant chemotherapy or chemoradiotherapy derived greater benefit than those receiving radiotherapy alone , radiotherapy alone may not be recommended as the best option for adjuvant therapy in gbc patients . fourth , patients with different tumor burden were included together in this study . despite performance of subgroup analyses by tumor stage and nodal status ( fig . 3 ) , those might be biased because psm was not done separately for the two variables . therefore , the association between the tumor burden and the efficacy of adjuvant therapy after r0 resection may not be confirmed in this study . given the results of previous reports and the subgroup analyses of our study ( without statistical significance ) ( fig . 3 ) favoring adjuvant therapy in patients with higher tumor stage and node - positive disease , further studies are warranted in these groups with r0 resection . this study suggests that fluoropyrimidine - based adjuvant chemotherapy and ccrt are not indicated in stage i - iii gbc patients who have undergone r0 resection . gemcitabine plus cisplatin is the current standard regimen in patients with locally advanced or metastatic biliary tract cancer . gemcitabine - based adjuvant therapy after curative resection recently showed promising results in patients with biliary tract cancer [ 10,19 - 22 ] . therefore , prospective , randomized studies of therapeutic strategies other than a fluoropyrimidine - based regimen are warranted to improve the prognosis of gbc patients following r0 resection .

purposethe purpose of this study is to assess the role of adjuvant therapy in stage i - iii gallbladder cancer ( gbc ) patients who have undergone r0 resection.materials and methodsclinical data were collected on 441 consecutive patients who underwent r0 resection for stage i - iii gbc . eligible patients were classified into adjuvant therapy and surveillance only groups . propensity score matching ( psm ) between the two groups was performed , adjusting clinical factors.resultsin total , 84 and 279 patients treated with adjuvant therapy and followed up with surveillance only , respectively , were included in the analysis . before psm , the 5-year relapse - free survival ( rfs ) rate was lower in the adjuvant therapy group than in the surveillance only group ( 50.8% vs. 74.8% , p < 0.001 ) , although there was no statistically significant difference in the 5-year overall survival ( os ) rate ( 66.2% vs. 79.5% , p=0.089 ) . after the psm , baseline characteristics became comparable and there were no differences in the 5-year rfs ( 50.8% vs. 64.8% , p=0.319 ) and os ( 66.2% vs. 70.4% , p=0.703 ) rates between the two groups.conclusionthe results suggest that fluoropyrimidine - based adjuvant therapy is not indicated in stage i - iii gbc patients who have undergone r0 resection .

Introduction Materials and Methods 1. Study population 2. Treatment 3. Data collection 4. Propensity score matching 5. Statistical analysis Results 1. Patient characteristics 2. Survival analysis in the unmatched cohort 3. Survival analysis in the matched cohort 4. Survival according to treatment modality in the adjuvant therapy group Discussion Conclusion

the prognosis of gbc is poor , with a 5-year overall survival ( os ) rate of 17% to 45% , even in patients treated with curative intent surgery [ 3 - 5 ] . advanced stage , high - grade histological differentiation , and a positive resection margin are known to be poor prognostic factors in gbc patients who have undergone surgical resection . nevertheless , there is controversy surrounding the role of adjuvant therapy in gbc . a recent study showed that gemcitabine - based adjuvant chemotherapy improved the survival of gbc patients after r1 or r2 resections . reported that gbc patients who have undergone non - curative resections may derive a slight benefit from systemic chemotherapy , but this was not observed in patients who underwent curative surgery in a subgroup analysis of the study , which combined patients with pancreatic , bile duct , and gallbladder cancers . however , the question of whether patients who have undergone r0 resection benefit from adjuvant therapy is controversial . in contrast , other studies did not show any survival benefit of adjuvant therapy in gbc patients who underwent r0 resection . a meta - analysis of 20 studies involving 6,712 patients reported that gbc patients who had undergone r1 resection , but not r0 resection , benefited from adjuvant therapy . another recent meta - analysis also reported similar results showing that gbc patients who underwent r0 resection did not benefit from adjuvant therapy compared with surgery alone . however , due to the potential selection bias caused by unbalanced clinical characteristics , which is an inherent drawback of a retrospective study , these two meta - analyses can not confirm the ineffectiveness of adjuvant therapy in gbc patients who have undergone r0 resection . therefore , we attempted to determine whether adjuvant therapy is necessary for non - metastatic gbc patients who have undergone r0 resection through a multicenter , case - control study with propensity score matching ( psm ) to reduce the selection bias . information on patients who consecutively underwent curative r0 resection for gbc from march 1999 to february 2014 at 6 tertiary medical centers was collected retrospectively . of the 441 patients who were initially screened , those in the following categories 1 ) : ( 1 ) stage iv disease ; ( 2 ) unknown histological subtype ; ( 3 ) minor histological subtype such as adenosquamous , mucinous , mucosal , or neuroendocrine carcinoma ; ( 4 ) follow - up duration less than 3 months ; ( 5 ) relapse within 1 month of surgical resection ; ( 6 ) adjuvant therapy with radiotherapy alone ; or ( 7 ) unknown preoperative serum carbohydrate antigen 19 - 9 ( ca 19 - 9 ) level . in patients treated with adjuvant chemotherapy alone , fluoropyrimidine - based chemotherapy , including 5-fluorouracil / leucovorin , doxifluridine , tegafur / uracil , or s-1 , was performed . , relapse and survival ) , were collected by medical chart review . relapse - free survival ( rfs ) was defined as the time from the date of surgery to the date of either the first relapse at any site or death . we hypothesized that patients with advanced disease may be more likely to be treated with adjuvant therapy at the discretion of the clinician . we therefore performed psm to reduce the selection bias between patients treated with adjuvant therapy and those managed with surveillance only using the matchit package in r statistical software ver . with the propensity score estimated , 84 pairs of patients in the adjuvant therapy group and the surveillance - only group were matched using a 1:1 nearest neighbor matching algorithm . information on patients who consecutively underwent curative r0 resection for gbc from march 1999 to february 2014 at 6 tertiary medical centers was collected retrospectively . of the 441 patients who were initially screened , those in the following categories 1 ) : ( 1 ) stage iv disease ; ( 2 ) unknown histological subtype ; ( 3 ) minor histological subtype such as adenosquamous , mucinous , mucosal , or neuroendocrine carcinoma ; ( 4 ) follow - up duration less than 3 months ; ( 5 ) relapse within 1 month of surgical resection ; ( 6 ) adjuvant therapy with radiotherapy alone ; or ( 7 ) unknown preoperative serum carbohydrate antigen 19 - 9 ( ca 19 - 9 ) level . in patients treated with adjuvant chemotherapy alone , fluoropyrimidine - based chemotherapy , including 5-fluorouracil / leucovorin , doxifluridine , tegafur / uracil , or s-1 , was performed . , relapse and survival ) , were collected by medical chart review . relapse - free survival ( rfs ) was defined as the time from the date of surgery to the date of either the first relapse at any site or death . we hypothesized that patients with advanced disease may be more likely to be treated with adjuvant therapy at the discretion of the clinician . we therefore performed psm to reduce the selection bias between patients treated with adjuvant therapy and those managed with surveillance only using the matchit package in r statistical software ver . with the propensity score estimated , 84 pairs of patients in the adjuvant therapy group and the surveillance - only group were matched using a 1:1 nearest neighbor matching algorithm . of the 363 patients included in the study , 84 patients were treated with adjuvant therapy ( chemotherapy , n=39 ; ccrt , n=45 ) and 279 were managed with surveillance only after surgical resection . in the unmatched cohort , the distributions of age , sex , and histological type were not significantly different between the two groups . however , the proportions of patients with stage iii disease ( 51.2% vs. 14.7% , p < 0.001 ) , node - positive disease ( 46.4% vs. 12.2% , p < 0.001 ) , and elevated ca 19 - 9 levels ( 29.8% vs. 18.6% , p=0.029 ) were higher in the adjuvant therapy group than in the surveillance - only group . there were no significant differences in the baseline characteristics between the two treatment groups in the matched cohort ( table 1 ) . thirty - seven ( 44.0% ) and 143 ( 51.3% ) patients were followed up for less than 5 years in the adjuvant therapy and surveillance only groups , respectively . follow - up was lost in 17 patients ( 20.2% ) in the adjuvant therapy group and in 46 patients ( 16.5% ) in the surveillance only group . of the 363 total patients , the 5-year rfs and os rates were 68.9% and 76.3% , respectively . in comparison of treatment outcomes between the two treatment groups , the adjuvant therapy group showed a shorter rfs compared with the surveillance - only group ( 3- and 5-year rfs rates , adjuvant therapy 58.0% and 50.8% vs. surveillance only 78.0% and 74.8% ; p < 0.001 ) ( fig . os also tended to be worse in the adjuvant therapy group than in the surveillance - only group ( 3- and 5-year os rates , adjuvant therapy 79.9% and 66.2% vs. surveillance only 85.5% and 79.5% ; p=0.089 ) ( fig . thirty - seven ( 44.0% ) and 44 ( 52.4% ) patients were followed up for less than 5 years in the adjuvant therapy and surveillance only groups , respectively . follow - up was lost in 17 patients ( 20.2% ) in the adjuvant therapy group and 15 patients ( 17.9% ) in the surveillance only group . in this cohort , rfs was not significantly different between the two treatment groups ( 3- and 5-year rfs rates , adjuvant therapy 58.0% and 50.8% vs. surveillance only 67.1% and 64.8% ; p=0.319 ) ( fig . there was also no significant difference in os between the two treatment groups ( 3- and 5-year os rates , adjuvant therapy 79.9% and 66.2% vs. surveillance only 74.8% and 70.4% ; p=0.703 ) ( fig . none of the variables , including age , sex , histological type , tnm stage , nodal status , and preoperative serum ca 19 - 9 level , were associated with the efficacy of adjuvant therapy ( fig . although there was a tendency in favor of adjuvant therapy in elderly patients , this result was biased by a higher proportion of stage iii disease in the surveillance - only group compared with the adjuvant therapy group ( 54.0% vs. 36.8% , p=0.110 ) among elderly patients . adjuvant therapy did not affect the prognosis of this cohort in the multivariate analysis ( table 2 ) . next , we evaluated the prognosis according to treatment modality among 84 patients treated with adjuvant therapy . in addition , patients treated with ccrt were more likely to have poor prognostic characteristics compared to those treated with chemotherapy , as follows : histological type other than well - differentiated grade ( 82.2% vs. 41.0% , p < 0.001 ) , stage iii disease ( 80.0% vs. 17.9% , p < 0.001 ) , and a higher preoperative serum ca 19 - 9 level ( 40.0% vs. 17.9% , p=0.027 ) ( table 3 ) . of the 363 patients included in the study , 84 patients were treated with adjuvant therapy ( chemotherapy , n=39 ; ccrt , n=45 ) and 279 were managed with surveillance only after surgical resection . in the unmatched cohort , the distributions of age , sex , and histological type were not significantly different between the two groups . however , the proportions of patients with stage iii disease ( 51.2% vs. 14.7% , p < 0.001 ) , node - positive disease ( 46.4% vs. 12.2% , p < 0.001 ) , and elevated ca 19 - 9 levels ( 29.8% vs. 18.6% , p=0.029 ) were higher in the adjuvant therapy group than in the surveillance - only group . there were no significant differences in the baseline characteristics between the two treatment groups in the matched cohort ( table 1 ) . in the unmatched cohort , the median follow - up durations were 60.4 and 53.4 months in all patients and in surviving patients at the time of analysis , respectively ( range , 5.3 to 177.6 months in both groups ) . thirty - seven ( 44.0% ) and 143 ( 51.3% ) patients were followed up for less than 5 years in the adjuvant therapy and surveillance only groups , respectively . follow - up was lost in 17 patients ( 20.2% ) in the adjuvant therapy group and in 46 patients ( 16.5% ) in the surveillance only group . of the 363 total patients , the 5-year rfs and os rates were 68.9% and 76.3% , respectively . in comparison of treatment outcomes between the two treatment groups , the adjuvant therapy group showed a shorter rfs compared with the surveillance - only group ( 3- and 5-year rfs rates , adjuvant therapy 58.0% and 50.8% vs. surveillance only 78.0% and 74.8% ; p < 0.001 ) ( fig . os also tended to be worse in the adjuvant therapy group than in the surveillance - only group ( 3- and 5-year os rates , adjuvant therapy 79.9% and 66.2% vs. surveillance only 85.5% and 79.5% ; p=0.089 ) ( fig . thirty - seven ( 44.0% ) and 44 ( 52.4% ) patients were followed up for less than 5 years in the adjuvant therapy and surveillance only groups , respectively . follow - up was lost in 17 patients ( 20.2% ) in the adjuvant therapy group and 15 patients ( 17.9% ) in the surveillance only group . in this cohort , rfs was not significantly different between the two treatment groups ( 3- and 5-year rfs rates , adjuvant therapy 58.0% and 50.8% vs. surveillance only 67.1% and 64.8% ; p=0.319 ) ( fig . there was also no significant difference in os between the two treatment groups ( 3- and 5-year os rates , adjuvant therapy 79.9% and 66.2% vs. surveillance only 74.8% and 70.4% ; p=0.703 ) ( fig . none of the variables , including age , sex , histological type , tnm stage , nodal status , and preoperative serum ca 19 - 9 level , were associated with the efficacy of adjuvant therapy ( fig . although there was a tendency in favor of adjuvant therapy in elderly patients , this result was biased by a higher proportion of stage iii disease in the surveillance - only group compared with the adjuvant therapy group ( 54.0% vs. 36.8% , p=0.110 ) among elderly patients . next , we evaluated the prognosis according to treatment modality among 84 patients treated with adjuvant therapy . patients treated with chemotherapy had better rfs than those treated with ccrt ( 3- and 5-year rfs rates , chemotherapy 70.3% and 57.0% vs. ccrt 47.3% and 47.3% ; p=0.027 ) ( fig . in addition , patients treated with ccrt were more likely to have poor prognostic characteristics compared to those treated with chemotherapy , as follows : histological type other than well - differentiated grade ( 82.2% vs. 41.0% , p < 0.001 ) , stage iii disease ( 80.0% vs. 17.9% , p < 0.001 ) , and a higher preoperative serum ca 19 - 9 level ( 40.0% vs. 17.9% , p=0.027 ) ( table 3 ) . upon initiating this study , we assessed which patients were likely to be treated with adjuvant therapy after r0 resection for gbc in clinical practice . patients with characteristics indicating high tumor burden , such as more advanced tnm stage , node - positive disease , and a higher preoperative serum ca 19 - 9 level , were more frequently treated with adjuvant therapy compared to those with a low tumor burden . however , adjuvant therapy failed to improve the prognosis of patients with a high tumor burden , resulting in shorter rfs and os rates compared with the prognosis of patients managed with surveillance only . however , even after the characteristics of patients were balanced via psm , there was no difference in survival , regardless of the postoperative treatment strategy . previous studies on adjuvant therapy in gbc patients who have undergone r0 resection are summarized in table 5 . in accordance with the results of our study , a few studies have suggested that adjuvant therapy is ineffective in gbc patients following r0 resection . a study of 279 gbc patients who had undergone surgery ( r0 resection in 164 patients ) found that neither adjuvant chemotherapy nor radiotherapy improved the os of patients following r0 resection . a subgroup analysis of 51 gbc patients who underwent curative resections in a phase iii trial showed that they did not benefit from adjuvant chemotherapy . the strengths of our study compared with previous studies are that the role of adjuvant therapy was assessed in selected patients ( r0 resection , no stage iv disease ) and the inevitable selection bias caused by the retrospective design was minimized by psm . in addition , to the best of our knowledge , our study included the largest number of cases among case - control studies of adjuvant therapy in gbc patients following r0 resection . two previous retrospective studies in gbc patients who had undergone r0 resection yielded opposite results from those obtained in our study . reported that adjuvant chemoradiotherapy reduced the risk of death by 70% compared with surgery alone in stage i - ii gbc patients who had undergone r0 resection . however , the study had a small sample size ( n=73 ) , and the prognosis of the surgery - only group was relatively worse compared with those reported in other studies . for example , in a large japanese study of 4,770 gbc patients , the 5-year survival rates among the patients in the surgery - only group with stage i and ii disease were 78% and 65% , respectively . similarly , in our study , the 5-year survival rate was 79.5% among the patients in the surgery - only group , 85.3% of whom had stage i or ii disease before psm . , the 5-year survival rate in the surgery - only group was approximately 40% , even though 80% of the patients had stage i disease . this result indicates that the survival difference between patients who underwent surgery alone and those who received adjuvant therapy observed in the study by gold et al . furthermore , in a previous western study of over 10,000 cases of gbc based on data from the national cancer database ( ncdb ) , the 5-year survival rates were only 50% and 29% among patients with stage i and ii disease , respectively , and these trends were identical in the subset of 7,462 patients who underwent potentially curative surgical resection . however , the data are difficult to interpret because the sample size was too small and there were few end - point events . based on both the lack of strong evidence supporting adjuvant therapy and on our findings , we recommend against the routine use of adjuvant therapy in stage i - iii gbc patients who have undergone r0 resection , particularly in asian patients . in this study , however , there was no difference in survival between patients treated with chemotherapy and those treated with ccrt . in addition , all patients enrolled in the study were treated with a fluoropyrimidine - based regimen , mainly due to the national insurance coverage policy in korea , although the specific dosage and the schedule of chemotherapy and radiotherapy varied . however , based on the two previous meta - analyses showing that patients receiving adjuvant chemotherapy or chemoradiotherapy derived greater benefit than those receiving radiotherapy alone , radiotherapy alone may not be recommended as the best option for adjuvant therapy in gbc patients . therefore , the association between the tumor burden and the efficacy of adjuvant therapy after r0 resection may not be confirmed in this study . 3 ) favoring adjuvant therapy in patients with higher tumor stage and node - positive disease , further studies are warranted in these groups with r0 resection . this study suggests that fluoropyrimidine - based adjuvant chemotherapy and ccrt are not indicated in stage i - iii gbc patients who have undergone r0 resection . gemcitabine - based adjuvant therapy after curative resection recently showed promising results in patients with biliary tract cancer [ 10,19 - 22 ] . therefore , prospective , randomized studies of therapeutic strategies other than a fluoropyrimidine - based regimen are warranted to improve the prognosis of gbc patients following r0 resection .

however , the incidence of gbc is relatively high in korea , where it accounts for 6.3 per 100,000 individuals and is the sixth leading cause of cancer - related death . the prognosis of gbc is poor , with a 5-year overall survival ( os ) rate of 17% to 45% , even in patients treated with curative intent surgery [ 3 - 5 ] . advanced stage , high - grade histological differentiation , and a positive resection margin are known to be poor prognostic factors in gbc patients who have undergone surgical resection . in addition , unlike cholangiocarcinoma , due to the limited clinical data available , the guidelines for gbc do not provide information on treatment strategy according to known prognostic factors , such as resection margin status . reported that gbc patients who have undergone non - curative resections may derive a slight benefit from systemic chemotherapy , but this was not observed in patients who underwent curative surgery in a subgroup analysis of the study , which combined patients with pancreatic , bile duct , and gallbladder cancers . a few studies reported that , compared with surgery alone , adjuvant chemoradiotherapy resulted in improved survival in gbc patients following r0 resection . however , due to the potential selection bias caused by unbalanced clinical characteristics , which is an inherent drawback of a retrospective study , these two meta - analyses can not confirm the ineffectiveness of adjuvant therapy in gbc patients who have undergone r0 resection . therefore , we attempted to determine whether adjuvant therapy is necessary for non - metastatic gbc patients who have undergone r0 resection through a multicenter , case - control study with propensity score matching ( psm ) to reduce the selection bias . of the 441 patients who were initially screened , those in the following categories 1 ) : ( 1 ) stage iv disease ; ( 2 ) unknown histological subtype ; ( 3 ) minor histological subtype such as adenosquamous , mucinous , mucosal , or neuroendocrine carcinoma ; ( 4 ) follow - up duration less than 3 months ; ( 5 ) relapse within 1 month of surgical resection ; ( 6 ) adjuvant therapy with radiotherapy alone ; or ( 7 ) unknown preoperative serum carbohydrate antigen 19 - 9 ( ca 19 - 9 ) level . in patients treated with adjuvant chemotherapy alone , fluoropyrimidine - based chemotherapy , including 5-fluorouracil / leucovorin , doxifluridine , tegafur / uracil , or s-1 , was performed . clinical data , including demographics , histological type , tnm stage based on the seventh edition of the american joint committee on cancer , preoperative serum level of ca 19 - 9 , treatment modality , and clinical outcomes ( e.g. relapse - free survival ( rfs ) was defined as the time from the date of surgery to the date of either the first relapse at any site or death . we hypothesized that patients with advanced disease may be more likely to be treated with adjuvant therapy at the discretion of the clinician . the propensity score was calculated from a logistic regression model that included age ( years ) , sex , histological type , tnm stage , and preoperative serum ca 19 - 9 level ( normal or elevated ) . with the propensity score estimated , 84 pairs of patients in the adjuvant therapy group and the surveillance - only group were matched using a 1:1 nearest neighbor matching algorithm . of the 441 patients who were initially screened , those in the following categories 1 ) : ( 1 ) stage iv disease ; ( 2 ) unknown histological subtype ; ( 3 ) minor histological subtype such as adenosquamous , mucinous , mucosal , or neuroendocrine carcinoma ; ( 4 ) follow - up duration less than 3 months ; ( 5 ) relapse within 1 month of surgical resection ; ( 6 ) adjuvant therapy with radiotherapy alone ; or ( 7 ) unknown preoperative serum carbohydrate antigen 19 - 9 ( ca 19 - 9 ) level . in patients treated with adjuvant chemotherapy alone , fluoropyrimidine - based chemotherapy , including 5-fluorouracil / leucovorin , doxifluridine , tegafur / uracil , or s-1 , was performed . clinical data , including demographics , histological type , tnm stage based on the seventh edition of the american joint committee on cancer , preoperative serum level of ca 19 - 9 , treatment modality , and clinical outcomes ( e.g. relapse - free survival ( rfs ) was defined as the time from the date of surgery to the date of either the first relapse at any site or death . we hypothesized that patients with advanced disease may be more likely to be treated with adjuvant therapy at the discretion of the clinician . the propensity score was calculated from a logistic regression model that included age ( years ) , sex , histological type , tnm stage , and preoperative serum ca 19 - 9 level ( normal or elevated ) . with the propensity score estimated , 84 pairs of patients in the adjuvant therapy group and the surveillance - only group were matched using a 1:1 nearest neighbor matching algorithm . of the 363 patients included in the study , 84 patients were treated with adjuvant therapy ( chemotherapy , n=39 ; ccrt , n=45 ) and 279 were managed with surveillance only after surgical resection . in the unmatched cohort , the distributions of age , sex , and histological type were not significantly different between the two groups . however , the proportions of patients with stage iii disease ( 51.2% vs. 14.7% , p < 0.001 ) , node - positive disease ( 46.4% vs. 12.2% , p < 0.001 ) , and elevated ca 19 - 9 levels ( 29.8% vs. 18.6% , p=0.029 ) were higher in the adjuvant therapy group than in the surveillance - only group . there were no significant differences in the baseline characteristics between the two treatment groups in the matched cohort ( table 1 ) . in the unmatched cohort , the median follow - up durations were 60.4 and 53.4 months in all patients and in surviving patients at the time of analysis , respectively ( range , 5.3 to 177.6 months in both groups ) . thirty - seven ( 44.0% ) and 143 ( 51.3% ) patients were followed up for less than 5 years in the adjuvant therapy and surveillance only groups , respectively . follow - up was lost in 17 patients ( 20.2% ) in the adjuvant therapy group and in 46 patients ( 16.5% ) in the surveillance only group . in comparison of treatment outcomes between the two treatment groups , the adjuvant therapy group showed a shorter rfs compared with the surveillance - only group ( 3- and 5-year rfs rates , adjuvant therapy 58.0% and 50.8% vs. surveillance only 78.0% and 74.8% ; p < 0.001 ) ( fig . os also tended to be worse in the adjuvant therapy group than in the surveillance - only group ( 3- and 5-year os rates , adjuvant therapy 79.9% and 66.2% vs. surveillance only 85.5% and 79.5% ; p=0.089 ) ( fig . the median follow - up durations were 62.1 and 53.4 months in all patients and in surviving patients at the time of analysis , respectively ( range , 4.9 to 177.1 months in both groups ) . thirty - seven ( 44.0% ) and 44 ( 52.4% ) patients were followed up for less than 5 years in the adjuvant therapy and surveillance only groups , respectively . follow - up was lost in 17 patients ( 20.2% ) in the adjuvant therapy group and 15 patients ( 17.9% ) in the surveillance only group . in this cohort , rfs was not significantly different between the two treatment groups ( 3- and 5-year rfs rates , adjuvant therapy 58.0% and 50.8% vs. surveillance only 67.1% and 64.8% ; p=0.319 ) ( fig . there was also no significant difference in os between the two treatment groups ( 3- and 5-year os rates , adjuvant therapy 79.9% and 66.2% vs. surveillance only 74.8% and 70.4% ; p=0.703 ) ( fig . none of the variables , including age , sex , histological type , tnm stage , nodal status , and preoperative serum ca 19 - 9 level , were associated with the efficacy of adjuvant therapy ( fig . although there was a tendency in favor of adjuvant therapy in elderly patients , this result was biased by a higher proportion of stage iii disease in the surveillance - only group compared with the adjuvant therapy group ( 54.0% vs. 36.8% , p=0.110 ) among elderly patients . in the matched cohort , advanced tnm stage , higher preoperative serum ca 19 - 9 level , and histological type other than well - differentiated grade disease were potential poor prognostic factors for os in the univariate analysis . next , we evaluated the prognosis according to treatment modality among 84 patients treated with adjuvant therapy . patients treated with chemotherapy had better rfs than those treated with ccrt ( 3- and 5-year rfs rates , chemotherapy 70.3% and 57.0% vs. ccrt 47.3% and 47.3% ; p=0.027 ) ( fig . however , os was not significantly different between patients treated with chemotherapy and those treated with ccrt ( 3- and 5-year os rates , chemotherapy 87.7% and 71.0% vs. ccrt 72.4% and 62.0% ; p=0.203 ) ( fig . in addition , patients treated with ccrt were more likely to have poor prognostic characteristics compared to those treated with chemotherapy , as follows : histological type other than well - differentiated grade ( 82.2% vs. 41.0% , p < 0.001 ) , stage iii disease ( 80.0% vs. 17.9% , p < 0.001 ) , and a higher preoperative serum ca 19 - 9 level ( 40.0% vs. 17.9% , p=0.027 ) ( table 3 ) . in a multivariate analysis adjusted for these poor prognostic markers , ccrt was not associated with worse rfs ( hr , 1.501 ; 95% ci , 0.685 to 3.286 ; p=0.310 ) as well as os ( hr , 1.062 ; 95% ci , 0.367 to 3.075 ; p=0.912 ) compared with chemotherapy ( table 4 ) . of the 363 patients included in the study , 84 patients were treated with adjuvant therapy ( chemotherapy , n=39 ; ccrt , n=45 ) and 279 were managed with surveillance only after surgical resection . however , the proportions of patients with stage iii disease ( 51.2% vs. 14.7% , p < 0.001 ) , node - positive disease ( 46.4% vs. 12.2% , p < 0.001 ) , and elevated ca 19 - 9 levels ( 29.8% vs. 18.6% , p=0.029 ) were higher in the adjuvant therapy group than in the surveillance - only group . in the unmatched cohort , the median follow - up durations were 60.4 and 53.4 months in all patients and in surviving patients at the time of analysis , respectively ( range , 5.3 to 177.6 months in both groups ) . follow - up was lost in 17 patients ( 20.2% ) in the adjuvant therapy group and in 46 patients ( 16.5% ) in the surveillance only group . in comparison of treatment outcomes between the two treatment groups , the adjuvant therapy group showed a shorter rfs compared with the surveillance - only group ( 3- and 5-year rfs rates , adjuvant therapy 58.0% and 50.8% vs. surveillance only 78.0% and 74.8% ; p < 0.001 ) ( fig . os also tended to be worse in the adjuvant therapy group than in the surveillance - only group ( 3- and 5-year os rates , adjuvant therapy 79.9% and 66.2% vs. surveillance only 85.5% and 79.5% ; p=0.089 ) ( fig . the median follow - up durations were 62.1 and 53.4 months in all patients and in surviving patients at the time of analysis , respectively ( range , 4.9 to 177.1 months in both groups ) . thirty - seven ( 44.0% ) and 44 ( 52.4% ) patients were followed up for less than 5 years in the adjuvant therapy and surveillance only groups , respectively . follow - up was lost in 17 patients ( 20.2% ) in the adjuvant therapy group and 15 patients ( 17.9% ) in the surveillance only group . in this cohort , rfs was not significantly different between the two treatment groups ( 3- and 5-year rfs rates , adjuvant therapy 58.0% and 50.8% vs. surveillance only 67.1% and 64.8% ; p=0.319 ) ( fig . there was also no significant difference in os between the two treatment groups ( 3- and 5-year os rates , adjuvant therapy 79.9% and 66.2% vs. surveillance only 74.8% and 70.4% ; p=0.703 ) ( fig . none of the variables , including age , sex , histological type , tnm stage , nodal status , and preoperative serum ca 19 - 9 level , were associated with the efficacy of adjuvant therapy ( fig . although there was a tendency in favor of adjuvant therapy in elderly patients , this result was biased by a higher proportion of stage iii disease in the surveillance - only group compared with the adjuvant therapy group ( 54.0% vs. 36.8% , p=0.110 ) among elderly patients . in the matched cohort , advanced tnm stage , higher preoperative serum ca 19 - 9 level , and histological type other than well - differentiated grade disease were potential poor prognostic factors for os in the univariate analysis . patients treated with chemotherapy had better rfs than those treated with ccrt ( 3- and 5-year rfs rates , chemotherapy 70.3% and 57.0% vs. ccrt 47.3% and 47.3% ; p=0.027 ) ( fig . however , os was not significantly different between patients treated with chemotherapy and those treated with ccrt ( 3- and 5-year os rates , chemotherapy 87.7% and 71.0% vs. ccrt 72.4% and 62.0% ; p=0.203 ) ( fig . in addition , patients treated with ccrt were more likely to have poor prognostic characteristics compared to those treated with chemotherapy , as follows : histological type other than well - differentiated grade ( 82.2% vs. 41.0% , p < 0.001 ) , stage iii disease ( 80.0% vs. 17.9% , p < 0.001 ) , and a higher preoperative serum ca 19 - 9 level ( 40.0% vs. 17.9% , p=0.027 ) ( table 3 ) . in a multivariate analysis adjusted for these poor prognostic markers , ccrt was not associated with worse rfs ( hr , 1.501 ; 95% ci , 0.685 to 3.286 ; p=0.310 ) as well as os ( hr , 1.062 ; 95% ci , 0.367 to 3.075 ; p=0.912 ) compared with chemotherapy ( table 4 ) . upon initiating this study , we assessed which patients were likely to be treated with adjuvant therapy after r0 resection for gbc in clinical practice . patients with characteristics indicating high tumor burden , such as more advanced tnm stage , node - positive disease , and a higher preoperative serum ca 19 - 9 level , were more frequently treated with adjuvant therapy compared to those with a low tumor burden . however , adjuvant therapy failed to improve the prognosis of patients with a high tumor burden , resulting in shorter rfs and os rates compared with the prognosis of patients managed with surveillance only . this paradoxical trend is similar to the results of a large , non - randomized japanese study in which , after surgical resection with or without a negative surgical margin , the prognosis of patients who received adjuvant chemotherapy was worse than that of patients who did not receive adjuvant chemotherapy . however , even after the characteristics of patients were balanced via psm , there was no difference in survival , regardless of the postoperative treatment strategy . the strengths of our study compared with previous studies are that the role of adjuvant therapy was assessed in selected patients ( r0 resection , no stage iv disease ) and the inevitable selection bias caused by the retrospective design was minimized by psm . in addition , to the best of our knowledge , our study included the largest number of cases among case - control studies of adjuvant therapy in gbc patients following r0 resection . however , the study had a small sample size ( n=73 ) , and the prognosis of the surgery - only group was relatively worse compared with those reported in other studies . for example , in a large japanese study of 4,770 gbc patients , the 5-year survival rates among the patients in the surgery - only group with stage i and ii disease were 78% and 65% , respectively . similarly , in our study , the 5-year survival rate was 79.5% among the patients in the surgery - only group , 85.3% of whom had stage i or ii disease before psm . may be attributed to the poorer prognosis of the surgery - only group compared with those in both the japanese study as well as our own study . furthermore , in a previous western study of over 10,000 cases of gbc based on data from the national cancer database ( ncdb ) , the 5-year survival rates were only 50% and 29% among patients with stage i and ii disease , respectively , and these trends were identical in the subset of 7,462 patients who underwent potentially curative surgical resection . based on both the lack of strong evidence supporting adjuvant therapy and on our findings , we recommend against the routine use of adjuvant therapy in stage i - iii gbc patients who have undergone r0 resection , particularly in asian patients . in addition , all patients enrolled in the study were treated with a fluoropyrimidine - based regimen , mainly due to the national insurance coverage policy in korea , although the specific dosage and the schedule of chemotherapy and radiotherapy varied . however , based on the two previous meta - analyses showing that patients receiving adjuvant chemotherapy or chemoradiotherapy derived greater benefit than those receiving radiotherapy alone , radiotherapy alone may not be recommended as the best option for adjuvant therapy in gbc patients . therefore , the association between the tumor burden and the efficacy of adjuvant therapy after r0 resection may not be confirmed in this study .

non - alcoholic fatty liver disease ( nafld ) is now regarded as the most common liver condition in the developed world , affecting up 30% of the population . it is closely linked with metabolic syndrome and its component parts including obesity , type ii diabetes mellitus , and dyslipidemia . numerous pharmacological interventions have been tested for the treatment of nafld with varying success . nonetheless , no current pharmacological therapy is recommended for the treatment of this condition . much focus has been given to the role of lifestyle intervention in nafld , including the role of prescribed exercise regimes and diets . this review aims to summarize the literature surrounding this approach , highlighting the impact and role these strategies have on nafld . we searched a public domain database ( pubmed ) with the following categories : disease ( nafld , fatty liver , non - alcoholic steatohepatitis [ nash ] , and simple steatosis ) and intervention ( lifestyle intervention , diet , exercise , and behavior ) with each possible combination . from this list , papers were considered for further review if they were written in english and involved adult patients only . the heterogeneity of the literature reviewed enabled us to collate studies together under broad headings , dealing with each of the main lifestyle intervention options . however , this heterogeneity also highlighted some salient issues , in particular standardization of a nafld phenotype . this classification is not always feasible or possible . where appropriate we have highlighted the descriptors used in defining the nafld phenotype . furthermore , we have included studies of features of metabolic syndrome , especially where liver health was considered and reviewed . nafld is defined as fat accumulation in the liver , greater than 5%10% by weight , in the absence of excessive alcohol consumption.1 it is a spectrum disease , ranging from simple steatosis without inflammation , to nash , progressing onwards to fibrosis and cirrhosis.2 nafld is closely associated with obesity and type ii diabetes mellitus ( t2 dm ) ; indeed , 75% of patients with nafld are obese , and it is frequently seen as the hepatic manifestation of metabolic syndrome.3 the pathophysiology of nafld is not completely understood , but insulin resistance is thought to play a part with increased lipolysis leading to an influx in free fatty acid into the liver.2 the gold standard for diagnosis of nafld is liver biopsy ; this is the only way of truly differentiating between the different stages of nafld . biopsy is an imperfect gold standard and has issues pertaining to morbidity , mortality , as well as intra and inter observer variability . ultrasound ( us ) is used in most cases ; the disadvantage of this is that fat is only picked up when it reaches levels of 33%3 and us is a poor detector of inflammation . nafld was relatively unknown prior to the early 1980s , but is fast becoming the most common chronic liver disease worldwide,4 with an estimated prevalence of 20%30% in western countries.5 the british transplant guidance website has also reported that in 2008 and 2009 12% of the patients on the waiting list for liver transplantation had nash cirrhosis.6 this increase in cases of nafld is strongly linked to the rising rate in obesity ; the national health service ( nhs ) database shows the percentage population with obesity has almost doubled in the last 8 years , with now roughly a quarter of the population having a raised body mass index ( bmi ) . changing lifestyles is ultimately to blame for this , including increased access to cheap food , eating on the go , and marketing of high calorific foods.7 given all the above mentioned , there has been increased interest in nafld and possible ways of managing and treating it . there have been many different suggestions for pharmaceuticals that could be used , including metformin , vitamin e , urodeoxycholic acid , to name a few.1 as of yet none of these treatments have shown any significant disease improvement and none are currently recommended . this paper reviews lifestyle modifications as a way of improving the outcome of patients with nafld . in this section , we describe studies which have assessed a combination of , or a comparison between , diet and exercise based interventions . in one of the few studies of lifestyle intervention using histological endpoints , promrat et al8 conducted a small study ( n=30 ) of patients randomly assigned to receive either diet , exercise , and behavioral strategies or a control group ( 2:1 ratio ) . the follow up period was 2 years , and liver biopsies were performed pre and post study period . the lifestyle intervention group had significant improvements in histology at 48 weeks , with 67% improving their nafld activity score to 2 . there was improvement in hepatic steatosis , lobular inflammation , parenchymal inflammation , and ballooning injury in the interventional group ; but results showed no improvement in fibrosis in either group . liver enzyme and one of the interesting observations from this study was the low attrition rate ; 28 of the 30 recruits completed the study period , including follow up biopsy . biopsy results were also used in a study by jin et al.9 one hundred and twenty living donors with nafld who had undergone a biopsy and were then encouraged to lose 5% of their body weight , by diet and exercise , were re - biopsied . there were no specific guidelines for how the patients should lose their weight , which was over an average period of 10 weeks . over this period 92 lost weight ( on average 3 kg ) , 15 gained weight and the remainder maintained their weight . one hundred and three biopsies showed improvement , 82 of these were in those who had lost weight . further analysis also showed that these improvements were greatest in those who were younger and had the largest decrease in bmi and cholesterol . the authors concluded that a target decrease in body weight should be 5% and decrease in cholesterol should be 10% in order to achieve a significant improvement in liver health . histological end points were also sought in a paper from bhat et al10 assessing whether lifestyle modifications can improve nafld , although they were not able to biopsy their entire cohort . sixty patients were required to carry out 45 minutes of exercise five times a week as well as an energy restricted diet ; 60% carbohydrate , 20% fat , 20% protein . at the end of a 6 month period 45 of the 60 were considered compliant of the intervention . eight patients had repeat liver biopsies in the compliant group and of these , six showed improvement in steatosis and necro - inflammatory score , with no change in fibrosis . unfortunately there are no biopsy results available for the non - compliant group , indeed there is no record if there were any taken . insulin resistance and liver function tests ( lft)s were also shown to decrease in the compliant group . lazo et al11 recruited 96 patients with t2 dm and magnetic resonance imaging ( mri ) evidence of hepatic steatosis . patients were allocated to either the intervention group ( calorie restriction and exercise ) or control , with a target weight loss of 10% from baseline . calorie restriction of 1,2001,500 calories / day for those with a weight of less than 141 kg and 1,5001,800 calories / day for those weighing greater then 141 kg was encouraged . also , an increase in exercise to 175 minutes of moderate intensity exercise per week was required and participants attended weekly and monthly counseling sessions . results were not available on how many achieved a 10% weight loss , but average percentage weight loss for the interventional group was 8.3% and 0.03% in the control group . although no significant difference was observed in liver enzymes , follow up mri showed a significant reduction in steatosis following intervention , as well as between the intervention and control groups . follow up mri scanning was performed after 12 months of intervention . in a similar study , shah et al12 assessed the additive effect of an exercise program to calorie restriction , using mri to assess hepatic fat . over a 6 month period , patients were either allocated to a calorie restriction alone group , or a calorie restriction plus exercise group . calorie restriction was of 5001,000 calories / day with 30% being fat , 50% carbohydrates , and 20% protein . exercise requirements were 90 minutes three times a week in an indoor facility supervised by a personal trainer . both groups showed improvements in steatosis ( measured by mri ) , liver enzymes , and insulin resistance from baseline ; however , no significant difference was observed between the groups . the study authors suggest that no additive benefit of exercise was seen , however conceded that the small number of recruits is a limitation of the study . larson - meyer et al13 compared results from computerized tomography ( ct ) and mri to assess the outcome of calorie restriction and exercise on a group of 46 overweight patients . all the patients in this sample were healthy with no underlying liver disease at baseline , and were recruited after 6 months either randomized to calorie restriction alone ( 25% energy from baseline ) or in combination with exercise ( 12.5% calorie restriction and 12.5% increase in exercise ) or a low calorie diet ( 15% weight loss then maintenance ) or a control group . the results showed that bmi , insulin resistance , alanine aminotransferase ( alt ) levels , and intrahepatic steatosis decrease in all interventional groups ( alt did not decrease in the low calorie diet group ) . mri and ct results correlated closely , but the paper found that mri was more reliable at imaging lower levels of hepatic fat . this study , although showing interesting results when it comes to imaging , actually may not be relevant as not only did no patients have a diagnosis of nafld , but patients were encouraged to drink less than 2 units of alcohol per day , some of whom had been drinking more prior to the trial . two of the papers we reviewed used us as a measurement of hepatic steatosis improvement over the trial period . scagiloni et al14 carried out a small trial , with twelve participants over a period of 3 months . all participants were encouraged to carry out a diet that had been personally tailored by a dietician , and to increase their physical activity , which was monitored by an armband ( comparisons made at baseline and 3 months ) . exercise over the 3 month period did not show any significant increase , although there was an average weight loss and bmi decrease of 7.34% . lfts also did not show any significant improvement but us showed a significant decrease in intrahepatic fat . in contrast , catalano et al15 used a much larger sample group of 50 over a period of 6 months . all of these participants were encouraged to lose 5% of their body weight over the trial period , although no specific guidelines were given . at the end of the 6 months , the average weight loss of 57% of the desired weight loss ( 5% ) was achieved . results showed that both bmi and insulin resistance decrease over the trial period and this correlated to a decrease in intrahepatic fat on us . on reviewing blood results , this trial also came to the conclusion that gamma - glutamyl transpeptidase ( ggt ) levels are the most reliable for assessing parallel modification of serological measurement and morphological changes . blood tests are by far the easiest and most accessible way of assessing liver function and this is represented by the number of papers that rely wholly on these as evidence for their trials . six trials in this section used blood results , and not imaging and histology , as a measureable outcome for their trials . sun et al16 is one of the larger trials included in this review : 1,006 patients were recruited into a 12 month trial where they were randomized to receive lifestyle interventions ( nafld education including dietary advice and exercise therapy ) or to a control group . alt levels and other markers of metabolic syndrome decreased over the trial period in the lifestyle group ( compared to those in the control group ) , all to a greater extent at 12 months than when reviewed at 6 months . straznicky et al17 recruited 58 patients with three or more markers of metabolic syndrome , rather than being exclusively diagnosed with nafld . nafld is often described as the hepatic manifestation of metabolic syndrome therefore this paper was included . over a 12 week period , the 58 patients were randomized to either weight loss by calorie restriction ( deficit of 400900 calories per day ) , weight loss by calorie restriction and exercise ( 40 minute bike ride on alternate days ) , and a control group , a design that is similar to larson - meyer et al13 and shah et al.12 the calorie restriction alone group lost on average 7.6% with a corresponding decrease in alt of 20% and ggt of 28% . the calorie restriction and exercise group lost 9.1% of their baseline weight on average and alt and ggt levels decreased by 24% and 33% , respectively . these results showed a significant decrease from baseline but no significant difference between the two groups : they do not support the additive effect of exercise . sreenivasa baba et al18 published another paper using blood tests as its primary follow up . fifty - nine patients , with us diagnosed nash , were recruited into a 3-month trial involving an exercise program ( 45 minutes of exercise five times a week ) , and a calorie controlled diet for those with a high bmi ( 25 calories per kg of their ideal body weight ) . forty - four patients were compliant to this exercise and diet regime , and those taking part in diet and exercise lost on average 3.1 kg , while those taking part in just the diet lost 0.2 kg , and no significant decrease in weight was seen in the non - compliant group . alt and aspartate aminotransferase ( ast ) levels decreased significantly in the compliant group ; therefore , this paper draws the conclusion that weight loss is beneficial regardless of exercise participation . this paper included 30 patients who had biopsies at baseline but did not follow these up at the end of the trial . a study by park et al19 had a similar set up to sreenivasa baba et al,18 though it was a much smaller trial . twenty - five obese adults with us diagnosed nafld were recruited for a 12 month trial . similar dietary advice of 2530 calories per kg of ideal body weight was prescribed , and generalized exercise and behavioral advice was given . unlike sreenivasa baba et al , participants in this trial were considered compliant if they showed a reduction in weight . again , alt and ast levels decreased in the compliant group , but in this trial , levels were shown to increase in the non - compliant patients who gained weight . kim et al20 used an internet program to encourage participants to improve their diet and increase their exercise . eighteen men were recruited for 8 weeks to use this program , which set individual goals and targets that could be monitored by a panel . at the end of the trial weight and some markers of metabolic syndrome were also shown to decrease , including diastolic blood pressure . although this paper does not mention nafld , as has been previously stated , nafld is considered the hepatic manifestation of metabolic syndrome , which is measured . this paper also shows a new innovative way of encouraging a population to lead a healthier lifestyle . the results of this paper therefore contradicts shah et al12 and straznicky et al,17 which stated that exercise did not play an important part in the improvement of liver steatosis markers , as in this trial exercise does seem to be an important factor . venditti et al21 used weight loss to assess whether lifestyle interventions were more effective the first or the second time around . the paper follows the diabetic prevention program22 in which participants had been randomized to either 16 sessions of lifestyle interventions , metformin , troflitazone ( which was withdrawn due to side effects ) , or placebo . after this initial trial , all participants were asked if they would continue on a further 6 months of lifestyle interventions and 2,808 agreed . those in the original lifestyle group were then compared to those who had been in the metformin and placebo groups . those who had not received lifestyle intervention before were more likely to join this secondary trial and to continue with it , compared to those on their secondary course . weight loss was therefore greater in the naive group and indeed correlated to the number of sessions attended in this group . although this paper does not mention nafld or liver function it shows the importance of lifestyle intervention leading to weight loss and the problems with compliance . this section includes the papers using diet as the only intervention ; seven papers in total fell under this category . ryan et al23 published a paper in 2006 , which was a post hoc analysis of 52 obese , insulin resistant adults who had taken part in a weight loss program . they had been randomized to receive either a low carbohydrate diet ( 40% carbohydrate/40% fat ) or a low fat diet ( 60% carbohydrate/25% fat ) for 16 weeks . both groups lost a significant amount of weight over the trial period but the alt levels decreased twice as much in those on the low carbohydrate diet compared to the low fat diet . alt levels were also found to decrease more in those with abnormal levels ( 30 units / l for men and 19 units / l for women ) at baseline than those with normal levels , irrespective of the diet type . insulin resistance levels were also shown to decrease in both groups with no significant differences between them . the authors draw the conclusion that low carbohydrate diets are more beneficial than low fat diets at reducing alt levels . this was again a post hoc analysis with data taken from a trial comparing a low calorie diet with topiramate ( an anti - epileptic drug ) or placebo . this paper focused on the low calorie diet group , and reviewed the follow up results from 32 and 60 weeks of 147 patients . there was a high drop out in this trial and as a result data were only available for 67 patients at 32 weeks and 68 patients at 60 weeks . on average , patients lost 12.1 kg , men losing more than women over the trial period . ast and alt levels were shown to decrease in men , while there was a transient increase in women . ggt decreased in men but not in woman and there was no significant change in alp in either group . these results are interesting , indicating that sex is a key determining factor in change in lft levels over a trial . de luis et al25 reports on a 3-month intervention of hypocaloric diet ( either low fat or low carbohydrate ) on basic anthropological and biochemical measures in an obese population . after 3 months of dietary intervention , all biochemical parameters improved , along with improvements in bmi and waist circumference . more so , the nafld phenotype ( n=30 ) showed improvements in alt and insulin resistance . although only small numbers were included in each group , and the nafld phenotype was loosely defined , the study reveals that even a short focused intervention ( 3 months ) has an impact on measurable markers associated with metabolic syndrome . rodrguez - hernndez et al26 again uses a low fat and low carbohydrate diet to assess their effect on aminotransferase , and this trial is nicely followed up by haufe et al.27 the rodrguez - hernndez et al26 trial included 54 women , with us diagnosed nafld , and randomized them to a low fat ( 25% protein , < 10% fat , 54% carbohydrate ) or low carbohydrate ( 27% protein , 28% fat , 45% carbohydrate ) diet for a period of 6 months . at the end of the trial , those on the low carbohydrate diet lost 5.7% of their body weight and those in the low fat group 5.5% , a non - significant result . alt and ast had both decreased in each group but again there was no significant difference . the study by haufe et al27 follows a similar design but with 102 patients , both male and female , over a 6-month period . in this trial , the diet design was low carbohydrate ( 90 g of carbohydrate and 0.8 g protein per kg weight , 30% fat ) and low fat ( 20% fat , 0.8 g of protein per kg , the remainder carbohydrate ) . the results were also similar in this trial : a non - significant decrease in weight and alt between both groups . this study went on to look at intrahepatic fat content by magnetic resonance spectroscopy and tomography . results showed that intrahepatic fat reduced by 47% in the low carbohydrate group and 42% in the low fat group , with no significant difference between the groups . this paper therefore neatly follows from rodrguez - hernndez et al , with the added evidence of imaging to support the results and a larger sample size . the small study by elias et al28 recruited 31 participants , with either ct or us diagnosed nafld , to carry out a hypocaloric diet ( reduction of 5001,000 calories / day ) for 6 months . seventeen patients were compliant with this diet and ended up losing 5% of their body weight ; with this , they also showed a significant decrease in ggt , alt , and insulin resistance . at the beginning of the trial , 45% of the patients had been diagnosed with metabolic syndrome and in the end there were only 10% , all in the non - adherent group . intrahepatic fat by ct was also shown to decrease in the adherent group compared to the non - adherent group . this was a small trial involving 16 participants who were required to carry out 3 weeks of over feeding followed by 6 months of a hypocaloric diet . subjects in this trial were also genotyped to assess the role of genetics in nafld . during the 3 week overfeeding liver fat , measured by mri , increased by 27% and then fell by 25% in the hypocaloric phase . this paper therefore concluded that the changes in liver fat during nafld are reversible by dieting . genetic markers of patatin - like phospholipase domain - containing protein 3 ( pnpla3)-148ii and pnpla3 - 148 mm were investigated in this study . those with pnpla3 - 148ii ( n=7 ) showed a significant increase in lipid fat content while this relationship was not seen in those with pnpla3 - 148 mm ( n=9 ) . the study hypothesized the reason behind this change but concluded that the sample size was too small to see a definite relationship . kistler et al30 produced the only paper in this section that used biopsy results as an outcome . the paper was a retrospective study looking at 813 biopsy results of patients with nafld and assessed if they were taking part in the recommended guidelines for exercise put forward by the united states department of health and human services . the guidelines stated that people should be taking part in more than 150 minutes of moderate intensity or 75 minutes of vigorous intensity exercise per week . participants in this trial were classified as inactive ; meeting the moderate intensity guidelines ; or meeting the vigorous intensity guidelines . results of a questionnaire showed that 20% of the participants met the moderate activity guidelines and 26% met the vigorous activity guidelines ; the majority of patients were inactive . analysis of the biopsies showed that meeting the vigorous activity guidelines decreases your odds ratio ( or ) of having nash ( or : 0.65 ) ; the same was not true for moderate activity . biopsies also showed that those meeting vigorous activity guidelines had significantly lower odds ratio fibrosis compared to no fibrosis ( or : 0.53 ) ; again , the same was not true for moderate activity . thus this paper concluded that only vigorous activity was enough to cause significant change in liver steatosis and fibrosis . hallsworth et al31 constructed a randomized control trial including 19 patients with mri diagnosed nafld . the patients were randomized to take part in 4560 minutes of resistance exercise three times a week for 8 weeks , or be in a control group . they were only included in the final results if they lost less than 5% of their body weight , therefore excluding the weight loss from affecting the results . participants were required to wear armbands 7 days prior to the intervention and for 7 days afterwards to assess the change . results showed that there was a 13% decrease in intrahepatic fat , recorded by mri , in the intervention group with no change in the control group . there was improvement in insulin resistance but no change in alt or blood lipid levels . similar to hallsworth et al,31 sullivan et al32 also recruited a small sample , 18 participants with nafld , to participate in a randomized control trial . although in this study , the intervention group was required to carry out 3060 minutes of exercise five times a week , one of these was under direct supervision . again , similarly , mri scans were used to determine the intrahepatic fat content , which showed a mild decrease in the intervention group compared to the control . the same was also true for alt values : the decrease in alt correlated with the decrease in intrahepatic fat in the intervention group . these trials have a similar design and they also both have a small sample size that may have led to bias . fealy et al33 carried out a small short lived trial to assess the effect of exercise training on markers of hepatocyte apoptosis . thirteen patients were recruited into the trial , where they were required to carry out 60 minutes of aerobic activity at 80%85% of their maximum heart rate for seven consecutive days . mri scans were performed on all participants pre and post - trial and these showed no change in intrahepatic fat during the intervention . blood tests did show some changes , those with raised alt at baseline had decreased levels after the trial , and blood glucose levels , and insulin sensitivity also showed improvements . this trial was very short lived which may explain the limited change in markers of nafld . the effect of a short prescribed program of endurance exercise was assessed for improvements in liver fat content in a cohort including lean and obese individuals by tarnopolsky et al.34 the study design precluded weight loss as an end point , instead focusing on participation in a moderate intensity program of endurance exercise ( stationary bike cycling ) and assessing its effect on liver fat content principally . after a 3-month period of endurance exercise , improvements in fitness ( maximal oxygen consumption ) and reduction in waist circumference were noted in both the lean and obese groups , regardless of sex . no change in bmi was seen . hepatic lipid content , as measured by ct ( liver attenuation ) , did not change post exercise , regardless of group or sex . this study is limited by the small numbers of patients included and by the apparent liver health of the group the obese men had a pre - exercise alt of 30.34.8 u / l and the obese women 16.82.2 no measurement of lipid profile was discussed ; early changes in triglyceride content may have supported and encouraged participation in a longer program . this study does however highlight the need for intervention programs designed to induce weight loss . this study was deliberately designed not to induce weight loss . with no effect on liver fat content , this supports the notion that loss of body weight is important for improving liver health in obese individuals . bae et al35 is one of the larger trials reviewed in this discussion , with a cross - sectional analysis taken on 72,359 korean adults . baseline results were collected from these patients at the kangbuk samsung hospital total healthcare centre , and the diagnosis of nafld was made by us . the participants were asked to fill out a questionnaire recording how much exercise they carried out on a weekly basis ; regular exercise was defined as 30 minutes more than three times a week for three consecutive months . there were 19,921 patients with nafld and this number was significantly higher in those who did not exercise regularly . the same was true for bmi : nafld occurrence was higher with those with a greater bmi and liver function levels were lower on average if the patient took part in regular exercise . both have a similar design with a low intensity lifestyle intervention group ( three counseling sessions throughout the trial ) , moderate intensity lifestyle intervention group ( six counseling sessions throughout the trial ) , a long term group ( receiving the same initial intervention as the moderate group but with further sessions up to 12 months ) , and a control group . all participants were encouraged to carry out the same diet with a reduction in energy intake of 17002400 kj over a 3 month period and to increase their exercise to 150200 minutes a week . the st george et al study that was published in october 200837 included 152 participants followed up for a 9-month period . exercise was shown to be significantly increased in both the low and moderate intensity exercise encouragement groups , with no change in the control group . weight loss was found to be highest in those receiving moderate intensity lifestyle interventions and lowest in the control group ; liver function tests were also found to correlate with weight loss . the moderate intensity group was also shown to have decreased markers of metabolic risk factors at the end of the trial period . the study by st george et al published in 200936 categorized 141 patients into whether they increased their physical activity by more than 60 minutes per week , maintained their physical activity , or decreased their physical activity . in the moderate intensity encouragement group 64% and 63% from the low intensity encouragement group increased their activity from base line . this was a significant difference from the control group with only 16% increasing their physical exercise . the greatest changes were in insulin resistance , metabolic risk factors , and lfts seen in those who increased their physical activity . gerber et al38 carried out a cross - sectional analysis of a population using data from two surveys carried out between 20032004 and 20052006 in the united states . the data from these surveys were mostly self - reported , collecting baseline characteristics , lifestyle choices , and medical backgrounds of the subjects . diagnosis of nafld was made using the fatty liver index,39 a simple scoring system that predicts the degree of hepatosteatosis using the baseline characteristics of the subjects . patients were asked to wear activity monitor arm bands , similar to the ones used in hallsworth et al,31 for seven consecutive days . those with nafld were found to be less likely to take part in regular exercise , and having diabetes as well , made this relationship stronger . results also showed that exercise did not correlate with aminotransferase levels . although this paper had a large sample size and an accurate way of recording exercise in participants , the trial period was very short , which may not reflect a true representation of the participants exercise levels . there are many theories about various medications that may improve the outcome of nafld ; this review paper includes three trials using a variety of medications . twenty - three women were recruited by mndez - snchez et al,40 for a double blinded control trial testing the effects of ursodeoxycholic acid on patients with nafld . patients were randomized to receive a healthy diet ( 1200 kcal / day of which 20% was fat , 60% carbohydrate , and 20% protein ) with either ursodeoxycholic acid or placebo . both groups lost weight over the trial with no significant difference between the two groups . alt decreased more in those taking ursodeoxycholic acid compared to those who were not , but other than that there was no significantly different blood results between the two groups . us was performed on the patients at the beginning and end of the trial , and fat liver content was shown to decrease in both groups but again no significant difference between the two groups was found . viusid ( catalysis , s.l . , madrid , spain ) , an antioxidant nutritional supplement , was investigated by vilar gomez et al41 in a randomized control trial . sixty patients , with biopsy proven nafld , were randomized to receive a hypocaloric diet and exercise with or without viusid . monitoring of the diet and exercise was done by self - reporting and biopsies were taken at baseline and at the end of the 24 week trial , which were examined by a pathologist . there were eleven dropouts over the trial for various reasons , and viusid was reported to have side effects including nausea and diarrhea in some cases . alt and ast decreased significantly over the trial with no significant difference between the two trial groups . the viusid group did show a significant improvement in liver histology , and improvements in fibrosis and steatosis , as did the control group . between group comparison showed significant improvements in the viusid group in all parameters of the nas score ( but not the fibrosis grade ) compared to the control . there was a relatively high dropout rate in this trial , and those who dropped out were not accurately followed up . the reason behind these dropouts were not reported ; these would have been interesting as they may have impacted on viusid s use in a clinical setting . krakoff et al42 used data from the diabetic prevention program , similar to venditti et al.21 venditti et al focused on the placebo group and followed them up after the trial , whereas krakoff et al compared a metformin group to a placebo group . in krakoff et al both groups received diet and exercise programs to induce weight loss , and the 2,153 had also been randomized to receive either placebo or metformin for an average trial period of 3.2 years . alt levels rose in both groups over the trial , although the rise was greatest in the placebo group . the study also showed that the incidence of abnormal alt decreased with increasing amount of weight loss . all participants in this trial had normal alt at baseline and nafld status was not known , which may explain why these results do not follow the same trends as other papers . akcam et al43 performed a small randomized double blind parallel group clinical trial assessing the effect of diet and exercise alone or in combination with either metformin or vitamin e on various markers of nafld , including ultrasonographically assessed liver steatosis , insulin resistance , lipid profile , and bmi . they recruited 67 adolescents with us defined nafld to three groups all of whom received personalized and prescribed diet and exercise regimes . one group also received metformin ( 850 mg once daily ) while another group received vitamin e ( 400 u daily ) . bmi improved in all groups , more so in the metformin and vitamin e groups , although this did not achieve statistical significance . unsurprisingly , insulin resistance was most improved in the metformin treated group , with only slight improvement seen in the other two groups . no direct markers of oxidative stress improvement were measured , although tumor necrosis factor ( tnf)-alpha did not improve significantly in any of the three groups . at the conclusion of the study period , repeat liver us was performed , with the metformin group showing the greatest improvement almost double that of the diet and exercise alone group and the vitamin e group . this well designed study compared pharmacological intervention with either metformin or vitamin e with diet and exercise alone . the short duration of the study is a limitation ; perhaps greater effects from all three interventions would be more apparent if the study period was extended . however , although the authors highlight the improvements seen with metformin in particular , the diet and exercise alone group did achieve an improvement in bmi supporting the role of this fundamental intervention in a young cohort . in this section , we describe studies which have assessed a combination of , or a comparison between , diet and exercise based interventions . in one of the few studies of lifestyle intervention using histological endpoints , promrat et al8 conducted a small study ( n=30 ) of patients randomly assigned to receive either diet , exercise , and behavioral strategies or a control group ( 2:1 ratio ) . the follow up period was 2 years , and liver biopsies were performed pre and post study period . the lifestyle intervention group had significant improvements in histology at 48 weeks , with 67% improving their nafld activity score to 2 . there was improvement in hepatic steatosis , lobular inflammation , parenchymal inflammation , and ballooning injury in the interventional group ; but results showed no improvement in fibrosis in either group . liver enzyme and weight loss were also significantly improved in the intervention group . one of the interesting observations from this study was the low attrition rate ; 28 of the 30 recruits completed the study period , including follow up biopsy . biopsy results were also used in a study by jin et al.9 one hundred and twenty living donors with nafld who had undergone a biopsy and were then encouraged to lose 5% of their body weight , by diet and exercise , were re - biopsied . there were no specific guidelines for how the patients should lose their weight , which was over an average period of 10 weeks . over this period 92 lost weight ( on average 3 kg ) , 15 gained weight and the remainder maintained their weight . one hundred and three biopsies showed improvement , 82 of these were in those who had lost weight . further analysis also showed that these improvements were greatest in those who were younger and had the largest decrease in bmi and cholesterol . the authors concluded that a target decrease in body weight should be 5% and decrease in cholesterol should be 10% in order to achieve a significant improvement in liver health . histological end points were also sought in a paper from bhat et al10 assessing whether lifestyle modifications can improve nafld , although they were not able to biopsy their entire cohort . sixty patients were required to carry out 45 minutes of exercise five times a week as well as an energy restricted diet ; 60% carbohydrate , 20% fat , 20% protein . at the end of a 6 month period 45 of the 60 eight patients had repeat liver biopsies in the compliant group and of these , six showed improvement in steatosis and necro - inflammatory score , with no change in fibrosis . unfortunately there are no biopsy results available for the non - compliant group , indeed there is no record if there were any taken . insulin resistance and liver function tests ( lft)s were also shown to decrease in the compliant group . lazo et al11 recruited 96 patients with t2 dm and magnetic resonance imaging ( mri ) evidence of hepatic steatosis . patients were allocated to either the intervention group ( calorie restriction and exercise ) or control , with a target weight loss of 10% from baseline . calorie restriction of 1,2001,500 calories / day for those with a weight of less than 141 kg and 1,5001,800 calories / day for those weighing greater then 141 kg was encouraged . also , an increase in exercise to 175 minutes of moderate intensity exercise per week was required and participants attended weekly and monthly counseling sessions . results were not available on how many achieved a 10% weight loss , but average percentage weight loss for the interventional group was 8.3% and 0.03% in the control group . although no significant difference was observed in liver enzymes , follow up mri showed a significant reduction in steatosis following intervention , as well as between the intervention and control groups . follow up mri scanning was performed after 12 months of intervention . in a similar study , shah et al12 assessed the additive effect of an exercise program to calorie restriction , using mri to assess hepatic fat . over a 6 month period , patients were either allocated to a calorie restriction alone group , or a calorie restriction plus exercise group . calorie restriction was of 5001,000 calories / day with 30% being fat , 50% carbohydrates , and 20% protein . exercise requirements were 90 minutes three times a week in an indoor facility supervised by a personal trainer . both groups showed improvements in steatosis ( measured by mri ) , liver enzymes , and insulin resistance from baseline ; however , no significant difference was observed between the groups . the study authors suggest that no additive benefit of exercise was seen , however conceded that the small number of recruits is a limitation of the study . larson - meyer et al13 compared results from computerized tomography ( ct ) and mri to assess the outcome of calorie restriction and exercise on a group of 46 overweight patients . all the patients in this sample were healthy with no underlying liver disease at baseline , and were recruited after 6 months either randomized to calorie restriction alone ( 25% energy from baseline ) or in combination with exercise ( 12.5% calorie restriction and 12.5% increase in exercise ) or a low calorie diet ( 15% weight loss then maintenance ) or a control group . the results showed that bmi , insulin resistance , alanine aminotransferase ( alt ) levels , and intrahepatic steatosis decrease in all interventional groups ( alt did not decrease in the low calorie diet group ) . mri and ct results correlated closely , but the paper found that mri was more reliable at imaging lower levels of hepatic fat . this study , although showing interesting results when it comes to imaging , actually may not be relevant as not only did no patients have a diagnosis of nafld , but patients were encouraged to drink less than 2 units of alcohol per day , some of whom had been drinking more prior to the trial . two of the papers we reviewed used us as a measurement of hepatic steatosis improvement over the trial period . scagiloni et al14 carried out a small trial , with twelve participants over a period of 3 months . all participants were encouraged to carry out a diet that had been personally tailored by a dietician , and to increase their physical activity , which was monitored by an armband ( comparisons made at baseline and 3 months ) . exercise over the 3 month period did not show any significant increase , although there was an average weight loss and bmi decrease of 7.34% . lfts also did not show any significant improvement but us showed a significant decrease in intrahepatic fat . in contrast , catalano et al15 used a much larger sample group of 50 over a period of 6 months . all of these participants were encouraged to lose 5% of their body weight over the trial period , although no specific guidelines were given . at the end of the 6 months , the average weight loss of 57% of the desired weight loss ( 5% ) was achieved . results showed that both bmi and insulin resistance decrease over the trial period and this correlated to a decrease in intrahepatic fat on us . on reviewing blood results , this trial also came to the conclusion that gamma - glutamyl transpeptidase ( ggt ) levels are the most reliable for assessing parallel modification of serological measurement and morphological changes . blood tests are by far the easiest and most accessible way of assessing liver function and this is represented by the number of papers that rely wholly on these as evidence for their trials . six trials in this section used blood results , and not imaging and histology , as a measureable outcome for their trials . sun et al16 is one of the larger trials included in this review : 1,006 patients were recruited into a 12 month trial where they were randomized to receive lifestyle interventions ( nafld education including dietary advice and exercise therapy ) or to a control group . alt levels and other markers of metabolic syndrome decreased over the trial period in the lifestyle group ( compared to those in the control group ) , all to a greater extent at 12 months than when reviewed at 6 months . straznicky et al17 recruited 58 patients with three or more markers of metabolic syndrome , rather than being exclusively diagnosed with nafld . nafld is often described as the hepatic manifestation of metabolic syndrome therefore this paper was included . over a 12 week period , the 58 patients were randomized to either weight loss by calorie restriction ( deficit of 400900 calories per day ) , weight loss by calorie restriction and exercise ( 40 minute bike ride on alternate days ) , and a control group , a design that is similar to larson - meyer et al13 and shah et al.12 the calorie restriction alone group lost on average 7.6% with a corresponding decrease in alt of 20% and ggt of 28% . the calorie restriction and exercise group lost 9.1% of their baseline weight on average and alt and ggt levels decreased by 24% and 33% , respectively . these results showed a significant decrease from baseline but no significant difference between the two groups : they do not support the additive effect of exercise . sreenivasa baba et al18 published another paper using blood tests as its primary follow up . fifty - nine patients , with us diagnosed nash , were recruited into a 3-month trial involving an exercise program ( 45 minutes of exercise five times a week ) , and a calorie controlled diet for those with a high bmi ( 25 calories per kg of their ideal body weight ) . forty - four patients were compliant to this exercise and diet regime , and those taking part in diet and exercise lost on average 3.1 kg , while those taking part in just the diet lost 0.2 kg , and no significant decrease in weight was seen in the non - compliant group . alt and aspartate aminotransferase ( ast ) levels decreased significantly in the compliant group ; therefore , this paper draws the conclusion that weight loss is beneficial regardless of exercise participation . this paper included 30 patients who had biopsies at baseline but did not follow these up at the end of the trial . a study by park et al19 had a similar set up to sreenivasa baba et al,18 though it was a much smaller trial . twenty - five obese adults with us diagnosed nafld were recruited for a 12 month trial . similar dietary advice of 2530 calories per kg of ideal body weight was prescribed , and generalized exercise and behavioral advice was given . unlike sreenivasa baba et al , participants in this trial were considered compliant if they showed a reduction in weight . again , alt and ast levels decreased in the compliant group , but in this trial , levels were shown to increase in the non - compliant patients who gained weight . kim et al20 used an internet program to encourage participants to improve their diet and increase their exercise . eighteen men were recruited for 8 weeks to use this program , which set individual goals and targets that could be monitored by a panel . at the end of the trial , weight and some markers of metabolic syndrome were also shown to decrease , including diastolic blood pressure . although this paper does not mention nafld , as has been previously stated , nafld is considered the hepatic manifestation of metabolic syndrome , which is measured . this paper also shows a new innovative way of encouraging a population to lead a healthier lifestyle . the results of this paper therefore contradicts shah et al12 and straznicky et al,17 which stated that exercise did not play an important part in the improvement of liver steatosis markers , as in this trial exercise does seem to be an important factor . venditti et al21 used weight loss to assess whether lifestyle interventions were more effective the first or the second time around . the paper follows the diabetic prevention program22 in which participants had been randomized to either 16 sessions of lifestyle interventions , metformin , troflitazone ( which was withdrawn due to side effects ) , or placebo . after this initial trial , all participants were asked if they would continue on a further 6 months of lifestyle interventions and 2,808 agreed . those in the original lifestyle group were then compared to those who had been in the metformin and placebo groups . those who had not received lifestyle intervention before were more likely to join this secondary trial and to continue with it , compared to those on their secondary course . weight loss was therefore greater in the naive group and indeed correlated to the number of sessions attended in this group . although this paper does not mention nafld or liver function it shows the importance of lifestyle intervention leading to weight loss and the problems with compliance . this section includes the papers using diet as the only intervention ; seven papers in total fell under this category . ryan et al23 published a paper in 2006 , which was a post hoc analysis of 52 obese , insulin resistant adults who had taken part in a weight loss program . they had been randomized to receive either a low carbohydrate diet ( 40% carbohydrate/40% fat ) or a low fat diet ( 60% carbohydrate/25% fat ) for 16 weeks . both groups lost a significant amount of weight over the trial period but the alt levels decreased twice as much in those on the low carbohydrate diet compared to the low fat diet . alt levels were also found to decrease more in those with abnormal levels ( 30 units / l for men and 19 units / l for women ) at baseline than those with normal levels , irrespective of the diet type . insulin resistance levels were also shown to decrease in both groups with no significant differences between them . the authors draw the conclusion that low carbohydrate diets are more beneficial than low fat diets at reducing alt levels . this was again a post hoc analysis with data taken from a trial comparing a low calorie diet with topiramate ( an anti - epileptic drug ) or placebo . this paper focused on the low calorie diet group , and reviewed the follow up results from 32 and 60 weeks of 147 patients . there was a high drop out in this trial and as a result data were only available for 67 patients at 32 weeks and 68 patients at 60 weeks . on average , patients lost 12.1 kg , men losing more than women over the trial period . ast and alt levels were shown to decrease in men , while there was a transient increase in women . ggt decreased in men but not in woman and there was no significant change in alp in either group . these results are interesting , indicating that sex is a key determining factor in change in lft levels over a trial . de luis et al25 reports on a 3-month intervention of hypocaloric diet ( either low fat or low carbohydrate ) on basic anthropological and biochemical measures in an obese population . after 3 months of dietary intervention , all biochemical parameters improved , along with improvements in bmi and waist circumference . more so , the nafld phenotype ( n=30 ) showed improvements in alt and insulin resistance . although only small numbers were included in each group , and the nafld phenotype was loosely defined , the study reveals that even a short focused intervention ( 3 months ) has an impact on measurable markers associated with metabolic syndrome . rodrguez - hernndez et al26 again uses a low fat and low carbohydrate diet to assess their effect on aminotransferase , and this trial is nicely followed up by haufe et al.27 the rodrguez - hernndez et al26 trial included 54 women , with us diagnosed nafld , and randomized them to a low fat ( 25% protein , < 10% fat , 54% carbohydrate ) or low carbohydrate ( 27% protein , 28% fat , 45% carbohydrate ) diet for a period of 6 months . at the end of the trial , those on the low carbohydrate diet lost 5.7% of their body weight and those in the low fat group 5.5% , a non - significant result . alt and ast had both decreased in each group but again there was no significant difference . the study by haufe et al27 follows a similar design but with 102 patients , both male and female , over a 6-month period . in this trial , the diet design was low carbohydrate ( 90 g of carbohydrate and 0.8 g protein per kg weight , 30% fat ) and low fat ( 20% fat , 0.8 g of protein per kg , the remainder carbohydrate ) . the results were also similar in this trial : a non - significant decrease in weight and alt between both groups . this study went on to look at intrahepatic fat content by magnetic resonance spectroscopy and tomography . results showed that intrahepatic fat reduced by 47% in the low carbohydrate group and 42% in the low fat group , with no significant difference between the groups . this paper therefore neatly follows from rodrguez - hernndez et al , with the added evidence of imaging to support the results and a larger sample size . the small study by elias et al28 recruited 31 participants , with either ct or us diagnosed nafld , to carry out a hypocaloric diet ( reduction of 5001,000 calories / day ) for 6 months . seventeen patients were compliant with this diet and ended up losing 5% of their body weight ; with this , they also showed a significant decrease in ggt , alt , and insulin resistance . at the beginning of the trial , 45% of the patients had been diagnosed with metabolic syndrome and in the end there were only 10% , all in the non - adherent group . intrahepatic fat by ct was also shown to decrease in the adherent group compared to the non - adherent group . this was a small trial involving 16 participants who were required to carry out 3 weeks of over feeding followed by 6 months of a hypocaloric diet . subjects in this trial were also genotyped to assess the role of genetics in nafld . during the 3 week overfeeding liver fat , measured by mri , increased by 27% and then fell by 25% in the hypocaloric phase . this paper therefore concluded that the changes in liver fat during nafld are reversible by dieting . genetic markers of patatin - like phospholipase domain - containing protein 3 ( pnpla3)-148ii and pnpla3 - 148 mm were investigated in this study . those with pnpla3 - 148ii ( n=7 ) showed a significant increase in lipid fat content while this relationship was not seen in those with pnpla3 - 148 mm ( n=9 ) . the study hypothesized the reason behind this change but concluded that the sample size was too small to see a definite relationship . kistler et al30 produced the only paper in this section that used biopsy results as an outcome . the paper was a retrospective study looking at 813 biopsy results of patients with nafld and assessed if they were taking part in the recommended guidelines for exercise put forward by the united states department of health and human services . the guidelines stated that people should be taking part in more than 150 minutes of moderate intensity or 75 minutes of vigorous intensity exercise per week . participants in this trial were classified as inactive ; meeting the moderate intensity guidelines ; or meeting the vigorous intensity guidelines . results of a questionnaire showed that 20% of the participants met the moderate activity guidelines and 26% met the vigorous activity guidelines ; the majority of patients were inactive . analysis of the biopsies showed that meeting the vigorous activity guidelines decreases your odds ratio ( or ) of having nash ( or : 0.65 ) ; the same was not true for moderate activity . biopsies also showed that those meeting vigorous activity guidelines had significantly lower odds ratio fibrosis compared to no fibrosis ( or : 0.53 ) ; again , the same was not true for moderate activity . thus this paper concluded that only vigorous activity was enough to cause significant change in liver steatosis and fibrosis . hallsworth et al31 constructed a randomized control trial including 19 patients with mri diagnosed nafld . the patients were randomized to take part in 4560 minutes of resistance exercise three times a week for 8 weeks , or be in a control group . they were only included in the final results if they lost less than 5% of their body weight , therefore excluding the weight loss from affecting the results . participants were required to wear armbands 7 days prior to the intervention and for 7 days afterwards to assess the change . results showed that there was a 13% decrease in intrahepatic fat , recorded by mri , in the intervention group with no change in the control group . there was improvement in insulin resistance but no change in alt or blood lipid levels . similar to hallsworth et al,31 sullivan et al32 also recruited a small sample , 18 participants with nafld , to participate in a randomized control trial . although in this study , the intervention group was required to carry out 3060 minutes of exercise five times a week , one of these was under direct supervision . again , similarly , mri scans were used to determine the intrahepatic fat content , which showed a mild decrease in the intervention group compared to the control . the same was also true for alt values : the decrease in alt correlated with the decrease in intrahepatic fat in the intervention group . these trials have a similar design and they also both have a small sample size that may have led to bias . fealy et al33 carried out a small short lived trial to assess the effect of exercise training on markers of hepatocyte apoptosis . thirteen patients were recruited into the trial , where they were required to carry out 60 minutes of aerobic activity at 80%85% of their maximum heart rate for seven consecutive days . mri scans were performed on all participants pre and post - trial and these showed no change in intrahepatic fat during the intervention . blood tests did show some changes , those with raised alt at baseline had decreased levels after the trial , and blood glucose levels , and insulin sensitivity also showed improvements . this trial was very short lived which may explain the limited change in markers of nafld . the effect of a short prescribed program of endurance exercise was assessed for improvements in liver fat content in a cohort including lean and obese individuals by tarnopolsky et al.34 the study design precluded weight loss as an end point , instead focusing on participation in a moderate intensity program of endurance exercise ( stationary bike cycling ) and assessing its effect on liver fat content principally . after a 3-month period of endurance exercise , improvements in fitness ( maximal oxygen consumption ) and reduction in waist circumference were noted in both the lean and obese groups , regardless of sex . no change in bmi was seen . hepatic lipid content , as measured by ct ( liver attenuation ) , did not change post exercise , regardless of group or sex . this study is limited by the small numbers of patients included and by the apparent liver health of the group the obese men had a pre - exercise alt of 30.34.8 u / l and the obese women 16.82.2 no measurement of lipid profile was discussed ; early changes in triglyceride content may have supported and encouraged participation in a longer program . this study does however highlight the need for intervention programs designed to induce weight loss . this study was deliberately designed not to induce weight loss . with no effect on liver fat content , this supports the notion that loss of body weight is important for improving liver health in obese individuals . bae et al35 is one of the larger trials reviewed in this discussion , with a cross - sectional analysis taken on 72,359 korean adults . baseline results were collected from these patients at the kangbuk samsung hospital total healthcare centre , and the diagnosis of nafld was made by us . the participants were asked to fill out a questionnaire recording how much exercise they carried out on a weekly basis ; regular exercise was defined as 30 minutes more than three times a week for three consecutive months . there were 19,921 patients with nafld and this number was significantly higher in those who did not exercise regularly . the same was true for bmi : nafld occurrence was higher with those with a greater bmi and liver function levels were lower on average if the patient took part in regular exercise . both have a similar design with a low intensity lifestyle intervention group ( three counseling sessions throughout the trial ) , moderate intensity lifestyle intervention group ( six counseling sessions throughout the trial ) , a long term group ( receiving the same initial intervention as the moderate group but with further sessions up to 12 months ) , and a control group . all participants were encouraged to carry out the same diet with a reduction in energy intake of 17002400 kj over a 3 month period and to increase their exercise to 150200 minutes a week . the st george et al study that was published in october 200837 included 152 participants followed up for a 9-month period . exercise was shown to be significantly increased in both the low and moderate intensity exercise encouragement groups , with no change in the control group . weight loss was found to be highest in those receiving moderate intensity lifestyle interventions and lowest in the control group ; liver function tests were also found to correlate with weight loss . the moderate intensity group was also shown to have decreased markers of metabolic risk factors at the end of the trial period . the study by st george et al published in 200936 categorized 141 patients into whether they increased their physical activity by more than 60 minutes per week , maintained their physical activity , or decreased their physical activity . in the moderate intensity encouragement group 64% and 63% from the low intensity encouragement group increased their activity from base line . this was a significant difference from the control group with only 16% increasing their physical exercise . the greatest changes were in insulin resistance , metabolic risk factors , and lfts seen in those who increased their physical activity . gerber et al38 carried out a cross - sectional analysis of a population using data from two surveys carried out between 20032004 and 20052006 in the united states . the data from these surveys were mostly self - reported , collecting baseline characteristics , lifestyle choices , and medical backgrounds of the subjects . diagnosis of nafld was made using the fatty liver index,39 a simple scoring system that predicts the degree of hepatosteatosis using the baseline characteristics of the subjects . patients were asked to wear activity monitor arm bands , similar to the ones used in hallsworth et al,31 for seven consecutive days . those with nafld were found to be less likely to take part in regular exercise , and having diabetes as well , made this relationship stronger . results also showed that exercise did not correlate with aminotransferase levels . although this paper had a large sample size and an accurate way of recording exercise in participants , the trial period was very short , which may not reflect a true representation of the participants exercise levels . there are many theories about various medications that may improve the outcome of nafld ; this review paper includes three trials using a variety of medications . twenty - three women were recruited by mndez - snchez et al,40 for a double blinded control trial testing the effects of ursodeoxycholic acid on patients with nafld . patients were randomized to receive a healthy diet ( 1200 kcal / day of which 20% was fat , 60% carbohydrate , and 20% protein ) with either ursodeoxycholic acid or placebo . both groups lost weight over the trial with no significant difference between the two groups . alt decreased more in those taking ursodeoxycholic acid compared to those who were not , but other than that there was no significantly different blood results between the two groups . us was performed on the patients at the beginning and end of the trial , and fat liver content was shown to decrease in both groups but again no significant difference between the two groups was found . viusid ( catalysis , s.l . , madrid , spain ) , an antioxidant nutritional supplement , was investigated by vilar gomez et al41 in a randomized control trial . sixty patients , with biopsy proven nafld , were randomized to receive a hypocaloric diet and exercise with or without viusid . monitoring of the diet and exercise was done by self - reporting and biopsies were taken at baseline and at the end of the 24 week trial , which were examined by a pathologist . there were eleven dropouts over the trial for various reasons , and viusid was reported to have side effects including nausea and diarrhea in some cases . alt and ast decreased significantly over the trial with no significant difference between the two trial groups . the viusid group did show a significant improvement in liver histology , and improvements in fibrosis and steatosis , as did the control group . between group comparison showed significant improvements in the viusid group in all parameters of the nas score ( but not the fibrosis grade ) compared to the control . there was a relatively high dropout rate in this trial , and those who dropped out were not accurately followed up . the reason behind these dropouts were not reported ; these would have been interesting as they may have impacted on viusid s use in a clinical setting . krakoff et al42 used data from the diabetic prevention program , similar to venditti et al.21 venditti et al focused on the placebo group and followed them up after the trial , whereas krakoff et al compared a metformin group to a placebo group . in krakoff et al both groups received diet and exercise programs to induce weight loss , and the 2,153 had also been randomized to receive either placebo or metformin for an average trial period of 3.2 years . alt levels rose in both groups over the trial , although the rise was greatest in the placebo group . the study also showed that the incidence of abnormal alt decreased with increasing amount of weight loss . all participants in this trial had normal alt at baseline and nafld status was not known , which may explain why these results do not follow the same trends as other papers . akcam et al43 performed a small randomized double blind parallel group clinical trial assessing the effect of diet and exercise alone or in combination with either metformin or vitamin e on various markers of nafld , including ultrasonographically assessed liver steatosis , insulin resistance , lipid profile , and bmi . they recruited 67 adolescents with us defined nafld to three groups all of whom received personalized and prescribed diet and exercise regimes . one group also received metformin ( 850 mg once daily ) while another group received vitamin e ( 400 u daily ) . bmi improved in all groups , more so in the metformin and vitamin e groups , although this did not achieve statistical significance . unsurprisingly , insulin resistance was most improved in the metformin treated group , with only slight improvement seen in the other two groups . no direct markers of oxidative stress improvement were measured , although tumor necrosis factor ( tnf)-alpha did not improve significantly in any of the three groups . at the conclusion of the study period , repeat liver us was performed , with the metformin group showing the greatest improvement almost double that of the diet and exercise alone group and the vitamin e group . this well designed study compared pharmacological intervention with either metformin or vitamin e with diet and exercise alone . the short duration of the study is a limitation ; perhaps greater effects from all three interventions would be more apparent if the study period was extended . however , although the authors highlight the improvements seen with metformin in particular , the diet and exercise alone group did achieve an improvement in bmi supporting the role of this fundamental intervention in a young cohort . overall , 34 studies were reviewed in this paper , 14 using diet and exercise interventions , seven with just diet interventions , nine with just exercise intervention , and four using medications as interventions . all of the 14 studies using diet and exercise as interventions showed improvements in their various measured outcomes . although there were a few , including shah et al12 and straznicky et al,17 who showed that exercise had no additive effect when used in conjunction with diet to improve liver steatosis markers . this was contradicted by kim et al20 and , indeed , the majority of the studies using just exercise interventions showed that exercise does improve liver markers of steatosis . of those in the exclusively exercise interventions all but one study showed a relationship between increased exercise and improvement of hepatic markers . tarnopolsky et al34 showed no improvement in hepatic fat content and blood results with increased exercise . this was concluded to be due to the relatively small sample size and short trial period , as well as participants being healthy at recruitment . fealy et al33 also showed no improvement in hepatic steatosis on us but did show improvement in blood results when participants increased their exercise . three of the studies looked at the comparison between different intensities of exercise , both st george et al36,37 studies showed improvements with moderate intensity exercise while kistler et al30 only showed improvements with vigorous activity . two of the studies were cross - sectional analyses , bae et al35 and gerber et al,38 both of these showed a strong relationship between the proportion of the population with nafld and the low level of activity that they took part in . seven studies used dietary interventions , all of which showed improvements in hepatic steatosis markers . two of the trials compared low carbohydrate and low fat diets , rodrguez - hernndez et al26 showed similar results between the two diets whereas ryan et al23 showed better results with those on the low carbohydrate diet . in the medication intervention group there were four studies : mndez - snchez et al40 using ursodeoxycholic acid , vilar gomez et al41 using viusid ( an antioxidant nutritional supplement ) , krakoff et al42 using metformin , and akcam et al43 compared metformin and vitamin e. all the studies apart from the one by vilar gomez et al41 concluded that there was no particular benefit from their intervention and that weight loss by diet and exercise was the most important factor . the vilar gomez et al study41 using viusid did show improvement , but there were issues with side effects from the drug which require further investigation before it can be endorsed . all papers used a variety of follow up methods , including biopsy , imaging and liver function tests . four studies used biopsies for follow up , three in the diet and exercise section , and one in the exercise section . there were also different imaging methods used , including mri , ct , and us . larson - meyer et al13 compared ct and mri results , concluding that mri produced better results with respect to lower levels of hepatic fat content . practice guidelines set forth by key stakeholders in nafld published in 2012 endorse the vital role of lifestyle intervention in the management of nafld.44 weight loss either by hypocaloric diet or in combination with increased physical activity to attain a 3%5% loss in body weight is required to improved steatosis , with up to 10% loss of body weight required to improve necroinflammation . this review concludes that lifestyle intervention is a vital and central component to the treatment of nafld and metabolic syndrome . the optimal strategy is not yet realized , although the importance of weight loss and exercise is paramount . sustainability of any intervention is key to success ; however , it is an elusive end point .

the burden of non - alcoholic fatty liver disease ( nafld ) worldwide is a significant clinical and public health issue , affecting approximately one third of the western population . this review assesses the effect and impact lifestyle interventions have on the treatment of this common condition . we review studies comparing the effect of calorie restriction and exercise programs , as well as comparison of lifestyle intervention with pharmaceutical intervention . both calorie restriction and exercise programs are shown to be beneficial in improving features of metabolic syndrome and surrogate markers of nafld . the paucity of studies using histological improvement hinders the ability to conclude a benefit on improvement of histological nafld , although this is shown in a small number of studies . there is a need to extend the intervention period to show a sustained improvement with intervention as most studies have a follow up period of 12 months of less .

Introduction Methods Results and discussion Diet and exercise focused regimes Diet only regimes Exercise only regimes Pharmacological interventions used concomitantly with or in comparison to lifestyle interventions Conclusion

non - alcoholic fatty liver disease ( nafld ) is now regarded as the most common liver condition in the developed world , affecting up 30% of the population . numerous pharmacological interventions have been tested for the treatment of nafld with varying success . nonetheless , no current pharmacological therapy is recommended for the treatment of this condition . much focus has been given to the role of lifestyle intervention in nafld , including the role of prescribed exercise regimes and diets . we searched a public domain database ( pubmed ) with the following categories : disease ( nafld , fatty liver , non - alcoholic steatohepatitis [ nash ] , and simple steatosis ) and intervention ( lifestyle intervention , diet , exercise , and behavior ) with each possible combination . furthermore , we have included studies of features of metabolic syndrome , especially where liver health was considered and reviewed . nafld is defined as fat accumulation in the liver , greater than 5%10% by weight , in the absence of excessive alcohol consumption.1 it is a spectrum disease , ranging from simple steatosis without inflammation , to nash , progressing onwards to fibrosis and cirrhosis.2 nafld is closely associated with obesity and type ii diabetes mellitus ( t2 dm ) ; indeed , 75% of patients with nafld are obese , and it is frequently seen as the hepatic manifestation of metabolic syndrome.3 the pathophysiology of nafld is not completely understood , but insulin resistance is thought to play a part with increased lipolysis leading to an influx in free fatty acid into the liver.2 the gold standard for diagnosis of nafld is liver biopsy ; this is the only way of truly differentiating between the different stages of nafld . biopsy is an imperfect gold standard and has issues pertaining to morbidity , mortality , as well as intra and inter observer variability . ultrasound ( us ) is used in most cases ; the disadvantage of this is that fat is only picked up when it reaches levels of 33%3 and us is a poor detector of inflammation . nafld was relatively unknown prior to the early 1980s , but is fast becoming the most common chronic liver disease worldwide,4 with an estimated prevalence of 20%30% in western countries.5 the british transplant guidance website has also reported that in 2008 and 2009 12% of the patients on the waiting list for liver transplantation had nash cirrhosis.6 this increase in cases of nafld is strongly linked to the rising rate in obesity ; the national health service ( nhs ) database shows the percentage population with obesity has almost doubled in the last 8 years , with now roughly a quarter of the population having a raised body mass index ( bmi ) . in one of the few studies of lifestyle intervention using histological endpoints , promrat et al8 conducted a small study ( n=30 ) of patients randomly assigned to receive either diet , exercise , and behavioral strategies or a control group ( 2:1 ratio ) . liver enzyme and one of the interesting observations from this study was the low attrition rate ; 28 of the 30 recruits completed the study period , including follow up biopsy . biopsy results were also used in a study by jin et al.9 one hundred and twenty living donors with nafld who had undergone a biopsy and were then encouraged to lose 5% of their body weight , by diet and exercise , were re - biopsied . histological end points were also sought in a paper from bhat et al10 assessing whether lifestyle modifications can improve nafld , although they were not able to biopsy their entire cohort . patients were allocated to either the intervention group ( calorie restriction and exercise ) or control , with a target weight loss of 10% from baseline . although no significant difference was observed in liver enzymes , follow up mri showed a significant reduction in steatosis following intervention , as well as between the intervention and control groups . follow up mri scanning was performed after 12 months of intervention . in a similar study , shah et al12 assessed the additive effect of an exercise program to calorie restriction , using mri to assess hepatic fat . the study authors suggest that no additive benefit of exercise was seen , however conceded that the small number of recruits is a limitation of the study . larson - meyer et al13 compared results from computerized tomography ( ct ) and mri to assess the outcome of calorie restriction and exercise on a group of 46 overweight patients . all the patients in this sample were healthy with no underlying liver disease at baseline , and were recruited after 6 months either randomized to calorie restriction alone ( 25% energy from baseline ) or in combination with exercise ( 12.5% calorie restriction and 12.5% increase in exercise ) or a low calorie diet ( 15% weight loss then maintenance ) or a control group . this study , although showing interesting results when it comes to imaging , actually may not be relevant as not only did no patients have a diagnosis of nafld , but patients were encouraged to drink less than 2 units of alcohol per day , some of whom had been drinking more prior to the trial . sun et al16 is one of the larger trials included in this review : 1,006 patients were recruited into a 12 month trial where they were randomized to receive lifestyle interventions ( nafld education including dietary advice and exercise therapy ) or to a control group . alt levels and other markers of metabolic syndrome decreased over the trial period in the lifestyle group ( compared to those in the control group ) , all to a greater extent at 12 months than when reviewed at 6 months . straznicky et al17 recruited 58 patients with three or more markers of metabolic syndrome , rather than being exclusively diagnosed with nafld . over a 12 week period , the 58 patients were randomized to either weight loss by calorie restriction ( deficit of 400900 calories per day ) , weight loss by calorie restriction and exercise ( 40 minute bike ride on alternate days ) , and a control group , a design that is similar to larson - meyer et al13 and shah et al.12 the calorie restriction alone group lost on average 7.6% with a corresponding decrease in alt of 20% and ggt of 28% . the calorie restriction and exercise group lost 9.1% of their baseline weight on average and alt and ggt levels decreased by 24% and 33% , respectively . these results showed a significant decrease from baseline but no significant difference between the two groups : they do not support the additive effect of exercise . at the end of the trial weight and some markers of metabolic syndrome were also shown to decrease , including diastolic blood pressure . although this paper does not mention nafld , as has been previously stated , nafld is considered the hepatic manifestation of metabolic syndrome , which is measured . the results of this paper therefore contradicts shah et al12 and straznicky et al,17 which stated that exercise did not play an important part in the improvement of liver steatosis markers , as in this trial exercise does seem to be an important factor . after this initial trial , all participants were asked if they would continue on a further 6 months of lifestyle interventions and 2,808 agreed . although this paper does not mention nafld or liver function it shows the importance of lifestyle intervention leading to weight loss and the problems with compliance . both groups lost a significant amount of weight over the trial period but the alt levels decreased twice as much in those on the low carbohydrate diet compared to the low fat diet . at the end of the trial , those on the low carbohydrate diet lost 5.7% of their body weight and those in the low fat group 5.5% , a non - significant result . at the beginning of the trial , 45% of the patients had been diagnosed with metabolic syndrome and in the end there were only 10% , all in the non - adherent group . similar to hallsworth et al,31 sullivan et al32 also recruited a small sample , 18 participants with nafld , to participate in a randomized control trial . fealy et al33 carried out a small short lived trial to assess the effect of exercise training on markers of hepatocyte apoptosis . the effect of a short prescribed program of endurance exercise was assessed for improvements in liver fat content in a cohort including lean and obese individuals by tarnopolsky et al.34 the study design precluded weight loss as an end point , instead focusing on participation in a moderate intensity program of endurance exercise ( stationary bike cycling ) and assessing its effect on liver fat content principally . both have a similar design with a low intensity lifestyle intervention group ( three counseling sessions throughout the trial ) , moderate intensity lifestyle intervention group ( six counseling sessions throughout the trial ) , a long term group ( receiving the same initial intervention as the moderate group but with further sessions up to 12 months ) , and a control group . the moderate intensity group was also shown to have decreased markers of metabolic risk factors at the end of the trial period . diagnosis of nafld was made using the fatty liver index,39 a simple scoring system that predicts the degree of hepatosteatosis using the baseline characteristics of the subjects . those with nafld were found to be less likely to take part in regular exercise , and having diabetes as well , made this relationship stronger . us was performed on the patients at the beginning and end of the trial , and fat liver content was shown to decrease in both groups but again no significant difference between the two groups was found . the viusid group did show a significant improvement in liver histology , and improvements in fibrosis and steatosis , as did the control group . in krakoff et al both groups received diet and exercise programs to induce weight loss , and the 2,153 had also been randomized to receive either placebo or metformin for an average trial period of 3.2 years . akcam et al43 performed a small randomized double blind parallel group clinical trial assessing the effect of diet and exercise alone or in combination with either metformin or vitamin e on various markers of nafld , including ultrasonographically assessed liver steatosis , insulin resistance , lipid profile , and bmi . no direct markers of oxidative stress improvement were measured , although tumor necrosis factor ( tnf)-alpha did not improve significantly in any of the three groups . the short duration of the study is a limitation ; perhaps greater effects from all three interventions would be more apparent if the study period was extended . however , although the authors highlight the improvements seen with metformin in particular , the diet and exercise alone group did achieve an improvement in bmi supporting the role of this fundamental intervention in a young cohort . in one of the few studies of lifestyle intervention using histological endpoints , promrat et al8 conducted a small study ( n=30 ) of patients randomly assigned to receive either diet , exercise , and behavioral strategies or a control group ( 2:1 ratio ) . histological end points were also sought in a paper from bhat et al10 assessing whether lifestyle modifications can improve nafld , although they were not able to biopsy their entire cohort . unfortunately there are no biopsy results available for the non - compliant group , indeed there is no record if there were any taken . patients were allocated to either the intervention group ( calorie restriction and exercise ) or control , with a target weight loss of 10% from baseline . although no significant difference was observed in liver enzymes , follow up mri showed a significant reduction in steatosis following intervention , as well as between the intervention and control groups . follow up mri scanning was performed after 12 months of intervention . in a similar study , shah et al12 assessed the additive effect of an exercise program to calorie restriction , using mri to assess hepatic fat . the study authors suggest that no additive benefit of exercise was seen , however conceded that the small number of recruits is a limitation of the study . larson - meyer et al13 compared results from computerized tomography ( ct ) and mri to assess the outcome of calorie restriction and exercise on a group of 46 overweight patients . all the patients in this sample were healthy with no underlying liver disease at baseline , and were recruited after 6 months either randomized to calorie restriction alone ( 25% energy from baseline ) or in combination with exercise ( 12.5% calorie restriction and 12.5% increase in exercise ) or a low calorie diet ( 15% weight loss then maintenance ) or a control group . this study , although showing interesting results when it comes to imaging , actually may not be relevant as not only did no patients have a diagnosis of nafld , but patients were encouraged to drink less than 2 units of alcohol per day , some of whom had been drinking more prior to the trial . blood tests are by far the easiest and most accessible way of assessing liver function and this is represented by the number of papers that rely wholly on these as evidence for their trials . sun et al16 is one of the larger trials included in this review : 1,006 patients were recruited into a 12 month trial where they were randomized to receive lifestyle interventions ( nafld education including dietary advice and exercise therapy ) or to a control group . alt levels and other markers of metabolic syndrome decreased over the trial period in the lifestyle group ( compared to those in the control group ) , all to a greater extent at 12 months than when reviewed at 6 months . straznicky et al17 recruited 58 patients with three or more markers of metabolic syndrome , rather than being exclusively diagnosed with nafld . over a 12 week period , the 58 patients were randomized to either weight loss by calorie restriction ( deficit of 400900 calories per day ) , weight loss by calorie restriction and exercise ( 40 minute bike ride on alternate days ) , and a control group , a design that is similar to larson - meyer et al13 and shah et al.12 the calorie restriction alone group lost on average 7.6% with a corresponding decrease in alt of 20% and ggt of 28% . the calorie restriction and exercise group lost 9.1% of their baseline weight on average and alt and ggt levels decreased by 24% and 33% , respectively . these results showed a significant decrease from baseline but no significant difference between the two groups : they do not support the additive effect of exercise . at the end of the trial , weight and some markers of metabolic syndrome were also shown to decrease , including diastolic blood pressure . although this paper does not mention nafld , as has been previously stated , nafld is considered the hepatic manifestation of metabolic syndrome , which is measured . the results of this paper therefore contradicts shah et al12 and straznicky et al,17 which stated that exercise did not play an important part in the improvement of liver steatosis markers , as in this trial exercise does seem to be an important factor . after this initial trial , all participants were asked if they would continue on a further 6 months of lifestyle interventions and 2,808 agreed . although this paper does not mention nafld or liver function it shows the importance of lifestyle intervention leading to weight loss and the problems with compliance . this paper focused on the low calorie diet group , and reviewed the follow up results from 32 and 60 weeks of 147 patients . at the end of the trial , those on the low carbohydrate diet lost 5.7% of their body weight and those in the low fat group 5.5% , a non - significant result . at the beginning of the trial , 45% of the patients had been diagnosed with metabolic syndrome and in the end there were only 10% , all in the non - adherent group . similar to hallsworth et al,31 sullivan et al32 also recruited a small sample , 18 participants with nafld , to participate in a randomized control trial . fealy et al33 carried out a small short lived trial to assess the effect of exercise training on markers of hepatocyte apoptosis . this trial was very short lived which may explain the limited change in markers of nafld . the effect of a short prescribed program of endurance exercise was assessed for improvements in liver fat content in a cohort including lean and obese individuals by tarnopolsky et al.34 the study design precluded weight loss as an end point , instead focusing on participation in a moderate intensity program of endurance exercise ( stationary bike cycling ) and assessing its effect on liver fat content principally . this study is limited by the small numbers of patients included and by the apparent liver health of the group the obese men had a pre - exercise alt of 30.34.8 u / l and the obese women 16.82.2 no measurement of lipid profile was discussed ; early changes in triglyceride content may have supported and encouraged participation in a longer program . both have a similar design with a low intensity lifestyle intervention group ( three counseling sessions throughout the trial ) , moderate intensity lifestyle intervention group ( six counseling sessions throughout the trial ) , a long term group ( receiving the same initial intervention as the moderate group but with further sessions up to 12 months ) , and a control group . exercise was shown to be significantly increased in both the low and moderate intensity exercise encouragement groups , with no change in the control group . the moderate intensity group was also shown to have decreased markers of metabolic risk factors at the end of the trial period . diagnosis of nafld was made using the fatty liver index,39 a simple scoring system that predicts the degree of hepatosteatosis using the baseline characteristics of the subjects . us was performed on the patients at the beginning and end of the trial , and fat liver content was shown to decrease in both groups but again no significant difference between the two groups was found . sixty patients , with biopsy proven nafld , were randomized to receive a hypocaloric diet and exercise with or without viusid . monitoring of the diet and exercise was done by self - reporting and biopsies were taken at baseline and at the end of the 24 week trial , which were examined by a pathologist . the viusid group did show a significant improvement in liver histology , and improvements in fibrosis and steatosis , as did the control group . in krakoff et al both groups received diet and exercise programs to induce weight loss , and the 2,153 had also been randomized to receive either placebo or metformin for an average trial period of 3.2 years . akcam et al43 performed a small randomized double blind parallel group clinical trial assessing the effect of diet and exercise alone or in combination with either metformin or vitamin e on various markers of nafld , including ultrasonographically assessed liver steatosis , insulin resistance , lipid profile , and bmi . no direct markers of oxidative stress improvement were measured , although tumor necrosis factor ( tnf)-alpha did not improve significantly in any of the three groups . the short duration of the study is a limitation ; perhaps greater effects from all three interventions would be more apparent if the study period was extended . however , although the authors highlight the improvements seen with metformin in particular , the diet and exercise alone group did achieve an improvement in bmi supporting the role of this fundamental intervention in a young cohort . all of the 14 studies using diet and exercise as interventions showed improvements in their various measured outcomes . this was contradicted by kim et al20 and , indeed , the majority of the studies using just exercise interventions showed that exercise does improve liver markers of steatosis . this was concluded to be due to the relatively small sample size and short trial period , as well as participants being healthy at recruitment . practice guidelines set forth by key stakeholders in nafld published in 2012 endorse the vital role of lifestyle intervention in the management of nafld.44 weight loss either by hypocaloric diet or in combination with increased physical activity to attain a 3%5% loss in body weight is required to improved steatosis , with up to 10% loss of body weight required to improve necroinflammation . this review concludes that lifestyle intervention is a vital and central component to the treatment of nafld and metabolic syndrome .

much focus has been given to the role of lifestyle intervention in nafld , including the role of prescribed exercise regimes and diets . this review aims to summarize the literature surrounding this approach , highlighting the impact and role these strategies have on nafld . we searched a public domain database ( pubmed ) with the following categories : disease ( nafld , fatty liver , non - alcoholic steatohepatitis [ nash ] , and simple steatosis ) and intervention ( lifestyle intervention , diet , exercise , and behavior ) with each possible combination . nafld is defined as fat accumulation in the liver , greater than 5%10% by weight , in the absence of excessive alcohol consumption.1 it is a spectrum disease , ranging from simple steatosis without inflammation , to nash , progressing onwards to fibrosis and cirrhosis.2 nafld is closely associated with obesity and type ii diabetes mellitus ( t2 dm ) ; indeed , 75% of patients with nafld are obese , and it is frequently seen as the hepatic manifestation of metabolic syndrome.3 the pathophysiology of nafld is not completely understood , but insulin resistance is thought to play a part with increased lipolysis leading to an influx in free fatty acid into the liver.2 the gold standard for diagnosis of nafld is liver biopsy ; this is the only way of truly differentiating between the different stages of nafld . nafld was relatively unknown prior to the early 1980s , but is fast becoming the most common chronic liver disease worldwide,4 with an estimated prevalence of 20%30% in western countries.5 the british transplant guidance website has also reported that in 2008 and 2009 12% of the patients on the waiting list for liver transplantation had nash cirrhosis.6 this increase in cases of nafld is strongly linked to the rising rate in obesity ; the national health service ( nhs ) database shows the percentage population with obesity has almost doubled in the last 8 years , with now roughly a quarter of the population having a raised body mass index ( bmi ) . changing lifestyles is ultimately to blame for this , including increased access to cheap food , eating on the go , and marketing of high calorific foods.7 given all the above mentioned , there has been increased interest in nafld and possible ways of managing and treating it . there have been many different suggestions for pharmaceuticals that could be used , including metformin , vitamin e , urodeoxycholic acid , to name a few.1 as of yet none of these treatments have shown any significant disease improvement and none are currently recommended . in one of the few studies of lifestyle intervention using histological endpoints , promrat et al8 conducted a small study ( n=30 ) of patients randomly assigned to receive either diet , exercise , and behavioral strategies or a control group ( 2:1 ratio ) . there was improvement in hepatic steatosis , lobular inflammation , parenchymal inflammation , and ballooning injury in the interventional group ; but results showed no improvement in fibrosis in either group . eight patients had repeat liver biopsies in the compliant group and of these , six showed improvement in steatosis and necro - inflammatory score , with no change in fibrosis . although no significant difference was observed in liver enzymes , follow up mri showed a significant reduction in steatosis following intervention , as well as between the intervention and control groups . larson - meyer et al13 compared results from computerized tomography ( ct ) and mri to assess the outcome of calorie restriction and exercise on a group of 46 overweight patients . all the patients in this sample were healthy with no underlying liver disease at baseline , and were recruited after 6 months either randomized to calorie restriction alone ( 25% energy from baseline ) or in combination with exercise ( 12.5% calorie restriction and 12.5% increase in exercise ) or a low calorie diet ( 15% weight loss then maintenance ) or a control group . the results showed that bmi , insulin resistance , alanine aminotransferase ( alt ) levels , and intrahepatic steatosis decrease in all interventional groups ( alt did not decrease in the low calorie diet group ) . this study , although showing interesting results when it comes to imaging , actually may not be relevant as not only did no patients have a diagnosis of nafld , but patients were encouraged to drink less than 2 units of alcohol per day , some of whom had been drinking more prior to the trial . at the end of the 6 months , the average weight loss of 57% of the desired weight loss ( 5% ) was achieved . on reviewing blood results , this trial also came to the conclusion that gamma - glutamyl transpeptidase ( ggt ) levels are the most reliable for assessing parallel modification of serological measurement and morphological changes . alt levels and other markers of metabolic syndrome decreased over the trial period in the lifestyle group ( compared to those in the control group ) , all to a greater extent at 12 months than when reviewed at 6 months . over a 12 week period , the 58 patients were randomized to either weight loss by calorie restriction ( deficit of 400900 calories per day ) , weight loss by calorie restriction and exercise ( 40 minute bike ride on alternate days ) , and a control group , a design that is similar to larson - meyer et al13 and shah et al.12 the calorie restriction alone group lost on average 7.6% with a corresponding decrease in alt of 20% and ggt of 28% . fifty - nine patients , with us diagnosed nash , were recruited into a 3-month trial involving an exercise program ( 45 minutes of exercise five times a week ) , and a calorie controlled diet for those with a high bmi ( 25 calories per kg of their ideal body weight ) . forty - four patients were compliant to this exercise and diet regime , and those taking part in diet and exercise lost on average 3.1 kg , while those taking part in just the diet lost 0.2 kg , and no significant decrease in weight was seen in the non - compliant group . alt and aspartate aminotransferase ( ast ) levels decreased significantly in the compliant group ; therefore , this paper draws the conclusion that weight loss is beneficial regardless of exercise participation . the results of this paper therefore contradicts shah et al12 and straznicky et al,17 which stated that exercise did not play an important part in the improvement of liver steatosis markers , as in this trial exercise does seem to be an important factor . the paper follows the diabetic prevention program22 in which participants had been randomized to either 16 sessions of lifestyle interventions , metformin , troflitazone ( which was withdrawn due to side effects ) , or placebo . alt levels were also found to decrease more in those with abnormal levels ( 30 units / l for men and 19 units / l for women ) at baseline than those with normal levels , irrespective of the diet type . although only small numbers were included in each group , and the nafld phenotype was loosely defined , the study reveals that even a short focused intervention ( 3 months ) has an impact on measurable markers associated with metabolic syndrome . rodrguez - hernndez et al26 again uses a low fat and low carbohydrate diet to assess their effect on aminotransferase , and this trial is nicely followed up by haufe et al.27 the rodrguez - hernndez et al26 trial included 54 women , with us diagnosed nafld , and randomized them to a low fat ( 25% protein , < 10% fat , 54% carbohydrate ) or low carbohydrate ( 27% protein , 28% fat , 45% carbohydrate ) diet for a period of 6 months . at the end of the trial , those on the low carbohydrate diet lost 5.7% of their body weight and those in the low fat group 5.5% , a non - significant result . in this trial , the diet design was low carbohydrate ( 90 g of carbohydrate and 0.8 g protein per kg weight , 30% fat ) and low fat ( 20% fat , 0.8 g of protein per kg , the remainder carbohydrate ) . at the beginning of the trial , 45% of the patients had been diagnosed with metabolic syndrome and in the end there were only 10% , all in the non - adherent group . those with pnpla3 - 148ii ( n=7 ) showed a significant increase in lipid fat content while this relationship was not seen in those with pnpla3 - 148 mm ( n=9 ) . the paper was a retrospective study looking at 813 biopsy results of patients with nafld and assessed if they were taking part in the recommended guidelines for exercise put forward by the united states department of health and human services . analysis of the biopsies showed that meeting the vigorous activity guidelines decreases your odds ratio ( or ) of having nash ( or : 0.65 ) ; the same was not true for moderate activity . biopsies also showed that those meeting vigorous activity guidelines had significantly lower odds ratio fibrosis compared to no fibrosis ( or : 0.53 ) ; again , the same was not true for moderate activity . the effect of a short prescribed program of endurance exercise was assessed for improvements in liver fat content in a cohort including lean and obese individuals by tarnopolsky et al.34 the study design precluded weight loss as an end point , instead focusing on participation in a moderate intensity program of endurance exercise ( stationary bike cycling ) and assessing its effect on liver fat content principally . after a 3-month period of endurance exercise , improvements in fitness ( maximal oxygen consumption ) and reduction in waist circumference were noted in both the lean and obese groups , regardless of sex . this study is limited by the small numbers of patients included and by the apparent liver health of the group the obese men had a pre - exercise alt of 30.34.8 u / l and the obese women 16.82.2 no measurement of lipid profile was discussed ; early changes in triglyceride content may have supported and encouraged participation in a longer program . both have a similar design with a low intensity lifestyle intervention group ( three counseling sessions throughout the trial ) , moderate intensity lifestyle intervention group ( six counseling sessions throughout the trial ) , a long term group ( receiving the same initial intervention as the moderate group but with further sessions up to 12 months ) , and a control group . us was performed on the patients at the beginning and end of the trial , and fat liver content was shown to decrease in both groups but again no significant difference between the two groups was found . monitoring of the diet and exercise was done by self - reporting and biopsies were taken at baseline and at the end of the 24 week trial , which were examined by a pathologist . between group comparison showed significant improvements in the viusid group in all parameters of the nas score ( but not the fibrosis grade ) compared to the control . akcam et al43 performed a small randomized double blind parallel group clinical trial assessing the effect of diet and exercise alone or in combination with either metformin or vitamin e on various markers of nafld , including ultrasonographically assessed liver steatosis , insulin resistance , lipid profile , and bmi . at the conclusion of the study period , repeat liver us was performed , with the metformin group showing the greatest improvement almost double that of the diet and exercise alone group and the vitamin e group . however , although the authors highlight the improvements seen with metformin in particular , the diet and exercise alone group did achieve an improvement in bmi supporting the role of this fundamental intervention in a young cohort . in one of the few studies of lifestyle intervention using histological endpoints , promrat et al8 conducted a small study ( n=30 ) of patients randomly assigned to receive either diet , exercise , and behavioral strategies or a control group ( 2:1 ratio ) . at the end of a 6 month period 45 of the 60 eight patients had repeat liver biopsies in the compliant group and of these , six showed improvement in steatosis and necro - inflammatory score , with no change in fibrosis . all the patients in this sample were healthy with no underlying liver disease at baseline , and were recruited after 6 months either randomized to calorie restriction alone ( 25% energy from baseline ) or in combination with exercise ( 12.5% calorie restriction and 12.5% increase in exercise ) or a low calorie diet ( 15% weight loss then maintenance ) or a control group . this study , although showing interesting results when it comes to imaging , actually may not be relevant as not only did no patients have a diagnosis of nafld , but patients were encouraged to drink less than 2 units of alcohol per day , some of whom had been drinking more prior to the trial . alt levels and other markers of metabolic syndrome decreased over the trial period in the lifestyle group ( compared to those in the control group ) , all to a greater extent at 12 months than when reviewed at 6 months . over a 12 week period , the 58 patients were randomized to either weight loss by calorie restriction ( deficit of 400900 calories per day ) , weight loss by calorie restriction and exercise ( 40 minute bike ride on alternate days ) , and a control group , a design that is similar to larson - meyer et al13 and shah et al.12 the calorie restriction alone group lost on average 7.6% with a corresponding decrease in alt of 20% and ggt of 28% . fifty - nine patients , with us diagnosed nash , were recruited into a 3-month trial involving an exercise program ( 45 minutes of exercise five times a week ) , and a calorie controlled diet for those with a high bmi ( 25 calories per kg of their ideal body weight ) . forty - four patients were compliant to this exercise and diet regime , and those taking part in diet and exercise lost on average 3.1 kg , while those taking part in just the diet lost 0.2 kg , and no significant decrease in weight was seen in the non - compliant group . alt and aspartate aminotransferase ( ast ) levels decreased significantly in the compliant group ; therefore , this paper draws the conclusion that weight loss is beneficial regardless of exercise participation . the results of this paper therefore contradicts shah et al12 and straznicky et al,17 which stated that exercise did not play an important part in the improvement of liver steatosis markers , as in this trial exercise does seem to be an important factor . the paper follows the diabetic prevention program22 in which participants had been randomized to either 16 sessions of lifestyle interventions , metformin , troflitazone ( which was withdrawn due to side effects ) , or placebo . rodrguez - hernndez et al26 again uses a low fat and low carbohydrate diet to assess their effect on aminotransferase , and this trial is nicely followed up by haufe et al.27 the rodrguez - hernndez et al26 trial included 54 women , with us diagnosed nafld , and randomized them to a low fat ( 25% protein , < 10% fat , 54% carbohydrate ) or low carbohydrate ( 27% protein , 28% fat , 45% carbohydrate ) diet for a period of 6 months . at the end of the trial , those on the low carbohydrate diet lost 5.7% of their body weight and those in the low fat group 5.5% , a non - significant result . in this trial , the diet design was low carbohydrate ( 90 g of carbohydrate and 0.8 g protein per kg weight , 30% fat ) and low fat ( 20% fat , 0.8 g of protein per kg , the remainder carbohydrate ) . at the beginning of the trial , 45% of the patients had been diagnosed with metabolic syndrome and in the end there were only 10% , all in the non - adherent group . the paper was a retrospective study looking at 813 biopsy results of patients with nafld and assessed if they were taking part in the recommended guidelines for exercise put forward by the united states department of health and human services . analysis of the biopsies showed that meeting the vigorous activity guidelines decreases your odds ratio ( or ) of having nash ( or : 0.65 ) ; the same was not true for moderate activity . biopsies also showed that those meeting vigorous activity guidelines had significantly lower odds ratio fibrosis compared to no fibrosis ( or : 0.53 ) ; again , the same was not true for moderate activity . the effect of a short prescribed program of endurance exercise was assessed for improvements in liver fat content in a cohort including lean and obese individuals by tarnopolsky et al.34 the study design precluded weight loss as an end point , instead focusing on participation in a moderate intensity program of endurance exercise ( stationary bike cycling ) and assessing its effect on liver fat content principally . after a 3-month period of endurance exercise , improvements in fitness ( maximal oxygen consumption ) and reduction in waist circumference were noted in both the lean and obese groups , regardless of sex . this study is limited by the small numbers of patients included and by the apparent liver health of the group the obese men had a pre - exercise alt of 30.34.8 u / l and the obese women 16.82.2 no measurement of lipid profile was discussed ; early changes in triglyceride content may have supported and encouraged participation in a longer program . both have a similar design with a low intensity lifestyle intervention group ( three counseling sessions throughout the trial ) , moderate intensity lifestyle intervention group ( six counseling sessions throughout the trial ) , a long term group ( receiving the same initial intervention as the moderate group but with further sessions up to 12 months ) , and a control group . us was performed on the patients at the beginning and end of the trial , and fat liver content was shown to decrease in both groups but again no significant difference between the two groups was found . monitoring of the diet and exercise was done by self - reporting and biopsies were taken at baseline and at the end of the 24 week trial , which were examined by a pathologist . akcam et al43 performed a small randomized double blind parallel group clinical trial assessing the effect of diet and exercise alone or in combination with either metformin or vitamin e on various markers of nafld , including ultrasonographically assessed liver steatosis , insulin resistance , lipid profile , and bmi . at the conclusion of the study period , repeat liver us was performed , with the metformin group showing the greatest improvement almost double that of the diet and exercise alone group and the vitamin e group . however , although the authors highlight the improvements seen with metformin in particular , the diet and exercise alone group did achieve an improvement in bmi supporting the role of this fundamental intervention in a young cohort . two of the studies were cross - sectional analyses , bae et al35 and gerber et al,38 both of these showed a strong relationship between the proportion of the population with nafld and the low level of activity that they took part in . in the medication intervention group there were four studies : mndez - snchez et al40 using ursodeoxycholic acid , vilar gomez et al41 using viusid ( an antioxidant nutritional supplement ) , krakoff et al42 using metformin , and akcam et al43 compared metformin and vitamin e. all the studies apart from the one by vilar gomez et al41 concluded that there was no particular benefit from their intervention and that weight loss by diet and exercise was the most important factor . practice guidelines set forth by key stakeholders in nafld published in 2012 endorse the vital role of lifestyle intervention in the management of nafld.44 weight loss either by hypocaloric diet or in combination with increased physical activity to attain a 3%5% loss in body weight is required to improved steatosis , with up to 10% loss of body weight required to improve necroinflammation .

type 1 diabetes mellitus ( t1 dm ) is identified by the progressive autoimmune destruction of pancreatic beta cells , which results in a dramatic decrease of insulin production and consequent metabolic complications . transplantation of human cadaveric pancreas or allogeneic islet cells could be considered therapeutic in this condition . in addition , replacement of the beta cell deficit along with regulation of autoimmune response to cells that express insulin is crucial for a t1 dm definitive cure . thus , in recent years , the usage of cell sources that modulate immune system along with islet cell replacement has received much attention . mesenchymal stem cells ( mscs ) represent a rare heterogeneous subset of multipotent stromal cells localized in many different adult and fetal tissues . they have self - renewal and multidifferentiation capacity that can give rise to diverse lineages of mesenchymal origin , including osteoblasts , adipocytes , and chondrocytes , and have also shown their potential for differentiating into nonmesodermal origin cells . due to these properties , mscs might be useful in tissue regeneration and cell - based therapies . although multipotent mscs are usually isolated from bone marrow ( bm ) , more recently , adipose tissue - derived mscs ( at - mscs ) due to more quantities , simple accessibility , and also the better immunomodulatory properties were represented as another alternative source for mscs [ 5 , 6 ] . numerous recent studies indicated that mscs possess immunomodulatory or immunosuppressive effects both in vitro and in vivo on several immune cells , not only t lymphocytes but also on b lymphocytes , dendritic cells ( dcs ) , and nk cells . in vitro studies have identified that the immunomodulatory function of mscs can be addressed by both cell - cell contact and soluble factors [ 9 , 10 ] . mscs can inhibit immune cells proliferation , reduce inflammatory cytokines secretion , and alter immune cell types to regulatory clones . they exert immune regulation by the secretion of anti - inflammatory factors , such as interleukin-10 ( il-10 ) , transforming growth factor- ( tgf- ) , indoleamine 2,3-dioxygenase ( ido ) [ 12 , 13 ] , nitric oxide , prostaglandin - e2 ( pge-2 ) , and human leukocyte antigen - g ( hla - g ) . in addition , mscs induce cell cycle arrest and apoptosis of t lymphocytes [ 11 , 16 ] and cause lymphocytes to secrete regulatory cytokines , especially il-10 . mesenchymal stem cells due to their low immunogenicity and immunomodulatory properties as well as high degree of differentiation and proliferation potential might be useful in inhibiting the autoimmunity and regenerating the insulin - secreting cells . furthermore , many studies declared that the regenerative role of mscs could be mediated by protective effects on functional islet cells and also differentiation potency to insulin - producing cells in vivo and in vitro . the ideal use of mscs in autoimmune diabetes regenerative therapy can only be obtained after we learn their immunomodulatory characteristics in detail . the aim of this research was to evaluate the immunomodulatory effects of adipose - derived mscs on autoimmune response besides islet protective function in streptozotocin- ( stz- ) induced diabetic mice model . this study may provide some basic information regarding application of human mscs in treating type 1 diabetes . female inbred c57bl/6 mice ( 68 weeks old ) were purchased from the pasteur institute , tehran , iran . diabetes was induced in the mice by intraperitoneal injection of multiple low - doses ( 50 mg / kg , 4 consecutive days ) of streptozotocin ( stz ) ( sigma , usa ) . stz was solubilized in the sodium citrate buffer , ph 4.5 , and injected within 10 min of preparation . abdominal adipose tissue was taken from c57bl/6 mice after sacrificing , washed 3 times with phosphate - buffered saline ( pbs ) , and minced . extracellular matrix was digested with 0.075% type i collagenase ( 37c and 5% co2 for 30 min ) and centrifuged at 500 g for 5 min ; then the pellet was cultured in high glucose dulbecco 's modified eagle 's medium ( dmem , gibco ) containing 10% fetal bovine serum ( fbs , gibco ) , 2 mm l - glutamine , penicillin , and streptomycin ( all from invitrogen ) as mscs culture media and incubated at 37c in 5% co2 . after 48 h , nonadherent cells were removed and fresh media were added . when adherent cells were confluent , they were trypsinized , harvested , and expanded . the cell surface markers on at - mscs were assessed using monoclonal antibodies against mouse cd73 , cd105 , cd29 , cd90 , cd31 , cd11b , cd45 , and cd34 ( all from ebioscience ) . the at - mscs at passage 3 were detached with 0.25% trypsin / edta and resuspended to 5 10 cells in pbs . the cells were incubated with the specific or isotype control antibodies ( mouse igg1-fitc and mouse igg1-pe , ebioscience ) in 100 l of 3% bovine serum albumin ( bsa , sigma ) in pbs for 1 hour at 4c . the cells were then fixed with 1% paraformaldehyde ( sigma ) and analyzed using a facs calibur flow cytometer ( bd biosciences , san diego , ca ) and cyflogic software ( cyflo ltd . ) . the at - mscs at passage 3 were analyzed for their ability to differentiate into osteoblast , adipocyte , and chondrocyte . for osteogenic differentiation , cells were cultured in medium containing 10 mm beta - glycerophosphate ( merck ) , 50 mg / ml ascorbic acid biphosphate ( sigma ) , and 100 nm dexamethasone ( sigma ) . after 3-week induction , cells were stained with alizarin red to assess mineralization . for adipogenic differentiation , cells were cultured in the presence of 250 nm dexamethasone ( sigma ) , 0.5 mm 3-isobutyl-1-methylxanthine ( sigma ) , 5 mm insulin ( sigma ) , and 100 mm indomethacin ( sigma ) . after 3 weeks oil red staining was used to determine the accumulation of oil droplets in the cytoplasm . for differentiation to chondrocytes , 1 10 cells were centrifuged to form a pelleted micromass and then treated with tgf - beta ( 10 ng / ml ; merck ) , ascorbic acid biphosphate ( 50 g / ml ) , dexamethasone ( 10 m ) , and insulin transferrin selenium ( its ) ( 50 g / ml ; sigma ) for 3 weeks . chondrocyte differentiation was assessed by alcian blue staining on sections obtained from micromasses . briefly , pancreas was excised from c57bl/6 mice under sterile conditions , inflated by collagenase type xi ( 1 mg / ml ; sigma ) for few moments , and minced into small pieces . the enzymatic digestion of the pancreatic tissue was fulfilled by collagenase type xi in 37c water bath for 20 min with interim agitation . then , digested contents were filtered through 500 and 100 m cell strainers , respectively , to capture the islets and allow the small exocrine cells to pass . to evaluate islet cell function , islets ( n = 10 ) were stimulated in rpmi-1640 culture solution with low glucose ( 5.6 mmol / l ) and incubated for 4 hours at 37c for detection of the total levels of insulin in the culture solution . the rpmi-1640 culture solution was then switched to high glucose ( 16.7 mmol / l ) and culture performed under the same condition ( 37c , 4 hours ) for insulin determination . islet cells lysate was prepared by freezing and thawing 10 islets in 0.5 ml of rpmi-1640 medium supplemented with 10% fbs ( assuming one islet contains 1000 single cells ) . the spleen was removed from the normal and diabetic mice and placed in cold rpmi-1640 media . splenocytes were extracted using a 5 ml syringe with a 23 g needle . cell suspensions were washed in cold rpmi-1640 media and counted and viability was assessed by 0.2% trypan blue . rpmi-1640 supplemented with 10% heat inactivated fbs , 100 mg / ml streptomycin , 100 units / ml penicillin , 2 mm l - glutamine , and 10 mm hepes was used as splenocyte culture medium . in proliferation assay , normal and diabetic splenocytes were cocultured with at - mscs in the mscs culture medium mixed 1 : 1 with fresh splenocyte culture medium ( mixed culture medium ) . prior to final plating , optimized concentration of splenocytes with or without phytohemagglutinin ( pha , gibco ) was determined at dilutions of 1 , 2 , 3 , 4 , and 5 10 cells in 96-well plate by mtt assay . final density of splenocytes was adjusted to 2.5 10 cells per well for coculture with at - mscs . at - mscs at passage 3 were harvested and adjusted to 2 10/ml , 1 10/ml , and 5 10/ml in mscs culture medium containing 10% fbs . a 100 l suspension of at - mscs was plated into 96-well plates and incubated for 24 h at 37c . after the at - mscs reached 70% confluence , the medium was removed , and 100 l of fresh medium containing 5 l of mitomycin - c ( 1 g/l ; sigma ) was added for 1 h at 37c to mitotically inactivate the at - mscs . after that medium was removed and inactivated at - mscs were washed twice with pbs . at - mscs were resuspended in 100 l of mixed culture medium , cocultured with 2.5 10 splenocytes , and stimulated by 5 g / ml pha ( 10 l ) or 10 l of islet cells lysate in control and test groups , respectively , for 48 h at 37c . the test and control groups in proliferation experiments were designed as in table 1 representing the content of each well in culture plate . three ratios of at - mscs to splenocytes were used : 1 : 50 , 1 : 250 , and 1 : 1250 . the suppressive effect of at - mscs on splenocytes proliferation in the absence and presence of pha ( as nonspecific stimulator ) and islet cells lysate ( as specific stimulator ) was determined by mtt [ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide ] assay . the cells , cultured in a 96-well plate , were incubated for 4 h in the presence of mtt ( 5 mg / ml ; sigma ) followed by addition of 0.1 ml dimethyl sulfoxide ( dmso ) . the formazan crystals were dissolved and the absorbance was read at 570 nm by elisa reader . the proliferation index was calculated using the following formula:(1)proliferation index = odmscs+splenocytesstimulatorodmscsodsplenocytes.in order to examine the proliferation of splenocytes ( in the presence of at - mscs ) , at - mscs monoculture was used as blank to subtract background absorbance of at - mscs . to provide the optimum condition , the mtt assay was repeated several times with different ratios of at - mscs to splenocytes , various concentrations of pha , and also different periods of coculture incubation . after being trypsinized , at - mscs were adjusted to 2.5 10/ml and plated ( 1 ml ) in 24-well plates . after the at - mscs reached 70% confluence , 10 l of mitomycin - c ( 1 g/l ) was added into each well . after 1 h at 37c and 5% co2 , the medium was removed and cells were washed twice with pbs . then 12.5 10 splenocytes in 1 ml of mixed culture medium were added and cocultured in the presence of 50 l of pha or 50 l islet lysate for 72 h at 37c . the supernatants of each group were collected and evaluated by multi - analyte elisarray kit ( mem-003a , sabiosciences ) for tgf- , il-10 , il-4 , and il-13 as regulatory cytokines and ifn- , il-2 , and il-17a as inflammatory cytokines . in order to examine cytokine production by splenocytes , at - mscs were used as blank to subtract background absorbance of the cytokines produced by at - mscs . at - mscs at passage 3 after reaching the ideal confluency , the medium was removed and cells were washed twice with pbs . then 2.5 10 normal and diabetic splenocytes were added and cocultured with 10 freshly isolated islets ( assuming one islet contains 1000 single cells ) in 100 l of mixed culture medium ( low glucose rpmi-1640 and high glucose dmem ) supplemented with 10% fbs for 24 h at 37c . after incubation period , the medium was removed slowly ; then the islets were challenged with 100 l stimulatory medium containing high glucose dmem supplemented with 5 mmol / l theophylline for a 10 min period . the supernatants of each group were collected and evaluated by mouse insulin elisa kit ( ezrmi-13k , millipore ) . the groups were designed as follows : islet cells ( positive control ) , islet cells + normal splenocytes , islet cells + diabetic splenocytes , islet cells + normal splenocytes + mscs , islet cells + diabetic splenocytes + mscs , and splenocytes ( negative control ) . statistical analysis was done using the one - way analysis of variance ( anova ) to compare results . in this study , diabetic mice model was developed by administration of multiple low - doses of stz . the blood glucose levels of 300 mg / dl were monitored within 1 week of stz treatment . in addition , the insulin levels of 4.95 0.52 ng / dl in normal mice decreased to < 0.5 ng / dl in diabetic mice and pancreatic islets destruction was confirmed by histopathological examination . mscs seeded to the culture flasks sparsely and the cells displayed a fibroblast - like morphology during the early days of incubation . after 68 days , the cells gradually grew to form small colonies that were termed colony - forming units . as growth continued , colonies gradually expanded in size and the adjacent ones interconnected with each other . these primary cells reached monolayer confluence after plating for 1012 days in their first passages . in later passages , mscs appeared to adopt a uniform fibroblast - like morphology . at - mscs at passage 3 the cells lacked cd11b , cd34 , cd45 , and cd31 whereas they were all positive for cd73 , cd90 , cd105 , and cd29 expression . moreover , at - mscs were able to differentiate toward osteogenic , adipogenic , and chondrogenic lineages . after 21 days , osteogenesis of at - mscs was demonstrated by mineralization of the extracellular matrix with alizarin red staining . additionally , lipid droplets were detectable by oil red o staining after three weeks of adipocytic induction . after 21 days of induction , chondrogenic differentiation of at - mscs was achieved . more than 80% of all cells stained positively with alcian blue showed the glucose amino glycan ( gag ) biosynthesis in the cell pellets ( a figure illustrating at - mscs characterization has been presented as supplementary data in supplementary material available online at http://dx.doi.org/10.1155/2015/878535 ) . the perfusion of pancreas through common bile duct is a complicated manipulation and needs professional experience . moreover , purification of islets by ficoll gradient method could have toxic side effects on islet cells . in this study , we modified the procedure by excluding the in situ pancreas perfusion and ficoll gradient steps . the islet count showed that the efficiency of isolation varied depending on animal age and strain . however , the duration of whole procedure decreased to 3040 min for each mouse . the purified islets showed intact morphology with smooth surface and were free of exocrine cells as confirmed with dtz staining . this experiment was designed to investigate whether at - mscs could inhibit proliferation of splenocytes that were triggered by pha as a nonspecific stimulator . firstly , to assess which concentration of splenocytes exerts optimized proliferation , we cultured resting cells at dilutions of 1 , 2 , 3 , 4 , and 5 10 in the absence or presence of pha . after drawing standard curve , the final density of normal splenocytes was adjusted to 2.5 10 cells for coculture with at - mscs . to evaluate antiproliferative effect of at - mscs , they were added to pha - stimulated splenocytes cultures with a ratio of at - mscs to splenocytes of 1 : 50 , 1 : 250 , and 1 : 1250 and cell proliferation was determined . as expected , the resting splenocytes showed a strong proliferation response in the presence of pha ( figure 1 ; p < 0.05 ) , whereas addition of at - msc to pha - stimulated splenocytes resulted in a significant reduction of splenocytes proliferation in a number - dependent manner ( figure 1 ; p < 0.05 ) . higher inhibition of splenocytes proliferation was shown at the lowest dilution tested ( 1 : 50 ) . the results also indicated that at - mscs could induce cell death of some splenocytes in the absence of mitogen ( figure 1 ; p < 0.05 ) . the greater cell death of splenocytes seemed to occur at the highest density of at - mscs ( 1 : 50 ) . in mitogen - induced proliferation experiment , no significant difference was detected between normal and diabetic splenocytes in the pattern of reduced proliferation and response to mitogen . after confirming that at - mscs could inhibit proliferation of mitogen - stimulated splenocytes , we then investigated whether at - mscs could inhibit proliferation of splenocytes triggered by specific stimulator . at - mscs were cocultured with islet lysate - stimulated splenocytes as previously described and cell proliferation was assessed . in contrast , diabetic cells showed a significant proliferation ( figure 2 ; p < 0.05 ) . again , the dose - dependent effect of at - mscs on diabetic splenocytes proliferation inhibition was observed in presence of islet lysate ( figure 2 ; p < 0.05 ) . at the lowest dilution tested ( 1 : 50 ) , more significant inhibition of splenocytes proliferation was observed . however , the results demonstrated that at - mscs could induce cell death in some of these splenocytes ( figure 2 ; p < 0.05 ) . to determine whether at - mscs modulate cytokine secretion by splenocytes , we prepared a coculture design with splenocytes supplemented by pha or islet cells lysate . analysis of cytokines production by resting at - mscs showed that they consistently secreted tgf- and il-6 in higher levels compared to other cytokines ( figure 3 ; p < 0.05 ) . as previously described , at - mscs were used as blank to subtract background absorbance of their cytokines production . the results demonstrated that addition of at - mscs to lysate - stimulated diabetic splenocytes significantly decreased ifn- , il-2 , and il-17 production by splenocytes ( figure 4 ; p < 0.05 ) . the similar results were obtained when at - mscs were added to pha - triggered splenocytes . in addition , this experiment indicated that at - mscs significantly increased tgf- , il-10 , il-4 , and il-13 production by splenocytes ( figure 5 ; p < 0.05 ) . although the increased production of these cytokines was also observed in the presence of pha , increasing levels were significantly prominent in at - mscs cocultured with lysate - stimulated diabetic splenocytes , particularly for il-13 ( figure 5 ) . to determine protective ability of at - mscs on islet cells function this experiment indicated that the crude islets alone secreted significant insulin levels when induced with stimulatory medium ( 6.45 0.69 ng / ml ) . however , diabetic splenocytes significantly decreased insulin secretion by islets ( 2.59 0.27 ng / ml ; p < 0.05 ) . in contrast , coculture with at - mscs led to a significant improvement in insulin secretion and the viability of islet cells ( 3.87 0.41 ng / ml versus 2.59 0.27 ng / ml ; p < 0.05 ) ( figure 6 ) . mscs also referred to as adult multipotent stem cells represent a fibroblast - like morphology that have not only the capacity to differentiate into several tissues , but also have immunomodulatory and anti - inflammatory effects in vivo and in vitro [ 3 , 21 ] . in this regard , previous studies have shown that mscs induce peripheral tolerance and migrate to injured tissues , where they can inhibit release of proinflammatory cytokines and promote the survival of damaged cells [ 2224 ] . it has also been reported that the underlying mechanism of mscs effects might be attributed by both cell - cell contact and soluble factors [ 9 , 10 ] . many studies that declared protective effects of mscs on islet mass and differentiation potency to insulin - producing cells in vivo and in vitro collaborate for the application of mscs regenerative role in t1 dm [ 2527 ] . although many researchers have demonstrated the immunomodulatory activities of mscs , in vitro potential assays able to predict or to correlate with therapeutic outcome of mscs are still partially known . thus , we assessedin vitro immunoregulatory effects of adipose - derived mscs on autoimmune response in experimental diabetic model . in this respect , we aimed at three main goals : firstly , to evaluate suppressive effect of at - mscs on autoreactive splenocytes proliferation ; secondly , to assess immunomodulatory effect of at - mscs on splenocytes cytokine patterns ; and finally , to evaluate protective effect of at - mscs on functional islets . in the current study , mscs were extracted from adipose tissue of healthy c57bl/6 mice and cultured to proliferate . diabetic c57bl/6 mouse model was prepared by administration of consecutive low - doses of stz and diabetic state was confirmed by serum glucose ( > 300 mg / dl ) , insulin levels , and pancreas histopathology . pancreatic islets were isolated from normal mice and splenocytes prepared from normal and diabetic mice . to evaluate antiproliferative effect of mscs , they were cocultured with diabetic splenocytes in the presence of islet lysate and proliferation assayed by mtt technique . the impact of mscs on pancreatic islet function was assayed by insulin measurement in direct cocultures of diabetic splenocytes with intact islets . the effect of mscs on cytokine patterns produced by splenocytes was assayed in cocultured splenocytes with mscs in the presence of islet lysate for 72 hours . inflammatory and regulatory cytokines were assayed in culture media by elisa technique . in our study , a stz - induced diabetic model was prepared . some strains of mice are th1-dominant ( c57bl/6 , dba/2 , akr , and cba ) , but the others are th2-dominant strains ( balb / c , bp2 , and a / j ) . many studies indicated that pathogenesis of t1 dm is an autoimmune disease mediated by th1-dependent inflammatory reaction [ 29 , 30 ] . therefore , in this study the selection of c57bl/6 mice as a suitable model for induction of autoimmune diabetes appears to be reasonable . high doses of stz selectively kill the insulin - producing beta cells , whereas multiple low - doses of stz induce expression of glutamic acid decarboxylase ( gad ) autoantigen and develop an autoimmune response . the shared immunophenotype and differential characteristics were identified in mscs isolated from different tissues . although some reports have shown that adipose - derived mscs due to more simple isolation and expansion and also relative better immunomodulatory effects are a novel promise in experimental and clinical application [ 5 , 32 ] , isolated at - mscs had fibroblast - like morphology and differential capacity that were consistent with previous studies . in addition , analysis of cell surface markers on at - mscs showed a lack of hematopoietic markers , such as cd34 and cd45 , and high expression of cd90 and cd29 . however , some reports have shown that expression of surface markers on at - mscs varied in different strains of mice . the biological characteristics similar to bone marrow mesenchymal stem cell ( bm - mscs ) indicate that at - mscs may present an alternative source for future application [ 34 , 35 ] . one of the main goals in this research was close to assessment of at - mscs suppressive effect on splenocytes proliferation in the presence of specific ( islet cells lysate ) and nonspecific ( pha ) stimulators . ( 1 ) the results indicated that at - mscs in a ratio of 1 : 50 , 1 : 250 , and 1 : 1250 to splenocytes could inhibit stimulated splenocytes proliferation in a dose - dependent manner with the most inhibition at the lowest dilution tested ( 1 : 50 ; p < 0.05 ) . however , at - mscs could induce cell death in some splenocytes in this concentration ( 1 : 50 ; p < 0.05 ) . the similar effects of at - mscs were observed in the presence of islet lysate and pha . in this respect , other studies have identified that mouse and human mscs harvested from different tissues can suppress t lymphocyte proliferation stimulated by allogeneic cells or mitogens such as pha [ 10 , 11 , 36 , 37 ] . in a recent study , it has been shown that mscs isolated from nod and balb / c mice could inhibit t cd4 proliferation in full mismatch mixed lymphocyte reaction ( mlr ) assay . it has also been clarified that , in an autoimmune fashion in vitro , when mscs are added to autoreactive t cd4 and allogeneic dendritic cells in the presence of islet peptides , they could significantly suppress autoreactive t cd4 proliferation . several reports have demonstrated that immunosuppressive effects of mscs occur in a dose - related manner in which it is considered independent of mhc interaction [ 3941 ] . although target cell - msc interactions may be an important factor , the immunosuppressive effect of mscs can also be mediated through the secretion of soluble molecules such as il-10 , tgf- , ido , no , pge-2 , and hla - g that are triggered following cross talk with target cells . ( 2 ) at - mscs caused some cell death in resting splenocytes without each type of stimulators ( p < 0.05 ) . this may suggest that mscs can induce cellular death through cell - cell contact as well as secretion of no and il-10 [ 9 , 11 ] ; also , they can drive splenocytes to produce il-10 . although , in accordance with previous studies , we suggest that mscs may induce cell death of splenocytes rather than cell cycle arrest , complementary studies are required to identify molecular mechanisms in detail . ( 3 ) in the absence of at - mscs , normal and diabetic splenocytes in response to nonspecific stimulator ( pha ) showed similar fashion . in contrast , in response to islet lysate normal splenocytes were not responsive , whereas a sharp proliferation was obtained in diabetic splenocytes ( p < 0.05 ) . in the next stage , we aimed at evaluating the effect of at - mscs on cytokine patterns produced by splenocytes . analysis of cytokines production by at - mscs alone showed that they consistently secreted tgf- and il-6 in higher levels compared to other cytokines ( p < 0.05 ) . several publications have confirmed spontaneous secretion of tgf-1 , il-6 , hepatocyte growth factor ( hgf ) , hla - g , and pge-2 that associated with immunomodulatory properties of mscs [ 42 , 43 ] . our in vitro experiment showed that at - mscs decreased production of ifn- , il-2 , and il-17 and increased production of tgf- , il-4 , il-10 , and il-13 by stimulated splenocytes in response to islet lysate or pha ( p < 0.05 ) . however , the increases in regulatory cytokines were significantly prominent in at - mscs cocultured with lysate - stimulated diabetic splenocytes , particularly for il-13 . pathogenesis of many autoimmune diseases was mediated by several types of helper t lymphocytes ( th ) such as th1 , th2 , and th17 identified by secretion of distinct cytokine profiles . th1 cells produced ifn- , il-2 , and tnf- and th2 cells secreted il-4 , il-5 , il-6 , il-10 , and il-13 . regulatory t cells and th3 cells produce tgf- and il10 and another t helper subtype ( th17 ) produces il-17 . it has been demonstrated that mscs have capacity to modulate immune reaction in vitro and in vivo , where they polarize proinflammatory state to anti - inflammatory state . this is mediated by a shift in the th1 to th2 cell balance or by inhibition of th17 differentiation and il-17 production . recent publications have reported the in vivo transplantation of mscs derived from bone marrow and adipose tissue along with a therapeutic approach , where they switch host immune response towards a th2 cytokine profile by downregulating th1 cytokines and/or upregulating th2 cytokines [ 22 , 4548 ] . finally , in this study we used other approaches of mscs properties to maintain in vitro viability and functionality of purified islets in the presence of reactive splenocytes . in this approach , it was found that at - mscs in coculture design had ability to provide islet functionality , proven by protected insulin secretion in coculture medium ( p < 0.05 ) . several studies examined in vivo immunoregulatory effects of mscs in cotransplantation with islets , which resulted in enhancing of islet allograft survival , insulin secretion , and sustained normoglycemia in stz - induced diabetic model [ 43 , 49 , 50 ] . in a recent study performed by karaoz et al . coculturing of normal and stz - injured rat pancreatic islets with rat bm - mscs has improved function and viability of islets . they showed that cocultivation of stz - injured islets and mscs increased expression of il-6 and tgf-1 in the culture medium besides the expression of antiapoptotic genes . they also demonstrated that cytoprotective , anti - inflammatory , and antiapoptotic effects of mscs were mediated through paracrine actions ( spontaneous production of il-6 , tgf-1 , and hgf ) . furthermore , effectiveness of mscs in prevention and treatment of diabetes in nod mice has also been reported . the protective effects of mscs may be dependent on both soluble mediators and also triggering of resident tissue precursor cells in vivo . however , many studies declared that the regenerative role of mscs could be mediated by differentiation potential to insulin - secreting cells in vivo and in vitro [ 18 , 25 ] . in brief , we have demonstrated that at - mscs in a dose - dependent manner inhibit splenocytes proliferative response to specific and nonspecific triggers that may be mediated by both cell - cell contact and soluble mediators such as tgf-1 and il-10 . at - mscs finally , our results indicated that at - mscs through cytoprotective and anti - inflammatory effects can effectively sustain viability as well as insulin secretion potential of islet cells in the presence of reactive splenocytes . although we note that adjunct in vivo studies are required to completely investigate the immunomodulatory functions of mscs in autoimmune diseases , this study may provide some basic information regarding application of human mscs in treating type 1 diabetes .

regenerative and immunomodulatory properties of mesenchymal stem cells ( mscs ) might be applied for type 1 diabetes mellitus ( t1 dm ) treatment . thus , we proposed in vitro assessment of adipose tissue - derived mscs ( at - mscs ) immunomodulation on autoimmune response along with beta cell protection in streptozotocin- ( stz- ) induced diabetic c57bl/6 mice model . mscs were extracted from abdominal adipose tissue of normal mice and cultured to proliferate . diabetic mice were prepared by administration of multiple low - doses of streptozotocin . pancreatic islets were isolated from normal mice and splenocytes prepared from normal and diabetic mice . proliferation , cytokine production , and insulin secretion assays were performed in coculture experiments . at - mscs inhibited splenocytes proliferative response to specific ( islet lysate ) and nonspecific ( pha ) triggers in a dose - dependent manner ( p < 0.05 ) . decreased production of proinflammatory cytokines , such as ifn- , il-2 , and il-17 , and increased secretion of regulatory cytokines such as tgf- , il-4 , il-10 , and il-13 by stimulated splenocytes were also shown in response to islet lysate or pha stimulants ( p < 0.05 ) . finally , we demonstrated that at - mscs could effectively sustain viability as well as insulin secretion potential of pancreatic islets in the presence of reactive splenocytes ( p < 0.05 ) . in conclusion , it seems that mscs may provide a new horizon for t1 dm cell therapy and islet transplantation in the future .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

type 1 diabetes mellitus ( t1 dm ) is identified by the progressive autoimmune destruction of pancreatic beta cells , which results in a dramatic decrease of insulin production and consequent metabolic complications . in addition , replacement of the beta cell deficit along with regulation of autoimmune response to cells that express insulin is crucial for a t1 dm definitive cure . mesenchymal stem cells ( mscs ) represent a rare heterogeneous subset of multipotent stromal cells localized in many different adult and fetal tissues . although multipotent mscs are usually isolated from bone marrow ( bm ) , more recently , adipose tissue - derived mscs ( at - mscs ) due to more quantities , simple accessibility , and also the better immunomodulatory properties were represented as another alternative source for mscs [ 5 , 6 ] . numerous recent studies indicated that mscs possess immunomodulatory or immunosuppressive effects both in vitro and in vivo on several immune cells , not only t lymphocytes but also on b lymphocytes , dendritic cells ( dcs ) , and nk cells . they exert immune regulation by the secretion of anti - inflammatory factors , such as interleukin-10 ( il-10 ) , transforming growth factor- ( tgf- ) , indoleamine 2,3-dioxygenase ( ido ) [ 12 , 13 ] , nitric oxide , prostaglandin - e2 ( pge-2 ) , and human leukocyte antigen - g ( hla - g ) . mesenchymal stem cells due to their low immunogenicity and immunomodulatory properties as well as high degree of differentiation and proliferation potential might be useful in inhibiting the autoimmunity and regenerating the insulin - secreting cells . the aim of this research was to evaluate the immunomodulatory effects of adipose - derived mscs on autoimmune response besides islet protective function in streptozotocin- ( stz- ) induced diabetic mice model . this study may provide some basic information regarding application of human mscs in treating type 1 diabetes . diabetes was induced in the mice by intraperitoneal injection of multiple low - doses ( 50 mg / kg , 4 consecutive days ) of streptozotocin ( stz ) ( sigma , usa ) . abdominal adipose tissue was taken from c57bl/6 mice after sacrificing , washed 3 times with phosphate - buffered saline ( pbs ) , and minced . the cell surface markers on at - mscs were assessed using monoclonal antibodies against mouse cd73 , cd105 , cd29 , cd90 , cd31 , cd11b , cd45 , and cd34 ( all from ebioscience ) . for adipogenic differentiation , cells were cultured in the presence of 250 nm dexamethasone ( sigma ) , 0.5 mm 3-isobutyl-1-methylxanthine ( sigma ) , 5 mm insulin ( sigma ) , and 100 mm indomethacin ( sigma ) . the spleen was removed from the normal and diabetic mice and placed in cold rpmi-1640 media . in proliferation assay , normal and diabetic splenocytes were cocultured with at - mscs in the mscs culture medium mixed 1 : 1 with fresh splenocyte culture medium ( mixed culture medium ) . at - mscs at passage 3 were harvested and adjusted to 2 10/ml , 1 10/ml , and 5 10/ml in mscs culture medium containing 10% fbs . after the at - mscs reached 70% confluence , the medium was removed , and 100 l of fresh medium containing 5 l of mitomycin - c ( 1 g/l ; sigma ) was added for 1 h at 37c to mitotically inactivate the at - mscs . after that medium was removed and inactivated at - mscs were washed twice with pbs . at - mscs were resuspended in 100 l of mixed culture medium , cocultured with 2.5 10 splenocytes , and stimulated by 5 g / ml pha ( 10 l ) or 10 l of islet cells lysate in control and test groups , respectively , for 48 h at 37c . three ratios of at - mscs to splenocytes were used : 1 : 50 , 1 : 250 , and 1 : 1250 . the suppressive effect of at - mscs on splenocytes proliferation in the absence and presence of pha ( as nonspecific stimulator ) and islet cells lysate ( as specific stimulator ) was determined by mtt [ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide ] assay . the cells , cultured in a 96-well plate , were incubated for 4 h in the presence of mtt ( 5 mg / ml ; sigma ) followed by addition of 0.1 ml dimethyl sulfoxide ( dmso ) . the proliferation index was calculated using the following formula:(1)proliferation index = odmscs+splenocytesstimulatorodmscsodsplenocytes.in order to examine the proliferation of splenocytes ( in the presence of at - mscs ) , at - mscs monoculture was used as blank to subtract background absorbance of at - mscs . after being trypsinized , at - mscs were adjusted to 2.5 10/ml and plated ( 1 ml ) in 24-well plates . then 12.5 10 splenocytes in 1 ml of mixed culture medium were added and cocultured in the presence of 50 l of pha or 50 l islet lysate for 72 h at 37c . the supernatants of each group were collected and evaluated by multi - analyte elisarray kit ( mem-003a , sabiosciences ) for tgf- , il-10 , il-4 , and il-13 as regulatory cytokines and ifn- , il-2 , and il-17a as inflammatory cytokines . in order to examine cytokine production by splenocytes , at - mscs were used as blank to subtract background absorbance of the cytokines produced by at - mscs . then 2.5 10 normal and diabetic splenocytes were added and cocultured with 10 freshly isolated islets ( assuming one islet contains 1000 single cells ) in 100 l of mixed culture medium ( low glucose rpmi-1640 and high glucose dmem ) supplemented with 10% fbs for 24 h at 37c . the groups were designed as follows : islet cells ( positive control ) , islet cells + normal splenocytes , islet cells + diabetic splenocytes , islet cells + normal splenocytes + mscs , islet cells + diabetic splenocytes + mscs , and splenocytes ( negative control ) . in this study , diabetic mice model was developed by administration of multiple low - doses of stz . in addition , the insulin levels of 4.95 0.52 ng / dl in normal mice decreased to < 0.5 ng / dl in diabetic mice and pancreatic islets destruction was confirmed by histopathological examination . moreover , at - mscs were able to differentiate toward osteogenic , adipogenic , and chondrogenic lineages . firstly , to assess which concentration of splenocytes exerts optimized proliferation , we cultured resting cells at dilutions of 1 , 2 , 3 , 4 , and 5 10 in the absence or presence of pha . after drawing standard curve , the final density of normal splenocytes was adjusted to 2.5 10 cells for coculture with at - mscs . to evaluate antiproliferative effect of at - mscs , they were added to pha - stimulated splenocytes cultures with a ratio of at - mscs to splenocytes of 1 : 50 , 1 : 250 , and 1 : 1250 and cell proliferation was determined . as expected , the resting splenocytes showed a strong proliferation response in the presence of pha ( figure 1 ; p < 0.05 ) , whereas addition of at - msc to pha - stimulated splenocytes resulted in a significant reduction of splenocytes proliferation in a number - dependent manner ( figure 1 ; p < 0.05 ) . the results also indicated that at - mscs could induce cell death of some splenocytes in the absence of mitogen ( figure 1 ; p < 0.05 ) . the greater cell death of splenocytes seemed to occur at the highest density of at - mscs ( 1 : 50 ) . in mitogen - induced proliferation experiment , no significant difference was detected between normal and diabetic splenocytes in the pattern of reduced proliferation and response to mitogen . after confirming that at - mscs could inhibit proliferation of mitogen - stimulated splenocytes , we then investigated whether at - mscs could inhibit proliferation of splenocytes triggered by specific stimulator . at - mscs were cocultured with islet lysate - stimulated splenocytes as previously described and cell proliferation was assessed . in contrast , diabetic cells showed a significant proliferation ( figure 2 ; p < 0.05 ) . again , the dose - dependent effect of at - mscs on diabetic splenocytes proliferation inhibition was observed in presence of islet lysate ( figure 2 ; p < 0.05 ) . however , the results demonstrated that at - mscs could induce cell death in some of these splenocytes ( figure 2 ; p < 0.05 ) . to determine whether at - mscs modulate cytokine secretion by splenocytes , we prepared a coculture design with splenocytes supplemented by pha or islet cells lysate . analysis of cytokines production by resting at - mscs showed that they consistently secreted tgf- and il-6 in higher levels compared to other cytokines ( figure 3 ; p < 0.05 ) . as previously described , at - mscs were used as blank to subtract background absorbance of their cytokines production . the results demonstrated that addition of at - mscs to lysate - stimulated diabetic splenocytes significantly decreased ifn- , il-2 , and il-17 production by splenocytes ( figure 4 ; p < 0.05 ) . in addition , this experiment indicated that at - mscs significantly increased tgf- , il-10 , il-4 , and il-13 production by splenocytes ( figure 5 ; p < 0.05 ) . although the increased production of these cytokines was also observed in the presence of pha , increasing levels were significantly prominent in at - mscs cocultured with lysate - stimulated diabetic splenocytes , particularly for il-13 ( figure 5 ) . however , diabetic splenocytes significantly decreased insulin secretion by islets ( 2.59 0.27 ng / ml ; p < 0.05 ) . in contrast , coculture with at - mscs led to a significant improvement in insulin secretion and the viability of islet cells ( 3.87 0.41 ng / ml versus 2.59 0.27 ng / ml ; p < 0.05 ) ( figure 6 ) . in this regard , previous studies have shown that mscs induce peripheral tolerance and migrate to injured tissues , where they can inhibit release of proinflammatory cytokines and promote the survival of damaged cells [ 2224 ] . thus , we assessedin vitro immunoregulatory effects of adipose - derived mscs on autoimmune response in experimental diabetic model . in this respect , we aimed at three main goals : firstly , to evaluate suppressive effect of at - mscs on autoreactive splenocytes proliferation ; secondly , to assess immunomodulatory effect of at - mscs on splenocytes cytokine patterns ; and finally , to evaluate protective effect of at - mscs on functional islets . in the current study , mscs were extracted from adipose tissue of healthy c57bl/6 mice and cultured to proliferate . diabetic c57bl/6 mouse model was prepared by administration of consecutive low - doses of stz and diabetic state was confirmed by serum glucose ( > 300 mg / dl ) , insulin levels , and pancreas histopathology . pancreatic islets were isolated from normal mice and splenocytes prepared from normal and diabetic mice . to evaluate antiproliferative effect of mscs , they were cocultured with diabetic splenocytes in the presence of islet lysate and proliferation assayed by mtt technique . the effect of mscs on cytokine patterns produced by splenocytes was assayed in cocultured splenocytes with mscs in the presence of islet lysate for 72 hours . high doses of stz selectively kill the insulin - producing beta cells , whereas multiple low - doses of stz induce expression of glutamic acid decarboxylase ( gad ) autoantigen and develop an autoimmune response . although some reports have shown that adipose - derived mscs due to more simple isolation and expansion and also relative better immunomodulatory effects are a novel promise in experimental and clinical application [ 5 , 32 ] , isolated at - mscs had fibroblast - like morphology and differential capacity that were consistent with previous studies . in addition , analysis of cell surface markers on at - mscs showed a lack of hematopoietic markers , such as cd34 and cd45 , and high expression of cd90 and cd29 . the biological characteristics similar to bone marrow mesenchymal stem cell ( bm - mscs ) indicate that at - mscs may present an alternative source for future application [ 34 , 35 ] . one of the main goals in this research was close to assessment of at - mscs suppressive effect on splenocytes proliferation in the presence of specific ( islet cells lysate ) and nonspecific ( pha ) stimulators . ( 1 ) the results indicated that at - mscs in a ratio of 1 : 50 , 1 : 250 , and 1 : 1250 to splenocytes could inhibit stimulated splenocytes proliferation in a dose - dependent manner with the most inhibition at the lowest dilution tested ( 1 : 50 ; p < 0.05 ) . however , at - mscs could induce cell death in some splenocytes in this concentration ( 1 : 50 ; p < 0.05 ) . the similar effects of at - mscs were observed in the presence of islet lysate and pha . in a recent study , it has been shown that mscs isolated from nod and balb / c mice could inhibit t cd4 proliferation in full mismatch mixed lymphocyte reaction ( mlr ) assay . it has also been clarified that , in an autoimmune fashion in vitro , when mscs are added to autoreactive t cd4 and allogeneic dendritic cells in the presence of islet peptides , they could significantly suppress autoreactive t cd4 proliferation . several reports have demonstrated that immunosuppressive effects of mscs occur in a dose - related manner in which it is considered independent of mhc interaction [ 3941 ] . although target cell - msc interactions may be an important factor , the immunosuppressive effect of mscs can also be mediated through the secretion of soluble molecules such as il-10 , tgf- , ido , no , pge-2 , and hla - g that are triggered following cross talk with target cells . ( 2 ) at - mscs caused some cell death in resting splenocytes without each type of stimulators ( p < 0.05 ) . this may suggest that mscs can induce cellular death through cell - cell contact as well as secretion of no and il-10 [ 9 , 11 ] ; also , they can drive splenocytes to produce il-10 . ( 3 ) in the absence of at - mscs , normal and diabetic splenocytes in response to nonspecific stimulator ( pha ) showed similar fashion . in contrast , in response to islet lysate normal splenocytes were not responsive , whereas a sharp proliferation was obtained in diabetic splenocytes ( p < 0.05 ) . in the next stage , we aimed at evaluating the effect of at - mscs on cytokine patterns produced by splenocytes . analysis of cytokines production by at - mscs alone showed that they consistently secreted tgf- and il-6 in higher levels compared to other cytokines ( p < 0.05 ) . several publications have confirmed spontaneous secretion of tgf-1 , il-6 , hepatocyte growth factor ( hgf ) , hla - g , and pge-2 that associated with immunomodulatory properties of mscs [ 42 , 43 ] . our in vitro experiment showed that at - mscs decreased production of ifn- , il-2 , and il-17 and increased production of tgf- , il-4 , il-10 , and il-13 by stimulated splenocytes in response to islet lysate or pha ( p < 0.05 ) . th1 cells produced ifn- , il-2 , and tnf- and th2 cells secreted il-4 , il-5 , il-6 , il-10 , and il-13 . it has been demonstrated that mscs have capacity to modulate immune reaction in vitro and in vivo , where they polarize proinflammatory state to anti - inflammatory state . finally , in this study we used other approaches of mscs properties to maintain in vitro viability and functionality of purified islets in the presence of reactive splenocytes . in this approach , it was found that at - mscs in coculture design had ability to provide islet functionality , proven by protected insulin secretion in coculture medium ( p < 0.05 ) . several studies examined in vivo immunoregulatory effects of mscs in cotransplantation with islets , which resulted in enhancing of islet allograft survival , insulin secretion , and sustained normoglycemia in stz - induced diabetic model [ 43 , 49 , 50 ] . coculturing of normal and stz - injured rat pancreatic islets with rat bm - mscs has improved function and viability of islets . they also demonstrated that cytoprotective , anti - inflammatory , and antiapoptotic effects of mscs were mediated through paracrine actions ( spontaneous production of il-6 , tgf-1 , and hgf ) . in brief , we have demonstrated that at - mscs in a dose - dependent manner inhibit splenocytes proliferative response to specific and nonspecific triggers that may be mediated by both cell - cell contact and soluble mediators such as tgf-1 and il-10 . at - mscs finally , our results indicated that at - mscs through cytoprotective and anti - inflammatory effects can effectively sustain viability as well as insulin secretion potential of islet cells in the presence of reactive splenocytes .

type 1 diabetes mellitus ( t1 dm ) is identified by the progressive autoimmune destruction of pancreatic beta cells , which results in a dramatic decrease of insulin production and consequent metabolic complications . in addition , replacement of the beta cell deficit along with regulation of autoimmune response to cells that express insulin is crucial for a t1 dm definitive cure . thus , in recent years , the usage of cell sources that modulate immune system along with islet cell replacement has received much attention . mesenchymal stem cells ( mscs ) represent a rare heterogeneous subset of multipotent stromal cells localized in many different adult and fetal tissues . they have self - renewal and multidifferentiation capacity that can give rise to diverse lineages of mesenchymal origin , including osteoblasts , adipocytes , and chondrocytes , and have also shown their potential for differentiating into nonmesodermal origin cells . although multipotent mscs are usually isolated from bone marrow ( bm ) , more recently , adipose tissue - derived mscs ( at - mscs ) due to more quantities , simple accessibility , and also the better immunomodulatory properties were represented as another alternative source for mscs [ 5 , 6 ] . numerous recent studies indicated that mscs possess immunomodulatory or immunosuppressive effects both in vitro and in vivo on several immune cells , not only t lymphocytes but also on b lymphocytes , dendritic cells ( dcs ) , and nk cells . in vitro studies have identified that the immunomodulatory function of mscs can be addressed by both cell - cell contact and soluble factors [ 9 , 10 ] . they exert immune regulation by the secretion of anti - inflammatory factors , such as interleukin-10 ( il-10 ) , transforming growth factor- ( tgf- ) , indoleamine 2,3-dioxygenase ( ido ) [ 12 , 13 ] , nitric oxide , prostaglandin - e2 ( pge-2 ) , and human leukocyte antigen - g ( hla - g ) . in addition , mscs induce cell cycle arrest and apoptosis of t lymphocytes [ 11 , 16 ] and cause lymphocytes to secrete regulatory cytokines , especially il-10 . mesenchymal stem cells due to their low immunogenicity and immunomodulatory properties as well as high degree of differentiation and proliferation potential might be useful in inhibiting the autoimmunity and regenerating the insulin - secreting cells . furthermore , many studies declared that the regenerative role of mscs could be mediated by protective effects on functional islet cells and also differentiation potency to insulin - producing cells in vivo and in vitro . the ideal use of mscs in autoimmune diabetes regenerative therapy can only be obtained after we learn their immunomodulatory characteristics in detail . the aim of this research was to evaluate the immunomodulatory effects of adipose - derived mscs on autoimmune response besides islet protective function in streptozotocin- ( stz- ) induced diabetic mice model . diabetes was induced in the mice by intraperitoneal injection of multiple low - doses ( 50 mg / kg , 4 consecutive days ) of streptozotocin ( stz ) ( sigma , usa ) . the cell surface markers on at - mscs were assessed using monoclonal antibodies against mouse cd73 , cd105 , cd29 , cd90 , cd31 , cd11b , cd45 , and cd34 ( all from ebioscience ) . for differentiation to chondrocytes , 1 10 cells were centrifuged to form a pelleted micromass and then treated with tgf - beta ( 10 ng / ml ; merck ) , ascorbic acid biphosphate ( 50 g / ml ) , dexamethasone ( 10 m ) , and insulin transferrin selenium ( its ) ( 50 g / ml ; sigma ) for 3 weeks . to evaluate islet cell function , islets ( n = 10 ) were stimulated in rpmi-1640 culture solution with low glucose ( 5.6 mmol / l ) and incubated for 4 hours at 37c for detection of the total levels of insulin in the culture solution . in proliferation assay , normal and diabetic splenocytes were cocultured with at - mscs in the mscs culture medium mixed 1 : 1 with fresh splenocyte culture medium ( mixed culture medium ) . prior to final plating , optimized concentration of splenocytes with or without phytohemagglutinin ( pha , gibco ) was determined at dilutions of 1 , 2 , 3 , 4 , and 5 10 cells in 96-well plate by mtt assay . after the at - mscs reached 70% confluence , the medium was removed , and 100 l of fresh medium containing 5 l of mitomycin - c ( 1 g/l ; sigma ) was added for 1 h at 37c to mitotically inactivate the at - mscs . at - mscs were resuspended in 100 l of mixed culture medium , cocultured with 2.5 10 splenocytes , and stimulated by 5 g / ml pha ( 10 l ) or 10 l of islet cells lysate in control and test groups , respectively , for 48 h at 37c . the suppressive effect of at - mscs on splenocytes proliferation in the absence and presence of pha ( as nonspecific stimulator ) and islet cells lysate ( as specific stimulator ) was determined by mtt [ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide ] assay . the proliferation index was calculated using the following formula:(1)proliferation index = odmscs+splenocytesstimulatorodmscsodsplenocytes.in order to examine the proliferation of splenocytes ( in the presence of at - mscs ) , at - mscs monoculture was used as blank to subtract background absorbance of at - mscs . to provide the optimum condition , the mtt assay was repeated several times with different ratios of at - mscs to splenocytes , various concentrations of pha , and also different periods of coculture incubation . the supernatants of each group were collected and evaluated by multi - analyte elisarray kit ( mem-003a , sabiosciences ) for tgf- , il-10 , il-4 , and il-13 as regulatory cytokines and ifn- , il-2 , and il-17a as inflammatory cytokines . the groups were designed as follows : islet cells ( positive control ) , islet cells + normal splenocytes , islet cells + diabetic splenocytes , islet cells + normal splenocytes + mscs , islet cells + diabetic splenocytes + mscs , and splenocytes ( negative control ) . in addition , the insulin levels of 4.95 0.52 ng / dl in normal mice decreased to < 0.5 ng / dl in diabetic mice and pancreatic islets destruction was confirmed by histopathological examination . mscs seeded to the culture flasks sparsely and the cells displayed a fibroblast - like morphology during the early days of incubation . at - mscs at passage 3 the cells lacked cd11b , cd34 , cd45 , and cd31 whereas they were all positive for cd73 , cd90 , cd105 , and cd29 expression . more than 80% of all cells stained positively with alcian blue showed the glucose amino glycan ( gag ) biosynthesis in the cell pellets ( a figure illustrating at - mscs characterization has been presented as supplementary data in supplementary material available online at http://dx.doi.org/10.1155/2015/878535 ) . in this study , we modified the procedure by excluding the in situ pancreas perfusion and ficoll gradient steps . firstly , to assess which concentration of splenocytes exerts optimized proliferation , we cultured resting cells at dilutions of 1 , 2 , 3 , 4 , and 5 10 in the absence or presence of pha . to evaluate antiproliferative effect of at - mscs , they were added to pha - stimulated splenocytes cultures with a ratio of at - mscs to splenocytes of 1 : 50 , 1 : 250 , and 1 : 1250 and cell proliferation was determined . as expected , the resting splenocytes showed a strong proliferation response in the presence of pha ( figure 1 ; p < 0.05 ) , whereas addition of at - msc to pha - stimulated splenocytes resulted in a significant reduction of splenocytes proliferation in a number - dependent manner ( figure 1 ; p < 0.05 ) . the results also indicated that at - mscs could induce cell death of some splenocytes in the absence of mitogen ( figure 1 ; p < 0.05 ) . in mitogen - induced proliferation experiment , no significant difference was detected between normal and diabetic splenocytes in the pattern of reduced proliferation and response to mitogen . after confirming that at - mscs could inhibit proliferation of mitogen - stimulated splenocytes , we then investigated whether at - mscs could inhibit proliferation of splenocytes triggered by specific stimulator . again , the dose - dependent effect of at - mscs on diabetic splenocytes proliferation inhibition was observed in presence of islet lysate ( figure 2 ; p < 0.05 ) . however , the results demonstrated that at - mscs could induce cell death in some of these splenocytes ( figure 2 ; p < 0.05 ) . to determine whether at - mscs modulate cytokine secretion by splenocytes , we prepared a coculture design with splenocytes supplemented by pha or islet cells lysate . analysis of cytokines production by resting at - mscs showed that they consistently secreted tgf- and il-6 in higher levels compared to other cytokines ( figure 3 ; p < 0.05 ) . the results demonstrated that addition of at - mscs to lysate - stimulated diabetic splenocytes significantly decreased ifn- , il-2 , and il-17 production by splenocytes ( figure 4 ; p < 0.05 ) . in addition , this experiment indicated that at - mscs significantly increased tgf- , il-10 , il-4 , and il-13 production by splenocytes ( figure 5 ; p < 0.05 ) . although the increased production of these cytokines was also observed in the presence of pha , increasing levels were significantly prominent in at - mscs cocultured with lysate - stimulated diabetic splenocytes , particularly for il-13 ( figure 5 ) . to determine protective ability of at - mscs on islet cells function this experiment indicated that the crude islets alone secreted significant insulin levels when induced with stimulatory medium ( 6.45 0.69 ng / ml ) . in contrast , coculture with at - mscs led to a significant improvement in insulin secretion and the viability of islet cells ( 3.87 0.41 ng / ml versus 2.59 0.27 ng / ml ; p < 0.05 ) ( figure 6 ) . mscs also referred to as adult multipotent stem cells represent a fibroblast - like morphology that have not only the capacity to differentiate into several tissues , but also have immunomodulatory and anti - inflammatory effects in vivo and in vitro [ 3 , 21 ] . many studies that declared protective effects of mscs on islet mass and differentiation potency to insulin - producing cells in vivo and in vitro collaborate for the application of mscs regenerative role in t1 dm [ 2527 ] . although many researchers have demonstrated the immunomodulatory activities of mscs , in vitro potential assays able to predict or to correlate with therapeutic outcome of mscs are still partially known . thus , we assessedin vitro immunoregulatory effects of adipose - derived mscs on autoimmune response in experimental diabetic model . in this respect , we aimed at three main goals : firstly , to evaluate suppressive effect of at - mscs on autoreactive splenocytes proliferation ; secondly , to assess immunomodulatory effect of at - mscs on splenocytes cytokine patterns ; and finally , to evaluate protective effect of at - mscs on functional islets . diabetic c57bl/6 mouse model was prepared by administration of consecutive low - doses of stz and diabetic state was confirmed by serum glucose ( > 300 mg / dl ) , insulin levels , and pancreas histopathology . to evaluate antiproliferative effect of mscs , they were cocultured with diabetic splenocytes in the presence of islet lysate and proliferation assayed by mtt technique . the effect of mscs on cytokine patterns produced by splenocytes was assayed in cocultured splenocytes with mscs in the presence of islet lysate for 72 hours . some strains of mice are th1-dominant ( c57bl/6 , dba/2 , akr , and cba ) , but the others are th2-dominant strains ( balb / c , bp2 , and a / j ) . therefore , in this study the selection of c57bl/6 mice as a suitable model for induction of autoimmune diabetes appears to be reasonable . high doses of stz selectively kill the insulin - producing beta cells , whereas multiple low - doses of stz induce expression of glutamic acid decarboxylase ( gad ) autoantigen and develop an autoimmune response . although some reports have shown that adipose - derived mscs due to more simple isolation and expansion and also relative better immunomodulatory effects are a novel promise in experimental and clinical application [ 5 , 32 ] , isolated at - mscs had fibroblast - like morphology and differential capacity that were consistent with previous studies . in addition , analysis of cell surface markers on at - mscs showed a lack of hematopoietic markers , such as cd34 and cd45 , and high expression of cd90 and cd29 . the biological characteristics similar to bone marrow mesenchymal stem cell ( bm - mscs ) indicate that at - mscs may present an alternative source for future application [ 34 , 35 ] . one of the main goals in this research was close to assessment of at - mscs suppressive effect on splenocytes proliferation in the presence of specific ( islet cells lysate ) and nonspecific ( pha ) stimulators . ( 1 ) the results indicated that at - mscs in a ratio of 1 : 50 , 1 : 250 , and 1 : 1250 to splenocytes could inhibit stimulated splenocytes proliferation in a dose - dependent manner with the most inhibition at the lowest dilution tested ( 1 : 50 ; p < 0.05 ) . it has also been clarified that , in an autoimmune fashion in vitro , when mscs are added to autoreactive t cd4 and allogeneic dendritic cells in the presence of islet peptides , they could significantly suppress autoreactive t cd4 proliferation . although target cell - msc interactions may be an important factor , the immunosuppressive effect of mscs can also be mediated through the secretion of soluble molecules such as il-10 , tgf- , ido , no , pge-2 , and hla - g that are triggered following cross talk with target cells . this may suggest that mscs can induce cellular death through cell - cell contact as well as secretion of no and il-10 [ 9 , 11 ] ; also , they can drive splenocytes to produce il-10 . ( 3 ) in the absence of at - mscs , normal and diabetic splenocytes in response to nonspecific stimulator ( pha ) showed similar fashion . in the next stage , we aimed at evaluating the effect of at - mscs on cytokine patterns produced by splenocytes . analysis of cytokines production by at - mscs alone showed that they consistently secreted tgf- and il-6 in higher levels compared to other cytokines ( p < 0.05 ) . several publications have confirmed spontaneous secretion of tgf-1 , il-6 , hepatocyte growth factor ( hgf ) , hla - g , and pge-2 that associated with immunomodulatory properties of mscs [ 42 , 43 ] . our in vitro experiment showed that at - mscs decreased production of ifn- , il-2 , and il-17 and increased production of tgf- , il-4 , il-10 , and il-13 by stimulated splenocytes in response to islet lysate or pha ( p < 0.05 ) . however , the increases in regulatory cytokines were significantly prominent in at - mscs cocultured with lysate - stimulated diabetic splenocytes , particularly for il-13 . recent publications have reported the in vivo transplantation of mscs derived from bone marrow and adipose tissue along with a therapeutic approach , where they switch host immune response towards a th2 cytokine profile by downregulating th1 cytokines and/or upregulating th2 cytokines [ 22 , 4548 ] . finally , in this study we used other approaches of mscs properties to maintain in vitro viability and functionality of purified islets in the presence of reactive splenocytes . in this approach , it was found that at - mscs in coculture design had ability to provide islet functionality , proven by protected insulin secretion in coculture medium ( p < 0.05 ) . several studies examined in vivo immunoregulatory effects of mscs in cotransplantation with islets , which resulted in enhancing of islet allograft survival , insulin secretion , and sustained normoglycemia in stz - induced diabetic model [ 43 , 49 , 50 ] . they showed that cocultivation of stz - injured islets and mscs increased expression of il-6 and tgf-1 in the culture medium besides the expression of antiapoptotic genes . however , many studies declared that the regenerative role of mscs could be mediated by differentiation potential to insulin - secreting cells in vivo and in vitro [ 18 , 25 ] . in brief , we have demonstrated that at - mscs in a dose - dependent manner inhibit splenocytes proliferative response to specific and nonspecific triggers that may be mediated by both cell - cell contact and soluble mediators such as tgf-1 and il-10 . at - mscs finally , our results indicated that at - mscs through cytoprotective and anti - inflammatory effects can effectively sustain viability as well as insulin secretion potential of islet cells in the presence of reactive splenocytes . although we note that adjunct in vivo studies are required to completely investigate the immunomodulatory functions of mscs in autoimmune diseases , this study may provide some basic information regarding application of human mscs in treating type 1 diabetes .

within the british national health service ( nhs ) , laparoscopic resection is recommended by the national institute for health and care excellence ( nice ) as an alternative to open surgery for people with colorectal cancer for whom both methods would be considered suitable , and if performed by a trained surgeon.1 a health technology assessment suggested that laparoscopic surgery was associated with higher costs , but was no more effective than open surgery . savings from shorter hospital stays after laparoscopic procedures did not fully offset higher operation costs and conversions to open methods due to complications.1 research concludes that there is no significant difference in disease free or overall survival at 3 years,1,2 or in health - related quality of life outcomes.36 however , few studies capture the short term benefits of laparoscopic surgery in the early postoperative period.1,7 the technology assessment concluded that , at the 30,000 cost per quality adjusted life year ( qaly ) threshold,8 laparoscopic surgery would need to result in a gain of 0.0090.010 qaly to be considered cost - effective , compared to open surgery.1,2 a study was undertaken to explore the qaly differences in the early postoperative recovery period,9 and costs.10 this paper reports the cost - effectiveness analysis that brings together the qaly and cost data for those patients who had a diagnosis of cancer or polyps . helped by the national training programme for laparoscopic colorectal surgery ( lapco ) , the uptake of laparoscopic surgery has increased in nhs hospitals , but it is still used in the minority of cases ( 40% in 2012).11 providing evidence on cost - effectiveness may encourage increased training of surgeons and wider use of laparoscopic methods.12 consecutive patients requiring elective colorectal resections ( over 18 years , without endometriosis , able to give consent ) in a district general hospital in south england were invited to join the study , 20062007 . patients were allocated to laparoscopic or open surgery by administrative staff , depending on availability of clinic appointments , or , in some cases , requests from referring general practitioners . following informed consent , baseline demographic information ( sex , age , operation type [ right or left side resection ] , american society of anesthesiologists ( asa ) physical status score ) was collected pre - operatively . a micro - costing study gathered treatment costs ( operative , in - hospital stay , community ) , in british pounds , 2006 , as described in full elsewhere.10 quality of life data were collected using euroqol 5d-3l instrument ( eq-5d ) . the five domains ( mobility , self - care , usual activities , pain / discomfort , anxiety / depression ) are scored on three levels ( 1 , no problem ; 2 , some problem ; 3 , serious / extreme problem / unable ) giving a theoretical maximum of 243 different health states which are converted to single index utility values ( range 0.59 for score 33333 to 1.0 [ perfect health ] for score 11111 ) , using uk tariff tables.1315 participants were asked to complete eq-5d on alternate days after surgery for 4 weeks , and at the end of the fifth and sixth week ( total of 16 times ) , in a diary that was returned at the 6 week outpatient follow - up clinic appointment . among 201 patients ( 131 laparoscopic , 70 open ) recruited , 129 ( 64.2% ) had a diagnosis of cancer or polyps ( 85 laparoscopic , 44 open ) . the sample was reduced to 98 ( 70 laparoscopic , 28 open ) by exclusion of patients undergoing a rectal resection because such procedures ( which typically result in higher costs and lower qaly due to requiring a stoma),7,10 were unevenly distributed between the groups ( n=15 , 17.6% laparoscopic ; n=16 , 36.4% open ) . a further three patients were lost because missing data were too extensive to be imputed . hence , the final sample included in the analysis was 95 ( 68 laparoscopic , 27 open ) , of which 17 ( 18.0% ) had a diagnosis of polyps , rather than cancer ; 14 ( 20.6% ) of laparoscopic procedures , 3 ( 11.1% ) of open . data were imported into stata12 ( stata statistical software , release 12 ; statacorp lp , college station , tx , usa ) . costs and quality of life data were assessed for missing data . since it was found that a complete case analysis would reduce the sample to 43 , and result in exclusion of some participants with complete eq-5d or cost data , multiple imputation ( monte carlo simulation ) was used to generate missing values.16 it was not possible to impute stable quality of life estimates up to 42 days , so the analysis focused on the first 28 days after surgery ( 14 eq-5d scores ) . an area under the curve ( auc ) method was employed to generate a qaly value for each patient . differences in mean qalys and costs between surgery groups were estimated simultaneously using a multivariate regression model applied to 20 imputed datasets with eq-5d at baseline , sex , age , and operation type ( left , right side ) included as covariates . asa score was strongly correlated with the eq-5d baseline score and therefore not included as a covariate . a cost - effectiveness ellipse and cost - effectiveness acceptability curve ( ceac ) were produced to indicate the probability that laparoscopic surgery is cost - effective compared to open for a given societal willingness - to - pay ( wtp ) threshold . a favorable ethical opinion was obtained prior to starting the research from the south west surrey local research ethics committee ( study 05/q1909/74 ) , and the hospital research and development committee . consecutive patients requiring elective colorectal resections ( over 18 years , without endometriosis , able to give consent ) in a district general hospital in south england were invited to join the study , 20062007 . patients were allocated to laparoscopic or open surgery by administrative staff , depending on availability of clinic appointments , or , in some cases , requests from referring general practitioners . following informed consent , baseline demographic information ( sex , age , operation type [ right or left side resection ] , american society of anesthesiologists ( asa ) physical status score ) was collected pre - operatively . a micro - costing study gathered treatment costs ( operative , in - hospital stay , community ) , in british pounds , 2006 , as described in full elsewhere.10 quality of life data were collected using euroqol 5d-3l instrument ( eq-5d ) . the five domains ( mobility , self - care , usual activities , pain / discomfort , anxiety / depression ) are scored on three levels ( 1 , no problem ; 2 , some problem ; 3 , serious / extreme problem / unable ) giving a theoretical maximum of 243 different health states which are converted to single index utility values ( range 0.59 for score 33333 to 1.0 [ perfect health ] for score 11111 ) , using uk tariff tables.1315 participants were asked to complete eq-5d on alternate days after surgery for 4 weeks , and at the end of the fifth and sixth week ( total of 16 times ) , in a diary that was returned at the 6 week outpatient follow - up clinic appointment . among 201 patients ( 131 laparoscopic , 70 open ) recruited , 129 ( 64.2% ) had a diagnosis of cancer or polyps ( 85 laparoscopic , 44 open ) . the sample was reduced to 98 ( 70 laparoscopic , 28 open ) by exclusion of patients undergoing a rectal resection because such procedures ( which typically result in higher costs and lower qaly due to requiring a stoma),7,10 were unevenly distributed between the groups ( n=15 , 17.6% laparoscopic ; n=16 , 36.4% open ) . a further three patients were lost because missing data were too extensive to be imputed . hence , the final sample included in the analysis was 95 ( 68 laparoscopic , 27 open ) , of which 17 ( 18.0% ) had a diagnosis of polyps , rather than cancer ; 14 ( 20.6% ) of laparoscopic procedures , 3 ( 11.1% ) of open . data were imported into stata12 ( stata statistical software , release 12 ; statacorp lp , college station , tx , usa ) . baseline characteristics of groups were compared . costs and quality of life data were assessed for missing data . since it was found that a complete case analysis would reduce the sample to 43 , and result in exclusion of some participants with complete eq-5d or cost data , multiple imputation ( monte carlo simulation ) was used to generate missing values.16 it was not possible to impute stable quality of life estimates up to 42 days , so the analysis focused on the first 28 days after surgery ( 14 eq-5d scores ) . an area under the curve ( auc ) method was employed to generate a qaly value for each patient . differences in mean qalys and costs between surgery groups were estimated simultaneously using a multivariate regression model applied to 20 imputed datasets with eq-5d at baseline , sex , age , and operation type ( left , right side ) included as covariates . asa score was strongly correlated with the eq-5d baseline score and therefore not included as a covariate . a cost - effectiveness ellipse and cost - effectiveness acceptability curve ( ceac ) were produced to indicate the probability that laparoscopic surgery is cost - effective compared to open for a given societal willingness - to - pay ( wtp ) threshold . a favorable ethical opinion was obtained prior to starting the research from the south west surrey local research ethics committee ( study 05/q1909/74 ) , and the hospital research and development committee . all participants gave informed consent prior to inclusion in the study . the multiple imputation regression estimates ( table 2 ) show the cost of the laparoscopic operations was 1,037 higher than open ( p<0.005 ) , due to equipment costs ; staff costs were 190 lower ( p=0.039 ) due to a shorter operating time ( table 3 ) . the open group had a longer mean length of hospital stay ( table 3 ) and incurred 897 higher bed day costs ( p=0.007 ) compared to laparoscopic . there was no significant difference in the total costs between the groups because the higher laparoscopic surgery costs were offset by shorter lengths of stay . compared to those undergoing open surgery , patients in the laparoscopic group gained an average of 0.011207 qalys over the first 28 days after surgery ( 95% confidence intervals 0.006 to 0.016 ) ( table 2 and figure 1 ) . at 28 days , the overall incremental cost - effectiveness ratio ( icer ) , calculated as the difference in adjusted mean costs divided by the difference in adjusted mean qalys , and showing the cost per qaly gained from laparoscopic , compared to open surgery , was 12,375 . uncertainty in the icer point estimates are represented , using confidence intervals , on the cost - effectiveness plane ( cep ) ( figure 2 ) . the cost - effectiveness ellipse is centered in the top right quadrant , indicating that laparoscopic surgery is more costly and more effective than open . the confidence intervals of the ellipse overlap the bottom right quadrant where laparoscopic is dominant ( less costly and more effective than open ) . the line bisecting the top right and lower left quadrants shows the threshold cost - effectiveness line , based on a wtp of 20,000 . the point estimate icer lies below this threshold , demonstrating laparoscopic surgery as cost - effective . the ceac illustrates the probability that laparoscopic treatment will be deemed cost - effective at different societal wtp per qaly gained ( figure 3 ) . at 28 days , the ceac indicates that , at wtp of 30,000 , the probability that laparoscopic surgery will be cost - effective , compared to open methods , exceeds 65% ( and this is also illustrated by the proportion of the 95% confidence interval below the threshold line in figure 2 ) . at the costs of equipment and supplies , operative medical staff , hospital bed days , and community costs were varied independently of one another between the minimum and maximum values of their 95% confidence intervals whilst all other variables were held constant at their mean point estimates . at their lowest level , where costs were most favorable toward laparoscopic surgery , the resulting icers were all dominant in favor of the laparoscopic procedure . where the differences in costs were considered at their highest ( in favor of open ) , the result was less clear . with operative staff costs and community costs at their highest level , laparoscopic surgery still appeared cost - effective at a wtp threshold of 30,000 ( with costs per qaly gained of 28,385 , and 17,219 respectively ) . when costs of equipment and supplies were at their highest level compared to open ( , 1477 more expensive ) , the resulting icer of 34,690 was marginally outside the cost - effective level ; the turning point was 1,424 . at its most expensive , where laparoscopic surgery achieved a reduction in hospital bed days costing only 247 compared to open , an icer of 70,332 was realized . the threshold , where an icer of 30,000 was achieved , was at a bed day cost reduction of 699 ; the mean cost reduction for bed days seen in the data exceeded this at 897 . varying the estimated difference in qaly between the lower ( 0.00603 ) and upper ( 0.01638 ) 95% confidence interval values had no effect on the cost - effectiveness at a wtp of 30,000 , achieving icer ranging from 22,994 to 8,466 . staff operative costs were lower in the laparoscopic group , reflecting a shorter operation time by mean of 47 minutes , ( which equates to a theatre staff and overhead cost of 169 ) . operation times may vary with surgeon experience and style , and have typically been shown to be higher in laparoscopic procedures than open.18 given the mean difference in qalys ( 0.011207 ) and costs ( 139 ) observed in this study , laparoscopic operation time could be increased by 55 minutes ( 197 ) , ie , 8 minutes longer than open , with all other factors held constant , and still achieve an icer 30,000 . at 28 days , the overall incremental cost - effectiveness ratio ( icer ) , calculated as the difference in adjusted mean costs divided by the difference in adjusted mean qalys , and showing the cost per qaly gained from laparoscopic , compared to open surgery , was 12,375 . uncertainty in the icer point estimates are represented , using confidence intervals , on the cost - effectiveness plane ( cep ) ( figure 2 ) . the cost - effectiveness ellipse is centered in the top right quadrant , indicating that laparoscopic surgery is more costly and more effective than open . the confidence intervals of the ellipse overlap the bottom right quadrant where laparoscopic is dominant ( less costly and more effective than open ) . the line bisecting the top right and lower left quadrants shows the threshold cost - effectiveness line , based on a wtp of 20,000 . the point estimate icer lies below this threshold , demonstrating laparoscopic surgery as cost - effective . the ceac illustrates the probability that laparoscopic treatment will be deemed cost - effective at different societal wtp per qaly gained ( figure 3 ) . at 28 days , the ceac indicates that , at wtp of 30,000 , the probability that laparoscopic surgery will be cost - effective , compared to open methods , exceeds 65% ( and this is also illustrated by the proportion of the 95% confidence interval below the threshold line in figure 2 ) . at the costs of equipment and supplies , operative medical staff , hospital bed days , and community costs were varied independently of one another between the minimum and maximum values of their 95% confidence intervals whilst all other variables were held constant at their mean point estimates . at their lowest level , where costs were most favorable toward laparoscopic surgery , the resulting icers were all dominant in favor of the laparoscopic procedure . where the differences in costs were considered at their highest ( in favor of open ) , the result was less clear . with operative staff costs and community costs at their highest level , laparoscopic surgery still appeared cost - effective at a wtp threshold of 30,000 ( with costs per qaly gained of 28,385 , and 17,219 respectively ) . when costs of equipment and supplies were at their highest level compared to open ( , 1477 more expensive ) , the resulting icer of 34,690 was marginally outside the cost - effective level ; the turning point was 1,424 . at its most expensive , where laparoscopic surgery achieved a reduction in hospital bed days costing only 247 compared to open , an icer of 70,332 was realized . the threshold , where an icer of 30,000 was achieved , was at a bed day cost reduction of 699 ; the mean cost reduction for bed days seen in the data exceeded this at 897 . varying the estimated difference in qaly between the lower ( 0.00603 ) and upper ( 0.01638 ) 95% confidence interval values had no effect on the cost - effectiveness at a wtp of 30,000 , achieving icer ranging from 22,994 to 8,466 . staff operative costs were lower in the laparoscopic group , reflecting a shorter operation time by mean of 47 minutes , ( which equates to a theatre staff and overhead cost of 169 ) . operation times may vary with surgeon experience and style , and have typically been shown to be higher in laparoscopic procedures than open.18 given the mean difference in qalys ( 0.011207 ) and costs ( 139 ) observed in this study , laparoscopic operation time could be increased by 55 minutes ( 197 ) , ie , 8 minutes longer than open , with all other factors held constant , and still achieve an icer 30,000 . this is the first cost - effectiveness analysis comparing laparoscopic and open colorectal surgery for colorectal resections to incorporate quality of life differences in the early postoperative recovery period . whilst previous studies , based on longer follow - up periods , suggest no difference in outcomes between laparoscopic and open surgery,36 this study provides robust evidence of improved health - related quality of life outcomes associated with laparoscopy in the first 6 weeks post - surgery , and demonstrates the cost - effectiveness of that approach , compared to open methods . improved health - related quality of life after laparoscopic surgery has been associated with less blood loss , better immune and inflammatory responses , less pain and analgesic requirements , faster postoperative recovery of bowel function , food intake , and physical activity.17,18 quality of life in this study was measured earlier in the recovery period and more intensively than in most other studies comparing laparoscopic and open methods for colorectal surgery . moreover , the eq-5d instrument was used to enable the calculation of qalys so that a cost - effectiveness analysis could be undertaken . health - related quality of life in most other studies has been measured using a variety of different generic and disease - specific instruments,4,5 and at later time points post - surgery , making direct comparison of the findings of different studies problematical . the analysis reported in this paper is based on those patients with a diagnosis of cancer or polyps in a larger study of patients undergoing colorectal surgery at a district general hospital in england,9 ie , some 65% of the whole sample , ( 47% cancer , 18% polyps ) . consistent with the parent study ( which included those with a diagnosis other than cancer or polyps , and rectal resections)10 and other evidence,19,20 the overall costs of laparoscopic and open approaches for this subgroup were not significantly different , with higher operation costs ( due to instrumentation ) for the laparoscopic group largely offset by a shorter length of hospital stay . the results demonstrate a significant qaly gain for laparoscopic surgery , compared to open , of 0.011207 at 28 days , which was similar to that observed for the whole sample in the parent study.7 this qaly gain exceeds that identified by the nice technology assessment panel ( 0.0090.010 ) as being required in order to deem laparoscopic surgery cost - effective for colorectal cancer.1,2 in the cost - effectiveness analysis , the point estimate cost per qaly gained was 12,375 . the probabilistic approach to analyze the uncertainty demonstrated that the laparoscopic procedure was close to clear dominance over open surgery and cost - effective at a wtp below the current threshold set by nice.8 the current guideline from nice11 is that no fixed wtp will be enforced , although the 2004 guidance was that a plausible threshold is 20,00030,000 . the sensitivity analysis showed the results to be relatively robust to variation in the main input variables . only the cost of hospital stay was independently capable of shifting the icer to a level above 30,000 , but at bed day savings associated with laparoscopic below those recorded in this study . the deterministic sensitivity analysis showed laparoscopic surgery to be cost - effective with operation times up to 8 minutes longer than open . the sample of cancer / polyp patients was drawn from a larger study of all patients undergoing colorectal resection , and statistical power was lost because the numbers in this subset was small . the sample size was further eroded by the removal of rectal resections , which was done to avoid bias due to the disproportionate number of rectal resections in the open group . there is no reason to believe that the imbalance was due to any factor other than chance . however , the lack of randomization of patients to study groups is a drawback . administrative staff , who were unconnected to the study were responsible for assigning patients to surgical teams at the point of referral by general practitioners . allocation was based on pragmatic factors , such as availability of appointments , but this is not a good substitute for randomization . the proportion of patients with a diagnosis of polyps , rather than cancer , was higher in the laparoscopic group than the open group ( 20.6% versus 11.1% ) . since the postoperative recovery of patients with cancer is potentially different from that of patients with polyps , we tested whether the sample imbalance might affect the results , by adding a diagnosis variable ( cancer versus polyps ) to the regression modelling . whilst this affected the coefficients by a very small amount , the diagnosis covariate was not significant ( p>0.5 ) , indicating that it had no material effect on the findings operative costs were based on observation and researchers had not been present during all surgeries.10 some participants , particularly those with higher asa grades at baseline , longer hospital stays and in higher intensity units,7 had not provided quality of life data . even using multiple imputation , it was not possible to extend the analysis from 28 to 42 days , due to missing information . multiple imputation was used in preference to a complete case analysis which would have reduced the sample size further . the impact of uncertainty in the sample data was accommodated in the sensitivity analysis and found to have little impact on the conclusions of the study . the results of the analysis using the imputed data sets were checked against those generated by a complete case analysis . the latter found laparoscopic surgery to be 258 more expensive , on average , than open ( versus 139 using multiple imputation ) , and achieving a qaly gain of 0.013523 ( versus 0.011207 ) ; icer point estimate of 19,106 ( versus 12,375 ) . no adjustment for inflation has been made , and in nominal terms the results may overstate the significance of the findings . since that time , the relative cost of the disposable instruments ( a major item for the laparoscopic procedure ) may have fallen due to increased uptake and advancements in technology . the cost of hospital stays ( predominantly affecting the open group ) has increased , but hospital policies for earlier discharge may have offset this to some extent . the study only focused on the first 6 weeks after surgery , and no subsequent follow - up of patients was conducted . this was because prior research had demonstrated no differences in survival or health - related quality of life in the longer term,16 and the research design deliberately focused on the early postoperative period where a gap in evidence had been identified.1,7 this study provides formal evidence of the cost - effectiveness of laparoscopic approaches based on quality of life gains , compared to open methods , in the early postoperative period . the findings support current guidelines that promote the use of laparoscopy where suitably trained surgeons are available.1 the national training programme lapco is enabling more hospitals to offer minimally invasive surgery options and patients to reap the benefits of faster recovery.21

backgroundavailable evidence that compares outcomes from laparoscopic and open surgery for colorectal cancer shows no difference in disease free or survival time , or in health - related quality of life outcomes , but does not capture the short term benefits of laparoscopic methods in the early postoperative period.aimto explore the cost - effectiveness of laparoscopic colorectal surgery , compared to open methods , using quality of life data gathered in the first 6 weeks after surgery.methodsparticipants were recruited in 20062007 in a district general hospital in the south of england ; those with a diagnosis of cancer or polyps were included in the analysis . quality of life data were collected using eq-5d , on alternate days after surgery for 4 weeks . costs per patient , from a national health service perspective ( in british pounds , 2006 ) comprised the sum of operative , hospital , and community costs . missing data were filled using multiple imputation methods . the difference in mean quality adjusted life years and costs between surgery groups were estimated simultaneously using a multivariate regression model applied to 20 imputed datasets . the probability that laparoscopic surgery is cost - effective compared to open surgery for a given societal willingness - to - pay threshold is illustrated using a cost - effectiveness acceptability curve.resultsthe sample comprised 68 laparoscopic and 27 open surgery patients . at 28 days , the incremental cost per quality adjusted life year gained from laparoscopic surgery was 12,375 . at a societal willingness - to - pay of 30,000 , the probability that laparoscopic surgery is cost - effective , exceeds 65% ( at 20,000 60% ) . in sensitivity analyses , laparoscopic surgery remained cost - effective compared to open surgery , provided it results in a saving 699 in hospital bed days and takes no more than 8 minutes longer to perform.conclusionthe study provides formal evidence of the cost - effectiveness of laparoscopic approaches and supports current guidelines that promote use of laparoscopy where suitably trained surgeons are available .

Introduction Methods Participants Data collection Sample Analysis Ethical considerations Results Cost-effectiveness Sensitivity analysis Discussion Conclusion

within the british national health service ( nhs ) , laparoscopic resection is recommended by the national institute for health and care excellence ( nice ) as an alternative to open surgery for people with colorectal cancer for whom both methods would be considered suitable , and if performed by a trained surgeon.1 a health technology assessment suggested that laparoscopic surgery was associated with higher costs , but was no more effective than open surgery . savings from shorter hospital stays after laparoscopic procedures did not fully offset higher operation costs and conversions to open methods due to complications.1 research concludes that there is no significant difference in disease free or overall survival at 3 years,1,2 or in health - related quality of life outcomes.36 however , few studies capture the short term benefits of laparoscopic surgery in the early postoperative period.1,7 the technology assessment concluded that , at the 30,000 cost per quality adjusted life year ( qaly ) threshold,8 laparoscopic surgery would need to result in a gain of 0.0090.010 qaly to be considered cost - effective , compared to open surgery.1,2 a study was undertaken to explore the qaly differences in the early postoperative recovery period,9 and costs.10 this paper reports the cost - effectiveness analysis that brings together the qaly and cost data for those patients who had a diagnosis of cancer or polyps . helped by the national training programme for laparoscopic colorectal surgery ( lapco ) , the uptake of laparoscopic surgery has increased in nhs hospitals , but it is still used in the minority of cases ( 40% in 2012).11 providing evidence on cost - effectiveness may encourage increased training of surgeons and wider use of laparoscopic methods.12 consecutive patients requiring elective colorectal resections ( over 18 years , without endometriosis , able to give consent ) in a district general hospital in south england were invited to join the study , 20062007 . patients were allocated to laparoscopic or open surgery by administrative staff , depending on availability of clinic appointments , or , in some cases , requests from referring general practitioners . a micro - costing study gathered treatment costs ( operative , in - hospital stay , community ) , in british pounds , 2006 , as described in full elsewhere.10 quality of life data were collected using euroqol 5d-3l instrument ( eq-5d ) . the five domains ( mobility , self - care , usual activities , pain / discomfort , anxiety / depression ) are scored on three levels ( 1 , no problem ; 2 , some problem ; 3 , serious / extreme problem / unable ) giving a theoretical maximum of 243 different health states which are converted to single index utility values ( range 0.59 for score 33333 to 1.0 [ perfect health ] for score 11111 ) , using uk tariff tables.1315 participants were asked to complete eq-5d on alternate days after surgery for 4 weeks , and at the end of the fifth and sixth week ( total of 16 times ) , in a diary that was returned at the 6 week outpatient follow - up clinic appointment . among 201 patients ( 131 laparoscopic , 70 open ) recruited , 129 ( 64.2% ) had a diagnosis of cancer or polyps ( 85 laparoscopic , 44 open ) . hence , the final sample included in the analysis was 95 ( 68 laparoscopic , 27 open ) , of which 17 ( 18.0% ) had a diagnosis of polyps , rather than cancer ; 14 ( 20.6% ) of laparoscopic procedures , 3 ( 11.1% ) of open . costs and quality of life data were assessed for missing data . since it was found that a complete case analysis would reduce the sample to 43 , and result in exclusion of some participants with complete eq-5d or cost data , multiple imputation ( monte carlo simulation ) was used to generate missing values.16 it was not possible to impute stable quality of life estimates up to 42 days , so the analysis focused on the first 28 days after surgery ( 14 eq-5d scores ) . differences in mean qalys and costs between surgery groups were estimated simultaneously using a multivariate regression model applied to 20 imputed datasets with eq-5d at baseline , sex , age , and operation type ( left , right side ) included as covariates . a cost - effectiveness ellipse and cost - effectiveness acceptability curve ( ceac ) were produced to indicate the probability that laparoscopic surgery is cost - effective compared to open for a given societal willingness - to - pay ( wtp ) threshold . consecutive patients requiring elective colorectal resections ( over 18 years , without endometriosis , able to give consent ) in a district general hospital in south england were invited to join the study , 20062007 . a micro - costing study gathered treatment costs ( operative , in - hospital stay , community ) , in british pounds , 2006 , as described in full elsewhere.10 quality of life data were collected using euroqol 5d-3l instrument ( eq-5d ) . the five domains ( mobility , self - care , usual activities , pain / discomfort , anxiety / depression ) are scored on three levels ( 1 , no problem ; 2 , some problem ; 3 , serious / extreme problem / unable ) giving a theoretical maximum of 243 different health states which are converted to single index utility values ( range 0.59 for score 33333 to 1.0 [ perfect health ] for score 11111 ) , using uk tariff tables.1315 participants were asked to complete eq-5d on alternate days after surgery for 4 weeks , and at the end of the fifth and sixth week ( total of 16 times ) , in a diary that was returned at the 6 week outpatient follow - up clinic appointment . among 201 patients ( 131 laparoscopic , 70 open ) recruited , 129 ( 64.2% ) had a diagnosis of cancer or polyps ( 85 laparoscopic , 44 open ) . hence , the final sample included in the analysis was 95 ( 68 laparoscopic , 27 open ) , of which 17 ( 18.0% ) had a diagnosis of polyps , rather than cancer ; 14 ( 20.6% ) of laparoscopic procedures , 3 ( 11.1% ) of open . costs and quality of life data were assessed for missing data . since it was found that a complete case analysis would reduce the sample to 43 , and result in exclusion of some participants with complete eq-5d or cost data , multiple imputation ( monte carlo simulation ) was used to generate missing values.16 it was not possible to impute stable quality of life estimates up to 42 days , so the analysis focused on the first 28 days after surgery ( 14 eq-5d scores ) . differences in mean qalys and costs between surgery groups were estimated simultaneously using a multivariate regression model applied to 20 imputed datasets with eq-5d at baseline , sex , age , and operation type ( left , right side ) included as covariates . a cost - effectiveness ellipse and cost - effectiveness acceptability curve ( ceac ) were produced to indicate the probability that laparoscopic surgery is cost - effective compared to open for a given societal willingness - to - pay ( wtp ) threshold . the multiple imputation regression estimates ( table 2 ) show the cost of the laparoscopic operations was 1,037 higher than open ( p<0.005 ) , due to equipment costs ; staff costs were 190 lower ( p=0.039 ) due to a shorter operating time ( table 3 ) . there was no significant difference in the total costs between the groups because the higher laparoscopic surgery costs were offset by shorter lengths of stay . compared to those undergoing open surgery , patients in the laparoscopic group gained an average of 0.011207 qalys over the first 28 days after surgery ( 95% confidence intervals 0.006 to 0.016 ) ( table 2 and figure 1 ) . at 28 days , the overall incremental cost - effectiveness ratio ( icer ) , calculated as the difference in adjusted mean costs divided by the difference in adjusted mean qalys , and showing the cost per qaly gained from laparoscopic , compared to open surgery , was 12,375 . uncertainty in the icer point estimates are represented , using confidence intervals , on the cost - effectiveness plane ( cep ) ( figure 2 ) . the cost - effectiveness ellipse is centered in the top right quadrant , indicating that laparoscopic surgery is more costly and more effective than open . the point estimate icer lies below this threshold , demonstrating laparoscopic surgery as cost - effective . the ceac illustrates the probability that laparoscopic treatment will be deemed cost - effective at different societal wtp per qaly gained ( figure 3 ) . at 28 days , the ceac indicates that , at wtp of 30,000 , the probability that laparoscopic surgery will be cost - effective , compared to open methods , exceeds 65% ( and this is also illustrated by the proportion of the 95% confidence interval below the threshold line in figure 2 ) . at the costs of equipment and supplies , operative medical staff , hospital bed days , and community costs were varied independently of one another between the minimum and maximum values of their 95% confidence intervals whilst all other variables were held constant at their mean point estimates . at their lowest level , where costs were most favorable toward laparoscopic surgery , the resulting icers were all dominant in favor of the laparoscopic procedure . with operative staff costs and community costs at their highest level , laparoscopic surgery still appeared cost - effective at a wtp threshold of 30,000 ( with costs per qaly gained of 28,385 , and 17,219 respectively ) . when costs of equipment and supplies were at their highest level compared to open ( , 1477 more expensive ) , the resulting icer of 34,690 was marginally outside the cost - effective level ; the turning point was 1,424 . at its most expensive , where laparoscopic surgery achieved a reduction in hospital bed days costing only 247 compared to open , an icer of 70,332 was realized . the threshold , where an icer of 30,000 was achieved , was at a bed day cost reduction of 699 ; the mean cost reduction for bed days seen in the data exceeded this at 897 . varying the estimated difference in qaly between the lower ( 0.00603 ) and upper ( 0.01638 ) 95% confidence interval values had no effect on the cost - effectiveness at a wtp of 30,000 , achieving icer ranging from 22,994 to 8,466 . operation times may vary with surgeon experience and style , and have typically been shown to be higher in laparoscopic procedures than open.18 given the mean difference in qalys ( 0.011207 ) and costs ( 139 ) observed in this study , laparoscopic operation time could be increased by 55 minutes ( 197 ) , ie , 8 minutes longer than open , with all other factors held constant , and still achieve an icer 30,000 . at 28 days , the overall incremental cost - effectiveness ratio ( icer ) , calculated as the difference in adjusted mean costs divided by the difference in adjusted mean qalys , and showing the cost per qaly gained from laparoscopic , compared to open surgery , was 12,375 . uncertainty in the icer point estimates are represented , using confidence intervals , on the cost - effectiveness plane ( cep ) ( figure 2 ) . the cost - effectiveness ellipse is centered in the top right quadrant , indicating that laparoscopic surgery is more costly and more effective than open . the point estimate icer lies below this threshold , demonstrating laparoscopic surgery as cost - effective . the ceac illustrates the probability that laparoscopic treatment will be deemed cost - effective at different societal wtp per qaly gained ( figure 3 ) . at 28 days , the ceac indicates that , at wtp of 30,000 , the probability that laparoscopic surgery will be cost - effective , compared to open methods , exceeds 65% ( and this is also illustrated by the proportion of the 95% confidence interval below the threshold line in figure 2 ) . at the costs of equipment and supplies , operative medical staff , hospital bed days , and community costs were varied independently of one another between the minimum and maximum values of their 95% confidence intervals whilst all other variables were held constant at their mean point estimates . at their lowest level , where costs were most favorable toward laparoscopic surgery , the resulting icers were all dominant in favor of the laparoscopic procedure . where the differences in costs were considered at their highest ( in favor of open ) , the result was less clear . with operative staff costs and community costs at their highest level , laparoscopic surgery still appeared cost - effective at a wtp threshold of 30,000 ( with costs per qaly gained of 28,385 , and 17,219 respectively ) . when costs of equipment and supplies were at their highest level compared to open ( , 1477 more expensive ) , the resulting icer of 34,690 was marginally outside the cost - effective level ; the turning point was 1,424 . at its most expensive , where laparoscopic surgery achieved a reduction in hospital bed days costing only 247 compared to open , an icer of 70,332 was realized . the threshold , where an icer of 30,000 was achieved , was at a bed day cost reduction of 699 ; the mean cost reduction for bed days seen in the data exceeded this at 897 . varying the estimated difference in qaly between the lower ( 0.00603 ) and upper ( 0.01638 ) 95% confidence interval values had no effect on the cost - effectiveness at a wtp of 30,000 , achieving icer ranging from 22,994 to 8,466 . operation times may vary with surgeon experience and style , and have typically been shown to be higher in laparoscopic procedures than open.18 given the mean difference in qalys ( 0.011207 ) and costs ( 139 ) observed in this study , laparoscopic operation time could be increased by 55 minutes ( 197 ) , ie , 8 minutes longer than open , with all other factors held constant , and still achieve an icer 30,000 . this is the first cost - effectiveness analysis comparing laparoscopic and open colorectal surgery for colorectal resections to incorporate quality of life differences in the early postoperative recovery period . whilst previous studies , based on longer follow - up periods , suggest no difference in outcomes between laparoscopic and open surgery,36 this study provides robust evidence of improved health - related quality of life outcomes associated with laparoscopy in the first 6 weeks post - surgery , and demonstrates the cost - effectiveness of that approach , compared to open methods . improved health - related quality of life after laparoscopic surgery has been associated with less blood loss , better immune and inflammatory responses , less pain and analgesic requirements , faster postoperative recovery of bowel function , food intake , and physical activity.17,18 quality of life in this study was measured earlier in the recovery period and more intensively than in most other studies comparing laparoscopic and open methods for colorectal surgery . moreover , the eq-5d instrument was used to enable the calculation of qalys so that a cost - effectiveness analysis could be undertaken . health - related quality of life in most other studies has been measured using a variety of different generic and disease - specific instruments,4,5 and at later time points post - surgery , making direct comparison of the findings of different studies problematical . the analysis reported in this paper is based on those patients with a diagnosis of cancer or polyps in a larger study of patients undergoing colorectal surgery at a district general hospital in england,9 ie , some 65% of the whole sample , ( 47% cancer , 18% polyps ) . consistent with the parent study ( which included those with a diagnosis other than cancer or polyps , and rectal resections)10 and other evidence,19,20 the overall costs of laparoscopic and open approaches for this subgroup were not significantly different , with higher operation costs ( due to instrumentation ) for the laparoscopic group largely offset by a shorter length of hospital stay . the results demonstrate a significant qaly gain for laparoscopic surgery , compared to open , of 0.011207 at 28 days , which was similar to that observed for the whole sample in the parent study.7 this qaly gain exceeds that identified by the nice technology assessment panel ( 0.0090.010 ) as being required in order to deem laparoscopic surgery cost - effective for colorectal cancer.1,2 in the cost - effectiveness analysis , the point estimate cost per qaly gained was 12,375 . the probabilistic approach to analyze the uncertainty demonstrated that the laparoscopic procedure was close to clear dominance over open surgery and cost - effective at a wtp below the current threshold set by nice.8 the current guideline from nice11 is that no fixed wtp will be enforced , although the 2004 guidance was that a plausible threshold is 20,00030,000 . only the cost of hospital stay was independently capable of shifting the icer to a level above 30,000 , but at bed day savings associated with laparoscopic below those recorded in this study . the deterministic sensitivity analysis showed laparoscopic surgery to be cost - effective with operation times up to 8 minutes longer than open . the sample of cancer / polyp patients was drawn from a larger study of all patients undergoing colorectal resection , and statistical power was lost because the numbers in this subset was small . the proportion of patients with a diagnosis of polyps , rather than cancer , was higher in the laparoscopic group than the open group ( 20.6% versus 11.1% ) . whilst this affected the coefficients by a very small amount , the diagnosis covariate was not significant ( p>0.5 ) , indicating that it had no material effect on the findings operative costs were based on observation and researchers had not been present during all surgeries.10 some participants , particularly those with higher asa grades at baseline , longer hospital stays and in higher intensity units,7 had not provided quality of life data . even using multiple imputation , it was not possible to extend the analysis from 28 to 42 days , due to missing information . the latter found laparoscopic surgery to be 258 more expensive , on average , than open ( versus 139 using multiple imputation ) , and achieving a qaly gain of 0.013523 ( versus 0.011207 ) ; icer point estimate of 19,106 ( versus 12,375 ) . since that time , the relative cost of the disposable instruments ( a major item for the laparoscopic procedure ) may have fallen due to increased uptake and advancements in technology . the study only focused on the first 6 weeks after surgery , and no subsequent follow - up of patients was conducted . this was because prior research had demonstrated no differences in survival or health - related quality of life in the longer term,16 and the research design deliberately focused on the early postoperative period where a gap in evidence had been identified.1,7 this study provides formal evidence of the cost - effectiveness of laparoscopic approaches based on quality of life gains , compared to open methods , in the early postoperative period . the findings support current guidelines that promote the use of laparoscopy where suitably trained surgeons are available.1 the national training programme lapco is enabling more hospitals to offer minimally invasive surgery options and patients to reap the benefits of faster recovery.21

within the british national health service ( nhs ) , laparoscopic resection is recommended by the national institute for health and care excellence ( nice ) as an alternative to open surgery for people with colorectal cancer for whom both methods would be considered suitable , and if performed by a trained surgeon.1 a health technology assessment suggested that laparoscopic surgery was associated with higher costs , but was no more effective than open surgery . savings from shorter hospital stays after laparoscopic procedures did not fully offset higher operation costs and conversions to open methods due to complications.1 research concludes that there is no significant difference in disease free or overall survival at 3 years,1,2 or in health - related quality of life outcomes.36 however , few studies capture the short term benefits of laparoscopic surgery in the early postoperative period.1,7 the technology assessment concluded that , at the 30,000 cost per quality adjusted life year ( qaly ) threshold,8 laparoscopic surgery would need to result in a gain of 0.0090.010 qaly to be considered cost - effective , compared to open surgery.1,2 a study was undertaken to explore the qaly differences in the early postoperative recovery period,9 and costs.10 this paper reports the cost - effectiveness analysis that brings together the qaly and cost data for those patients who had a diagnosis of cancer or polyps . helped by the national training programme for laparoscopic colorectal surgery ( lapco ) , the uptake of laparoscopic surgery has increased in nhs hospitals , but it is still used in the minority of cases ( 40% in 2012).11 providing evidence on cost - effectiveness may encourage increased training of surgeons and wider use of laparoscopic methods.12 consecutive patients requiring elective colorectal resections ( over 18 years , without endometriosis , able to give consent ) in a district general hospital in south england were invited to join the study , 20062007 . a micro - costing study gathered treatment costs ( operative , in - hospital stay , community ) , in british pounds , 2006 , as described in full elsewhere.10 quality of life data were collected using euroqol 5d-3l instrument ( eq-5d ) . the five domains ( mobility , self - care , usual activities , pain / discomfort , anxiety / depression ) are scored on three levels ( 1 , no problem ; 2 , some problem ; 3 , serious / extreme problem / unable ) giving a theoretical maximum of 243 different health states which are converted to single index utility values ( range 0.59 for score 33333 to 1.0 [ perfect health ] for score 11111 ) , using uk tariff tables.1315 participants were asked to complete eq-5d on alternate days after surgery for 4 weeks , and at the end of the fifth and sixth week ( total of 16 times ) , in a diary that was returned at the 6 week outpatient follow - up clinic appointment . the sample was reduced to 98 ( 70 laparoscopic , 28 open ) by exclusion of patients undergoing a rectal resection because such procedures ( which typically result in higher costs and lower qaly due to requiring a stoma),7,10 were unevenly distributed between the groups ( n=15 , 17.6% laparoscopic ; n=16 , 36.4% open ) . hence , the final sample included in the analysis was 95 ( 68 laparoscopic , 27 open ) , of which 17 ( 18.0% ) had a diagnosis of polyps , rather than cancer ; 14 ( 20.6% ) of laparoscopic procedures , 3 ( 11.1% ) of open . since it was found that a complete case analysis would reduce the sample to 43 , and result in exclusion of some participants with complete eq-5d or cost data , multiple imputation ( monte carlo simulation ) was used to generate missing values.16 it was not possible to impute stable quality of life estimates up to 42 days , so the analysis focused on the first 28 days after surgery ( 14 eq-5d scores ) . differences in mean qalys and costs between surgery groups were estimated simultaneously using a multivariate regression model applied to 20 imputed datasets with eq-5d at baseline , sex , age , and operation type ( left , right side ) included as covariates . a cost - effectiveness ellipse and cost - effectiveness acceptability curve ( ceac ) were produced to indicate the probability that laparoscopic surgery is cost - effective compared to open for a given societal willingness - to - pay ( wtp ) threshold . consecutive patients requiring elective colorectal resections ( over 18 years , without endometriosis , able to give consent ) in a district general hospital in south england were invited to join the study , 20062007 . a micro - costing study gathered treatment costs ( operative , in - hospital stay , community ) , in british pounds , 2006 , as described in full elsewhere.10 quality of life data were collected using euroqol 5d-3l instrument ( eq-5d ) . the five domains ( mobility , self - care , usual activities , pain / discomfort , anxiety / depression ) are scored on three levels ( 1 , no problem ; 2 , some problem ; 3 , serious / extreme problem / unable ) giving a theoretical maximum of 243 different health states which are converted to single index utility values ( range 0.59 for score 33333 to 1.0 [ perfect health ] for score 11111 ) , using uk tariff tables.1315 participants were asked to complete eq-5d on alternate days after surgery for 4 weeks , and at the end of the fifth and sixth week ( total of 16 times ) , in a diary that was returned at the 6 week outpatient follow - up clinic appointment . the sample was reduced to 98 ( 70 laparoscopic , 28 open ) by exclusion of patients undergoing a rectal resection because such procedures ( which typically result in higher costs and lower qaly due to requiring a stoma),7,10 were unevenly distributed between the groups ( n=15 , 17.6% laparoscopic ; n=16 , 36.4% open ) . hence , the final sample included in the analysis was 95 ( 68 laparoscopic , 27 open ) , of which 17 ( 18.0% ) had a diagnosis of polyps , rather than cancer ; 14 ( 20.6% ) of laparoscopic procedures , 3 ( 11.1% ) of open . since it was found that a complete case analysis would reduce the sample to 43 , and result in exclusion of some participants with complete eq-5d or cost data , multiple imputation ( monte carlo simulation ) was used to generate missing values.16 it was not possible to impute stable quality of life estimates up to 42 days , so the analysis focused on the first 28 days after surgery ( 14 eq-5d scores ) . differences in mean qalys and costs between surgery groups were estimated simultaneously using a multivariate regression model applied to 20 imputed datasets with eq-5d at baseline , sex , age , and operation type ( left , right side ) included as covariates . a cost - effectiveness ellipse and cost - effectiveness acceptability curve ( ceac ) were produced to indicate the probability that laparoscopic surgery is cost - effective compared to open for a given societal willingness - to - pay ( wtp ) threshold . a favorable ethical opinion was obtained prior to starting the research from the south west surrey local research ethics committee ( study 05/q1909/74 ) , and the hospital research and development committee . the multiple imputation regression estimates ( table 2 ) show the cost of the laparoscopic operations was 1,037 higher than open ( p<0.005 ) , due to equipment costs ; staff costs were 190 lower ( p=0.039 ) due to a shorter operating time ( table 3 ) . the open group had a longer mean length of hospital stay ( table 3 ) and incurred 897 higher bed day costs ( p=0.007 ) compared to laparoscopic . there was no significant difference in the total costs between the groups because the higher laparoscopic surgery costs were offset by shorter lengths of stay . compared to those undergoing open surgery , patients in the laparoscopic group gained an average of 0.011207 qalys over the first 28 days after surgery ( 95% confidence intervals 0.006 to 0.016 ) ( table 2 and figure 1 ) . at 28 days , the overall incremental cost - effectiveness ratio ( icer ) , calculated as the difference in adjusted mean costs divided by the difference in adjusted mean qalys , and showing the cost per qaly gained from laparoscopic , compared to open surgery , was 12,375 . uncertainty in the icer point estimates are represented , using confidence intervals , on the cost - effectiveness plane ( cep ) ( figure 2 ) . the cost - effectiveness ellipse is centered in the top right quadrant , indicating that laparoscopic surgery is more costly and more effective than open . the line bisecting the top right and lower left quadrants shows the threshold cost - effectiveness line , based on a wtp of 20,000 . the ceac illustrates the probability that laparoscopic treatment will be deemed cost - effective at different societal wtp per qaly gained ( figure 3 ) . at 28 days , the ceac indicates that , at wtp of 30,000 , the probability that laparoscopic surgery will be cost - effective , compared to open methods , exceeds 65% ( and this is also illustrated by the proportion of the 95% confidence interval below the threshold line in figure 2 ) . at the costs of equipment and supplies , operative medical staff , hospital bed days , and community costs were varied independently of one another between the minimum and maximum values of their 95% confidence intervals whilst all other variables were held constant at their mean point estimates . at their lowest level , where costs were most favorable toward laparoscopic surgery , the resulting icers were all dominant in favor of the laparoscopic procedure . with operative staff costs and community costs at their highest level , laparoscopic surgery still appeared cost - effective at a wtp threshold of 30,000 ( with costs per qaly gained of 28,385 , and 17,219 respectively ) . when costs of equipment and supplies were at their highest level compared to open ( , 1477 more expensive ) , the resulting icer of 34,690 was marginally outside the cost - effective level ; the turning point was 1,424 . the threshold , where an icer of 30,000 was achieved , was at a bed day cost reduction of 699 ; the mean cost reduction for bed days seen in the data exceeded this at 897 . varying the estimated difference in qaly between the lower ( 0.00603 ) and upper ( 0.01638 ) 95% confidence interval values had no effect on the cost - effectiveness at a wtp of 30,000 , achieving icer ranging from 22,994 to 8,466 . staff operative costs were lower in the laparoscopic group , reflecting a shorter operation time by mean of 47 minutes , ( which equates to a theatre staff and overhead cost of 169 ) . operation times may vary with surgeon experience and style , and have typically been shown to be higher in laparoscopic procedures than open.18 given the mean difference in qalys ( 0.011207 ) and costs ( 139 ) observed in this study , laparoscopic operation time could be increased by 55 minutes ( 197 ) , ie , 8 minutes longer than open , with all other factors held constant , and still achieve an icer 30,000 . at 28 days , the overall incremental cost - effectiveness ratio ( icer ) , calculated as the difference in adjusted mean costs divided by the difference in adjusted mean qalys , and showing the cost per qaly gained from laparoscopic , compared to open surgery , was 12,375 . uncertainty in the icer point estimates are represented , using confidence intervals , on the cost - effectiveness plane ( cep ) ( figure 2 ) . the cost - effectiveness ellipse is centered in the top right quadrant , indicating that laparoscopic surgery is more costly and more effective than open . the ceac illustrates the probability that laparoscopic treatment will be deemed cost - effective at different societal wtp per qaly gained ( figure 3 ) . at 28 days , the ceac indicates that , at wtp of 30,000 , the probability that laparoscopic surgery will be cost - effective , compared to open methods , exceeds 65% ( and this is also illustrated by the proportion of the 95% confidence interval below the threshold line in figure 2 ) . at the costs of equipment and supplies , operative medical staff , hospital bed days , and community costs were varied independently of one another between the minimum and maximum values of their 95% confidence intervals whilst all other variables were held constant at their mean point estimates . at their lowest level , where costs were most favorable toward laparoscopic surgery , the resulting icers were all dominant in favor of the laparoscopic procedure . with operative staff costs and community costs at their highest level , laparoscopic surgery still appeared cost - effective at a wtp threshold of 30,000 ( with costs per qaly gained of 28,385 , and 17,219 respectively ) . when costs of equipment and supplies were at their highest level compared to open ( , 1477 more expensive ) , the resulting icer of 34,690 was marginally outside the cost - effective level ; the turning point was 1,424 . varying the estimated difference in qaly between the lower ( 0.00603 ) and upper ( 0.01638 ) 95% confidence interval values had no effect on the cost - effectiveness at a wtp of 30,000 , achieving icer ranging from 22,994 to 8,466 . staff operative costs were lower in the laparoscopic group , reflecting a shorter operation time by mean of 47 minutes , ( which equates to a theatre staff and overhead cost of 169 ) . operation times may vary with surgeon experience and style , and have typically been shown to be higher in laparoscopic procedures than open.18 given the mean difference in qalys ( 0.011207 ) and costs ( 139 ) observed in this study , laparoscopic operation time could be increased by 55 minutes ( 197 ) , ie , 8 minutes longer than open , with all other factors held constant , and still achieve an icer 30,000 . this is the first cost - effectiveness analysis comparing laparoscopic and open colorectal surgery for colorectal resections to incorporate quality of life differences in the early postoperative recovery period . whilst previous studies , based on longer follow - up periods , suggest no difference in outcomes between laparoscopic and open surgery,36 this study provides robust evidence of improved health - related quality of life outcomes associated with laparoscopy in the first 6 weeks post - surgery , and demonstrates the cost - effectiveness of that approach , compared to open methods . improved health - related quality of life after laparoscopic surgery has been associated with less blood loss , better immune and inflammatory responses , less pain and analgesic requirements , faster postoperative recovery of bowel function , food intake , and physical activity.17,18 quality of life in this study was measured earlier in the recovery period and more intensively than in most other studies comparing laparoscopic and open methods for colorectal surgery . moreover , the eq-5d instrument was used to enable the calculation of qalys so that a cost - effectiveness analysis could be undertaken . health - related quality of life in most other studies has been measured using a variety of different generic and disease - specific instruments,4,5 and at later time points post - surgery , making direct comparison of the findings of different studies problematical . the analysis reported in this paper is based on those patients with a diagnosis of cancer or polyps in a larger study of patients undergoing colorectal surgery at a district general hospital in england,9 ie , some 65% of the whole sample , ( 47% cancer , 18% polyps ) . consistent with the parent study ( which included those with a diagnosis other than cancer or polyps , and rectal resections)10 and other evidence,19,20 the overall costs of laparoscopic and open approaches for this subgroup were not significantly different , with higher operation costs ( due to instrumentation ) for the laparoscopic group largely offset by a shorter length of hospital stay . the results demonstrate a significant qaly gain for laparoscopic surgery , compared to open , of 0.011207 at 28 days , which was similar to that observed for the whole sample in the parent study.7 this qaly gain exceeds that identified by the nice technology assessment panel ( 0.0090.010 ) as being required in order to deem laparoscopic surgery cost - effective for colorectal cancer.1,2 in the cost - effectiveness analysis , the point estimate cost per qaly gained was 12,375 . the probabilistic approach to analyze the uncertainty demonstrated that the laparoscopic procedure was close to clear dominance over open surgery and cost - effective at a wtp below the current threshold set by nice.8 the current guideline from nice11 is that no fixed wtp will be enforced , although the 2004 guidance was that a plausible threshold is 20,00030,000 . only the cost of hospital stay was independently capable of shifting the icer to a level above 30,000 , but at bed day savings associated with laparoscopic below those recorded in this study . the proportion of patients with a diagnosis of polyps , rather than cancer , was higher in the laparoscopic group than the open group ( 20.6% versus 11.1% ) . since the postoperative recovery of patients with cancer is potentially different from that of patients with polyps , we tested whether the sample imbalance might affect the results , by adding a diagnosis variable ( cancer versus polyps ) to the regression modelling . whilst this affected the coefficients by a very small amount , the diagnosis covariate was not significant ( p>0.5 ) , indicating that it had no material effect on the findings operative costs were based on observation and researchers had not been present during all surgeries.10 some participants , particularly those with higher asa grades at baseline , longer hospital stays and in higher intensity units,7 had not provided quality of life data . the impact of uncertainty in the sample data was accommodated in the sensitivity analysis and found to have little impact on the conclusions of the study . the latter found laparoscopic surgery to be 258 more expensive , on average , than open ( versus 139 using multiple imputation ) , and achieving a qaly gain of 0.013523 ( versus 0.011207 ) ; icer point estimate of 19,106 ( versus 12,375 ) . since that time , the relative cost of the disposable instruments ( a major item for the laparoscopic procedure ) may have fallen due to increased uptake and advancements in technology . this was because prior research had demonstrated no differences in survival or health - related quality of life in the longer term,16 and the research design deliberately focused on the early postoperative period where a gap in evidence had been identified.1,7 this study provides formal evidence of the cost - effectiveness of laparoscopic approaches based on quality of life gains , compared to open methods , in the early postoperative period . the findings support current guidelines that promote the use of laparoscopy where suitably trained surgeons are available.1 the national training programme lapco is enabling more hospitals to offer minimally invasive surgery options and patients to reap the benefits of faster recovery.21

the present analyses were performed in the rotterdam study , an ongoing population - based study on the determinants of disease and disability among persons 55 years and older . a trained investigator obtained information on health status , medical history , medication use and lifestyle . subsequently , participants were invited to the study center where they underwent an extensive clinical examination . the medical ethics committee of the erasmus medical center approved the study protocol and all participants provided written informed consent . from 10,275 eligible subjects , 7,983 ( 78% ) individuals agreed to participate in the study and were examined at baseline . the clinical examination included weight , height , systolic and diastolic blood pressure , serum glucose , total serum cholesterol and high - density lipoprotein ( hdl ) cholesterol levels . bmi was computed as weight ( kg ) divided by height squared ( m ) . participants were classified as underweight ( bmi < 18.5 kg / m ) , normal weight ( 18.5 kg / m bmi < 25 kg / m ) , overweight ( 25 kg / m bmi < 30 kg / m ) or obese ( bmi 30 kg / m ) . the waist and hip circumferences were measured , and the waist - hip - ratio ( whr ) was calculated as indirect assessment of abdominal fat . systolic and diastolic blood pressures were measured twice in a sitting position after 5 min rest using a random - zero sphygmomanometer . hypertension was defined as systolic blood pressure higher than 160 mmhg , diastolic blood pressure higher than 100 mmhg , or the use of antihypertensive medication indicated to treat high blood pressure ( hypertension grades 2 and 3 ) . diabetes was diagnosed based on a random or post - load glucose level higher than 11.0 mmol / l and/or the use of anti - diabetic medication . total serum cholesterol and hdl - cholesterol hypercholesterolemia was defined as a total serum cholesterol level above 6.2 mmol / l . the apoe genotype was determined on dna samples using a polymerase chain reaction followed by enzymatic digestion using methods previously described . the frequencies of apoe genotypes were apoe 2/2 0.8% , apoe 2/3 12.8% , apoe 2/4 2.7% , apoe 3/3 58.4% , apoe 3/4 22.9% and apoe 4/4 2.4% . the proportions of the apoe alleles and genotypes were in hardy - weinberg equilibrium ( p = 0.71 ) . information on the vital status of the participants was obtained at regular intervals from the municipal population registry . causes of death were obtained from the general practitioners by means of a standardized questionnaire relating to the circumstances of death , most likely cause of death and time and place of death . two independent research physicians coded all events according to the international classification of diseases , 10th edition ( icd-10 ) . chd mortality was defined as death from diseases coded i20i25 , i46 , i50 or r96 . of the 7,983 individuals who participated at baseline , 5,817 ( 73% ) had apoe genotyped successfully and had complete information on bmi , whr and cholesterol levels . because persons with an extremely low bmi may suffer from life - threatening diseases such as cancer , individuals with a bmi below 18.5 kg / m ( n = 51 ) were excluded from the analyses . also , apoe 2/4 individuals were excluded from the analyses ( n = 158 ) to distinguish the effect of the apoe alleles unambiguously . differences in baseline characteristics by apoe genotypes were tested using the chi - squared statistic ( categorical variables ) or anova ( continuous variables ) . p for trend was obtained by testing the linearity of the sum of squares from the anova analyses . multiple linear regression analysis was used to assess the interaction between apoe genotype and bmi on total serum cholesterol and hdl - cholesterol levels . differences in survival probabilities were examined by comparing kaplan - meier survival curves and tested using the log rank test . kaplan - meier plots were constructed using age as the time scale to take proper account of the effect of age [ 31 , 32 ] . risks of mortality were quantified as hazard ratios ( hrs ) using cox proportional hazards analyses with age as the time scale . because the proportionality assumption was not met , hrs were calculated for early and late mortality using 80 years as the cut - off age . this cut - off age was selected because it led to proportionality of the models below and above the cut - off age . hrs were calculated adjusted for gender , smoking status , education level , total serum cholesterol , hdl - cholesterol , waist to hip ratio hypertension and diabetes mellitus . the present analyses were performed in the rotterdam study , an ongoing population - based study on the determinants of disease and disability among persons 55 years and older . a trained investigator obtained information on health status , medical history , medication use and lifestyle . subsequently , participants were invited to the study center where they underwent an extensive clinical examination . the medical ethics committee of the erasmus medical center approved the study protocol and all participants provided written informed consent . from 10,275 eligible subjects , 7,983 ( 78% ) individuals agreed to participate in the study and were examined at baseline . the clinical examination included weight , height , systolic and diastolic blood pressure , serum glucose , total serum cholesterol and high - density lipoprotein ( hdl ) cholesterol levels . bmi was computed as weight ( kg ) divided by height squared ( m ) . participants were classified as underweight ( bmi < 18.5 kg / m ) , normal weight ( 18.5 kg / m bmi < 25 kg / m ) , overweight ( 25 kg / m bmi < 30 kg / m ) or obese ( bmi 30 the waist - hip - ratio ( whr ) was calculated as indirect assessment of abdominal fat . systolic and diastolic blood pressures were measured twice in a sitting position after 5 min rest using a random - zero sphygmomanometer . hypertension was defined as systolic blood pressure higher than 160 mmhg , diastolic blood pressure higher than 100 mmhg , or the use of antihypertensive medication indicated to treat high blood pressure ( hypertension grades 2 and 3 ) . diabetes was diagnosed based on a random or post - load glucose level higher than 11.0 mmol / l and/or the use of anti - diabetic medication . hypercholesterolemia was defined as a total serum cholesterol level above 6.2 mmol / l . the apoe genotype was determined on dna samples using a polymerase chain reaction followed by enzymatic digestion using methods previously described . the frequencies of apoe genotypes were apoe 2/2 0.8% , apoe 2/3 12.8% , apoe 2/4 2.7% , apoe 3/3 58.4% , apoe 3/4 22.9% and apoe 4/4 2.4% . the proportions of the apoe alleles and genotypes were in hardy - weinberg equilibrium ( p = 0.71 ) . information on the vital status of the participants was obtained at regular intervals from the municipal population registry . causes of death were obtained from the general practitioners by means of a standardized questionnaire relating to the circumstances of death , most likely cause of death and time and place of death . two independent research physicians coded all events according to the international classification of diseases , 10th edition ( icd-10 ) . chd mortality was defined as death from diseases coded i20i25 , i46 , i50 or r96 . the clinical examination included weight , height , systolic and diastolic blood pressure , serum glucose , total serum cholesterol and high - density lipoprotein ( hdl ) cholesterol levels . bmi was computed as weight ( kg ) divided by height squared ( m ) . participants were classified as underweight ( bmi < 18.5 kg / m ) , normal weight ( 18.5 kg / m bmi < 25 kg / m ) , overweight ( 25 kg / m bmi < 30 kg / m ) or obese ( bmi 30 the waist - hip - ratio ( whr ) was calculated as indirect assessment of abdominal fat . systolic and diastolic blood pressures were measured twice in a sitting position after 5 min rest using a random - zero sphygmomanometer . hypertension was defined as systolic blood pressure higher than 160 mmhg , diastolic blood pressure higher than 100 mmhg , or the use of antihypertensive medication indicated to treat high blood pressure ( hypertension grades 2 and 3 ) . diabetes was diagnosed based on a random or post - load glucose level higher than 11.0 mmol / l and/or the use of anti - diabetic medication . hypercholesterolemia was defined as a total serum cholesterol level above 6.2 mmol / l . the apoe genotype was determined on dna samples using a polymerase chain reaction followed by enzymatic digestion using methods previously described . the frequencies of apoe genotypes were apoe 2/2 0.8% , apoe 2/3 12.8% , apoe 2/4 2.7% , apoe 3/3 58.4% , apoe 3/4 22.9% and apoe 4/4 2.4% . the proportions of the apoe alleles and genotypes were in hardy - weinberg equilibrium ( p = 0.71 ) . information on the vital status of the participants was obtained at regular intervals from the municipal population registry . causes of death were obtained from the general practitioners by means of a standardized questionnaire relating to the circumstances of death , most likely cause of death and time and place of death . two independent research physicians coded all events according to the international classification of diseases , 10th edition ( icd-10 ) . chd mortality was defined as death from diseases coded i20i25 , i46 , i50 or r96 . of the 7,983 individuals who participated at baseline , 5,817 ( 73% ) had apoe genotyped successfully and had complete information on bmi , whr and cholesterol levels . because persons with an extremely low bmi may suffer from life - threatening diseases such as cancer , individuals with a bmi below 18.5 kg / m ( n = 51 ) also , apoe 2/4 individuals were excluded from the analyses ( n = 158 ) to distinguish the effect of the apoe alleles unambiguously . differences in baseline characteristics by apoe genotypes were tested using the chi - squared statistic ( categorical variables ) or anova ( continuous variables ) . p for trend was obtained by testing the linearity of the sum of squares from the anova analyses . multiple linear regression analysis was used to assess the interaction between apoe genotype and bmi on total serum cholesterol and hdl - cholesterol levels . differences in survival probabilities were examined by comparing kaplan - meier survival curves and tested using the log rank test . kaplan - meier plots were constructed using age as the time scale to take proper account of the effect of age [ 31 , 32 ] . risks of mortality were quantified as hazard ratios ( hrs ) using cox proportional hazards analyses with age as the time scale . because the proportionality assumption was not met , hrs were calculated for early and late mortality using 80 years as the cut - off age . this cut - off age was selected because it led to proportionality of the models below and above the cut - off age . hrs were calculated adjusted for gender , smoking status , education level , total serum cholesterol , hdl - cholesterol , waist to hip ratio hypertension and diabetes mellitus . the mean age at entry was 68.7 years ( sd = 8.7 years ) and 41.8% of the participants were men . the mean follow up time was 11.1 years ( sd = 3.8 years ) . baseline characteristics of apoe*2 carriers , apoe*3 homozygotes and apoe*4 carriers are presented in table 1 . the prevalence of hypercholesterolaemia was lowest among apoe*2 carriers ( 49% ) and highest among apoe*4 carriers ( 66% ) . furthermore , there were statistically significant differences in bmi scores between the genotype groups , with apoe*2 carriers having the highest mean bmi ( 26.7 3.6 kg / m ) and apoe*4 carriers the lowest ( 26.1 3.4 kg / m ) . table 1baseline characteristics by apoe genotype groupsapoe*2 carriersapoe 3/3apoe*4 carrierspn7823,3691,457sex ( men)3842420.05age at entry ( years)68.8 ( 8.8)68.9 ( 8.7)68.3 ( 8.5)0.14body mass index ( kg / m)26.7 ( 3.6)26.4 ( 3.6)26.1 ( 3.4)<0.01normal weight343940overweight494747<0.05obese171413waist - to - hip ratio0.9 ( 0.1)0.9 ( 0.1)0.9 ( 0.1)0.18systolic blood pressure ( mmhg)140 ( 22)140 ( 23)138 ( 2)0.04diastolic blood pressure ( mmhg)74 ( 11)74 ( 12)73 ( 11)0.06hypertension3634330.25total serum cholesterol ( mmol / l)6.4 ( 1.3)6.6 ( 1.2)6.8 ( 1.2)<0.001hdl - cholesterol ( mmol / l)1.4 ( 0.4)1.3 ( 0.3)1.3 ( 0.4)<0.001cholesterol lowering medication3230.71hypercholesterolemia496066<0.001smoking status ( current)3935330.02education level lower434345 intermediate4342400.21 higher141515diabetes mellitus101090.75values are means ( standard deviations ) for continuous variables and percentages for categorical variables . p values are obtained by anova for continuous variables and by for categorical variables baseline characteristics by apoe genotype groups values are means ( standard deviations ) for continuous variables and percentages for categorical variables . p values are obtained by anova for continuous variables and by for categorical variables figure 1 presents mean total serum cholesterol and hdl - cholesterol levels by apoe genotype in normal weight , overweight and obese persons . in all bmi strata , apoe*2 carriers had the lowest and apoe*4 carriers the highest total serum cholesterol ( pfor trend < 0.01 ) . the association between hdl - cholesterol and apoe genotype was in the opposite direction ( pfor trend < 0.01 ) . note that even though apoe*2 carriers had the lowest total serum cholesterol levels in each bmi group , still 50% of the overweight and 57% of the obese apoe*2 carriers had hypercholesterolaemia compared to 41% of the normal weight apoe*2 carriers ( p = 0.003 ) . there was no significant evidence for interaction between apoe genotype and bmi in relation to total serum cholesterol ( p = 0.26 ) and hdl - cholesterol ( p = 0.25 ) . however , when bmi was analyzed as a continuous trait , then the p value for interaction was 0.04 for total serum cholesterol and less than 0.001 for hdl - cholesterol . 1mean total serum cholesterol and hdl - cholesterol levels by apoe genotypes for normal weight , overweight and obese individuals . hdl : high - density lipoproteins mean total serum cholesterol and hdl - cholesterol levels by apoe genotypes for normal weight , overweight and obese individuals . hdl : high - density lipoproteins a total of 1,918 deaths occurred during follow - up . of those , 257 occurred in 782 apoe*2 carriers ( 33% ) , 1,160 in 3,369 apoe*3 homozygotes ( 33% ) and 501 in 1,457 apoe*4 carriers ( 34% ) . differences between the kaplan - meier survival curves of the apoe*2 carriers , apoe*3 homozygotes and apoe*4 carriers were small but statistically significant ( p = 0.03 ) with apoe*2 carriers having slightly lower mortality risks ( fig . 2 ) . after adjustment for other cardiovascular risk factors , apoe genotype was not associated with all - cause mortality before or after 80 years of age in the overall population ( table 2 ) . the differences between the curves were only statistically significant in normal weight individuals ( p = 0.04 ; fig . 3 ) . normal weight carriers of the apoe*2 allele had a significantly lower risk of mortality before age 80 years ( hr [ 95% ci ] = 0.7 [ 0.50.9 ] ) , but not after age 80 years ( crude hr [ 95% ci ] = 1.0 [ 0.81.4 ] ) . there were no significant differences in the risk of mortality between genotype groups among overweight or obese persons ( table 2 ) . 2kaplan meier survival curves for all - cause mortality by apoe genotypestable 2incidence rates and hazard ratios for all - cause mortality by apoe genotypes and bmi categoriesfollow - upincidence ratehazard ratiofollow - upincidence ratehazard ratiodeaths(py)deaths/1,000 py ( 95% ci)(95% ci)deaths(py)deaths/1,000 py ( 95% ci)(95% ci)before 80 years of ageafter 80 years of ageallapoe*2 carriers1016,78214.9 ( 12.318.1)0.9 ( 0.71.1)15621,7647.2 ( 6.18.4)0.9 ( 0.81.1)apoe 3/348128,70316.8 ( 15.318.3)67995,8087.1 ( 6.67.6)apoe*4 carriers19812,69315.6 ( 13.617.9)1.0 ( 0.81.2)30341,6377.3 ( 6.58.1)1.2 ( 1.01.4)total78048,17816.2 ( 15.117.4)1,138159,2097.1 ( 6.77.6)normal weightapoe*2 carriers282,37011.8 ( 8.217.1)0.7 ( 0.50.9)577,2897.8 ( 6.010.1)1.0 ( 0.81.4)apoe 3/320611,31418.2 ( 15.920.9)25638,0456.7 ( 6.07.6)apoe*4 carriers785,08015.4 ( 12.319.2)1.0 ( 0.71.3)12717,3057.3 ( 6.28.7)1.3 ( 0.91.7)total31218,76416.6 ( 14.918.6)44062,6397.0 ( 6.47.7)overweightapoe*2 carriers593,30517.9 ( 13.823.0)1.1 ( 0.91.5)6511,0645.9 ( 4.67.5)0.9 ( 0.71.1)apoe 3/321713,57316.0 ( 14.018.3)31045,3666.8 ( 6.17.6)apoe*4 carriers906,04714.9 ( 12.118.3)0.9 ( 0.71.2)13719,3987.1 ( 6.08.4)1.2 ( 1.01.5)total36622,92416.0 ( 14.417.7)51275,8276.8 ( 6.27.4)obeseapoe*2 carriers141,10712.6 ( 7.521.4)1.0 ( 0.51.8)343,41110.0 ( 7.114.0)1.0 ( 0.61.5)apoe 3/3583,81615.2 ( 11.719.7)11312,3989.1 ( 7.611.0)apoe*4 carriers301,56619.2 ( 13.427.4)1.3 ( 0.92.0)394,9347.9 ( 5.810.8)1.1 ( 0.81.6)total1026,48915.7 ( 12.919.1)18620,7429.0 ( 7.810.4)hazard ratios adjusted for sex , waist - hip - ratio , smoking , education level , total serum cholesterol , hdl cholesterol hypertension and diabetes ; py : person - years ; ci : confidence intervalfig . 3kaplan meier survival curves for all - cause mortality by apoe genotypes for normal weight , overweight and obese individuals kaplan meier survival curves for all - cause mortality by apoe genotypes incidence rates and hazard ratios for all - cause mortality by apoe genotypes and bmi categories hazard ratios adjusted for sex , waist - hip - ratio , smoking , education level , total serum cholesterol , hdl cholesterol hypertension and diabetes ; py : person - years ; ci : confidence interval kaplan meier survival curves for all - cause mortality by apoe genotypes for normal weight , overweight and obese individuals to investigate whether the lower risk of mortality observed among normal weight apoe*2 carriers was due to a lower risk of mortality from chd , we calculated the hrs separately for chd mortality . analysis within bmi strata showed that normal weight apoe*2 carriers tended to have lower risks of chd mortality ( adjusted hr [ 95% ci ] = 0.5 [ 0.21.2 ] ) . no differences in the risk of chd mortality between genotype groups were found in overweight or obese participants ( table 3 ) . table 3incidence rates and hazard ratios for chd mortality by apoe genotypes and bmi categoriesfollow - upincidence ratehazard ratiofollow - upincidence ratehazard ratiodeaths(py)deaths/1,000 py ( 95% ci)(95% ci)deaths(py)deaths/1,000 py ( 95% ci)(95% ci)before 80 years of ageafter 80 years of ageallapoe*2 carriers266,7823.8 ( 2.65.6)1.0 ( 0.61.5)412,10319.5 ( 14.426.5)1.1 ( 0.81.5)apoe 3/312628,7034.4 ( 3.75.2)1718,74419.6 ( 16.822.7)apoe*4 carriers5912,6934.6 ( 3.66.0)1.1 ( 0.81.4)663,47219.0 ( 14.924.2)1.1 ( 0.81.4)total21148,1784.4 ( 3.85.0)27814,31919.4 ( 17.321.8)normal weightapoe*2 carriers52,3702.1 ( 0.95.1)0.5 ( 0.21.2)1566322.6 ( 13.637.5)1.6 ( 0.92.9)apoe 3/35611,3144.9 ( 3.86.4)483,07515.6 ( 11.820.7)apoe*4 carriers265,0805.1 ( 3.57.5)1.2 ( 0.71.9)211,28616.3 ( 10.625.1)1.1 ( 0.61.8)total8718,7644.6 ( 3.85.7)845,02416.7 ( 13.520.7)overweightapoe*2 carriers163,3054.8 ( 3.07.9)1.4 ( 0.82.4)1598915.2 ( 9.125.2)0.7 ( 0.41.3)apoe 3/35113,5733.8 ( 2.94.9)924,03522.8 ( 18.628.0)apoe*4 carriers216,0473.5 ( 2.35.3)0.8 ( 0.51.4)351,61921.6 ( 15.530.1)1.0 ( 0.71.5)total8822,9243.8 ( 3.14.7)1426,64321.4 ( 18.125.2)obeseapoe*2 carriers51,1074.5 ( 1.910.8)1.1 ( 0.43.4)1145024.4 ( 13.544.1)1.4 ( 0.73.1)apoe 3/3193,8165.0 ( 3.27.8)311,63419.0 ( 13.327.0)apoe*4 carriers121,5667.7 ( 4.413.5)1.6 ( 0.83.5)1056817.6 ( 9.532.7)1.1 ( 0.52.2)total366,4895.5 ( 4.07.7)522,65119.6 ( 14.925.7)hazard ratios adjusted for sex , waist - hip - ratio , smoking , education level , total serum cholesterol , hdl cholesterol hypertension and diabetes ; py : person - years ; ci : confidence interval incidence rates and hazard ratios for chd mortality by apoe genotypes and bmi categories hazard ratios adjusted for sex , waist - hip - ratio , smoking , education level , total serum cholesterol , hdl cholesterol hypertension and diabetes ; py : person - years ; ci : confidence interval analyses within bmi strata demonstrated that this genetic advantage of apoe*2 was observed only in normal weight individuals , but not in overweight and obese apoe*2 carriers . the results of the present analyses may seem to contradict our earlier work in this population . we previously reported that the apoe gene was not significantly related to mortality . while our findings come from the same population , the mean follow - up time of the participants has increased from 5.4 years to 11.1 years and the number of deaths increased by 53% from 18% to 34% . this extended follow - up has improved the statistical power of our study to show a modest effect of apoe genotype on overall mortality . research on the relationship between apoe and mortality has merely provided conflicting results [ 619 , 34 ] . three out of eleven studies showed a protective effect of apoe*2 [ 6 , 14 , 18 ] . and three a deleterious effect of apoe*4 [ 810 ] . the latter effect may partly mediated by the increased risk of alzheimer disease in association with apoe*4 and mortality [ 10 , 11 ] . note that our analysis showed small significant difference in the kaplan - meier survival curves , demonstrating a protective effect of apoe*2 before age 80 . this may be due to the effect of age that becomes apparent when performing survival analysis with age as time scale , as we did in the present paper . such finding is compatible with previous reports suggesting that genes affecting human lifespan might be age - specific . genetic and environmental interactions at older ages vary from those at early ages and therefore explain the differences in the association between apoe and mortality before and after 80 years of age . a previous report showed that among nonagenarians , apoe genotype has no effect on cognitive function , cognitive decline or survival . in line with previous studies [ 2022 ] , we found that the total serum cholesterol levels associated with apoe genotype differed between normal weight , overweight and obese individuals . as expected , mean levels of total serum cholesterol increased with bmi and apoe*2 carriers had the lowest mean total serum cholesterol in each bmi group . apoe*4 carriers had higher cholesterol levels and lower hdl - cholesterol concentrations than apoe*2 carriers despite the presence of a lower bmi and equal whr . previous reports showed that apoe genotype , bmi and whr determine together the lipid levels [ 2022 , 37 ] . however , in our study abdominal fat , as measured by whr , did not have any effect on the lipid levels by apoe genotype , while bmi was an important determinant . the significant interaction between apoe genotype and bmi in relation to total serum cholesterol found previously , was also supported by this study . although the mechanism of such interaction has not been yet elucidated , evidence suggests that obesity and abdominal fat increase low density lipoprotein cholesterol and therefore total cholesterol at a higher degree among apoe*4 carriers [ 2022 , 37 ] . this is in line with our observation that the deleterious effect of increased bmi prevails over the beneficial effects of the apoe*2 allele . furthermore , previous research in the rotterdam study and the dutch population indicated that among apoe*2 homozygous carriers , the expression of hyperlipoproteinemia type iii is determined to a great extent by hyperinsulinemia and the presence of insulin resistance syndrome , conferring and increased risk of chd through accelerated atherosclerosis . although the association between high bmi and high cholesterol levels is well known and the apoe gene is associated with cholesterol levels as previously described , this is the first study that examined the effect of apoe genotype on mortality in bmi strata . we found that presence of the apoe*2 allele was associated with a decreased risk of mortality only in normal weight individuals and that this lower risk was partly explained by lower risk of chd mortality . these results were in line with the lower total cholesterol and higher hdl levels among normal weight apoe*2 carriers . our finding that the protective effect of apoe*2 was only found in normal weight individuals suggests that the negative influences of increasing bmi outweigh the positive effects of genetic predisposition . when these results are confirmed by other population - based studies , a challenging question remains whether weight loss in overweight and obese apoe*2 carriers restores the genetic advantage of their apoe status .

objective to investigate the relationship between the apolipoprotein e ( apoe ) gene and the risk of mortality in normal weight , overweight and obese individuals . methods and results in a population - based study of 7,983 individuals aged 55 years and older , we compared the risks of all - cause and coronary heart disease ( chd ) mortality by apoe genotype , both overall and in subgroups defined by body mass index ( bmi ) . we found significant evidence for interaction between apoe and bmi in relation to total cholesterol ( p = 0.04 ) and hdl cholesterol ( p < 0.001 ) . overall , apoe*2 carriers showed a decreased risk of all - cause mortality . analyses within bmi strata showed a beneficial effect of apoe*2 only in normal weight persons ( adjusted hazard ratio ( hr ) 0.7[95% ci 0.50.9 ] ) . apoe*2 was not associated with a lower risk of all - cause mortality in overweight or obese persons . the effect of apoe*2 in normal weight individuals tended to be due to the risk of chd mortality ( adjusted hr 0.5 [ 95% ci 0.21.2 ] ) . conclusion the apoe*2 allele confers a lower risk of all - cause mortality only to normal weight individuals .

Methods Study population and procedures Data collection Clinical and laboratory assessments Mortality data Statistical analyses Results Discussion

the present analyses were performed in the rotterdam study , an ongoing population - based study on the determinants of disease and disability among persons 55 years and older . participants were classified as underweight ( bmi < 18.5 kg / m ) , normal weight ( 18.5 kg / m bmi < 25 kg / m ) , overweight ( 25 kg / m bmi < 30 kg / m ) or obese ( bmi 30 kg / m ) . the waist and hip circumferences were measured , and the waist - hip - ratio ( whr ) was calculated as indirect assessment of abdominal fat . the proportions of the apoe alleles and genotypes were in hardy - weinberg equilibrium ( p = 0.71 ) . also , apoe 2/4 individuals were excluded from the analyses ( n = 158 ) to distinguish the effect of the apoe alleles unambiguously . multiple linear regression analysis was used to assess the interaction between apoe genotype and bmi on total serum cholesterol and hdl - cholesterol levels . the present analyses were performed in the rotterdam study , an ongoing population - based study on the determinants of disease and disability among persons 55 years and older . participants were classified as underweight ( bmi < 18.5 kg / m ) , normal weight ( 18.5 kg / m bmi < 25 kg / m ) , overweight ( 25 kg / m bmi < 30 kg / m ) or obese ( bmi 30 the waist - hip - ratio ( whr ) was calculated as indirect assessment of abdominal fat . the proportions of the apoe alleles and genotypes were in hardy - weinberg equilibrium ( p = 0.71 ) . participants were classified as underweight ( bmi < 18.5 kg / m ) , normal weight ( 18.5 kg / m bmi < 25 kg / m ) , overweight ( 25 kg / m bmi < 30 kg / m ) or obese ( bmi 30 the waist - hip - ratio ( whr ) was calculated as indirect assessment of abdominal fat . the proportions of the apoe alleles and genotypes were in hardy - weinberg equilibrium ( p = 0.71 ) . because persons with an extremely low bmi may suffer from life - threatening diseases such as cancer , individuals with a bmi below 18.5 kg / m ( n = 51 ) also , apoe 2/4 individuals were excluded from the analyses ( n = 158 ) to distinguish the effect of the apoe alleles unambiguously . multiple linear regression analysis was used to assess the interaction between apoe genotype and bmi on total serum cholesterol and hdl - cholesterol levels . baseline characteristics of apoe*2 carriers , apoe*3 homozygotes and apoe*4 carriers are presented in table 1 . furthermore , there were statistically significant differences in bmi scores between the genotype groups , with apoe*2 carriers having the highest mean bmi ( 26.7 3.6 kg / m ) and apoe*4 carriers the lowest ( 26.1 3.4 kg / m ) . table 1baseline characteristics by apoe genotype groupsapoe*2 carriersapoe 3/3apoe*4 carrierspn7823,3691,457sex ( men)3842420.05age at entry ( years)68.8 ( 8.8)68.9 ( 8.7)68.3 ( 8.5)0.14body mass index ( kg / m)26.7 ( 3.6)26.4 ( 3.6)26.1 ( 3.4)<0.01normal weight343940overweight494747<0.05obese171413waist - to - hip ratio0.9 ( 0.1)0.9 ( 0.1)0.9 ( 0.1)0.18systolic blood pressure ( mmhg)140 ( 22)140 ( 23)138 ( 2)0.04diastolic blood pressure ( mmhg)74 ( 11)74 ( 12)73 ( 11)0.06hypertension3634330.25total serum cholesterol ( mmol / l)6.4 ( 1.3)6.6 ( 1.2)6.8 ( 1.2)<0.001hdl - cholesterol ( mmol / l)1.4 ( 0.4)1.3 ( 0.3)1.3 ( 0.4)<0.001cholesterol lowering medication3230.71hypercholesterolemia496066<0.001smoking status ( current)3935330.02education level lower434345 intermediate4342400.21 higher141515diabetes mellitus101090.75values are means ( standard deviations ) for continuous variables and percentages for categorical variables . p values are obtained by anova for continuous variables and by for categorical variables figure 1 presents mean total serum cholesterol and hdl - cholesterol levels by apoe genotype in normal weight , overweight and obese persons . in all bmi strata , apoe*2 carriers had the lowest and apoe*4 carriers the highest total serum cholesterol ( pfor trend < 0.01 ) . note that even though apoe*2 carriers had the lowest total serum cholesterol levels in each bmi group , still 50% of the overweight and 57% of the obese apoe*2 carriers had hypercholesterolaemia compared to 41% of the normal weight apoe*2 carriers ( p = 0.003 ) . there was no significant evidence for interaction between apoe genotype and bmi in relation to total serum cholesterol ( p = 0.26 ) and hdl - cholesterol ( p = 0.25 ) . however , when bmi was analyzed as a continuous trait , then the p value for interaction was 0.04 for total serum cholesterol and less than 0.001 for hdl - cholesterol . 1mean total serum cholesterol and hdl - cholesterol levels by apoe genotypes for normal weight , overweight and obese individuals . hdl : high - density lipoproteins mean total serum cholesterol and hdl - cholesterol levels by apoe genotypes for normal weight , overweight and obese individuals . differences between the kaplan - meier survival curves of the apoe*2 carriers , apoe*3 homozygotes and apoe*4 carriers were small but statistically significant ( p = 0.03 ) with apoe*2 carriers having slightly lower mortality risks ( fig . after adjustment for other cardiovascular risk factors , apoe genotype was not associated with all - cause mortality before or after 80 years of age in the overall population ( table 2 ) . the differences between the curves were only statistically significant in normal weight individuals ( p = 0.04 ; fig . normal weight carriers of the apoe*2 allele had a significantly lower risk of mortality before age 80 years ( hr [ 95% ci ] = 0.7 [ 0.50.9 ] ) , but not after age 80 years ( crude hr [ 95% ci ] = 1.0 [ 0.81.4 ] ) . there were no significant differences in the risk of mortality between genotype groups among overweight or obese persons ( table 2 ) . 2kaplan meier survival curves for all - cause mortality by apoe genotypestable 2incidence rates and hazard ratios for all - cause mortality by apoe genotypes and bmi categoriesfollow - upincidence ratehazard ratiofollow - upincidence ratehazard ratiodeaths(py)deaths/1,000 py ( 95% ci)(95% ci)deaths(py)deaths/1,000 py ( 95% ci)(95% ci)before 80 years of ageafter 80 years of ageallapoe*2 carriers1016,78214.9 ( 12.318.1)0.9 ( 0.71.1)15621,7647.2 ( 6.18.4)0.9 ( 0.81.1)apoe 3/348128,70316.8 ( 15.318.3)67995,8087.1 ( 6.67.6)apoe*4 carriers19812,69315.6 ( 13.617.9)1.0 ( 0.81.2)30341,6377.3 ( 6.58.1)1.2 ( 1.01.4)total78048,17816.2 ( 15.117.4)1,138159,2097.1 ( 6.77.6)normal weightapoe*2 carriers282,37011.8 ( 8.217.1)0.7 ( 0.50.9)577,2897.8 ( 6.010.1)1.0 ( 0.81.4)apoe 3/320611,31418.2 ( 15.920.9)25638,0456.7 ( 6.07.6)apoe*4 carriers785,08015.4 ( 12.319.2)1.0 ( 0.71.3)12717,3057.3 ( 6.28.7)1.3 ( 0.91.7)total31218,76416.6 ( 14.918.6)44062,6397.0 ( 6.47.7)overweightapoe*2 carriers593,30517.9 ( 13.823.0)1.1 ( 0.91.5)6511,0645.9 ( 4.67.5)0.9 ( 0.71.1)apoe 3/321713,57316.0 ( 14.018.3)31045,3666.8 ( 6.17.6)apoe*4 carriers906,04714.9 ( 12.118.3)0.9 ( 0.71.2)13719,3987.1 ( 6.08.4)1.2 ( 1.01.5)total36622,92416.0 ( 14.417.7)51275,8276.8 ( 6.27.4)obeseapoe*2 carriers141,10712.6 ( 7.521.4)1.0 ( 0.51.8)343,41110.0 ( 7.114.0)1.0 ( 0.61.5)apoe 3/3583,81615.2 ( 11.719.7)11312,3989.1 ( 7.611.0)apoe*4 carriers301,56619.2 ( 13.427.4)1.3 ( 0.92.0)394,9347.9 ( 5.810.8)1.1 ( 0.81.6)total1026,48915.7 ( 12.919.1)18620,7429.0 ( 7.810.4)hazard ratios adjusted for sex , waist - hip - ratio , smoking , education level , total serum cholesterol , hdl cholesterol hypertension and diabetes ; py : person - years ; ci : confidence intervalfig . 3kaplan meier survival curves for all - cause mortality by apoe genotypes for normal weight , overweight and obese individuals kaplan meier survival curves for all - cause mortality by apoe genotypes incidence rates and hazard ratios for all - cause mortality by apoe genotypes and bmi categories hazard ratios adjusted for sex , waist - hip - ratio , smoking , education level , total serum cholesterol , hdl cholesterol hypertension and diabetes ; py : person - years ; ci : confidence interval kaplan meier survival curves for all - cause mortality by apoe genotypes for normal weight , overweight and obese individuals to investigate whether the lower risk of mortality observed among normal weight apoe*2 carriers was due to a lower risk of mortality from chd , we calculated the hrs separately for chd mortality . analysis within bmi strata showed that normal weight apoe*2 carriers tended to have lower risks of chd mortality ( adjusted hr [ 95% ci ] = 0.5 [ 0.21.2 ] ) . no differences in the risk of chd mortality between genotype groups were found in overweight or obese participants ( table 3 ) . table 3incidence rates and hazard ratios for chd mortality by apoe genotypes and bmi categoriesfollow - upincidence ratehazard ratiofollow - upincidence ratehazard ratiodeaths(py)deaths/1,000 py ( 95% ci)(95% ci)deaths(py)deaths/1,000 py ( 95% ci)(95% ci)before 80 years of ageafter 80 years of ageallapoe*2 carriers266,7823.8 ( 2.65.6)1.0 ( 0.61.5)412,10319.5 ( 14.426.5)1.1 ( 0.81.5)apoe 3/312628,7034.4 ( 3.75.2)1718,74419.6 ( 16.822.7)apoe*4 carriers5912,6934.6 ( 3.66.0)1.1 ( 0.81.4)663,47219.0 ( 14.924.2)1.1 ( 0.81.4)total21148,1784.4 ( 3.85.0)27814,31919.4 ( 17.321.8)normal weightapoe*2 carriers52,3702.1 ( 0.95.1)0.5 ( 0.21.2)1566322.6 ( 13.637.5)1.6 ( 0.92.9)apoe 3/35611,3144.9 ( 3.86.4)483,07515.6 ( 11.820.7)apoe*4 carriers265,0805.1 ( 3.57.5)1.2 ( 0.71.9)211,28616.3 ( 10.625.1)1.1 ( 0.61.8)total8718,7644.6 ( 3.85.7)845,02416.7 ( 13.520.7)overweightapoe*2 carriers163,3054.8 ( 3.07.9)1.4 ( 0.82.4)1598915.2 ( 9.125.2)0.7 ( 0.41.3)apoe 3/35113,5733.8 ( 2.94.9)924,03522.8 ( 18.628.0)apoe*4 carriers216,0473.5 ( 2.35.3)0.8 ( 0.51.4)351,61921.6 ( 15.530.1)1.0 ( 0.71.5)total8822,9243.8 ( 3.14.7)1426,64321.4 ( 18.125.2)obeseapoe*2 carriers51,1074.5 ( 1.910.8)1.1 ( 0.43.4)1145024.4 ( 13.544.1)1.4 ( 0.73.1)apoe 3/3193,8165.0 ( 3.27.8)311,63419.0 ( 13.327.0)apoe*4 carriers121,5667.7 ( 4.413.5)1.6 ( 0.83.5)1056817.6 ( 9.532.7)1.1 ( 0.52.2)total366,4895.5 ( 4.07.7)522,65119.6 ( 14.925.7)hazard ratios adjusted for sex , waist - hip - ratio , smoking , education level , total serum cholesterol , hdl cholesterol hypertension and diabetes ; py : person - years ; ci : confidence interval incidence rates and hazard ratios for chd mortality by apoe genotypes and bmi categories hazard ratios adjusted for sex , waist - hip - ratio , smoking , education level , total serum cholesterol , hdl cholesterol hypertension and diabetes ; py : person - years ; ci : confidence interval analyses within bmi strata demonstrated that this genetic advantage of apoe*2 was observed only in normal weight individuals , but not in overweight and obese apoe*2 carriers . while our findings come from the same population , the mean follow - up time of the participants has increased from 5.4 years to 11.1 years and the number of deaths increased by 53% from 18% to 34% . research on the relationship between apoe and mortality has merely provided conflicting results [ 619 , 34 ] . three out of eleven studies showed a protective effect of apoe*2 [ 6 , 14 , 18 ] . this may be due to the effect of age that becomes apparent when performing survival analysis with age as time scale , as we did in the present paper . in line with previous studies [ 2022 ] , we found that the total serum cholesterol levels associated with apoe genotype differed between normal weight , overweight and obese individuals . however , in our study abdominal fat , as measured by whr , did not have any effect on the lipid levels by apoe genotype , while bmi was an important determinant . the significant interaction between apoe genotype and bmi in relation to total serum cholesterol found previously , was also supported by this study . this is in line with our observation that the deleterious effect of increased bmi prevails over the beneficial effects of the apoe*2 allele . furthermore , previous research in the rotterdam study and the dutch population indicated that among apoe*2 homozygous carriers , the expression of hyperlipoproteinemia type iii is determined to a great extent by hyperinsulinemia and the presence of insulin resistance syndrome , conferring and increased risk of chd through accelerated atherosclerosis . although the association between high bmi and high cholesterol levels is well known and the apoe gene is associated with cholesterol levels as previously described , this is the first study that examined the effect of apoe genotype on mortality in bmi strata . we found that presence of the apoe*2 allele was associated with a decreased risk of mortality only in normal weight individuals and that this lower risk was partly explained by lower risk of chd mortality . these results were in line with the lower total cholesterol and higher hdl levels among normal weight apoe*2 carriers . our finding that the protective effect of apoe*2 was only found in normal weight individuals suggests that the negative influences of increasing bmi outweigh the positive effects of genetic predisposition . when these results are confirmed by other population - based studies , a challenging question remains whether weight loss in overweight and obese apoe*2 carriers restores the genetic advantage of their apoe status .

the present analyses were performed in the rotterdam study , an ongoing population - based study on the determinants of disease and disability among persons 55 years and older . the clinical examination included weight , height , systolic and diastolic blood pressure , serum glucose , total serum cholesterol and high - density lipoprotein ( hdl ) cholesterol levels . participants were classified as underweight ( bmi < 18.5 kg / m ) , normal weight ( 18.5 kg / m bmi < 25 kg / m ) , overweight ( 25 kg / m bmi < 30 kg / m ) or obese ( bmi 30 kg / m ) . the waist and hip circumferences were measured , and the waist - hip - ratio ( whr ) was calculated as indirect assessment of abdominal fat . the frequencies of apoe genotypes were apoe 2/2 0.8% , apoe 2/3 12.8% , apoe 2/4 2.7% , apoe 3/3 58.4% , apoe 3/4 22.9% and apoe 4/4 2.4% . the proportions of the apoe alleles and genotypes were in hardy - weinberg equilibrium ( p = 0.71 ) . causes of death were obtained from the general practitioners by means of a standardized questionnaire relating to the circumstances of death , most likely cause of death and time and place of death . of the 7,983 individuals who participated at baseline , 5,817 ( 73% ) had apoe genotyped successfully and had complete information on bmi , whr and cholesterol levels . multiple linear regression analysis was used to assess the interaction between apoe genotype and bmi on total serum cholesterol and hdl - cholesterol levels . risks of mortality were quantified as hazard ratios ( hrs ) using cox proportional hazards analyses with age as the time scale . hrs were calculated adjusted for gender , smoking status , education level , total serum cholesterol , hdl - cholesterol , waist to hip ratio hypertension and diabetes mellitus . the present analyses were performed in the rotterdam study , an ongoing population - based study on the determinants of disease and disability among persons 55 years and older . from 10,275 eligible subjects , 7,983 ( 78% ) individuals agreed to participate in the study and were examined at baseline . the clinical examination included weight , height , systolic and diastolic blood pressure , serum glucose , total serum cholesterol and high - density lipoprotein ( hdl ) cholesterol levels . participants were classified as underweight ( bmi < 18.5 kg / m ) , normal weight ( 18.5 kg / m bmi < 25 kg / m ) , overweight ( 25 kg / m bmi < 30 kg / m ) or obese ( bmi 30 the waist - hip - ratio ( whr ) was calculated as indirect assessment of abdominal fat . the frequencies of apoe genotypes were apoe 2/2 0.8% , apoe 2/3 12.8% , apoe 2/4 2.7% , apoe 3/3 58.4% , apoe 3/4 22.9% and apoe 4/4 2.4% . causes of death were obtained from the general practitioners by means of a standardized questionnaire relating to the circumstances of death , most likely cause of death and time and place of death . the clinical examination included weight , height , systolic and diastolic blood pressure , serum glucose , total serum cholesterol and high - density lipoprotein ( hdl ) cholesterol levels . participants were classified as underweight ( bmi < 18.5 kg / m ) , normal weight ( 18.5 kg / m bmi < 25 kg / m ) , overweight ( 25 kg / m bmi < 30 kg / m ) or obese ( bmi 30 the waist - hip - ratio ( whr ) was calculated as indirect assessment of abdominal fat . the frequencies of apoe genotypes were apoe 2/2 0.8% , apoe 2/3 12.8% , apoe 2/4 2.7% , apoe 3/3 58.4% , apoe 3/4 22.9% and apoe 4/4 2.4% . causes of death were obtained from the general practitioners by means of a standardized questionnaire relating to the circumstances of death , most likely cause of death and time and place of death . because persons with an extremely low bmi may suffer from life - threatening diseases such as cancer , individuals with a bmi below 18.5 kg / m ( n = 51 ) also , apoe 2/4 individuals were excluded from the analyses ( n = 158 ) to distinguish the effect of the apoe alleles unambiguously . multiple linear regression analysis was used to assess the interaction between apoe genotype and bmi on total serum cholesterol and hdl - cholesterol levels . furthermore , there were statistically significant differences in bmi scores between the genotype groups , with apoe*2 carriers having the highest mean bmi ( 26.7 3.6 kg / m ) and apoe*4 carriers the lowest ( 26.1 3.4 kg / m ) . table 1baseline characteristics by apoe genotype groupsapoe*2 carriersapoe 3/3apoe*4 carrierspn7823,3691,457sex ( men)3842420.05age at entry ( years)68.8 ( 8.8)68.9 ( 8.7)68.3 ( 8.5)0.14body mass index ( kg / m)26.7 ( 3.6)26.4 ( 3.6)26.1 ( 3.4)<0.01normal weight343940overweight494747<0.05obese171413waist - to - hip ratio0.9 ( 0.1)0.9 ( 0.1)0.9 ( 0.1)0.18systolic blood pressure ( mmhg)140 ( 22)140 ( 23)138 ( 2)0.04diastolic blood pressure ( mmhg)74 ( 11)74 ( 12)73 ( 11)0.06hypertension3634330.25total serum cholesterol ( mmol / l)6.4 ( 1.3)6.6 ( 1.2)6.8 ( 1.2)<0.001hdl - cholesterol ( mmol / l)1.4 ( 0.4)1.3 ( 0.3)1.3 ( 0.4)<0.001cholesterol lowering medication3230.71hypercholesterolemia496066<0.001smoking status ( current)3935330.02education level lower434345 intermediate4342400.21 higher141515diabetes mellitus101090.75values are means ( standard deviations ) for continuous variables and percentages for categorical variables . note that even though apoe*2 carriers had the lowest total serum cholesterol levels in each bmi group , still 50% of the overweight and 57% of the obese apoe*2 carriers had hypercholesterolaemia compared to 41% of the normal weight apoe*2 carriers ( p = 0.003 ) . there was no significant evidence for interaction between apoe genotype and bmi in relation to total serum cholesterol ( p = 0.26 ) and hdl - cholesterol ( p = 0.25 ) . however , when bmi was analyzed as a continuous trait , then the p value for interaction was 0.04 for total serum cholesterol and less than 0.001 for hdl - cholesterol . differences between the kaplan - meier survival curves of the apoe*2 carriers , apoe*3 homozygotes and apoe*4 carriers were small but statistically significant ( p = 0.03 ) with apoe*2 carriers having slightly lower mortality risks ( fig . after adjustment for other cardiovascular risk factors , apoe genotype was not associated with all - cause mortality before or after 80 years of age in the overall population ( table 2 ) . normal weight carriers of the apoe*2 allele had a significantly lower risk of mortality before age 80 years ( hr [ 95% ci ] = 0.7 [ 0.50.9 ] ) , but not after age 80 years ( crude hr [ 95% ci ] = 1.0 [ 0.81.4 ] ) . there were no significant differences in the risk of mortality between genotype groups among overweight or obese persons ( table 2 ) . 2kaplan meier survival curves for all - cause mortality by apoe genotypestable 2incidence rates and hazard ratios for all - cause mortality by apoe genotypes and bmi categoriesfollow - upincidence ratehazard ratiofollow - upincidence ratehazard ratiodeaths(py)deaths/1,000 py ( 95% ci)(95% ci)deaths(py)deaths/1,000 py ( 95% ci)(95% ci)before 80 years of ageafter 80 years of ageallapoe*2 carriers1016,78214.9 ( 12.318.1)0.9 ( 0.71.1)15621,7647.2 ( 6.18.4)0.9 ( 0.81.1)apoe 3/348128,70316.8 ( 15.318.3)67995,8087.1 ( 6.67.6)apoe*4 carriers19812,69315.6 ( 13.617.9)1.0 ( 0.81.2)30341,6377.3 ( 6.58.1)1.2 ( 1.01.4)total78048,17816.2 ( 15.117.4)1,138159,2097.1 ( 6.77.6)normal weightapoe*2 carriers282,37011.8 ( 8.217.1)0.7 ( 0.50.9)577,2897.8 ( 6.010.1)1.0 ( 0.81.4)apoe 3/320611,31418.2 ( 15.920.9)25638,0456.7 ( 6.07.6)apoe*4 carriers785,08015.4 ( 12.319.2)1.0 ( 0.71.3)12717,3057.3 ( 6.28.7)1.3 ( 0.91.7)total31218,76416.6 ( 14.918.6)44062,6397.0 ( 6.47.7)overweightapoe*2 carriers593,30517.9 ( 13.823.0)1.1 ( 0.91.5)6511,0645.9 ( 4.67.5)0.9 ( 0.71.1)apoe 3/321713,57316.0 ( 14.018.3)31045,3666.8 ( 6.17.6)apoe*4 carriers906,04714.9 ( 12.118.3)0.9 ( 0.71.2)13719,3987.1 ( 6.08.4)1.2 ( 1.01.5)total36622,92416.0 ( 14.417.7)51275,8276.8 ( 6.27.4)obeseapoe*2 carriers141,10712.6 ( 7.521.4)1.0 ( 0.51.8)343,41110.0 ( 7.114.0)1.0 ( 0.61.5)apoe 3/3583,81615.2 ( 11.719.7)11312,3989.1 ( 7.611.0)apoe*4 carriers301,56619.2 ( 13.427.4)1.3 ( 0.92.0)394,9347.9 ( 5.810.8)1.1 ( 0.81.6)total1026,48915.7 ( 12.919.1)18620,7429.0 ( 7.810.4)hazard ratios adjusted for sex , waist - hip - ratio , smoking , education level , total serum cholesterol , hdl cholesterol hypertension and diabetes ; py : person - years ; ci : confidence intervalfig . 3kaplan meier survival curves for all - cause mortality by apoe genotypes for normal weight , overweight and obese individuals kaplan meier survival curves for all - cause mortality by apoe genotypes incidence rates and hazard ratios for all - cause mortality by apoe genotypes and bmi categories hazard ratios adjusted for sex , waist - hip - ratio , smoking , education level , total serum cholesterol , hdl cholesterol hypertension and diabetes ; py : person - years ; ci : confidence interval kaplan meier survival curves for all - cause mortality by apoe genotypes for normal weight , overweight and obese individuals to investigate whether the lower risk of mortality observed among normal weight apoe*2 carriers was due to a lower risk of mortality from chd , we calculated the hrs separately for chd mortality . table 3incidence rates and hazard ratios for chd mortality by apoe genotypes and bmi categoriesfollow - upincidence ratehazard ratiofollow - upincidence ratehazard ratiodeaths(py)deaths/1,000 py ( 95% ci)(95% ci)deaths(py)deaths/1,000 py ( 95% ci)(95% ci)before 80 years of ageafter 80 years of ageallapoe*2 carriers266,7823.8 ( 2.65.6)1.0 ( 0.61.5)412,10319.5 ( 14.426.5)1.1 ( 0.81.5)apoe 3/312628,7034.4 ( 3.75.2)1718,74419.6 ( 16.822.7)apoe*4 carriers5912,6934.6 ( 3.66.0)1.1 ( 0.81.4)663,47219.0 ( 14.924.2)1.1 ( 0.81.4)total21148,1784.4 ( 3.85.0)27814,31919.4 ( 17.321.8)normal weightapoe*2 carriers52,3702.1 ( 0.95.1)0.5 ( 0.21.2)1566322.6 ( 13.637.5)1.6 ( 0.92.9)apoe 3/35611,3144.9 ( 3.86.4)483,07515.6 ( 11.820.7)apoe*4 carriers265,0805.1 ( 3.57.5)1.2 ( 0.71.9)211,28616.3 ( 10.625.1)1.1 ( 0.61.8)total8718,7644.6 ( 3.85.7)845,02416.7 ( 13.520.7)overweightapoe*2 carriers163,3054.8 ( 3.07.9)1.4 ( 0.82.4)1598915.2 ( 9.125.2)0.7 ( 0.41.3)apoe 3/35113,5733.8 ( 2.94.9)924,03522.8 ( 18.628.0)apoe*4 carriers216,0473.5 ( 2.35.3)0.8 ( 0.51.4)351,61921.6 ( 15.530.1)1.0 ( 0.71.5)total8822,9243.8 ( 3.14.7)1426,64321.4 ( 18.125.2)obeseapoe*2 carriers51,1074.5 ( 1.910.8)1.1 ( 0.43.4)1145024.4 ( 13.544.1)1.4 ( 0.73.1)apoe 3/3193,8165.0 ( 3.27.8)311,63419.0 ( 13.327.0)apoe*4 carriers121,5667.7 ( 4.413.5)1.6 ( 0.83.5)1056817.6 ( 9.532.7)1.1 ( 0.52.2)total366,4895.5 ( 4.07.7)522,65119.6 ( 14.925.7)hazard ratios adjusted for sex , waist - hip - ratio , smoking , education level , total serum cholesterol , hdl cholesterol hypertension and diabetes ; py : person - years ; ci : confidence interval incidence rates and hazard ratios for chd mortality by apoe genotypes and bmi categories hazard ratios adjusted for sex , waist - hip - ratio , smoking , education level , total serum cholesterol , hdl cholesterol hypertension and diabetes ; py : person - years ; ci : confidence interval analyses within bmi strata demonstrated that this genetic advantage of apoe*2 was observed only in normal weight individuals , but not in overweight and obese apoe*2 carriers . while our findings come from the same population , the mean follow - up time of the participants has increased from 5.4 years to 11.1 years and the number of deaths increased by 53% from 18% to 34% . genetic and environmental interactions at older ages vary from those at early ages and therefore explain the differences in the association between apoe and mortality before and after 80 years of age . although the mechanism of such interaction has not been yet elucidated , evidence suggests that obesity and abdominal fat increase low density lipoprotein cholesterol and therefore total cholesterol at a higher degree among apoe*4 carriers [ 2022 , 37 ] . furthermore , previous research in the rotterdam study and the dutch population indicated that among apoe*2 homozygous carriers , the expression of hyperlipoproteinemia type iii is determined to a great extent by hyperinsulinemia and the presence of insulin resistance syndrome , conferring and increased risk of chd through accelerated atherosclerosis . although the association between high bmi and high cholesterol levels is well known and the apoe gene is associated with cholesterol levels as previously described , this is the first study that examined the effect of apoe genotype on mortality in bmi strata . we found that presence of the apoe*2 allele was associated with a decreased risk of mortality only in normal weight individuals and that this lower risk was partly explained by lower risk of chd mortality . our finding that the protective effect of apoe*2 was only found in normal weight individuals suggests that the negative influences of increasing bmi outweigh the positive effects of genetic predisposition .

vitreous body is the clear gel that fills the vitreous chamber or posterior chamber ( pc ) of the eyeball , the space between the lens and the eyewall , whose inner layer is the neurosensorial tissue that receives and transmits the image to the central nervous system called the retina . its functions are to give volume to the eye , to support the retina attached , and to maintain its transparency to allow light beams to reach onto the retina . unlike the fluid in the anterior segment of the eye ( aqueous humour ) , which is continuously replaced , vitreous humor is stagnant , and its composition remains quite constant throughout life . the vitreous gel is avascular , composed mainly of water ( 98 - 99% ) , and 0.9% of inorganic salts ( sodium , potassium , and chloride ) . most of the protein is forming fibrils composed of a small collagen type v / xi core wrapped in a thick layer of collagen type ii ( 75% of the fibril by mass ) [ 1 , 2 ] . it also contains very few cells , mostly phagocytes , whose function is to remove undesired cellular debris from the visual field , as well as hyalocytes of the surface of vitreous , which act as macrophages [ 3 , 4 ] . the vitreous is feebly antigenic and is characterized by the absence of gamma - globulins and immunocompetent cells . because it only exhibits phagocytosis , this represents an incomplete and primitive immunological system , reacting like an embryonic tissue . the immune privilege , also , a physiologic mechanism characteristic of the internal compartments of the eye , is designed to provide protection against pathogens , protecting the delicate visual axis from the sight - destroying effect of immunogenic inflammation . at the same time , there is a sustained suppressive microenvironment in the pc of the eye that inhibits the local expression of preexisting systemic immunity and participates in modifying the primary immune responses to ocular antigen [ 7 , 8 ] . in the vitreous cavity it would develop a deviant form of immunity , similar to that in the anterior chamber , in which antigen - specific suppressor t cells are generated and delayed hypersensitivity reactivity is selectively impaired . both the vitreous and the retina supply immunosuppressive molecules to the pc , but it has been suggested that retinal cells contribute more significantly to the immune suppressive microenvironment than vitreous cells : tgf is produced by retinal astrocytes and retinal pigment epithelium ; and muller 's cells ( glia ) of the retina suppress t cell proliferation by a direct contact mechanism . in addition , the retinal vascular endothelium , bruch 's membrane , and the pigment epithelium together form the so - called ocular - blood barrier . vitreous has a major role in the origin and the triggering of several ocular pathologies . posterior vitreous liquefaction developed through years by means of dissolution of collagen fibers yields to several primary degenerative pathologies in vitreoretinal junction . other diseases are easily diagnosable in the fundus eye and only affect the vitreous in late stages , like retinal vasculopathies as diabetic retinopathy and other vitreoretinal proliferations . also , certain eye diseases have their beginning in other more hidden structures of the eye but may secrete molecules or even cells to the vitreous chamber , causing symptoms and helping in the diagnosis , since the vitreous is more accessible to study than other posterior pole structures . the latter is the case of uveitis , a wide term that actually comprises a large group of diverse diseases affecting the retina , optic nerve , and also the vitreous compartment . these diseases may affect , in addition , several territories of the eye simultaneously and can have in major or minor degree manifestation in the vitreous humor , specially those concerning the posterior chamber . cases are limited to the eye ( organ specific ) , whereas the remainder forms part of more generalized diseases , so that the pathophysiology of uveitis depends on the specific etiology , but in all types there is a breach in the ocular - blood barrier that normally prevents cells and large proteins from entering the eye . these cells then recognize antigens ( autoantigen or foreign antigen ) presented on the cell surface of antigen - presenting cells ( apcs , like dendritic cells or macrophages ) , and activation and clonal expansion will take place , which results in increased production of il-2 , interferon- , and tnf- . only certain responses are capable of overcoming the condition of immune privilege of the eye and unleashing the inflammatory cell accumulation and the tisular damage . in most cases , the clinical appearance is sufficient for diagnosis , but since the majority of these patients have an unknown etiology for the intraocular inflammation , and this can be in addition related to a primarily nondiagnosed systemic disease , could be infectious , inflammatory , or even tumoral , the correct diagnosis can prove difficult . thus , the intraocular inflammation is associated with the increased expression and action of several cytokines and growth factors , which can be determined in the vitreous and can help in the diagnosis . several molecules have been identified along the last decades in the vitreous humor , which may be the key in the physiopathology of certain ocular diseases . in most cases this determination has investigational purposes , in others is helpful for the prognosis of the patients , and in a minority of cases its finding constitutes an indispensable diagnostic tool . many patients with uveitis have such characteristic ocular signs and symptoms , associated systemic disorders , and laboratory abnormalities that a satisfactory clinical diagnosis can be established without the need for invasive intraocular studies . most other patients have mild , self - limited , and/or readily controllable disease that does not warrant aggressive invasive testing . in contrast , some patients have atypical ophthalmic and/or systemic features or do not respond to conventional anti - inflammatory therapies . several techniques have been employed for diagnostic purposes in these cases : aqueous aspiration , vitreous aspiration , diagnostic vitrectomy , fine - needle aspiration biopsy , controlled aspiration of subretinal fluid , incisional chorio - retinal biopsy , and diagnostic enucleation . the indications of anterior chamber aspiration may be varied , but most common situations are patients with anterior chamber inflammation with suspicion of masquerade syndromes , a hypopion suspicious of infection , endophthalmitis , lens - induced uveitis and for cytopathologic examination . a fine needle ( 30 , 27 , or 25 gauge ) is introduced with the bevel up through clear cornea over the iris stroma , with optimal visualization by means of slit lamp or the surgical microscope , taking care to avoid the lens . a 0.1 - 0.2 ml of aqueous humor is withdrawn into a 3 ml syringe in a sterile technique , and then balanced salt solution may be used to reform the chamber . the cytospin technique or others can be used to increase the sensitivity of the cytology specimen . in the aqueous humor many other techniques can be performed for the diagnosis of infectious posterior uveitis , as polymerase chain reaction ( pcr ) and pathogen - specific antibody production for herpes simplex virus ( hsv ) , varicella zoster virus ( vzv ) , cytomegalovirus , epstein - barr virus ( ebv ) , human immunodeficiency virus ( hiv ) , propionibacterium acnes , and toxoplasma gondii . further studies have demonstrated for pcr of aqueous humor to yield a diagnosis in one - third of patients with posterior infectious uveitis with a sensitivity of 82% and specificity of 100% , equal or better than vitreous biopsy . the advantage of anterior chamber aspiration is that it can be performed in an outpatient setting , but the disadvantage is that it retrieves a limited sample volume of 100 to 200 l per procedure , which limits the number of molecular examinations that can be performed on the sample . the indications for this technique are basically in posterior uveitis unresponsiveness to treatment when malignancy needs to be ruled out ; when intraocular infection is considered the primary cause of inflammation in the absence or insignificant amount of vitreous cells that would preclude diagnosis through pars plana vitrectomy ( ppv ) [ 17 , 18 ] . the major indication for vitreous aspiration would be when intraocular lymphoma is a significant diagnostic possibility . the technique of vitreous aspiration is similar to that of anterior chamber paracentesis and is currently performed through the pars plana under local anesthesia with a large caliber needle , such as 21-gauge ( g ) hollow needle or fine , like 23 or 25-g mounted on a 1-ml syringe as an aspirating device , permitting to aspirate a volume of 100 to 250 l of vitreous humor while the needle is directed posteriorly towards the optic nerve head . with vitreous biopsy , specimens obtained using a needle and syringe were positive in 54% of infected eyes compared with 75% of the specimens collected with vitrectomy procedures , although posteriorly , in the endophthalmitis vitrectomy study , no significant difference was shown between needle tap versus mechanized vitreous biopsy with respect to microbiologic yield . the main complications associated with the technique are retinal tears and endophthalmitis ; however the risk is low , but both more common than after vitrectomy . other advantages of vitreous biopsies over vitrectomy are quickness , can be done in the outpatient setting without inpatient admission , can be repeated , and are less traumatic to the eye . another variation of the technique is the fine - needle aspiration biopsy ( fnab ) , in which a fine - needle gauge is directed to the localized suspected areas of intraocular tumor or lesion . aspiration is performed automatic or manually using a 25-g to 30-g needle connected to an aspirating syringe ( figure 1 ) . in case of intraocular tumor , the aspirated block obtained is likely to have a higher concentration of neoplastic cells than any of the adjacent intraocular fluids , decreasing the possibility of inconclusive cytological diagnoses , which often occur in vitrectomy specimens . for aqueous or vitreous humor the technique turns into an effective method for cytology that generally is able to obtain a sufficient amount of cells ( 100 to 500 l of ocular fluid ) to perform routine analysis like microbiologic , cytomorphological evaluation of papanicolaou or haematoxylin- and eosin - stained cells , immunocytochemical analysis , and other applications . the fine - needle aspiration technique is less invasive and has fewer complications than others , being in certain centers the preferred method for diagnostic purposes . this technique may be the better option in selected cases , such as when vitreous removal is considered to be not only diagnostic but also therapeutic ( e.g. , endophthalmitis , intraocular bleeding with suspected malignant origin , and for the treatment of complications of chronic uveitis ) , and when the eye is inflamed and thereby patients may experience substantial discomfort during the vitreous biopsy tap . some authors recommend that vitrectomy - assisted biopsy should be considered only in cases in which fnab fails , or multiple tests are needed and therefore requiring several punctures . a diagnostic standard three - port pars plana vitrectomy ( vpp ) provides a large amount of vitreous , retina , or choroid ( though diluted ) , but always requires an operation theater under sterile conditions and direct visualization of the vitrectomy instruments . in order to obtain an undiluted vitreous sample , the infusion cannula of the system must be closed and the vitreous specimen is collected through undiluted lines using the vitreous cutter connected directly to a 3 ml syringe until the eye is noted to soften visibly . at least 1.5 ml of undiluted vitreous can be reliably obtained with this technique . with perfluorocarbon - perfused vitrectomy , in which aspirated vitreous is compensated with perfluorocarbon liquid entry during vitreous aspiration , other authors were able to obtain an average of 2.4 ml of undiluted vitreous . there is controversy whether using classic 20-g ports vpp needing suture , or the newest microincisional systems with 23- , 25- , or 27-g systems , but with anyone of these , the overall diagnostic yield of vpp varies considerably in different published studies from 14.3 to 61.5% [ 2631 ] , and the success for the procedure was greater when an intraocular infection was suspected compared with an intraocular malignancy , and greater for detecting primary vitreoretinal lymphoma than for detecting metastatic disease . biopsies have been performed to investigate uncertain uveitis , choroiditis , and retinal and choroidal masses . the indications for biopsy included major diagnostic uncertainty , suspected cancer metastasis to the choroid without other evidence of systemic malignancy , and patient insistence on biopsy confirmation of the diagnosis prior to treatment . the limited performance of intraocular biopsy is explained by the risks for dissemination of malignant cells , eye complications ( mainly hemorrhage , retinal detachment , and infection ) , and fears of misdiagnosis , although the literature gives little support to these [ 34 , 35 ] . however , on the other hand , several authors have claimed that identifying patients with aggressive disease and a high risk for dissemination in malignant processes should be a priority and histopathological diagnosis should be mandatory [ 36 , 37 ] . various techniques have been developed to minimize the risks aforementioned . in the classic , transscleral approach , a sharp blade then incises the choroid , and the biopsy tissue is grasped with forceps . a retinal specimen may also be obtained with the choroidal specimen if a chorioretinal sample is the subject of study . several modifications have been later described to facilitate the biopsy procedure and also reduce the risk of complications ; vpp is now often performed before creating the scleral flap , and another modification is the use of cyanoacrylate glue to provide increased stability to the tissue . in the transvitreal or ab interno approach , a retinochoroidectomy down to the sclera is performed after vitrectomy . the risk for complications is high , mostly due to hemorrhage and retinal detachment . the tip of the needle may be bent , facilitating entry into shallow choroidal lesions . studies have shown increased levels of il-6 ( t - cell cytokine ) in the vitreous fluid of patients with active intermediate or posterior uveitis , although it did not correlate with a specific uveitis type , suggesting that il-6 is an inflammatory mediator common in various uveitis etiologies . il-12 , produced by monocytes , macrophages , b cells , and connective tissue - type mast cells has , also been found increased in aqueous humor and vitreous fluid of patients with low - grade intraocular inflammation and in uveitis in clinical remission for as long as 2 years . intraocular inflammation that fails to respond to immunosuppressive treatment raises suspicion for another different process . since diagnostic analysis of vitreous fluid in patients with uveitis uveitis masquerade syndrome ( ums ) is a group of disorders that mimic intraocular inflammation , but cells seen may be of noninflammatory origin ( e.g. , pigment , blood or malignant cells ) or are inflammatory but secondary to another disorder [ 42 , 43 ] . theodore in 1967 was the first author who described a conjunctival carcinoma manifesting as a chronic conjunctivitis and named it masquerade syndrome . the frequency of ums among the patients with uveitis in a tertiary ophthalmologic center was 5% . the causes of ums may be variate , such as malignant , including hematologic malignancies , retinoblastoma , melanoma , and lung cancer metastasis ; or nonmalignant , like ocular toxoplasmosis , diabetic retinopathy , hypertension , retinal detachment or degeneration , intraocular trauma , and radiation retinopathy [ 42 , 43 , 4569 ] ( table 1 ) . they are often misdiagnosed as a chronic idiopathic uveitis , but they can present in any location of the eye manifesting as panuveitis , pars planitis , vitreitis , papillitis , anterior segment cells , hypopyon or vitreal , and/or chorioretinal infiltrates ( figure 2 ) . although they constitute rare presentations of uncommon diseases in the eye , the ophthalmologist must be aware because many of the ums etiologies are malignancies with deleterious effects for the patient , for what early diagnosis and prompt treatment are mandatory . the study of vitreous body can be of great help specially in these cases , since the achievement of a small sample in doubtful cases can provide us the diagnosis . although both hodgkin 's lymphoma and non - hodgkin 's lymphoma ( nhl ) can present as intraocular inflammation , in the case of hodgkin 's lymphoma ocular involvement generally is rare and often occurs late in the course of the disease , whereas nhl affects more commonly the eye . nhl can be divided in two clinically different entities : systemic nhl with metastases to the eye , and nhl of the central nervous system ( nhl - cns ) . recently , coupland and co - workers proposed an anatomical classification according to the localization of the disease in the eye ; retinal lymphomas are high - grade b - cell malignancies associated with a poor prognosis , whereas primary uveal lymphomas are typically low - grade b - cell tumours derived from the postgerminal centre ( memory ) b cell . the variant with major ophthalmic repercussion is the primary vitreoretinal lymphoma ( pvrl ) , a subtype of primary central ( cns ) lymphoma , typically classified as a diffuse large b - cell lymphoma and most frequently develops in elderly populations . over 15% of primary cns lymphoma patients develop intraocular lymphoma , usually occurring in the retina and/or vitreous , and conversely , 65%90% of pvrl patients develop cns lymphoma . consequently , pvrl is often fatal because of ultimate cns association , that can appear from 1 month to 10 years after [ 72 , 73 ] . both retinal and uveal lymphoma can manifest as any form of uveitis , but pvrl typical clinical findings include vitreous cellular infiltration ( lymphoma and inflammatory cells ) and subretinal tumor infiltration . choroid is the predominant location for primary uveal lymphomas and most often manifests as recurrent episodes of blurred vision and metamorphopsia secondary to exudative retinal detachment affecting the fovea . a classic finding is the presence of solitary or multiple yellow , creamy choroidal infiltrates with clear vitreous , that can evolve to diffuse thickening of the uveal tract and in some cases , to episcleral extension appearing as a nonmobile orange to yellow or salmon patch . the clinical suspicion is very important given the potential lethality if an uncorrect diagnosis is made and a proper systemic treatment is applied . currently , pvrl is most often diagnosed using citology ( the gold standard ) or vitrectomy to identify lymphoma cells in the vitreous or retina . in order to prevent degeneration of lymphoma cells , vitreous specimens are placed into a tube containing culture medium like rpmi ( roswell park memorial institute ) , whereas others prefer immediate placement in normal saline , taking care not fixing with alcohol with the aim to not alter the identification of pvrl cells in the vitreous sample . citologyas lymphoma cells are fragile , the general consensus recommends sending the samples immediately to an experienced cytopathologist to distinguish the malignant cells ( usually b lymphocytes ) from the reactive lymphocytes ( t cells ) . the malignant b cells of pvrl exhibit characteristic features with papanicolaou , giemsa , or diff - quick stains [ 26 , 76 ] : large round or oval nuclei , frequently segmented and often containing prominent nucleoli , surrounded by scant basophilic cytoplasm . samples often are negative because of poor biopsy samples , with a reported effectivity of only 48.3% of lymphoma cases for ppv , although other authors have found for fnab a diagnostic effectivity in 87.5% of the suspicious intraocular lymphoma cases . other authors advocate for the fixation of the samples with cytolit or hope solution ( herpes - glutamic acid buffer mediated organic solvent protection effect ) in order to facilitate the transportation from the theater to the laboratory . as lymphoma cells are fragile , the general consensus recommends sending the samples immediately to an experienced cytopathologist to distinguish the malignant cells ( usually b lymphocytes ) from the reactive lymphocytes ( t cells ) . the malignant b cells of pvrl exhibit characteristic features with papanicolaou , giemsa , or diff - quick stains [ 26 , 76 ] : large round or oval nuclei , frequently segmented and often containing prominent nucleoli , surrounded by scant basophilic cytoplasm . samples often are negative because of poor biopsy samples , with a reported effectivity of only 48.3% of lymphoma cases for ppv , although other authors have found for fnab a diagnostic effectivity in 87.5% of the suspicious intraocular lymphoma cases . other authors advocate for the fixation of the samples with cytolit or hope solution ( herpes - glutamic acid buffer mediated organic solvent protection effect ) in order to facilitate the transportation from the theater to the laboratory . molecular analysisflow cytometric immunophenotyping ( fci ) can be done in diluted samples , allows for the analysis of several different cell surface markers simultaneously , and offers a quantitative method of determining the percentage of a particular cellular phenotype , increasing the efficiency of a biopsy specimen . dilute vitreous is centrifuged and resuspended in cell culture medium , and cells are counted and stained with antibodies to detect markers that identify leukocytes , t lymphocytes , b lymphocytes ( including cd19 , cd20 , cd22 , , and light chain markers ) , monocyte / macrophages , and lymphocyte activation . the test relies on the finding that the majority of pvrl have restricted expression of or chains , with the most sensitive marker being a : ratio 3 or 0.6 ( 80% ) , whereas cd22 and cd20 markers are not very sensitive for lymphoma ( 50 and 33% , resp . ) , although they are quite specific ( 94 and 89% , resp . ) . for patients with possible t - cell lymphoma , cell surface markers more commonly searched are cd3 , cd8 , cd4 , cd7 , cd2 , cd25 , and cd52 . flow cytometric immunophenotyping ( fci ) can be done in diluted samples , allows for the analysis of several different cell surface markers simultaneously , and offers a quantitative method of determining the percentage of a particular cellular phenotype , increasing the efficiency of a biopsy specimen . dilute vitreous is centrifuged and resuspended in cell culture medium , and cells are counted and stained with antibodies to detect markers that identify leukocytes , t lymphocytes , b lymphocytes ( including cd19 , cd20 , cd22 , , and light chain markers ) , monocyte / macrophages , and lymphocyte activation . the test relies on the finding that the majority of pvrl have restricted expression of or chains , with the most sensitive marker being a : ratio 3 or 0.6 ( 80% ) , whereas cd22 and cd20 markers are not very sensitive for lymphoma ( 50 and 33% , resp . ) , although they are quite specific ( 94 and 89% , resp . ) . for patients with possible t - cell lymphoma , cell surface markers more commonly searched are cd3 , cd8 , cd4 , cd7 , cd2 , cd25 , and cd52 . microdissection allows for the selection of only few or poorly preserved malignant or atypical lymphoid cells that would have been nondiagnostic for pvrl by routine cytological techniques . pcr can determine monoclonality by immunoglobulin heavy chain ( igh ) rearrangement and t(14 ; 18 ) translocation of the bcl-2 gene that promote cell survival and predict a more aggressive tumor course in b - cell lymphoma [ 75 , 79 , 80 ] . pcr has been found to be 64% sensitive for pvrl , and is being used to study the genotypic classification of pvrl with the goal of identifying prognostic factors ; patients with a translocation in the bcl-2 gene are significantly younger than patients who lacked the translocation , suggesting that younger patients with the translocation may need to be treated aggressively . some authors have advocated an inhibition of b - lymphocyte chemoattractants ( bca-1 , cxcl13 , and scyb13 ) and their ligands cxcr4 and cxcr5 could be a future strategy for the treatment of this disease with limited side - effects profile . for margolis , if the quality of the cytology finally is poor , then a second vitrectomy may be necessary , but because cell numbers are likely to be low in a vitrectomized eye , a retinochoroidal biopsy may be performed at the time of vitrectomy surgery . vitreous biomarkers of intraocular lymphomapossibly , pvrl is the best example of ocular disease in which intravitreal cytokines are more useful for the diagnosis . increased concentration of il-10 , a growth and differentiation factor for activated b lymphocytes , has been found increased in vitreous fluids of pvrl patients , in contrast with the increased concentration of il-6 characteristic of uveitis , for which many authors have indicated that an il-10/il-6 ratio greater than 1.0 is useful for the diagnosis of pvrl . cytokine analysis can be useful adjunctive tests in corroborating suspicion of pvrl and determining whether there is a significant response to treatment [ 75 , 8087 ] , but can not be used only to make the diagnosis , as some studies have reported false positive or false negative results . the il-10/il-6 ratio greater than 1.0 in suspected cases of pvrl was associated with a sensitivity and specificity of 74.3 and 75.0% , respectively , and cassoux and co - workers found in 51 vitrectomies performed in patients with proven pvrl that an il-10 cut - off value of 400 pg / ml was associated with 80% sensitivity and 99% specificity . possibly , pvrl is the best example of ocular disease in which intravitreal cytokines are more useful for the diagnosis . increased concentration of il-10 , a growth and differentiation factor for activated b lymphocytes , has been found increased in vitreous fluids of pvrl patients , in contrast with the increased concentration of il-6 characteristic of uveitis , for which many authors have indicated that an il-10/il-6 ratio greater than 1.0 is useful for the diagnosis of pvrl . cytokine analysis can be useful adjunctive tests in corroborating suspicion of pvrl and determining whether there is a significant response to treatment [ 75 , 8087 ] , but can not be used only to make the diagnosis , as some studies have reported false positive or false negative results . the il-10/il-6 ratio greater than 1.0 in suspected cases of pvrl was associated with a sensitivity and specificity of 74.3 and 75.0% , respectively , and cassoux and co - workers found in 51 vitrectomies performed in patients with proven pvrl that an il-10 cut - off value of 400 pg / ml was associated with 80% sensitivity and 99% specificity . the diagnosis of intraocular lymphoma from vitreous specimens depends on proper handling of the specimens , methods of aspiration , concentration , fixation , and staining [ 90 , 91 ] . addition of culture medium with fetal calf serum can improve the survival and viability of the malignant cell . prior treatment of patients with steroids reduces the number of viable lymphoma cells , which are known to be cytolytic , so that discontinuing systemic and topical corticosteroids is strongly recommended before biopsy to increase the profitability of these cells . leukemia has increased the variability of ocular presentations associated , due to the improvement in the survival after the new era of effective antileukemic therapy . leukemia may involve almost every ocular tissue , with the retina being the most frequent affected structure ( up to 69% of all patients show fundus changes at some point in the course disease ) . hemorrhages , infiltrates , and aggregates of leukemic cells are found at all levels [ 48 , 92 ] , and generally the internal limiting membrane acts as a barrier ; however , cells occasionally invade the vitreous possibly emerging from the optic nerve head and these cases can be diagnosed by examination of the specimens obtained from the vitreous [ 93 , 94 ] . nevertheless , primary presentation of these diseases are rarely ophthalmological and more frequently occur in patients with advanced systemic disease . as relapsing uveitis or hyphemas can be related to leukemia , are in these cases when cytological sample must be obtained , thus allowing us to ascertain the inflammatory origin or reactivation of the disease . funduscopy combined with ultrasonography actually gives an accurate diagnosis in almost 95% of the patients , but there are , however , some cases difficult to diagnose due to atypical ocular manifestations or accompanying intraocular changes , such as extensive retinal detachment , vitreous hemorrhage , or others . in these cases cytological tests using modified shorr 's or others stains have been capable of diagnosing cells with intracytoplasmic melanin pigment granules from samples obtained in eyes harbouring choroidal or metastatic cutaneous melanomas [ 95 , 96 ] . a recent study shows that 5-s - cysteinyldopa ( 5-s - cd ) , a metabolite generated during pheomelanin synthesis , may reflect a direct secretion from the tumor into the vitreous or an alteration of dynamics of intraocular fluids , because its concentration is increased in vitreous fluid from um patients . but the diagnosis from vitreous samples in um probably would not become extensible in the future due to the unknown exact role of this biomarker , the possibility of extraocular dissemination of um implicit in the surgical intervention , and to the efficiency of other simpler diagnostic methods . intraocular metastases often appear in the choroid as solitary or multiple mass in a patient with history of systemic malignancy , although in 34% of the cases had no known primary site . vitrectomy has helped to diagnose metastatic cutaneous melanoma in difficult cases like nonpigmented vitreous clumps and thickened posterior vitreous membranes , and moreover can be therapeutic in these cases . carcinomas also can metastasize directly in the vitreous or indirectly by means of vitreal seeds from an underlying choroidal , retinal , or optic nerve infiltration , and both vitrectomy or fine - needle aspiration cytology can help in the diagnosis [ 102 , 103 ] . vitreous body constitutes a little - known intraocular structure , but we are increasingly supporting our diagnostic searches in it thanks to the recent advantages in collection , handling , and analysis of vitreous samples . inflammation is not always the cause of apparent inflammatory diseases and sometimes the origin is degenerative , traumatic , vascular , infectious , or even neoplastic . vitreous cells in hands of experienced cytologists can be sufficient , but an accurate diagnosis needs the employment of sophisticated molecular analysis such as flow cytometric immunophenotyping ( fci ) , microdissection and polymerase chain reaction ( pcr ) , or cytokine analysis like il-10/il-6 ratio . the major diagnostic use of vitreous sampling would be the masquerade syndromes , in which a devastating neoplastic disease can be behind a few vague and slightly specific ocular signs , and possibly the biggest representative of this group is the primary vitreoretinal lymphoma and some metastatic intraocular lesions . even though , little information exists nowadays on the number and the specific role of different molecules acting in the pathophysiology of diseases that represent a challenge for our daily practice . further investigation is needed to increase our knowledge on the molecular pathogenic mechanisms underliying neoplastic diseases , with which we could interact to create new targeted and powerful therapeutic pathways or at least alternatives to the current ones .

vitreous body is an intraocular structure , origin of diverse pathologies , but is also the place where cells and inflammatory mediators are released coming from several pathologic processes . these inflammatory reactions can happen in any other ocular location like choroid , retina , optic nerve , or ciliary body and vitreous humor constitutes a stagnant reservoir for these resulting substances and debris . through the recent techniques of vitreous collecting , handling , and analysis , increasingly more sophisticated and with fewer complications , cellularity and molecules in the vitreous of challenging pathologies for the ophthalmologist can now be studied . the most usefulness for vitreous diagnosis would be the masquerade syndromes , and the best exponent in this group is the primary vitreoretinal lymphoma ( pvrl ) , in which cytology and an il-10/il-6 ratio more than 1 is fundamental for the diagnosis .

1. Introduction 2. Ocular Diseases with Clinical Repercussion in Vitreous Body 3. Diagnostic Techniques in Atypical 4. Vitreal Biomarkers of Uveitis 5. Uveitis Masquerade Syndromes 6. Conclusion

vitreous body is the clear gel that fills the vitreous chamber or posterior chamber ( pc ) of the eyeball , the space between the lens and the eyewall , whose inner layer is the neurosensorial tissue that receives and transmits the image to the central nervous system called the retina . unlike the fluid in the anterior segment of the eye ( aqueous humour ) , which is continuously replaced , vitreous humor is stagnant , and its composition remains quite constant throughout life . the vitreous gel is avascular , composed mainly of water ( 98 - 99% ) , and 0.9% of inorganic salts ( sodium , potassium , and chloride ) . at the same time , there is a sustained suppressive microenvironment in the pc of the eye that inhibits the local expression of preexisting systemic immunity and participates in modifying the primary immune responses to ocular antigen [ 7 , 8 ] . in the vitreous cavity it would develop a deviant form of immunity , similar to that in the anterior chamber , in which antigen - specific suppressor t cells are generated and delayed hypersensitivity reactivity is selectively impaired . both the vitreous and the retina supply immunosuppressive molecules to the pc , but it has been suggested that retinal cells contribute more significantly to the immune suppressive microenvironment than vitreous cells : tgf is produced by retinal astrocytes and retinal pigment epithelium ; and muller 's cells ( glia ) of the retina suppress t cell proliferation by a direct contact mechanism . in addition , the retinal vascular endothelium , bruch 's membrane , and the pigment epithelium together form the so - called ocular - blood barrier . vitreous has a major role in the origin and the triggering of several ocular pathologies . also , certain eye diseases have their beginning in other more hidden structures of the eye but may secrete molecules or even cells to the vitreous chamber , causing symptoms and helping in the diagnosis , since the vitreous is more accessible to study than other posterior pole structures . the latter is the case of uveitis , a wide term that actually comprises a large group of diverse diseases affecting the retina , optic nerve , and also the vitreous compartment . these diseases may affect , in addition , several territories of the eye simultaneously and can have in major or minor degree manifestation in the vitreous humor , specially those concerning the posterior chamber . cases are limited to the eye ( organ specific ) , whereas the remainder forms part of more generalized diseases , so that the pathophysiology of uveitis depends on the specific etiology , but in all types there is a breach in the ocular - blood barrier that normally prevents cells and large proteins from entering the eye . these cells then recognize antigens ( autoantigen or foreign antigen ) presented on the cell surface of antigen - presenting cells ( apcs , like dendritic cells or macrophages ) , and activation and clonal expansion will take place , which results in increased production of il-2 , interferon- , and tnf- . in most cases , the clinical appearance is sufficient for diagnosis , but since the majority of these patients have an unknown etiology for the intraocular inflammation , and this can be in addition related to a primarily nondiagnosed systemic disease , could be infectious , inflammatory , or even tumoral , the correct diagnosis can prove difficult . thus , the intraocular inflammation is associated with the increased expression and action of several cytokines and growth factors , which can be determined in the vitreous and can help in the diagnosis . several molecules have been identified along the last decades in the vitreous humor , which may be the key in the physiopathology of certain ocular diseases . in most cases this determination has investigational purposes , in others is helpful for the prognosis of the patients , and in a minority of cases its finding constitutes an indispensable diagnostic tool . the indications of anterior chamber aspiration may be varied , but most common situations are patients with anterior chamber inflammation with suspicion of masquerade syndromes , a hypopion suspicious of infection , endophthalmitis , lens - induced uveitis and for cytopathologic examination . in the aqueous humor many other techniques can be performed for the diagnosis of infectious posterior uveitis , as polymerase chain reaction ( pcr ) and pathogen - specific antibody production for herpes simplex virus ( hsv ) , varicella zoster virus ( vzv ) , cytomegalovirus , epstein - barr virus ( ebv ) , human immunodeficiency virus ( hiv ) , propionibacterium acnes , and toxoplasma gondii . the indications for this technique are basically in posterior uveitis unresponsiveness to treatment when malignancy needs to be ruled out ; when intraocular infection is considered the primary cause of inflammation in the absence or insignificant amount of vitreous cells that would preclude diagnosis through pars plana vitrectomy ( ppv ) [ 17 , 18 ] . the major indication for vitreous aspiration would be when intraocular lymphoma is a significant diagnostic possibility . the technique of vitreous aspiration is similar to that of anterior chamber paracentesis and is currently performed through the pars plana under local anesthesia with a large caliber needle , such as 21-gauge ( g ) hollow needle or fine , like 23 or 25-g mounted on a 1-ml syringe as an aspirating device , permitting to aspirate a volume of 100 to 250 l of vitreous humor while the needle is directed posteriorly towards the optic nerve head . with vitreous biopsy , specimens obtained using a needle and syringe were positive in 54% of infected eyes compared with 75% of the specimens collected with vitrectomy procedures , although posteriorly , in the endophthalmitis vitrectomy study , no significant difference was shown between needle tap versus mechanized vitreous biopsy with respect to microbiologic yield . other advantages of vitreous biopsies over vitrectomy are quickness , can be done in the outpatient setting without inpatient admission , can be repeated , and are less traumatic to the eye . another variation of the technique is the fine - needle aspiration biopsy ( fnab ) , in which a fine - needle gauge is directed to the localized suspected areas of intraocular tumor or lesion . for aqueous or vitreous humor the technique turns into an effective method for cytology that generally is able to obtain a sufficient amount of cells ( 100 to 500 l of ocular fluid ) to perform routine analysis like microbiologic , cytomorphological evaluation of papanicolaou or haematoxylin- and eosin - stained cells , immunocytochemical analysis , and other applications . , endophthalmitis , intraocular bleeding with suspected malignant origin , and for the treatment of complications of chronic uveitis ) , and when the eye is inflamed and thereby patients may experience substantial discomfort during the vitreous biopsy tap . a diagnostic standard three - port pars plana vitrectomy ( vpp ) provides a large amount of vitreous , retina , or choroid ( though diluted ) , but always requires an operation theater under sterile conditions and direct visualization of the vitrectomy instruments . in order to obtain an undiluted vitreous sample , the infusion cannula of the system must be closed and the vitreous specimen is collected through undiluted lines using the vitreous cutter connected directly to a 3 ml syringe until the eye is noted to soften visibly . with perfluorocarbon - perfused vitrectomy , in which aspirated vitreous is compensated with perfluorocarbon liquid entry during vitreous aspiration , other authors were able to obtain an average of 2.4 ml of undiluted vitreous . there is controversy whether using classic 20-g ports vpp needing suture , or the newest microincisional systems with 23- , 25- , or 27-g systems , but with anyone of these , the overall diagnostic yield of vpp varies considerably in different published studies from 14.3 to 61.5% [ 2631 ] , and the success for the procedure was greater when an intraocular infection was suspected compared with an intraocular malignancy , and greater for detecting primary vitreoretinal lymphoma than for detecting metastatic disease . the indications for biopsy included major diagnostic uncertainty , suspected cancer metastasis to the choroid without other evidence of systemic malignancy , and patient insistence on biopsy confirmation of the diagnosis prior to treatment . the limited performance of intraocular biopsy is explained by the risks for dissemination of malignant cells , eye complications ( mainly hemorrhage , retinal detachment , and infection ) , and fears of misdiagnosis , although the literature gives little support to these [ 34 , 35 ] . in the classic , transscleral approach , a sharp blade then incises the choroid , and the biopsy tissue is grasped with forceps . several modifications have been later described to facilitate the biopsy procedure and also reduce the risk of complications ; vpp is now often performed before creating the scleral flap , and another modification is the use of cyanoacrylate glue to provide increased stability to the tissue . studies have shown increased levels of il-6 ( t - cell cytokine ) in the vitreous fluid of patients with active intermediate or posterior uveitis , although it did not correlate with a specific uveitis type , suggesting that il-6 is an inflammatory mediator common in various uveitis etiologies . il-12 , produced by monocytes , macrophages , b cells , and connective tissue - type mast cells has , also been found increased in aqueous humor and vitreous fluid of patients with low - grade intraocular inflammation and in uveitis in clinical remission for as long as 2 years . since diagnostic analysis of vitreous fluid in patients with uveitis uveitis masquerade syndrome ( ums ) is a group of disorders that mimic intraocular inflammation , but cells seen may be of noninflammatory origin ( e.g. although they constitute rare presentations of uncommon diseases in the eye , the ophthalmologist must be aware because many of the ums etiologies are malignancies with deleterious effects for the patient , for what early diagnosis and prompt treatment are mandatory . the study of vitreous body can be of great help specially in these cases , since the achievement of a small sample in doubtful cases can provide us the diagnosis . although both hodgkin 's lymphoma and non - hodgkin 's lymphoma ( nhl ) can present as intraocular inflammation , in the case of hodgkin 's lymphoma ocular involvement generally is rare and often occurs late in the course of the disease , whereas nhl affects more commonly the eye . the variant with major ophthalmic repercussion is the primary vitreoretinal lymphoma ( pvrl ) , a subtype of primary central ( cns ) lymphoma , typically classified as a diffuse large b - cell lymphoma and most frequently develops in elderly populations . over 15% of primary cns lymphoma patients develop intraocular lymphoma , usually occurring in the retina and/or vitreous , and conversely , 65%90% of pvrl patients develop cns lymphoma . both retinal and uveal lymphoma can manifest as any form of uveitis , but pvrl typical clinical findings include vitreous cellular infiltration ( lymphoma and inflammatory cells ) and subretinal tumor infiltration . currently , pvrl is most often diagnosed using citology ( the gold standard ) or vitrectomy to identify lymphoma cells in the vitreous or retina . in order to prevent degeneration of lymphoma cells , vitreous specimens are placed into a tube containing culture medium like rpmi ( roswell park memorial institute ) , whereas others prefer immediate placement in normal saline , taking care not fixing with alcohol with the aim to not alter the identification of pvrl cells in the vitreous sample . molecular analysisflow cytometric immunophenotyping ( fci ) can be done in diluted samples , allows for the analysis of several different cell surface markers simultaneously , and offers a quantitative method of determining the percentage of a particular cellular phenotype , increasing the efficiency of a biopsy specimen . dilute vitreous is centrifuged and resuspended in cell culture medium , and cells are counted and stained with antibodies to detect markers that identify leukocytes , t lymphocytes , b lymphocytes ( including cd19 , cd20 , cd22 , , and light chain markers ) , monocyte / macrophages , and lymphocyte activation . the test relies on the finding that the majority of pvrl have restricted expression of or chains , with the most sensitive marker being a : ratio 3 or 0.6 ( 80% ) , whereas cd22 and cd20 markers are not very sensitive for lymphoma ( 50 and 33% , resp . ) flow cytometric immunophenotyping ( fci ) can be done in diluted samples , allows for the analysis of several different cell surface markers simultaneously , and offers a quantitative method of determining the percentage of a particular cellular phenotype , increasing the efficiency of a biopsy specimen . dilute vitreous is centrifuged and resuspended in cell culture medium , and cells are counted and stained with antibodies to detect markers that identify leukocytes , t lymphocytes , b lymphocytes ( including cd19 , cd20 , cd22 , , and light chain markers ) , monocyte / macrophages , and lymphocyte activation . the test relies on the finding that the majority of pvrl have restricted expression of or chains , with the most sensitive marker being a : ratio 3 or 0.6 ( 80% ) , whereas cd22 and cd20 markers are not very sensitive for lymphoma ( 50 and 33% , resp . ) pcr has been found to be 64% sensitive for pvrl , and is being used to study the genotypic classification of pvrl with the goal of identifying prognostic factors ; patients with a translocation in the bcl-2 gene are significantly younger than patients who lacked the translocation , suggesting that younger patients with the translocation may need to be treated aggressively . some authors have advocated an inhibition of b - lymphocyte chemoattractants ( bca-1 , cxcl13 , and scyb13 ) and their ligands cxcr4 and cxcr5 could be a future strategy for the treatment of this disease with limited side - effects profile . vitreous biomarkers of intraocular lymphomapossibly , pvrl is the best example of ocular disease in which intravitreal cytokines are more useful for the diagnosis . increased concentration of il-10 , a growth and differentiation factor for activated b lymphocytes , has been found increased in vitreous fluids of pvrl patients , in contrast with the increased concentration of il-6 characteristic of uveitis , for which many authors have indicated that an il-10/il-6 ratio greater than 1.0 is useful for the diagnosis of pvrl . cytokine analysis can be useful adjunctive tests in corroborating suspicion of pvrl and determining whether there is a significant response to treatment [ 75 , 8087 ] , but can not be used only to make the diagnosis , as some studies have reported false positive or false negative results . the il-10/il-6 ratio greater than 1.0 in suspected cases of pvrl was associated with a sensitivity and specificity of 74.3 and 75.0% , respectively , and cassoux and co - workers found in 51 vitrectomies performed in patients with proven pvrl that an il-10 cut - off value of 400 pg / ml was associated with 80% sensitivity and 99% specificity . possibly , pvrl is the best example of ocular disease in which intravitreal cytokines are more useful for the diagnosis . increased concentration of il-10 , a growth and differentiation factor for activated b lymphocytes , has been found increased in vitreous fluids of pvrl patients , in contrast with the increased concentration of il-6 characteristic of uveitis , for which many authors have indicated that an il-10/il-6 ratio greater than 1.0 is useful for the diagnosis of pvrl . cytokine analysis can be useful adjunctive tests in corroborating suspicion of pvrl and determining whether there is a significant response to treatment [ 75 , 8087 ] , but can not be used only to make the diagnosis , as some studies have reported false positive or false negative results . the il-10/il-6 ratio greater than 1.0 in suspected cases of pvrl was associated with a sensitivity and specificity of 74.3 and 75.0% , respectively , and cassoux and co - workers found in 51 vitrectomies performed in patients with proven pvrl that an il-10 cut - off value of 400 pg / ml was associated with 80% sensitivity and 99% specificity . hemorrhages , infiltrates , and aggregates of leukemic cells are found at all levels [ 48 , 92 ] , and generally the internal limiting membrane acts as a barrier ; however , cells occasionally invade the vitreous possibly emerging from the optic nerve head and these cases can be diagnosed by examination of the specimens obtained from the vitreous [ 93 , 94 ] . funduscopy combined with ultrasonography actually gives an accurate diagnosis in almost 95% of the patients , but there are , however , some cases difficult to diagnose due to atypical ocular manifestations or accompanying intraocular changes , such as extensive retinal detachment , vitreous hemorrhage , or others . a recent study shows that 5-s - cysteinyldopa ( 5-s - cd ) , a metabolite generated during pheomelanin synthesis , may reflect a direct secretion from the tumor into the vitreous or an alteration of dynamics of intraocular fluids , because its concentration is increased in vitreous fluid from um patients . but the diagnosis from vitreous samples in um probably would not become extensible in the future due to the unknown exact role of this biomarker , the possibility of extraocular dissemination of um implicit in the surgical intervention , and to the efficiency of other simpler diagnostic methods . carcinomas also can metastasize directly in the vitreous or indirectly by means of vitreal seeds from an underlying choroidal , retinal , or optic nerve infiltration , and both vitrectomy or fine - needle aspiration cytology can help in the diagnosis [ 102 , 103 ] . vitreous body constitutes a little - known intraocular structure , but we are increasingly supporting our diagnostic searches in it thanks to the recent advantages in collection , handling , and analysis of vitreous samples . vitreous cells in hands of experienced cytologists can be sufficient , but an accurate diagnosis needs the employment of sophisticated molecular analysis such as flow cytometric immunophenotyping ( fci ) , microdissection and polymerase chain reaction ( pcr ) , or cytokine analysis like il-10/il-6 ratio . the major diagnostic use of vitreous sampling would be the masquerade syndromes , in which a devastating neoplastic disease can be behind a few vague and slightly specific ocular signs , and possibly the biggest representative of this group is the primary vitreoretinal lymphoma and some metastatic intraocular lesions . even though , little information exists nowadays on the number and the specific role of different molecules acting in the pathophysiology of diseases that represent a challenge for our daily practice .

vitreous body is the clear gel that fills the vitreous chamber or posterior chamber ( pc ) of the eyeball , the space between the lens and the eyewall , whose inner layer is the neurosensorial tissue that receives and transmits the image to the central nervous system called the retina . unlike the fluid in the anterior segment of the eye ( aqueous humour ) , which is continuously replaced , vitreous humor is stagnant , and its composition remains quite constant throughout life . most of the protein is forming fibrils composed of a small collagen type v / xi core wrapped in a thick layer of collagen type ii ( 75% of the fibril by mass ) [ 1 , 2 ] . the immune privilege , also , a physiologic mechanism characteristic of the internal compartments of the eye , is designed to provide protection against pathogens , protecting the delicate visual axis from the sight - destroying effect of immunogenic inflammation . at the same time , there is a sustained suppressive microenvironment in the pc of the eye that inhibits the local expression of preexisting systemic immunity and participates in modifying the primary immune responses to ocular antigen [ 7 , 8 ] . in the vitreous cavity it would develop a deviant form of immunity , similar to that in the anterior chamber , in which antigen - specific suppressor t cells are generated and delayed hypersensitivity reactivity is selectively impaired . both the vitreous and the retina supply immunosuppressive molecules to the pc , but it has been suggested that retinal cells contribute more significantly to the immune suppressive microenvironment than vitreous cells : tgf is produced by retinal astrocytes and retinal pigment epithelium ; and muller 's cells ( glia ) of the retina suppress t cell proliferation by a direct contact mechanism . other diseases are easily diagnosable in the fundus eye and only affect the vitreous in late stages , like retinal vasculopathies as diabetic retinopathy and other vitreoretinal proliferations . also , certain eye diseases have their beginning in other more hidden structures of the eye but may secrete molecules or even cells to the vitreous chamber , causing symptoms and helping in the diagnosis , since the vitreous is more accessible to study than other posterior pole structures . cases are limited to the eye ( organ specific ) , whereas the remainder forms part of more generalized diseases , so that the pathophysiology of uveitis depends on the specific etiology , but in all types there is a breach in the ocular - blood barrier that normally prevents cells and large proteins from entering the eye . these cells then recognize antigens ( autoantigen or foreign antigen ) presented on the cell surface of antigen - presenting cells ( apcs , like dendritic cells or macrophages ) , and activation and clonal expansion will take place , which results in increased production of il-2 , interferon- , and tnf- . in most cases , the clinical appearance is sufficient for diagnosis , but since the majority of these patients have an unknown etiology for the intraocular inflammation , and this can be in addition related to a primarily nondiagnosed systemic disease , could be infectious , inflammatory , or even tumoral , the correct diagnosis can prove difficult . thus , the intraocular inflammation is associated with the increased expression and action of several cytokines and growth factors , which can be determined in the vitreous and can help in the diagnosis . several molecules have been identified along the last decades in the vitreous humor , which may be the key in the physiopathology of certain ocular diseases . several techniques have been employed for diagnostic purposes in these cases : aqueous aspiration , vitreous aspiration , diagnostic vitrectomy , fine - needle aspiration biopsy , controlled aspiration of subretinal fluid , incisional chorio - retinal biopsy , and diagnostic enucleation . the indications of anterior chamber aspiration may be varied , but most common situations are patients with anterior chamber inflammation with suspicion of masquerade syndromes , a hypopion suspicious of infection , endophthalmitis , lens - induced uveitis and for cytopathologic examination . in the aqueous humor many other techniques can be performed for the diagnosis of infectious posterior uveitis , as polymerase chain reaction ( pcr ) and pathogen - specific antibody production for herpes simplex virus ( hsv ) , varicella zoster virus ( vzv ) , cytomegalovirus , epstein - barr virus ( ebv ) , human immunodeficiency virus ( hiv ) , propionibacterium acnes , and toxoplasma gondii . further studies have demonstrated for pcr of aqueous humor to yield a diagnosis in one - third of patients with posterior infectious uveitis with a sensitivity of 82% and specificity of 100% , equal or better than vitreous biopsy . the indications for this technique are basically in posterior uveitis unresponsiveness to treatment when malignancy needs to be ruled out ; when intraocular infection is considered the primary cause of inflammation in the absence or insignificant amount of vitreous cells that would preclude diagnosis through pars plana vitrectomy ( ppv ) [ 17 , 18 ] . the technique of vitreous aspiration is similar to that of anterior chamber paracentesis and is currently performed through the pars plana under local anesthesia with a large caliber needle , such as 21-gauge ( g ) hollow needle or fine , like 23 or 25-g mounted on a 1-ml syringe as an aspirating device , permitting to aspirate a volume of 100 to 250 l of vitreous humor while the needle is directed posteriorly towards the optic nerve head . with vitreous biopsy , specimens obtained using a needle and syringe were positive in 54% of infected eyes compared with 75% of the specimens collected with vitrectomy procedures , although posteriorly , in the endophthalmitis vitrectomy study , no significant difference was shown between needle tap versus mechanized vitreous biopsy with respect to microbiologic yield . in case of intraocular tumor , the aspirated block obtained is likely to have a higher concentration of neoplastic cells than any of the adjacent intraocular fluids , decreasing the possibility of inconclusive cytological diagnoses , which often occur in vitrectomy specimens . for aqueous or vitreous humor the technique turns into an effective method for cytology that generally is able to obtain a sufficient amount of cells ( 100 to 500 l of ocular fluid ) to perform routine analysis like microbiologic , cytomorphological evaluation of papanicolaou or haematoxylin- and eosin - stained cells , immunocytochemical analysis , and other applications . , endophthalmitis , intraocular bleeding with suspected malignant origin , and for the treatment of complications of chronic uveitis ) , and when the eye is inflamed and thereby patients may experience substantial discomfort during the vitreous biopsy tap . there is controversy whether using classic 20-g ports vpp needing suture , or the newest microincisional systems with 23- , 25- , or 27-g systems , but with anyone of these , the overall diagnostic yield of vpp varies considerably in different published studies from 14.3 to 61.5% [ 2631 ] , and the success for the procedure was greater when an intraocular infection was suspected compared with an intraocular malignancy , and greater for detecting primary vitreoretinal lymphoma than for detecting metastatic disease . the limited performance of intraocular biopsy is explained by the risks for dissemination of malignant cells , eye complications ( mainly hemorrhage , retinal detachment , and infection ) , and fears of misdiagnosis , although the literature gives little support to these [ 34 , 35 ] . studies have shown increased levels of il-6 ( t - cell cytokine ) in the vitreous fluid of patients with active intermediate or posterior uveitis , although it did not correlate with a specific uveitis type , suggesting that il-6 is an inflammatory mediator common in various uveitis etiologies . il-12 , produced by monocytes , macrophages , b cells , and connective tissue - type mast cells has , also been found increased in aqueous humor and vitreous fluid of patients with low - grade intraocular inflammation and in uveitis in clinical remission for as long as 2 years . since diagnostic analysis of vitreous fluid in patients with uveitis uveitis masquerade syndrome ( ums ) is a group of disorders that mimic intraocular inflammation , but cells seen may be of noninflammatory origin ( e.g. the causes of ums may be variate , such as malignant , including hematologic malignancies , retinoblastoma , melanoma , and lung cancer metastasis ; or nonmalignant , like ocular toxoplasmosis , diabetic retinopathy , hypertension , retinal detachment or degeneration , intraocular trauma , and radiation retinopathy [ 42 , 43 , 4569 ] ( table 1 ) . they are often misdiagnosed as a chronic idiopathic uveitis , but they can present in any location of the eye manifesting as panuveitis , pars planitis , vitreitis , papillitis , anterior segment cells , hypopyon or vitreal , and/or chorioretinal infiltrates ( figure 2 ) . although they constitute rare presentations of uncommon diseases in the eye , the ophthalmologist must be aware because many of the ums etiologies are malignancies with deleterious effects for the patient , for what early diagnosis and prompt treatment are mandatory . although both hodgkin 's lymphoma and non - hodgkin 's lymphoma ( nhl ) can present as intraocular inflammation , in the case of hodgkin 's lymphoma ocular involvement generally is rare and often occurs late in the course of the disease , whereas nhl affects more commonly the eye . nhl can be divided in two clinically different entities : systemic nhl with metastases to the eye , and nhl of the central nervous system ( nhl - cns ) . recently , coupland and co - workers proposed an anatomical classification according to the localization of the disease in the eye ; retinal lymphomas are high - grade b - cell malignancies associated with a poor prognosis , whereas primary uveal lymphomas are typically low - grade b - cell tumours derived from the postgerminal centre ( memory ) b cell . the variant with major ophthalmic repercussion is the primary vitreoretinal lymphoma ( pvrl ) , a subtype of primary central ( cns ) lymphoma , typically classified as a diffuse large b - cell lymphoma and most frequently develops in elderly populations . over 15% of primary cns lymphoma patients develop intraocular lymphoma , usually occurring in the retina and/or vitreous , and conversely , 65%90% of pvrl patients develop cns lymphoma . a classic finding is the presence of solitary or multiple yellow , creamy choroidal infiltrates with clear vitreous , that can evolve to diffuse thickening of the uveal tract and in some cases , to episcleral extension appearing as a nonmobile orange to yellow or salmon patch . in order to prevent degeneration of lymphoma cells , vitreous specimens are placed into a tube containing culture medium like rpmi ( roswell park memorial institute ) , whereas others prefer immediate placement in normal saline , taking care not fixing with alcohol with the aim to not alter the identification of pvrl cells in the vitreous sample . the malignant b cells of pvrl exhibit characteristic features with papanicolaou , giemsa , or diff - quick stains [ 26 , 76 ] : large round or oval nuclei , frequently segmented and often containing prominent nucleoli , surrounded by scant basophilic cytoplasm . samples often are negative because of poor biopsy samples , with a reported effectivity of only 48.3% of lymphoma cases for ppv , although other authors have found for fnab a diagnostic effectivity in 87.5% of the suspicious intraocular lymphoma cases . the malignant b cells of pvrl exhibit characteristic features with papanicolaou , giemsa , or diff - quick stains [ 26 , 76 ] : large round or oval nuclei , frequently segmented and often containing prominent nucleoli , surrounded by scant basophilic cytoplasm . samples often are negative because of poor biopsy samples , with a reported effectivity of only 48.3% of lymphoma cases for ppv , although other authors have found for fnab a diagnostic effectivity in 87.5% of the suspicious intraocular lymphoma cases . molecular analysisflow cytometric immunophenotyping ( fci ) can be done in diluted samples , allows for the analysis of several different cell surface markers simultaneously , and offers a quantitative method of determining the percentage of a particular cellular phenotype , increasing the efficiency of a biopsy specimen . the test relies on the finding that the majority of pvrl have restricted expression of or chains , with the most sensitive marker being a : ratio 3 or 0.6 ( 80% ) , whereas cd22 and cd20 markers are not very sensitive for lymphoma ( 50 and 33% , resp . ) for patients with possible t - cell lymphoma , cell surface markers more commonly searched are cd3 , cd8 , cd4 , cd7 , cd2 , cd25 , and cd52 . flow cytometric immunophenotyping ( fci ) can be done in diluted samples , allows for the analysis of several different cell surface markers simultaneously , and offers a quantitative method of determining the percentage of a particular cellular phenotype , increasing the efficiency of a biopsy specimen . the test relies on the finding that the majority of pvrl have restricted expression of or chains , with the most sensitive marker being a : ratio 3 or 0.6 ( 80% ) , whereas cd22 and cd20 markers are not very sensitive for lymphoma ( 50 and 33% , resp . ) for patients with possible t - cell lymphoma , cell surface markers more commonly searched are cd3 , cd8 , cd4 , cd7 , cd2 , cd25 , and cd52 . pcr can determine monoclonality by immunoglobulin heavy chain ( igh ) rearrangement and t(14 ; 18 ) translocation of the bcl-2 gene that promote cell survival and predict a more aggressive tumor course in b - cell lymphoma [ 75 , 79 , 80 ] . pcr has been found to be 64% sensitive for pvrl , and is being used to study the genotypic classification of pvrl with the goal of identifying prognostic factors ; patients with a translocation in the bcl-2 gene are significantly younger than patients who lacked the translocation , suggesting that younger patients with the translocation may need to be treated aggressively . for margolis , if the quality of the cytology finally is poor , then a second vitrectomy may be necessary , but because cell numbers are likely to be low in a vitrectomized eye , a retinochoroidal biopsy may be performed at the time of vitrectomy surgery . increased concentration of il-10 , a growth and differentiation factor for activated b lymphocytes , has been found increased in vitreous fluids of pvrl patients , in contrast with the increased concentration of il-6 characteristic of uveitis , for which many authors have indicated that an il-10/il-6 ratio greater than 1.0 is useful for the diagnosis of pvrl . cytokine analysis can be useful adjunctive tests in corroborating suspicion of pvrl and determining whether there is a significant response to treatment [ 75 , 8087 ] , but can not be used only to make the diagnosis , as some studies have reported false positive or false negative results . the il-10/il-6 ratio greater than 1.0 in suspected cases of pvrl was associated with a sensitivity and specificity of 74.3 and 75.0% , respectively , and cassoux and co - workers found in 51 vitrectomies performed in patients with proven pvrl that an il-10 cut - off value of 400 pg / ml was associated with 80% sensitivity and 99% specificity . increased concentration of il-10 , a growth and differentiation factor for activated b lymphocytes , has been found increased in vitreous fluids of pvrl patients , in contrast with the increased concentration of il-6 characteristic of uveitis , for which many authors have indicated that an il-10/il-6 ratio greater than 1.0 is useful for the diagnosis of pvrl . cytokine analysis can be useful adjunctive tests in corroborating suspicion of pvrl and determining whether there is a significant response to treatment [ 75 , 8087 ] , but can not be used only to make the diagnosis , as some studies have reported false positive or false negative results . the il-10/il-6 ratio greater than 1.0 in suspected cases of pvrl was associated with a sensitivity and specificity of 74.3 and 75.0% , respectively , and cassoux and co - workers found in 51 vitrectomies performed in patients with proven pvrl that an il-10 cut - off value of 400 pg / ml was associated with 80% sensitivity and 99% specificity . prior treatment of patients with steroids reduces the number of viable lymphoma cells , which are known to be cytolytic , so that discontinuing systemic and topical corticosteroids is strongly recommended before biopsy to increase the profitability of these cells . hemorrhages , infiltrates , and aggregates of leukemic cells are found at all levels [ 48 , 92 ] , and generally the internal limiting membrane acts as a barrier ; however , cells occasionally invade the vitreous possibly emerging from the optic nerve head and these cases can be diagnosed by examination of the specimens obtained from the vitreous [ 93 , 94 ] . funduscopy combined with ultrasonography actually gives an accurate diagnosis in almost 95% of the patients , but there are , however , some cases difficult to diagnose due to atypical ocular manifestations or accompanying intraocular changes , such as extensive retinal detachment , vitreous hemorrhage , or others . a recent study shows that 5-s - cysteinyldopa ( 5-s - cd ) , a metabolite generated during pheomelanin synthesis , may reflect a direct secretion from the tumor into the vitreous or an alteration of dynamics of intraocular fluids , because its concentration is increased in vitreous fluid from um patients . but the diagnosis from vitreous samples in um probably would not become extensible in the future due to the unknown exact role of this biomarker , the possibility of extraocular dissemination of um implicit in the surgical intervention , and to the efficiency of other simpler diagnostic methods . vitreous cells in hands of experienced cytologists can be sufficient , but an accurate diagnosis needs the employment of sophisticated molecular analysis such as flow cytometric immunophenotyping ( fci ) , microdissection and polymerase chain reaction ( pcr ) , or cytokine analysis like il-10/il-6 ratio . the major diagnostic use of vitreous sampling would be the masquerade syndromes , in which a devastating neoplastic disease can be behind a few vague and slightly specific ocular signs , and possibly the biggest representative of this group is the primary vitreoretinal lymphoma and some metastatic intraocular lesions .

primary and secondary cancers are amongst the leading causes of morbidity and mortality . along with the lung and liver , the bony skeleton is one of the most common sites for metastases , with most cases occurring in the spine.13 skeletal metastases frequently originate from carcinoma of the breast , prostate , and lung , and if they continue to grow and destroy bone result in pain and pathologic fracture , impaired ambulatory ability and poorer quality of life.27 the process of cancer metastases following tumor growth at the primary site of origin involves intravasation and survival in the bloodstream , arrest , extravasation and finally establishment by invasion and angiogenesis at a distant site.2,3,8 the ability of metastatic cancer to grow within and invade bone is largely mediated by tumor - induced growth factors and cytokines that result in imbalance between the bone - forming osteoblasts and bone - degrading osteoclasts of bone marrow that normally regulate bone turnover.2,3,9 animal models of cancer metastases are essential in order to better understand the molecular pathways behind metastatic spread and local growth and invasion at distant sites , enable analysis of host and cancer cell interactions , identify barriers to the metastatic process , and to provide potential platforms to develop and test novel therapeutic agents . the ideal model should be clinically relevant , reproducible and representative of the human condition . the broad approaches to animal model studies include spontaneous , chemically or genetically - induced , syngeneic , and xenograft cancer models . although spontaneous models resemble what occurs when humans develop cancer , the spontaneous development of primary and metastatic cancer in animals is extremely rare.10 similarly , the metastasis rate of genetically - induced tumors , either by inducing expression of oncogenes or deletion of tumor suppressor genes , is generally very low and too variable and unpredictable in terms of spreading to bone to provide useful skeletal metastasis models . syngeneic models are those in which tumor cells and host are of the same origin and involve introduction of tumor cells isolated from spontaneously occurring rodent or small animal cancers , whereas xenograft models involve the introduction of human tumor cell lines into an immunodeficient host . important considerations include the host animal , method of inoculation of tumor cells , type of cancer for investigation , analysis of the developing tumors and clinical application of the model . this paper reviews these considerations , with a specific focus on in vivo animal models of cancer metastases to the bony skeleton . considerations for the choice of host animal to be used for studying cancer and metastasis include animal availability , cost , handling , desired cancer type for investigation , technique of introduction of cancer cells , and whether surgical procedures are required for tumor implantation or treatment , as well as how the resulting tumors and tissues are to be imaged and analyzed . established animal models used for studying skeletal metastases are summarized in table 1 . to date , the rodent has been the most popular host animal used in the study of bone metastases.8 rodents have a high degree of gene sequence homology with humans , similar anatomical organs , are easy to handle and maintain , readily available and relatively cheap , and can be manipulated for investigation of specific cancer pathways by generating knockout , transgenic , or over - expressing strains.11,12 immune - deficient animals such as balb / c athymic nude mice and severe combined immunodeficiency ( scid ) mice are commonly used in xenograft models as they are unable to immunologically reject transplanted tissue or cells , thus enabling the study of human cancer cell lines . as scid mice are more severely immunocompromised than nude mice , it has been suggested that some cancer cell lines may grow more rapidly or produce a higher incidence of metastases in scid compared with nude mice.13 strube et al described a mouse model of human renal cell carcinoma metastasizing to bone following intracardiac inoculation of human 786-o / luciferase cells into nude mice , resulting in aggressive osteolytic bone lesions involving the hindlimbs , forelimbs , pelvis , and spine.14 similarly , garcia et al injected a bone metastasizing - only subclone ( b02 ) from the mda - mb-231 human breast cancer cell line into the tail vein of nude mice , resulting in osteolytic bone metastases of the hindlimb.5 one of the disadvantages of using immunodeficient animals in models of cancer metastases is that they preclude investigation into the important role of the immune system in combating cancer.11 this may be overcome by the use of syngeneic models , in which cancer cell lines of the same species as the host are introduced into immunocompetent animals . lelelakis et al described a mouse model of breast cancer metastases to bone using a clonal tumor cell line from a spontaneously arising murine mammary cancer , in which metastases to the femur and spine followed intracardiac and mammary pad inoculation of the cancer cells.15 another disadvantage of rodent cancer models includes the small size of rodent bone , potentially making direct implantation of cancer cells , as well as investigation of therapeutic modalities such as local drug delivery devices and invasive image - guided - therapies , more technically challenging.12,16,17 the use of rabbits may be less technically demanding for surgical procedures and may enable easier analysis and preclinical testing of skeletal metastases . however , experimental immunodeficient rabbits are currently not available ; thus , species - specific cancer cell lines are required in rabbit syngeneic cancer models and these are extremely limited in availability.11,16,17 despite this , vx2 carcinoma cells derived from a virus induced papilloma of the rabbit have been used in two rabbit models of spinal tumors to successfully replicate the clinical , radiological , and histopathologic characteristics of the human condition.16,17 orthotopic implantation of the vx2 carcinoma cells into the third lumbar vertebra or the lower thoracic vertebra of healthy rabbits after surgical exposure led to lower limb paralysis in most rabbits by four weeks post - implantation . larger - sized animals that can potentially be used in cancer models include the cat and dog . although the cat has not yet been established as a useful syngeneic or xenograft animal model for the study of skeletal metastasis , spontaneous metastases to the digits of the bony skeleton and in soft tissues of the proximal limbs in cats have been observed secondary to primary pulmonary adenocarcinoma.18,19 in a larger syngeneic orthotopic animal model , a dog model of prostate cancer metastasis , anidjar et al demonstrated bone metastases following direct inoculation of dpc-1 poorly differentiated canine prostate adenocarcinoma cells into the prostate gland.20 histopathology of the mixed osteoblastic and osteolytic pelvic bone lesions showed similarities to human prostate cancer skeletal metastases.20 a potential advantage of the larger canine model over rabbits or rodents is that dogs may be more amenable to diagnostic and surgical procedures with equipment used in human clinical practice ; however , the cost and restricted availability of the animals and species - specific cancer cell lines remain limitations . as described above , since the spontaneous development of metastatic cancer in animals is rare and unpredictable , animal models of skeletal metastases typically require physical introduction of cancer cells . commonly used methods of cancer cell inoculation include systemic intracardiac or tail vein injection , injection into the arterial circulation , direct inoculation into the primary cancer site such as the mammary fat pad or prostate gland , or direct orthotopic inoculation into bone , reproducing the secondary skeletal deposit in isolation ( table 1 ) . models involving systemic dissemination of tumor cells permit investigation of the key elements of the metastatic cascade from survival within the bloodstream , to selection of a distal site , and eventually to extravasation , establishment and growth of distant metastases , thus allowing analysis of the molecular mechanisms involved in growth and proliferation , migration , invasion and angiogenesis of metastatic cancer cells.11,15 intracardiac injection into the left ventricle results in systemic arterial circulatory dissemination of tumor cells , with the potential of generating widespread metastases . arguello et al described a reproducible murine model of skeletal vertebral and long bone metastasis following intracardiac injection of b16 ( sub - line g3.26 ) murine melanoma cells.21 however , the sites of metastatic deposition and number and size of the metastases were unpredictable , extremely variable , and dependent on the amount of cancer cells administered . tail vein inoculation similarly results in hematogenous dissemination of cancer cells , although cells may be filtered and cleared by the pulmonary system during the first passage through circulation.22 passineau et al described tail vein injection of a20 murine lymphoma cells to generate a syngeneic mouse model to mimic disseminated human b cell lymphoma , with bony deposits to the femur , pelvis and vertebral column , eventually leading to bone destruction and nerve compression.23 biesalski et al described intra - arterial injection of walker 256 mammary carcinoma cells directly into the circulation of the hindlimbs in rats via the superficial caudal epigastric artery , a branch of the femoral artery , resulting in metastases to the distal femur or proximal tibia / fibula in 9 of 11 animals at 3 weeks post - inoculation.24 limitations of systemic administration of tumor cells include variability and unpredictability in site , size , number , and time to development of metastasis.10,11 this may also lead to excessive tumor burden and morbidity in the experimental animal , therefore precluding the ability to investigate potential therapeutic interventions . direct inoculation of tumor cells into primary sites such as the mammary fat pad also has the potential to create a complete model of the metastatic process , since the tumor must first establish in the primary site before seeding to distant sites.15 schubert et al described a mouse model of breast cancer metastasis to the femur and lung after mda - mb-435 human breast cancer cells were injected into the mammary fat pad of nude mice.25 the ability of gonadotropin - releasing hormone ( gnrh ) analogs to significantly reduce formation and growth of breast cancer metastases in this model suggested the multi - focal role of gnrh throughout the metastatic process of breast cancer , including epithelial - mesenchymal transition , migration , invasion , and biology of circulating cells . direct orthotopic inoculation into bone has the potential to produce reproducible , efficient , and site - specific models of cancer and metastasis since a known quantity of cancer cells can be directly administered into the target bone of choice . direct inoculation into bone bypasses the metastatic cascade and is not a true model of metastases , nevertheless it permits targeted investigation of the cellular processes involved within a particular anatomical site and the local effects of the tumor itself . mann et al described a mouse model of tumor - induced osteolysis following orthotopic injection of f10 human breast cancer cells into the distal femoral metaphysis to assess the ability of non - invasive imaging and computational techniques for predicting the strength of bones with osteolytic lesions.4 choi et al used direct orthotopic inoculation of vx2 carcinoma tissue fragments into the tibial shaft of rabbits to create a model for magnetic resonance imaging ( mri ) and histopathologic evaluation of the evolution of metastatic bone tumor.26 orthotopic inoculation may be a relatively straightforward procedure if prominent and subcutaneous bones are selected , such as the distal femur or proximal tibia , which are also common sites for primary and secondary bone cancers . in our own laboratory , we recently established a novel in vivo model of spinal cancer that causes a reproducible , evolving paraplegia following orthotopic inoculation of mda-231 human breast cancer cells or pc-3 human prostate cancer cells into the vertebral body at the thoracolumbar junction of balb - c athymic nude mice ( fig . the progressive neurological decline in the animals , from gait asymmetry and unilateral hindlimb weakness , to complete unilateral hindlimb paralysis and finally to complete bilateral hindlimb paralysis , closely resembles the natural history of untreated metastatic epidural spinal cord compression and potentially enables closer analysis of the temporospatial pattern of cancer growth within the spine and the molecular mechanisms behind this devastating condition . amongst the commonest primary cancers that metastasize to bone are breast , prostate , lung , and renal , carcinoma . since the biological behavior and response to therapy of bone metastases is largely dependent on the primary cancer type , there is a need to develop investigative models of varying cell lines . for example , prostate cancer bone metastases in humans are generally more osteoblastic or sclerotic in nature , while renal cell carcinoma bone metastases are often more lytic and vascular compared with other cancer types.2,3,14 indeed , a sub - line of the 786-o human renal cell carcinoma cell line with increased bone metastatic potential showed higher expression of the pro - angiogenic factors vegf and basic fibroblast growth factor ( bfgf ) compared with the parental cell line.14 immortalized cancer cell lines that are generated through isolation of tumor cells from these spontaneously occurring primary or secondary cancer types in humans or animals are ideal for use in in vivo cancer models as they have already demonstrated ability to form , grow , invade , and spread within the primary organ or metastatic site of choice . human cancer cells lines with a propensity for localization to bone and that are well - established for use in animal cancer metastasis models of metastases include the mda - mb-231 human breast cancer cell line isolated from the pleural effusion of a patient following breast cancer metastasis to the lungs,5,22,2731 and pc-3 human prostate cancer cells derived from a bone metastasis.8,9,32 the use of cancer cell lines of human origin ensures that molecular and histologic analysis is as comparable to the human condition as possible ; however , one important limitation is the requirement for use of an immunocompromised animal , which precludes investigation of the host immune response to the development of cancer and metastases.11 the b16 murine melanoma cell line , derived from a melanoma from the skin of a c57bl/6 strain mouse,6,21,33 and the vx2 rabbit carcinoma cell line , originated from a carcinoma induced by the shope cottontail rabbit papilloma virus,16,17,26 are common animal cell lines used in models of skeletal metastasis models that exhibit similar molecular characteristics to human cancer cells . cell sub - lines with site - specific metastatic properties , such as the propensity to metastasize to the skeleton , are often generated from primary cell lines through serial culture and repeated injection of cells cultured from metastatic lesions at the site of interest . for example , zadnik et al derived a novel cell sub - line , rbc-3 , from human mda - mb-231 breast cancer metastatic spinal lesions in a rat.27 parent mda - mb-231 human breast adenocarcinoma cells were introduced by intracardiac injection into rats to induce metastatic spinal lesions that caused neurologic deterioration and were confirmed on imaging . affected animals were then euthanized , tumor tissue was excised , and tumor cells were harvested through a process of tissue digestion , cell filtration , centrifugation , and incubation . the selected cell sub - line ( rbc-3 ) was observed to have a growth pattern consistent with that of the parent line , and following intracardiac injection into a host animal , resulted in a statistically significant increase in tumor burden in comparison to the parent mda - mb-231 breast cancer cells.27 similar findings were observed in a mouse model of human breast cancer metastasis , whereby the selected cell sub - line ( b02 ) injected into the tail vein demonstrated a propensity to form rapidly growing osteolytic hindlimb bone metastases.5 these cancer cell lines may potentially be investigated in vitro to identify molecular events important in cellular proliferation , survival , paracrine , and autocrine factors involved in angiogenesis and bone breakdown as well as response to treatment . real - time imaging is useful for the detection , quantification , staging , and longitudinal monitoring of established tumors and response to treatment in in vivo cancer models.10,27 however , accurate delineation of tumor shape , size , and relationship to surrounding tissues remains a challenge due to the relatively small size of the subject matter . plain radiographs are cheap , easy to use , and readily available , but can only detect large osteolytic lesions within bone.16 nuclear medicine bone scans are sensitive for detecting osteoblastic bone metastases , but may miss small lesions less than one centimeter in diameter and lack specificity due to tracer accumulation in any area of increased bone turnover.33 optical imaging is a popular imaging modality currently used in in vivo cancer models and is based on the detection of photon emissions from within living tissues . prior to inoculation , cancer cells are transfected with genes encoding fluorescent or bioluminescent reporter proteins ; however , these differ in generating light emitting photons . fluorescent sources emit light at a particular wavelength in response to an external excitation light source , whereas bioluminescent sources produce light as a result of a chemical reaction between a systemically distributed substrate , such as injection of luciferin , and the enzyme activity of a reporter protein encoded into cancer cells , such as the firefly luciferase protein.10,34 in a mouse model of breast cancer skeletal metastases following intracardiac injection of luciferase transfected mda-231-b cells , bioluminescence imaging detected bone metastases of approximately 0.5 mm in volume.22 real - time identification of tumors at their early stages of growth potentially enables monitoring of disease progression and investigation of efficacy of therapeutic interventions . computed tomography ( ct ) and mri provide a higher degree of spatial resolution and anatomical definition compared with plain radiographs and are the gold standard for anatomical imaging of bone and soft tissue cancers in human patients . micro - ct provides quantitative analysis of bone volume , density , and surface area , and has been utilized in vivo for longitudinal analysis of tumors in animal models of skeletal metastases and to monitor the effects of potential therapies.4,29,31 live animal micro - ct was capable of performing reproducible quantitative analyses of bone volume in an anti - resorptive drug treatment mouse model of breast cancer metastasis to bone.31 dedicated small animal mri scanners use higher strength magnetic - field gradients and higher - sensitivity radiofrequency coils in order to approach the sensitivity and resolution required for imaging small animals.33 in a mouse model of malignant melanoma , the smallest micro metastasis detectable by animal mri measured 0.3 mm.6 further advances in imaging technology may enable the combination of bioluminescence or fluorescence with ct or mri to create a three - dimensional , quantitative , cancer - specific image . potential advantages and disadvantages of the current imaging modalities used for analysis of animal models of skeletal metastasis are summarized in table 2 . histopathologic assessment may be used in analysis of skeletal metastases models to complement the clinical and radiological imaging observations and to better understand the pathogenesis of metastasis by more specifically defining the location and extent of tumor growth , morphologic changes of bone and alterations in gene and protein expression.12,2830 histopathologic correlation can be utilized to confirm similarities between animal and human cell lines with regards to cytologic appearance of tumor cells and progression of tumor growth.9,20,23 furthermore , the mechanisms by which cancer metastases cause clinical symptoms in the experimental animals may be determined through histologic correlation . vertebral histologic cross - sections in a rat model of intraosseous spinal metastases showed aggressive infiltration and marked osteolytic activity of the tumor cells invading through the vertebral body and compressing the spinal cord , consistent with the clinically observed progressive neurological dysfunction.36 in a rabbit model of paraplegia caused by spinal tumors , histologic analysis of spine tumor cross - sections from animals with complete paralysis showed a marked reduction in both grey matter and blood vessels to the spinal cord , suggesting that paraplegia may be caused by both direct compressive and ischemic vascular effects to the spinal cord.16 immunohistochemistry and immunotyping through methods such as enzyme - linked immunosorbent assay ( elisa ) , flow cytometry and polymerase chain reaction may be used to identify markers that contribute to the metastatic potential of tumor cells.14,30,35 in the development of a mouse model of renal cell carcinoma bone metastases , elisa analysis of the selected cell subline with increased bone metastatic potential demonstrated greater production of chemokines and growth factors such as vascular endothelial growth factor ( vegf ) involved in tumor progression and osteoclast activation than the parent cell line.14 further detailed molecular analysis of tumor sections at the metastatic site will provide improved understanding of the temporospatial pattern and pathological mechanisms behind cancer spread to bone and its subsequent growth and invasion into surrounding soft tissues . animal models are essential for investigating the important molecular mechanisms involved in the metastatic spread of cancer to bone and the testing of promising novel therapies prior to clinical application in human patients.10,37 the bone marrow microenvironment , incorporating local tissue and tumor - expressed factors , is known to be important in the initial establishment and proliferation of cancer cells in bone.10,15 factors involved in physiological and pathological bone turnover and angiogenesis such as parathyroid hormone - related protein ( pthrp ) , receptor activator of nfkb ligand ( rankl ) , vegf , tumor necrosis factor- , transforming growth factor , prostaglandins , and interleukins , have been shown to be expressed in many primary and secondary cancers and have been extensively investigated in animal models of skeletal metastases.11,14,15 lelekakis et al found high expression of pthrp in both primary tumors and bone lesions in a murine breast carcinoma model.15 bone metastases induced by parental mda-231 human breast cancer cells in a xenograft mouse model were shown to highly express bone resorbing and pro - angiogenic factors such as pthrp , macrophage colony stimulating factor , and vegf , highlighting the importance of these factors in the bone metastatic cascade.22 consequently , the angiogenesis and osteoclastic bone - degradation pathways are effective targets for current and emerging therapies for skeletal metastases . an important pro - osteoclastogenic factor required for the development and function of bone - resorbing osteoclasts is rankl . blocking rankl has been shown to inhibit breast and prostate cancer - induced osteoclastogenesis and tumor development , growth and progression in bone.14 bisphosphonates have also been shown to potently inhibit osteoclast - mediated bone resorption and can reduce skeletal tumor burden and inhibit the formation of bone metastases in vivo , as well as demonstrating clinical application in the treatment of patients with osteolytic bone metastases.31,34,38,39 photodynamic therapy , which causes oxygen toxicity and tumor cell necrosis by light activation of an administered photosensitizer accumulated in tumor tissue , has been shown to inhibit tumor growth in several rat models of human breast cancer vertebral metastases , with additional improvement in the structural integrity of vertebral bone when used in conjunction with bisphosphonates.3840 in a metastatic mouse breast cancer model , takahashi et al demonstrated that expression of the chemokine ccl2 negatively regulated bone metastasis and that ccl2 may act as a negative upstream regulator of intracellular adhesion molecule-1 expression , which is important for determining the capacity for growth , invasion , and metastasis.35 overexpression of the av3 integrin , a transmembrane protein that integrates intra- and extra - cellular activities and induces tumor cell migration , proliferation and differentiation has been observed in several experimental models of bone metastasis.30,34 treatment with cilengitide , a small molecule inhibitor of av3 and av5 integrins , resulted in pronounced anti - resorptive and anti - tumor effects in a rat model of breast cancer bone metastasis.30 , further development and characterization of animal models of skeletal metastasis are necessary to provide suitable platforms on which to identify and test novel therapies . metastatic spread of cancer to the skeleton has the potential to cause severe morbidity , deterioration in function and mobility , and impaired quality of life . to date , there are many established in vivo animal models of skeletal metastasis , varying in host animal , type of cancer investigated , method of tumor inoculation and metastatic potential . further characterization of these models and development of new , novel animal models focusing on specific cancer types and metastatic sites are essential for better understanding the mechanisms behind why and how particular cancers metastasize to , establish within , and invade bone . ultimately , the identification of key targets for therapeutic intervention , development of novel therapies , and testing of these agents in clinically relevant animal models may be translated into improved treatment , quality of life , and survival of patients with metastatic cancer .

the bony skeleton is one of the most common sites of metastatic spread of cancer and is a significant source of morbidity in cancer patients , causing pain and pathologic fracture , impaired ambulatory ability , and poorer quality of life . animal cancer models of skeletal metastases are essential for better understanding of the molecular pathways behind metastatic spread and local growth and invasion of bone , to enable analysis of host - tumor cell interactions , identify barriers to the metastatic process , and to provide platforms to develop and test novel therapies prior to clinical application in human patients . thus , the ideal model should be clinically relevant , reproducible and representative of the human condition . this review summarizes the current in vivo animal models used in the study of cancer metastases of the skeleton .

Introduction Host Animal for Cancer Metastasis Models Inoculation of Cancer Cells Cancer Cell Lines Analysis of Animal Cancer Metastasis Models Clinical Application of Animal Cancer Metastasis Models Conclusion

along with the lung and liver , the bony skeleton is one of the most common sites for metastases , with most cases occurring in the spine.13 skeletal metastases frequently originate from carcinoma of the breast , prostate , and lung , and if they continue to grow and destroy bone result in pain and pathologic fracture , impaired ambulatory ability and poorer quality of life.27 the process of cancer metastases following tumor growth at the primary site of origin involves intravasation and survival in the bloodstream , arrest , extravasation and finally establishment by invasion and angiogenesis at a distant site.2,3,8 the ability of metastatic cancer to grow within and invade bone is largely mediated by tumor - induced growth factors and cytokines that result in imbalance between the bone - forming osteoblasts and bone - degrading osteoclasts of bone marrow that normally regulate bone turnover.2,3,9 animal models of cancer metastases are essential in order to better understand the molecular pathways behind metastatic spread and local growth and invasion at distant sites , enable analysis of host and cancer cell interactions , identify barriers to the metastatic process , and to provide potential platforms to develop and test novel therapeutic agents . the ideal model should be clinically relevant , reproducible and representative of the human condition . important considerations include the host animal , method of inoculation of tumor cells , type of cancer for investigation , analysis of the developing tumors and clinical application of the model . this paper reviews these considerations , with a specific focus on in vivo animal models of cancer metastases to the bony skeleton . considerations for the choice of host animal to be used for studying cancer and metastasis include animal availability , cost , handling , desired cancer type for investigation , technique of introduction of cancer cells , and whether surgical procedures are required for tumor implantation or treatment , as well as how the resulting tumors and tissues are to be imaged and analyzed . established animal models used for studying skeletal metastases are summarized in table 1 . to date , the rodent has been the most popular host animal used in the study of bone metastases.8 rodents have a high degree of gene sequence homology with humans , similar anatomical organs , are easy to handle and maintain , readily available and relatively cheap , and can be manipulated for investigation of specific cancer pathways by generating knockout , transgenic , or over - expressing strains.11,12 immune - deficient animals such as balb / c athymic nude mice and severe combined immunodeficiency ( scid ) mice are commonly used in xenograft models as they are unable to immunologically reject transplanted tissue or cells , thus enabling the study of human cancer cell lines . as scid mice are more severely immunocompromised than nude mice , it has been suggested that some cancer cell lines may grow more rapidly or produce a higher incidence of metastases in scid compared with nude mice.13 strube et al described a mouse model of human renal cell carcinoma metastasizing to bone following intracardiac inoculation of human 786-o / luciferase cells into nude mice , resulting in aggressive osteolytic bone lesions involving the hindlimbs , forelimbs , pelvis , and spine.14 similarly , garcia et al injected a bone metastasizing - only subclone ( b02 ) from the mda - mb-231 human breast cancer cell line into the tail vein of nude mice , resulting in osteolytic bone metastases of the hindlimb.5 one of the disadvantages of using immunodeficient animals in models of cancer metastases is that they preclude investigation into the important role of the immune system in combating cancer.11 this may be overcome by the use of syngeneic models , in which cancer cell lines of the same species as the host are introduced into immunocompetent animals . lelelakis et al described a mouse model of breast cancer metastases to bone using a clonal tumor cell line from a spontaneously arising murine mammary cancer , in which metastases to the femur and spine followed intracardiac and mammary pad inoculation of the cancer cells.15 another disadvantage of rodent cancer models includes the small size of rodent bone , potentially making direct implantation of cancer cells , as well as investigation of therapeutic modalities such as local drug delivery devices and invasive image - guided - therapies , more technically challenging.12,16,17 the use of rabbits may be less technically demanding for surgical procedures and may enable easier analysis and preclinical testing of skeletal metastases . however , experimental immunodeficient rabbits are currently not available ; thus , species - specific cancer cell lines are required in rabbit syngeneic cancer models and these are extremely limited in availability.11,16,17 despite this , vx2 carcinoma cells derived from a virus induced papilloma of the rabbit have been used in two rabbit models of spinal tumors to successfully replicate the clinical , radiological , and histopathologic characteristics of the human condition.16,17 orthotopic implantation of the vx2 carcinoma cells into the third lumbar vertebra or the lower thoracic vertebra of healthy rabbits after surgical exposure led to lower limb paralysis in most rabbits by four weeks post - implantation . although the cat has not yet been established as a useful syngeneic or xenograft animal model for the study of skeletal metastasis , spontaneous metastases to the digits of the bony skeleton and in soft tissues of the proximal limbs in cats have been observed secondary to primary pulmonary adenocarcinoma.18,19 in a larger syngeneic orthotopic animal model , a dog model of prostate cancer metastasis , anidjar et al demonstrated bone metastases following direct inoculation of dpc-1 poorly differentiated canine prostate adenocarcinoma cells into the prostate gland.20 histopathology of the mixed osteoblastic and osteolytic pelvic bone lesions showed similarities to human prostate cancer skeletal metastases.20 a potential advantage of the larger canine model over rabbits or rodents is that dogs may be more amenable to diagnostic and surgical procedures with equipment used in human clinical practice ; however , the cost and restricted availability of the animals and species - specific cancer cell lines remain limitations . as described above , since the spontaneous development of metastatic cancer in animals is rare and unpredictable , animal models of skeletal metastases typically require physical introduction of cancer cells . models involving systemic dissemination of tumor cells permit investigation of the key elements of the metastatic cascade from survival within the bloodstream , to selection of a distal site , and eventually to extravasation , establishment and growth of distant metastases , thus allowing analysis of the molecular mechanisms involved in growth and proliferation , migration , invasion and angiogenesis of metastatic cancer cells.11,15 intracardiac injection into the left ventricle results in systemic arterial circulatory dissemination of tumor cells , with the potential of generating widespread metastases . arguello et al described a reproducible murine model of skeletal vertebral and long bone metastasis following intracardiac injection of b16 ( sub - line g3.26 ) murine melanoma cells.21 however , the sites of metastatic deposition and number and size of the metastases were unpredictable , extremely variable , and dependent on the amount of cancer cells administered . tail vein inoculation similarly results in hematogenous dissemination of cancer cells , although cells may be filtered and cleared by the pulmonary system during the first passage through circulation.22 passineau et al described tail vein injection of a20 murine lymphoma cells to generate a syngeneic mouse model to mimic disseminated human b cell lymphoma , with bony deposits to the femur , pelvis and vertebral column , eventually leading to bone destruction and nerve compression.23 biesalski et al described intra - arterial injection of walker 256 mammary carcinoma cells directly into the circulation of the hindlimbs in rats via the superficial caudal epigastric artery , a branch of the femoral artery , resulting in metastases to the distal femur or proximal tibia / fibula in 9 of 11 animals at 3 weeks post - inoculation.24 limitations of systemic administration of tumor cells include variability and unpredictability in site , size , number , and time to development of metastasis.10,11 this may also lead to excessive tumor burden and morbidity in the experimental animal , therefore precluding the ability to investigate potential therapeutic interventions . direct inoculation of tumor cells into primary sites such as the mammary fat pad also has the potential to create a complete model of the metastatic process , since the tumor must first establish in the primary site before seeding to distant sites.15 schubert et al described a mouse model of breast cancer metastasis to the femur and lung after mda - mb-435 human breast cancer cells were injected into the mammary fat pad of nude mice.25 the ability of gonadotropin - releasing hormone ( gnrh ) analogs to significantly reduce formation and growth of breast cancer metastases in this model suggested the multi - focal role of gnrh throughout the metastatic process of breast cancer , including epithelial - mesenchymal transition , migration , invasion , and biology of circulating cells . direct orthotopic inoculation into bone has the potential to produce reproducible , efficient , and site - specific models of cancer and metastasis since a known quantity of cancer cells can be directly administered into the target bone of choice . the progressive neurological decline in the animals , from gait asymmetry and unilateral hindlimb weakness , to complete unilateral hindlimb paralysis and finally to complete bilateral hindlimb paralysis , closely resembles the natural history of untreated metastatic epidural spinal cord compression and potentially enables closer analysis of the temporospatial pattern of cancer growth within the spine and the molecular mechanisms behind this devastating condition . since the biological behavior and response to therapy of bone metastases is largely dependent on the primary cancer type , there is a need to develop investigative models of varying cell lines . for example , prostate cancer bone metastases in humans are generally more osteoblastic or sclerotic in nature , while renal cell carcinoma bone metastases are often more lytic and vascular compared with other cancer types.2,3,14 indeed , a sub - line of the 786-o human renal cell carcinoma cell line with increased bone metastatic potential showed higher expression of the pro - angiogenic factors vegf and basic fibroblast growth factor ( bfgf ) compared with the parental cell line.14 immortalized cancer cell lines that are generated through isolation of tumor cells from these spontaneously occurring primary or secondary cancer types in humans or animals are ideal for use in in vivo cancer models as they have already demonstrated ability to form , grow , invade , and spread within the primary organ or metastatic site of choice . human cancer cells lines with a propensity for localization to bone and that are well - established for use in animal cancer metastasis models of metastases include the mda - mb-231 human breast cancer cell line isolated from the pleural effusion of a patient following breast cancer metastasis to the lungs,5,22,2731 and pc-3 human prostate cancer cells derived from a bone metastasis.8,9,32 the use of cancer cell lines of human origin ensures that molecular and histologic analysis is as comparable to the human condition as possible ; however , one important limitation is the requirement for use of an immunocompromised animal , which precludes investigation of the host immune response to the development of cancer and metastases.11 the b16 murine melanoma cell line , derived from a melanoma from the skin of a c57bl/6 strain mouse,6,21,33 and the vx2 rabbit carcinoma cell line , originated from a carcinoma induced by the shope cottontail rabbit papilloma virus,16,17,26 are common animal cell lines used in models of skeletal metastasis models that exhibit similar molecular characteristics to human cancer cells . real - time imaging is useful for the detection , quantification , staging , and longitudinal monitoring of established tumors and response to treatment in in vivo cancer models.10,27 however , accurate delineation of tumor shape , size , and relationship to surrounding tissues remains a challenge due to the relatively small size of the subject matter . plain radiographs are cheap , easy to use , and readily available , but can only detect large osteolytic lesions within bone.16 nuclear medicine bone scans are sensitive for detecting osteoblastic bone metastases , but may miss small lesions less than one centimeter in diameter and lack specificity due to tracer accumulation in any area of increased bone turnover.33 optical imaging is a popular imaging modality currently used in in vivo cancer models and is based on the detection of photon emissions from within living tissues . computed tomography ( ct ) and mri provide a higher degree of spatial resolution and anatomical definition compared with plain radiographs and are the gold standard for anatomical imaging of bone and soft tissue cancers in human patients . micro - ct provides quantitative analysis of bone volume , density , and surface area , and has been utilized in vivo for longitudinal analysis of tumors in animal models of skeletal metastases and to monitor the effects of potential therapies.4,29,31 live animal micro - ct was capable of performing reproducible quantitative analyses of bone volume in an anti - resorptive drug treatment mouse model of breast cancer metastasis to bone.31 dedicated small animal mri scanners use higher strength magnetic - field gradients and higher - sensitivity radiofrequency coils in order to approach the sensitivity and resolution required for imaging small animals.33 in a mouse model of malignant melanoma , the smallest micro metastasis detectable by animal mri measured 0.3 mm.6 further advances in imaging technology may enable the combination of bioluminescence or fluorescence with ct or mri to create a three - dimensional , quantitative , cancer - specific image . potential advantages and disadvantages of the current imaging modalities used for analysis of animal models of skeletal metastasis are summarized in table 2 . histopathologic assessment may be used in analysis of skeletal metastases models to complement the clinical and radiological imaging observations and to better understand the pathogenesis of metastasis by more specifically defining the location and extent of tumor growth , morphologic changes of bone and alterations in gene and protein expression.12,2830 histopathologic correlation can be utilized to confirm similarities between animal and human cell lines with regards to cytologic appearance of tumor cells and progression of tumor growth.9,20,23 furthermore , the mechanisms by which cancer metastases cause clinical symptoms in the experimental animals may be determined through histologic correlation . vertebral histologic cross - sections in a rat model of intraosseous spinal metastases showed aggressive infiltration and marked osteolytic activity of the tumor cells invading through the vertebral body and compressing the spinal cord , consistent with the clinically observed progressive neurological dysfunction.36 in a rabbit model of paraplegia caused by spinal tumors , histologic analysis of spine tumor cross - sections from animals with complete paralysis showed a marked reduction in both grey matter and blood vessels to the spinal cord , suggesting that paraplegia may be caused by both direct compressive and ischemic vascular effects to the spinal cord.16 immunohistochemistry and immunotyping through methods such as enzyme - linked immunosorbent assay ( elisa ) , flow cytometry and polymerase chain reaction may be used to identify markers that contribute to the metastatic potential of tumor cells.14,30,35 in the development of a mouse model of renal cell carcinoma bone metastases , elisa analysis of the selected cell subline with increased bone metastatic potential demonstrated greater production of chemokines and growth factors such as vascular endothelial growth factor ( vegf ) involved in tumor progression and osteoclast activation than the parent cell line.14 further detailed molecular analysis of tumor sections at the metastatic site will provide improved understanding of the temporospatial pattern and pathological mechanisms behind cancer spread to bone and its subsequent growth and invasion into surrounding soft tissues . animal models are essential for investigating the important molecular mechanisms involved in the metastatic spread of cancer to bone and the testing of promising novel therapies prior to clinical application in human patients.10,37 the bone marrow microenvironment , incorporating local tissue and tumor - expressed factors , is known to be important in the initial establishment and proliferation of cancer cells in bone.10,15 factors involved in physiological and pathological bone turnover and angiogenesis such as parathyroid hormone - related protein ( pthrp ) , receptor activator of nfkb ligand ( rankl ) , vegf , tumor necrosis factor- , transforming growth factor , prostaglandins , and interleukins , have been shown to be expressed in many primary and secondary cancers and have been extensively investigated in animal models of skeletal metastases.11,14,15 lelekakis et al found high expression of pthrp in both primary tumors and bone lesions in a murine breast carcinoma model.15 bone metastases induced by parental mda-231 human breast cancer cells in a xenograft mouse model were shown to highly express bone resorbing and pro - angiogenic factors such as pthrp , macrophage colony stimulating factor , and vegf , highlighting the importance of these factors in the bone metastatic cascade.22 consequently , the angiogenesis and osteoclastic bone - degradation pathways are effective targets for current and emerging therapies for skeletal metastases . blocking rankl has been shown to inhibit breast and prostate cancer - induced osteoclastogenesis and tumor development , growth and progression in bone.14 bisphosphonates have also been shown to potently inhibit osteoclast - mediated bone resorption and can reduce skeletal tumor burden and inhibit the formation of bone metastases in vivo , as well as demonstrating clinical application in the treatment of patients with osteolytic bone metastases.31,34,38,39 photodynamic therapy , which causes oxygen toxicity and tumor cell necrosis by light activation of an administered photosensitizer accumulated in tumor tissue , has been shown to inhibit tumor growth in several rat models of human breast cancer vertebral metastases , with additional improvement in the structural integrity of vertebral bone when used in conjunction with bisphosphonates.3840 in a metastatic mouse breast cancer model , takahashi et al demonstrated that expression of the chemokine ccl2 negatively regulated bone metastasis and that ccl2 may act as a negative upstream regulator of intracellular adhesion molecule-1 expression , which is important for determining the capacity for growth , invasion , and metastasis.35 overexpression of the av3 integrin , a transmembrane protein that integrates intra- and extra - cellular activities and induces tumor cell migration , proliferation and differentiation has been observed in several experimental models of bone metastasis.30,34 treatment with cilengitide , a small molecule inhibitor of av3 and av5 integrins , resulted in pronounced anti - resorptive and anti - tumor effects in a rat model of breast cancer bone metastasis.30 , further development and characterization of animal models of skeletal metastasis are necessary to provide suitable platforms on which to identify and test novel therapies . metastatic spread of cancer to the skeleton has the potential to cause severe morbidity , deterioration in function and mobility , and impaired quality of life . to date , there are many established in vivo animal models of skeletal metastasis , varying in host animal , type of cancer investigated , method of tumor inoculation and metastatic potential . further characterization of these models and development of new , novel animal models focusing on specific cancer types and metastatic sites are essential for better understanding the mechanisms behind why and how particular cancers metastasize to , establish within , and invade bone . ultimately , the identification of key targets for therapeutic intervention , development of novel therapies , and testing of these agents in clinically relevant animal models may be translated into improved treatment , quality of life , and survival of patients with metastatic cancer .

along with the lung and liver , the bony skeleton is one of the most common sites for metastases , with most cases occurring in the spine.13 skeletal metastases frequently originate from carcinoma of the breast , prostate , and lung , and if they continue to grow and destroy bone result in pain and pathologic fracture , impaired ambulatory ability and poorer quality of life.27 the process of cancer metastases following tumor growth at the primary site of origin involves intravasation and survival in the bloodstream , arrest , extravasation and finally establishment by invasion and angiogenesis at a distant site.2,3,8 the ability of metastatic cancer to grow within and invade bone is largely mediated by tumor - induced growth factors and cytokines that result in imbalance between the bone - forming osteoblasts and bone - degrading osteoclasts of bone marrow that normally regulate bone turnover.2,3,9 animal models of cancer metastases are essential in order to better understand the molecular pathways behind metastatic spread and local growth and invasion at distant sites , enable analysis of host and cancer cell interactions , identify barriers to the metastatic process , and to provide potential platforms to develop and test novel therapeutic agents . although spontaneous models resemble what occurs when humans develop cancer , the spontaneous development of primary and metastatic cancer in animals is extremely rare.10 similarly , the metastasis rate of genetically - induced tumors , either by inducing expression of oncogenes or deletion of tumor suppressor genes , is generally very low and too variable and unpredictable in terms of spreading to bone to provide useful skeletal metastasis models . considerations for the choice of host animal to be used for studying cancer and metastasis include animal availability , cost , handling , desired cancer type for investigation , technique of introduction of cancer cells , and whether surgical procedures are required for tumor implantation or treatment , as well as how the resulting tumors and tissues are to be imaged and analyzed . to date , the rodent has been the most popular host animal used in the study of bone metastases.8 rodents have a high degree of gene sequence homology with humans , similar anatomical organs , are easy to handle and maintain , readily available and relatively cheap , and can be manipulated for investigation of specific cancer pathways by generating knockout , transgenic , or over - expressing strains.11,12 immune - deficient animals such as balb / c athymic nude mice and severe combined immunodeficiency ( scid ) mice are commonly used in xenograft models as they are unable to immunologically reject transplanted tissue or cells , thus enabling the study of human cancer cell lines . as scid mice are more severely immunocompromised than nude mice , it has been suggested that some cancer cell lines may grow more rapidly or produce a higher incidence of metastases in scid compared with nude mice.13 strube et al described a mouse model of human renal cell carcinoma metastasizing to bone following intracardiac inoculation of human 786-o / luciferase cells into nude mice , resulting in aggressive osteolytic bone lesions involving the hindlimbs , forelimbs , pelvis , and spine.14 similarly , garcia et al injected a bone metastasizing - only subclone ( b02 ) from the mda - mb-231 human breast cancer cell line into the tail vein of nude mice , resulting in osteolytic bone metastases of the hindlimb.5 one of the disadvantages of using immunodeficient animals in models of cancer metastases is that they preclude investigation into the important role of the immune system in combating cancer.11 this may be overcome by the use of syngeneic models , in which cancer cell lines of the same species as the host are introduced into immunocompetent animals . lelelakis et al described a mouse model of breast cancer metastases to bone using a clonal tumor cell line from a spontaneously arising murine mammary cancer , in which metastases to the femur and spine followed intracardiac and mammary pad inoculation of the cancer cells.15 another disadvantage of rodent cancer models includes the small size of rodent bone , potentially making direct implantation of cancer cells , as well as investigation of therapeutic modalities such as local drug delivery devices and invasive image - guided - therapies , more technically challenging.12,16,17 the use of rabbits may be less technically demanding for surgical procedures and may enable easier analysis and preclinical testing of skeletal metastases . however , experimental immunodeficient rabbits are currently not available ; thus , species - specific cancer cell lines are required in rabbit syngeneic cancer models and these are extremely limited in availability.11,16,17 despite this , vx2 carcinoma cells derived from a virus induced papilloma of the rabbit have been used in two rabbit models of spinal tumors to successfully replicate the clinical , radiological , and histopathologic characteristics of the human condition.16,17 orthotopic implantation of the vx2 carcinoma cells into the third lumbar vertebra or the lower thoracic vertebra of healthy rabbits after surgical exposure led to lower limb paralysis in most rabbits by four weeks post - implantation . although the cat has not yet been established as a useful syngeneic or xenograft animal model for the study of skeletal metastasis , spontaneous metastases to the digits of the bony skeleton and in soft tissues of the proximal limbs in cats have been observed secondary to primary pulmonary adenocarcinoma.18,19 in a larger syngeneic orthotopic animal model , a dog model of prostate cancer metastasis , anidjar et al demonstrated bone metastases following direct inoculation of dpc-1 poorly differentiated canine prostate adenocarcinoma cells into the prostate gland.20 histopathology of the mixed osteoblastic and osteolytic pelvic bone lesions showed similarities to human prostate cancer skeletal metastases.20 a potential advantage of the larger canine model over rabbits or rodents is that dogs may be more amenable to diagnostic and surgical procedures with equipment used in human clinical practice ; however , the cost and restricted availability of the animals and species - specific cancer cell lines remain limitations . models involving systemic dissemination of tumor cells permit investigation of the key elements of the metastatic cascade from survival within the bloodstream , to selection of a distal site , and eventually to extravasation , establishment and growth of distant metastases , thus allowing analysis of the molecular mechanisms involved in growth and proliferation , migration , invasion and angiogenesis of metastatic cancer cells.11,15 intracardiac injection into the left ventricle results in systemic arterial circulatory dissemination of tumor cells , with the potential of generating widespread metastases . tail vein inoculation similarly results in hematogenous dissemination of cancer cells , although cells may be filtered and cleared by the pulmonary system during the first passage through circulation.22 passineau et al described tail vein injection of a20 murine lymphoma cells to generate a syngeneic mouse model to mimic disseminated human b cell lymphoma , with bony deposits to the femur , pelvis and vertebral column , eventually leading to bone destruction and nerve compression.23 biesalski et al described intra - arterial injection of walker 256 mammary carcinoma cells directly into the circulation of the hindlimbs in rats via the superficial caudal epigastric artery , a branch of the femoral artery , resulting in metastases to the distal femur or proximal tibia / fibula in 9 of 11 animals at 3 weeks post - inoculation.24 limitations of systemic administration of tumor cells include variability and unpredictability in site , size , number , and time to development of metastasis.10,11 this may also lead to excessive tumor burden and morbidity in the experimental animal , therefore precluding the ability to investigate potential therapeutic interventions . direct inoculation of tumor cells into primary sites such as the mammary fat pad also has the potential to create a complete model of the metastatic process , since the tumor must first establish in the primary site before seeding to distant sites.15 schubert et al described a mouse model of breast cancer metastasis to the femur and lung after mda - mb-435 human breast cancer cells were injected into the mammary fat pad of nude mice.25 the ability of gonadotropin - releasing hormone ( gnrh ) analogs to significantly reduce formation and growth of breast cancer metastases in this model suggested the multi - focal role of gnrh throughout the metastatic process of breast cancer , including epithelial - mesenchymal transition , migration , invasion , and biology of circulating cells . mann et al described a mouse model of tumor - induced osteolysis following orthotopic injection of f10 human breast cancer cells into the distal femoral metaphysis to assess the ability of non - invasive imaging and computational techniques for predicting the strength of bones with osteolytic lesions.4 choi et al used direct orthotopic inoculation of vx2 carcinoma tissue fragments into the tibial shaft of rabbits to create a model for magnetic resonance imaging ( mri ) and histopathologic evaluation of the evolution of metastatic bone tumor.26 orthotopic inoculation may be a relatively straightforward procedure if prominent and subcutaneous bones are selected , such as the distal femur or proximal tibia , which are also common sites for primary and secondary bone cancers . in our own laboratory , we recently established a novel in vivo model of spinal cancer that causes a reproducible , evolving paraplegia following orthotopic inoculation of mda-231 human breast cancer cells or pc-3 human prostate cancer cells into the vertebral body at the thoracolumbar junction of balb - c athymic nude mice ( fig . the progressive neurological decline in the animals , from gait asymmetry and unilateral hindlimb weakness , to complete unilateral hindlimb paralysis and finally to complete bilateral hindlimb paralysis , closely resembles the natural history of untreated metastatic epidural spinal cord compression and potentially enables closer analysis of the temporospatial pattern of cancer growth within the spine and the molecular mechanisms behind this devastating condition . for example , prostate cancer bone metastases in humans are generally more osteoblastic or sclerotic in nature , while renal cell carcinoma bone metastases are often more lytic and vascular compared with other cancer types.2,3,14 indeed , a sub - line of the 786-o human renal cell carcinoma cell line with increased bone metastatic potential showed higher expression of the pro - angiogenic factors vegf and basic fibroblast growth factor ( bfgf ) compared with the parental cell line.14 immortalized cancer cell lines that are generated through isolation of tumor cells from these spontaneously occurring primary or secondary cancer types in humans or animals are ideal for use in in vivo cancer models as they have already demonstrated ability to form , grow , invade , and spread within the primary organ or metastatic site of choice . human cancer cells lines with a propensity for localization to bone and that are well - established for use in animal cancer metastasis models of metastases include the mda - mb-231 human breast cancer cell line isolated from the pleural effusion of a patient following breast cancer metastasis to the lungs,5,22,2731 and pc-3 human prostate cancer cells derived from a bone metastasis.8,9,32 the use of cancer cell lines of human origin ensures that molecular and histologic analysis is as comparable to the human condition as possible ; however , one important limitation is the requirement for use of an immunocompromised animal , which precludes investigation of the host immune response to the development of cancer and metastases.11 the b16 murine melanoma cell line , derived from a melanoma from the skin of a c57bl/6 strain mouse,6,21,33 and the vx2 rabbit carcinoma cell line , originated from a carcinoma induced by the shope cottontail rabbit papilloma virus,16,17,26 are common animal cell lines used in models of skeletal metastasis models that exhibit similar molecular characteristics to human cancer cells . for example , zadnik et al derived a novel cell sub - line , rbc-3 , from human mda - mb-231 breast cancer metastatic spinal lesions in a rat.27 parent mda - mb-231 human breast adenocarcinoma cells were introduced by intracardiac injection into rats to induce metastatic spinal lesions that caused neurologic deterioration and were confirmed on imaging . the selected cell sub - line ( rbc-3 ) was observed to have a growth pattern consistent with that of the parent line , and following intracardiac injection into a host animal , resulted in a statistically significant increase in tumor burden in comparison to the parent mda - mb-231 breast cancer cells.27 similar findings were observed in a mouse model of human breast cancer metastasis , whereby the selected cell sub - line ( b02 ) injected into the tail vein demonstrated a propensity to form rapidly growing osteolytic hindlimb bone metastases.5 these cancer cell lines may potentially be investigated in vitro to identify molecular events important in cellular proliferation , survival , paracrine , and autocrine factors involved in angiogenesis and bone breakdown as well as response to treatment . fluorescent sources emit light at a particular wavelength in response to an external excitation light source , whereas bioluminescent sources produce light as a result of a chemical reaction between a systemically distributed substrate , such as injection of luciferin , and the enzyme activity of a reporter protein encoded into cancer cells , such as the firefly luciferase protein.10,34 in a mouse model of breast cancer skeletal metastases following intracardiac injection of luciferase transfected mda-231-b cells , bioluminescence imaging detected bone metastases of approximately 0.5 mm in volume.22 real - time identification of tumors at their early stages of growth potentially enables monitoring of disease progression and investigation of efficacy of therapeutic interventions . micro - ct provides quantitative analysis of bone volume , density , and surface area , and has been utilized in vivo for longitudinal analysis of tumors in animal models of skeletal metastases and to monitor the effects of potential therapies.4,29,31 live animal micro - ct was capable of performing reproducible quantitative analyses of bone volume in an anti - resorptive drug treatment mouse model of breast cancer metastasis to bone.31 dedicated small animal mri scanners use higher strength magnetic - field gradients and higher - sensitivity radiofrequency coils in order to approach the sensitivity and resolution required for imaging small animals.33 in a mouse model of malignant melanoma , the smallest micro metastasis detectable by animal mri measured 0.3 mm.6 further advances in imaging technology may enable the combination of bioluminescence or fluorescence with ct or mri to create a three - dimensional , quantitative , cancer - specific image . histopathologic assessment may be used in analysis of skeletal metastases models to complement the clinical and radiological imaging observations and to better understand the pathogenesis of metastasis by more specifically defining the location and extent of tumor growth , morphologic changes of bone and alterations in gene and protein expression.12,2830 histopathologic correlation can be utilized to confirm similarities between animal and human cell lines with regards to cytologic appearance of tumor cells and progression of tumor growth.9,20,23 furthermore , the mechanisms by which cancer metastases cause clinical symptoms in the experimental animals may be determined through histologic correlation . vertebral histologic cross - sections in a rat model of intraosseous spinal metastases showed aggressive infiltration and marked osteolytic activity of the tumor cells invading through the vertebral body and compressing the spinal cord , consistent with the clinically observed progressive neurological dysfunction.36 in a rabbit model of paraplegia caused by spinal tumors , histologic analysis of spine tumor cross - sections from animals with complete paralysis showed a marked reduction in both grey matter and blood vessels to the spinal cord , suggesting that paraplegia may be caused by both direct compressive and ischemic vascular effects to the spinal cord.16 immunohistochemistry and immunotyping through methods such as enzyme - linked immunosorbent assay ( elisa ) , flow cytometry and polymerase chain reaction may be used to identify markers that contribute to the metastatic potential of tumor cells.14,30,35 in the development of a mouse model of renal cell carcinoma bone metastases , elisa analysis of the selected cell subline with increased bone metastatic potential demonstrated greater production of chemokines and growth factors such as vascular endothelial growth factor ( vegf ) involved in tumor progression and osteoclast activation than the parent cell line.14 further detailed molecular analysis of tumor sections at the metastatic site will provide improved understanding of the temporospatial pattern and pathological mechanisms behind cancer spread to bone and its subsequent growth and invasion into surrounding soft tissues . animal models are essential for investigating the important molecular mechanisms involved in the metastatic spread of cancer to bone and the testing of promising novel therapies prior to clinical application in human patients.10,37 the bone marrow microenvironment , incorporating local tissue and tumor - expressed factors , is known to be important in the initial establishment and proliferation of cancer cells in bone.10,15 factors involved in physiological and pathological bone turnover and angiogenesis such as parathyroid hormone - related protein ( pthrp ) , receptor activator of nfkb ligand ( rankl ) , vegf , tumor necrosis factor- , transforming growth factor , prostaglandins , and interleukins , have been shown to be expressed in many primary and secondary cancers and have been extensively investigated in animal models of skeletal metastases.11,14,15 lelekakis et al found high expression of pthrp in both primary tumors and bone lesions in a murine breast carcinoma model.15 bone metastases induced by parental mda-231 human breast cancer cells in a xenograft mouse model were shown to highly express bone resorbing and pro - angiogenic factors such as pthrp , macrophage colony stimulating factor , and vegf , highlighting the importance of these factors in the bone metastatic cascade.22 consequently , the angiogenesis and osteoclastic bone - degradation pathways are effective targets for current and emerging therapies for skeletal metastases . blocking rankl has been shown to inhibit breast and prostate cancer - induced osteoclastogenesis and tumor development , growth and progression in bone.14 bisphosphonates have also been shown to potently inhibit osteoclast - mediated bone resorption and can reduce skeletal tumor burden and inhibit the formation of bone metastases in vivo , as well as demonstrating clinical application in the treatment of patients with osteolytic bone metastases.31,34,38,39 photodynamic therapy , which causes oxygen toxicity and tumor cell necrosis by light activation of an administered photosensitizer accumulated in tumor tissue , has been shown to inhibit tumor growth in several rat models of human breast cancer vertebral metastases , with additional improvement in the structural integrity of vertebral bone when used in conjunction with bisphosphonates.3840 in a metastatic mouse breast cancer model , takahashi et al demonstrated that expression of the chemokine ccl2 negatively regulated bone metastasis and that ccl2 may act as a negative upstream regulator of intracellular adhesion molecule-1 expression , which is important for determining the capacity for growth , invasion , and metastasis.35 overexpression of the av3 integrin , a transmembrane protein that integrates intra- and extra - cellular activities and induces tumor cell migration , proliferation and differentiation has been observed in several experimental models of bone metastasis.30,34 treatment with cilengitide , a small molecule inhibitor of av3 and av5 integrins , resulted in pronounced anti - resorptive and anti - tumor effects in a rat model of breast cancer bone metastasis.30 , further development and characterization of animal models of skeletal metastasis are necessary to provide suitable platforms on which to identify and test novel therapies .

neuroblastoma ( nb ) is a neuroendocrine tumor that arises from neural crest elements of the sympathetic nervous system . it is the most common extracranial solid tumor of childhood , with a long - term survival rate of only 15% . nearly all of the 45% of nb patients diagnosed with high - risk tumors present with metastases , including those diagnosed at any age with amplification of the mycn oncogene and those older than 18 months with non - mycn - amplified tumors.2 , 3 , 4 , 5 although patient outcomes have steadily improved over the past 20 years , 3-year event - free survival rate is only 40% for patients who have high - risk , stage 4 metastatic disease . the current standard of care for these patients consists of : ( 1 ) intensive induction chemotherapy , ( 2 ) myeloablative consolidation chemoradiotherapy and autologous hematopoietic stem cell transplantation , and ( 3 ) 13-cis retinoic acid with immunotherapy that targets disialoganglioside ( gd2).6 , 7 , 8 , 9 treatment failures occur at both primary and metastatic sites , and particularly in metastases to the bone and bone marrow , suggesting that minimal residual disease is an important cause of recurrence . current regimens of dose - intensive chemotherapy and irradiation are likely at the limit of both anti - tumor efficacy and patient tolerance , and post - consolidation therapy does not eradicate minimal residual disease ( mrd ) in many patients.6 , 7 , 8 , 9 therefore , there is a critical need for improved , less toxic therapeutic approaches for tumor cyto - reduction ( induction and consolidation phases ) and eradication of mrd ( post - consolidation phase ) to improve clinical outcome in children with this disease . selective activation of the prodrug irinotecan ( cpt-11 ; camptosar ; 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin ) to its 1,000-fold more cytotoxic active metabolite sn-38 ( 7-ethyl-10-hydroxycamptothecin ) , a topoisomerase-1 inhibitor , can be achieved with carboxylesterases ( ces ) . this activation results in increased cytotoxicity and improved antitumor response in human tumor xenograft models of nb . irinotecan is currently being used in front - line treatment for nb and tested in combination with other drugs in a phase i clinical trial for this disease , as well as colon cancer.12 , 13 clinical trials with this agent are also under way in variety of other solid malignancies ( e.g. , sarcomas and non - small - cell lung cancer).14 , 15 , 16 , 17 , 18 neural stem cells ( nscs ) are inherently tumor tropic and selectively localize to solid tumor foci in multiple organs after intravenous administration in several metastatic tumor models , including breast cancer,19 , 20 , 21 ovarian cancer , lung cancer , and nb.24 , 25 , 26 nsc - mediated enzyme and prodrug therapy has been shown to be effective in several tumor models including glioma , medulloblastoma , melanoma brain metastases , and metastatic breast cancer.19 , 27 , 28 , 29 we previously showed proof of concept for increased therapeutic efficacy in mouse models of metastatic nb , using tumor - tropic nscs expressing a modified rabbit carboxylesterase ( rce ) to convert the prodrug irinotecan to sn-38.10 , 19 , 30 we now present data using a well - characterized , clonal human nsc line ( hb1.f3.cd clone 21 ) that has demonstrated clinical safety and proof of concept for brain tumor localized , nsc - expressed enzyme - mediated conversion of a prodrug ( 5-fluocytosine ) to its active chemotherapeutic ( 5-fluorouracil ) ( investigational new drug [ ind ] application 14041 ; nct01172964 ) . we transduced this nsc line with replication - deficient adenovirus designed to allow high - level , transient expression and secretion of a modified human ce1 ( hce1m6 ) . the hce1m6-expression vector was generated from the human liver ce hce1 , specifically to allow for efficient conversion of irinotecan to sn-38 , and has demonstrated functional equivalence to rce , both in vitro and in vivo . this nsc - secreted form of ce accumulates at tumor foci ( because of nsc tropism to tumor sites ) , where it can convert administered irinotecan to sn-38 and can generate a greater therapeutic radius of tumor kill around each nsc ( the bystander effect ) , as compared with irinotecan alone.34 , 35 toward clinical translation , our goal was to maximize therapeutic efficacy by determining the optimal clinically relevant dose and schedule of hce1m6-nscs + irinotecan in a mouse model of metastatic nb . repeat treatment of mice bearing nb with intravenously administered hce1m6-nscs and irinotecan resulted in both a significant decrease in tumor burden ( 1.4-fold , p = 0.0093 ) and increased long - term survival versus treatment with irinotecan alone . these studies support further development of nsc - mediated gene therapy for improved clinical outcome in nb patients . to determine whether hce1m6 expressed by nscs could enhance the cell - killing effects of irinotecan on nb cells , we measured the half - maximal inhibitory drug concentration ( ic50 ) for the drug in the presence and absence of nsc - secreted hce1m6 . the cytotoxicity of irinotecan and sn-38 was determined for a panel of human - derived nb cell lines ( sms - kcnr , chla-255 , chla-136 , and sk - n - as ) ( figure 1 ; table 1 ) . in brief , nb cells were incubated for 4 hr in cell culture medium containing irinotecan ( 0.0011,000 m ) or sn-38 ( 1100 nm ) , or in conditioned media ( cm ) harvested from nscs expressing hce1m6 to which irinotecan ( 1100 m ) was added . all nb cell lines were significantly more sensitive to irinotecan in the presence of hce1m6 as compared with irinotecan alone ( figures 1a1d ) ( table 1 ) , because of conversion of irinotecan to sn-38 , as demonstrated before . for the most sensitive cell line , sms - kcnr , the cytotoxicity of irinotecan ( ic50 = 7.95 m ) was increased up to 900-fold by addition of hce1m6-nsc cm ( ic50 = 0.009 m ) . although the change in sensitivity was not as dramatic in the other cell lines , addition of hce1m6-nsc cm led to an increase of at least 139-fold ( table 1 ) . these data suggest that hce1m6 produced by nscs can effectively convert irinotecan to the active metabolite sn-38 , reaching therapeutic ic50 levels similar to those observed for sn-38 alone . important for clinical translation , only nanomolar levels of sn-38 are needed for significant efficacy ( table 1 ) . before translating use of hce1m6-nscs to a therapeutic approach , it will be necessary to ensure that these cells do not localize to non - tumor - harboring tissues and organs . to assess this , we performed in vivo time - dependent nsc biodistribution or clearance studies . for all experiments , plasma - esterase - deficient mice ( es1/scid ) were used to more closely represent human plasma esterase levels . this allows for assessment of clinically relevant human doses of irinotecan in these mice . because the hce1m6-nscs have a single copy of the v - myc gene per cell , we used qpcr ( taqman qpcr ) to quantify the relative amounts of nscs in the circulation and major organs . one hour after intravenous administration into non - tumor - bearing es1/scid mice , nscs were detected in the lungs , liver , spleen , blood , and brain ( figure 2a ) . however , hce1m6-nscs were undetectable by pcr at 24 hr , 48 hr , and 5 days post - injection ( figure 2a , 1 and 24 hr time points are shown ) . at the 1 hr time point , the relative rank of v - myc - positive cells per 100,000 normal tissue cells was lung > blood > liver > spleen > brain . statistically significant differences were found when numbers of nscs in lung were compared with blood ( p = 0.02 ) , liver ( p = 0.003 ) , spleen ( p = 0.003 ) , and brain ( p = 0.007 ) , because it is likely that the majority of stem cells are initially trapped in the lungs . no detectable v - myc dna was observed 24 hr after nsc injection , indicating essentially total clearance of nscs by this time point . brain , liver , lung , and kidneys harvested at 1 and 24 hr after nsc administration were further analyzed by prussian blue staining and histological examination to detect iron - labeled hce1m6-nscs ( positive control for prussian blue staining of molday iron - labeled hce1m6-nscs was subcutaneous sknas tumor sections with nscs ; data not shown ) . we did not see any cells that were positive for prussian blue staining in any of the sample tissue sections examined ( figure 2b ) , indicating the retention of exogenous hce1m6-nscs in normal tissues is a rare event and , given the assays used , detectable only by qpcr . to confirm that hce1m6-nscs could localize to nb tumors , we developed subcutaneous xenografts of all four human nb cell lines in es1/scid mice . when tumors became palpable , mice were intravenously injected with 2 10 hce1m6-nscs . forty - eight hours later , tumors were excised and analyzed for the presence of cells containing v - myc . all nb tumors contained nscs ( figure 3 ) . the average nsc tumor distributions for chla-136 , sms - kcnr , chla-255 , and sk - n - as nb xenografts were 269 340 , 153 312 , 124 223 , and 38 27 nscs per 100,000 tumor cells , respectively . the levels of v - myc among the samples , both within the same tumor samples or among mouse tumors , were highly variable , and averages were not significantly different ( as assessed by analysis of variance ) . this was similar to previous observations for which nsc distribution was heterogeneous in other solid tumor models.27 , 30 following establishment of subcutaneous chla-255 or chla-136 nb xenografts in es1/scid mice , we intravenously injected tumor - bearing mice with 2 10 hce1m6-nscs on day 0 and with irinotecan ( 15 mg / kg ) on day 2 . one hour after drug administration , mice were sacrificed and nb tumors were excised and analyzed for drug ( irinotecan and sn-38 ) levels by high - performance liquid chromatography - ms / ms . sn-38 levels in both chla-255 and chla-136 nb tumors in mice that received hce1m6-nscs were highest when irinotecan was given 2 days after nsc administration ( figure 4 ) . we saw an approximately 2-fold increase in sn-38 concentrations in tumors from mice that received hce1m6-nscs on day 2 after nsc injection versus treatment with irinotecan alone ( chla-255 : 445.1 69.0 versus 217.7 60.7 ng / ml [ 2.0-fold difference , p = 0.0001 ] ; chla-136 : 766.7 154.1 versus 460.9 87.1 ng / ml [ 1.7-fold difference , p = 0.0069 ) . in contrast , plasma concentrations of sn-38 were similar in all mice , regardless of whether they received hce1m6-nscs . no significant differences in sn-38 concentrations were observed at 3 or 4 days after hce1m6-nsc injection as compared with irinotecan alone in all mice ( data not shown ) . to assess the efficacy of treatment of hce1m6 nscs and irinotecan , we intravenously injected 2 10 chla-136 nb cells expressing firefly luciferase ffluc into es1/scid mice on day 0 , and treatment was initiated on day 10 . tumor growth was monitored by bioluminescent imaging , and mean signal intensities were obtained from these scans at multiple times points for each mouse . two days after intravenous administration of hce1m6-nscs , mice were treated with three rounds of irinotecan using an established clinical dosing regimen . treatment groups included : group a , tumor only ; group b , irinotecan only ( 7.5 mg / kg , three times a week ) ; group c , hce1m6-nscs + irinotecan ( 7.5 mg / kg , three times a week ) ; group d , irinotecan only ( 15 mg / kg , three times a week ) ; and group e , hce1m6-nscs + irinotecan at ( 15 mg / kg , three times a week ) . two weeks of treatment was followed by 1 week of rest ( considered one cycle of therapy ) . mice that received hce1m6-nscs in combination with irinotecan ( 15 mg / kg , approximately equivalent to human dose of 50 mg / m ) , demonstrated a significant delay in tumor growth as compared with those that received irinotecan alone ( figure 5a ) , resulting in a 1.7-fold log scale decrease in luciferase signal intensity at day 88 ( p = 0.0001 ) . we saw no difference in tumor growth between mice that did or did not receive nscs when 7.5 mg / kg irinotecan was used . the observed delay in tumor growth was associated with a significant extension of median survival in mice treated with hce1m6-nscs and irinotecan ( 15 mg / kg ) ( 137 days ; 95% confidence interval [ ci ] , 126139 days ) versus drug alone ( 117 days ; 95% ci , 112126 days ; p = 0.034 ) ( figure 5b ) . similar studies using the chla-255 tumor line also demonstrated a significant delay in tumor growth in mice treated with hce1m6-nscs and irinotecan ( 15 mg / kg ) treatment versus drug alone ( 0.74-fold decrease in tumor growth at day 42 , p = 0.0080 ) . to further analyze the effect of this nsc - mediated gene therapy , we histologically examined whether metastatic foci were present in the liver , lungs , and kidneys ( figure 6 ) . mice treated with hce1m6-nscs and irinotecan ( 15 mg / kg ) in group e showed a significant reduction in tumor burden in the liver , with only two of eight animals demonstrating tumors in this organ as compared with all ( 8/8 ) control mice ( no treatment , tumor only ; p = 0.0070 ) ( figure 6 ; table 2 ) . we also saw fewer tumor foci in the irinotecan alone ( 15 mg / kg ) treatment group ( 5/7 ) ; however , this result was not significant ( p = 0.31 ; table 2 ) . nb lesions were infrequently seen in the lungs of untreated mice ( 3/8 ) , and none was observed in mice that underwent high - dose ( 15 mg / kg ) irinotecan alone therapy . no differences were seen in the presence of tumors in the kidney in any of the cohorts ( table 2 ) . the data presented here support further efforts toward clinical translation studies of this ce - irinotecan enzyme - prodrug gene therapy approach for high - risk nb patients using an nsc line that has demonstrated clinical safety in brain tumor patients ( nct01172964 ) . these cells have been transduced ex vivo with an adenovirus designed to express a modified human ce ( hce1m6 ) , an enzyme highly proficient at irinotecan activation . ex vivo transduction of nscs with replication - deficient adenovirus offers several advantages , including : ( 1 ) high , transient secretion of hce1m6 , providing an expanded radius of action via the enzyme - prodrug bystander effect34 , 35 ; ( 2 ) non - integration into the dna of the parent nsc line , avoiding the possibility of insertional mutagenesis ; and ( 3 ) the potential to increase the effectiveness of a clinically approved chemotherapeutic agent by significantly increasing intratumoral concentrations of the active drug . treatment of several human - derived nb lines with media harvested from nscs secreting hce1m6 and irinotecan demonstrated 139- to 883-fold lower ic50 values when compared with treatment with drug alone . this is consistent with the enhanced hydrolysis of irinotecan in the media , resulting in significantly higher concentrations of sn-38 , leading to increased cytotoxicity . because only nanomolar levels of sn-38 are needed for tumor cytotoxicity , increased drug activation at tumor foci using nscs can be achieved using this strategy . in vivo studies in es1/scid mice demonstrated that hce1m6-nscs are cleared from normal organs within 24 hr of intravenous administration . this is an advantage over mesenchymal stem cells ( mscs ) , which are larger and can get trapped in the lungs . because clearance of nscs from normal tissues will be important for limiting off - target effects , based on these results , we decided to wait at least 2 days after nsc administration before treating with irinotecan in in vivo studies . we also confirmed ( by qpcr for v - myc ) that hce1m6-nscs selectively localize to nb tumors , although v - myc expression among the samples , both within the same tumor and between tumors , was highly variable . this finding is consistent with other results showing heterogeneous nsc distribution within metastatic tumor foci in other metastatic solid tumor models.27 , 28 , 39 additional studies that may enhance nsc - tumor tropism would allow for further optimization , but this would require a detailed understanding of the mechanism by which these cells target tumors in vivo . because such information is currently lacking , efforts to further improve nsc - directed tumor targeting will likely require further studies to dissect the homing and signaling cascades in this process . however , it is possible that administering the nscs in multiple doses ( such as two to four times in one day ) rather than as a single bolus dose may improve biodistribution within and among tumor foci . again , detailed preclinical studies will be necessary to validate this hypothesis . in vivo pharmacokinetic studies indicated that the optimal timing for irinotecan administration was 2 days after hce1m6-nscs . in these experiments , approximately 2-fold higher sn-38 concentrations were seen in the presence of hce1m6-nscs on day 2 versus irinotecan treatment alone . no significant increase in sn-38 levels were observed at days 3 and 4 ( as compared with non - nsc - injected animals ) , and the exact reasons for this are unclear . it may result from reduced expression of hce1m6 from the nscs in vivo , diffusion of the secreted ce away from the tumor xenografts , and/or enhanced proteolysis of the secreted enzyme . however , based upon these results , therapeutic efficacy studies were conducted in nb metastatic models , using clinically relevant concentrations of irinotecan given 2 days post - nsc administration . this resulted in significantly delayed progression of tumor growth in mice that received hce1m6-nscs in combination with the drug ( 15 mg / kg ) . in addition , there was a significant delay in tumor growth in these mice as compared with animals that received irinotecan alone . although this establishes proof of concept for this approach , further studies are warranted to optimize the conditions for maximal therapeutic efficacy . for example , cell dosing strategies that lead to an increase in both the number and the coverage of nscs in tumors would be expected to result in even greater intratumoral conversion of the irinotecan to sn-38 and may further prolong survival . furthermore , increasing tumor biodistribution of nscs by fractionating the dose of cells may further prolong survival . finally , mice treated with hce1m6-nscs and irinotecan ( 15 mg / kg ) demonstrated significantly less tumor burden in the liver as compared with control animals ( 6/8 had no tumor ) . minimizing liver metastases interestingly , mice with liver tumors in the irinotecan - treated groups ( either with or without hce1m6-nscs ) had fewer large , consolidated tumors , as compared with higher numbers of small tumors distributed throughout this organ in control animals . additional studies are currently ongoing in other mouse models of liver metastases to determine whether there is an organ - site - specific effect of the nscs , and if so , what the mechanism of this effect might be . collectively , the studies described herein demonstrate that hce1m6-nscs can increase sn-38 levels at tumor sites as compared with irinotecan treatment alone and have a marked effect on nb progression . in the future , the addition of temozolomide may further enhance the activity of treatment with hce1m6-nscs plus irinotecan by creating topoisomerase 1-dna complexes that may augment the activity of irinotecan . furthermore , patients with other cancers that are often treated with irinotecan , such as metastatic colon cancer that involves the liver , may also benefit from this therapy . as a consequence , further studies that seek to develop this nsc - mediated gene therapy strategy are in progress for facile translation to the clinic . chla-255 , chla-136 , and sms - kcnr human nb lines were transduced with the firefly luciferase ( fluc ) gene using a lentivirus vector . chla-255-fluc and chla-136-fluc were cultured in iscove s modified dulbecco s medium ( imdm ) supplemented with 10% fetal bovine serum ( fbs ) , 1% l - glutamine ( 100 , 25030 - 164 ; gibco ) and 1% penicillin - streptomycin ( catalog no . sms - kcnr - fluc cells were cultured in rpmi 1640 supplemented with 10% fbs , 1% l - glutamine ( 100 , catalog no . 25030 - 164 ; gibco ) and 1% penicillin - streptomycin for cytotoxicity assays , nb cells were seeded into each well of 96-well white opaque culture plates ( catalog no . 353296 ; falcon / corning ) and grown overnight ( chla-255-fluc , 4e3 cells / well ; chla-136-fluc , 5e3 cells / well ) or for 3 days ( sms - kcnr - fluc , 1e4 cells / well ) . nb cells were then exposed for 4 hr to : ( 1 ) sn-38 ( 0.0011 nm ) in cell culture media , ( 2 ) irinotecan ( 0.0011,000 m ) in cell culture media , or 3 ) irinotecan ( 0.0011000 nm ) in conditioned media from hce1m6-nscs . the media were then replaced with fresh culture media and cells were incubated for 5 days . e1605 ; promega ) was then added to each well , and cells were incubated for 5 min in the dark , after which cell viability was measured as ffluc luminescence using a glomax multi - detection system ( model e8032 ; promega ) . statistical analyses were performed using sas ( statistical analysis system ) version 9.4 and prism version 6 . values for each cell line were normalized relative to average baseline ( no drug control ) viability . statistical significance at an alpha level of 0.05 was ascertained by non - overlapping confidence intervals . all in vivo and in vitro studies used a clonal v - myc immortalized human nsc line , hb1.f3.cd clone 21 ( hb1.f3.cd21 ; established as a master cell bank [ mcb ] at passage 20 at the city of hope ) . nscs from the mcb were thawed and expanded in t-175 tissue culture flasks in dmem ( 10313 - 021 ; invitrogen ) supplemented with 10% heat - inactivated fbs ( sh30070.03 ; hyclone ) and 2 mm l - glutamine ( 25030 - 081 ; invitrogen ) as described previously . nscs were then transduced overnight with a clinical - grade recombinant replication - deficient adenovirus encoding hce1m6 ( adv.hce1m6 ) , resulting in transient secretion hce1m6 . all mouse studies were conducted under a city of hope institutional animal care and use committee ( iacuc)-approved protocol ( iacuc protocols used chla 353 and st . non - tumor - bearing naive es1/scid mice , both male and female , 812 weeks old , were intravenously injected with hce1m6-nscs ( 2 10 cells/200 l ) , pre - labeled with feraheme ( fe ) as previously described . mice were euthanized 1 hr , 24 hr , 48 hr , or 5 days after hce1m6-nsc administration . mouse tissues ( lung , liver , spleen , brain , and blood ) were snap frozen , and genomic dna was isolated for taqman qpcr analysis to identify the presence of nscs . the primer sequences and probe were designed to target gag - v - myc that is unique to our nsc line.19 , 20 the limit of detection is two copies of v - myc per 1 g starting dna . the limit of quantitation ( i.e. , the lowest standard ) is eight copies per 1 g of tissue . gapdh was also quantified as an internal control gene by using real - time qpcr with sybr green supermix ( bio - rad ) for dna from mouse tissues . lung , liver , brain , and whole - blood samples were analyzed by qpcr for the presence of v - myc to detect nscs over time . each organ was divided into one to three pieces for pcr analysis , depending on the starting size of the tissue , and each tissue sample was run in triplicate on a 96-well plate . averages were compared using analysis of variance with bonferroni adjustment for pairwise comparisons of other organs to lung ( graphpad prism version 6 ) . subcutaneous xenograft models of chla-255 and chla-136 ffluc - labeled human nb tumors ( 5 10 cells ) were established in 6- to 12-week - old male and female es1/scid mice ( n = 5 per nb type ) to determine the biodistribution of intravenously administered hce1m6-nscs at the tumor site . all mice were pre - treated with rat anti - mouse cd122 antibody ( 200 g / mouse , injected intraperitoneally ) and total body irradiation at 200 cgy before nb cell injection . once tumors were detectable ( generally 23 weeks after nb cell injection ) , all mice were intravenously injected with hce1m6-nscs ( 2 10 cells/200 l pbs ) . tumor tissues were snap frozen and genomic dna was isolated for qpcr analysis to identify the presence of nscs . each tumor was divided into four quarters , dna was isolated from each quarter , and two to four dna samples per tumor were analyzed for v - myc using qpcr as described above . averages were compared using anova with bonferroni correction for pairwise comparisons ( graphpad prism version 6 ) . subcutaneous nb tumors were established as described above . when tumors became palpable ( 5 mm in diameter ) , mice were intravenously injected with hce1m6-nscs ( 2 10 cells ) , followed 2 days later by intravenous irinotecan injections ( 15 mg / kg ) . one to six replicate measurements of drug concentrations were taken in each mouse , and the maximum concentration was used for analyses . fold differences in concentrations by nsc administration were reported , and averages were compared using unpaired t tests ( graphpad prism version 6 ) . adult es1/scid mice , 612 weeks old , both male and female , were used in all experiments . eight mice per group were injected intravenously with 2 10 nb cells ( chla-136 , chla-255 ) on day 0 . two days after administration of hce1m6-nscs ( 2 10 nscs were intravenously administered to mice on day 10 after nb cell injection ) , mice were treated with three rounds of irinotecan using an established clinical dosing regimen . treatment groups included : group a , tumor only ; group b , irinotecan only ( 7.5 mg / kg , three times a week ) ; group c , hce1m6-nscs + irinotecan ( 7.5 mg / kg , three times a week ) ; group d , irinotecan only ( 15 mg / kg , three times a week ) ; and group e , hce1m6-nscs + irinotecan ( 15 mg / kg , three times a week ) . two weeks of treatment was followed by 1 week of rest ( considered one cycle of therapy ) . tumor growth was monitored by bioluminescent imaging : mean signal intensities were obtained from scans at multiple time points for each mouse . results were analyzed using random intercept and slope regression models with intensities analyzed on a log scale . t tests on raw data were used to confirm earliest time point of difference . estimated differences from model curves kaplan - meier curves were created and median survival with 95% confidence intervals were estimated . developed protocols and conducted in vitro and in vivo experiments , acquired data , analyzed data , and created figures .

despite improved survival for children with newly diagnosed neuroblastoma ( nb ) , recurrent disease is a significant problem , with treatment options limited by anti - tumor efficacy , patient drug tolerance , and cumulative toxicity . we previously demonstrated that neural stem cells ( nscs ) expressing a modified rabbit carboxylesterase ( rce ) can distribute to metastatic nb tumor foci in multiple organs in mice and convert the prodrug irinotecan ( cpt-11 ) to the 1,000-fold more toxic topoisomerase-1 inhibitor sn-38 , resulting in significant therapeutic efficacy . we sought to extend these studies by using a clinically relevant nsc line expressing a modified human ce ( hce1m6-nscs ) to establish proof of concept and identify an intravenous dose and treatment schedule that gave maximal efficacy . human - derived nb cell lines were significantly more sensitive to treatment with hce1m6-nscs and irinotecan as compared with drug alone . this was supported by pharmacokinetic studies in subcutaneous nb mouse models demonstrating tumor - specific conversion of irinotecan to sn-38 . furthermore , nb - bearing mice that received repeat treatment with intravenous hce1m6-nscs and irinotecan showed significantly lower tumor burden ( 1.4-fold , p = 0.0093 ) and increased long - term survival compared with mice treated with drug alone . these studies support the continued development of nsc - mediated gene therapy for improved clinical outcome in nb patients .

Introduction Results Discussion Materials and Methods Author Contributions Conflicts of Interest

neuroblastoma ( nb ) is a neuroendocrine tumor that arises from neural crest elements of the sympathetic nervous system . it is the most common extracranial solid tumor of childhood , with a long - term survival rate of only 15% . the current standard of care for these patients consists of : ( 1 ) intensive induction chemotherapy , ( 2 ) myeloablative consolidation chemoradiotherapy and autologous hematopoietic stem cell transplantation , and ( 3 ) 13-cis retinoic acid with immunotherapy that targets disialoganglioside ( gd2).6 , 7 , 8 , 9 treatment failures occur at both primary and metastatic sites , and particularly in metastases to the bone and bone marrow , suggesting that minimal residual disease is an important cause of recurrence . current regimens of dose - intensive chemotherapy and irradiation are likely at the limit of both anti - tumor efficacy and patient tolerance , and post - consolidation therapy does not eradicate minimal residual disease ( mrd ) in many patients.6 , 7 , 8 , 9 therefore , there is a critical need for improved , less toxic therapeutic approaches for tumor cyto - reduction ( induction and consolidation phases ) and eradication of mrd ( post - consolidation phase ) to improve clinical outcome in children with this disease . selective activation of the prodrug irinotecan ( cpt-11 ; camptosar ; 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin ) to its 1,000-fold more cytotoxic active metabolite sn-38 ( 7-ethyl-10-hydroxycamptothecin ) , a topoisomerase-1 inhibitor , can be achieved with carboxylesterases ( ces ) . , sarcomas and non - small - cell lung cancer).14 , 15 , 16 , 17 , 18 neural stem cells ( nscs ) are inherently tumor tropic and selectively localize to solid tumor foci in multiple organs after intravenous administration in several metastatic tumor models , including breast cancer,19 , 20 , 21 ovarian cancer , lung cancer , and nb.24 , 25 , 26 nsc - mediated enzyme and prodrug therapy has been shown to be effective in several tumor models including glioma , medulloblastoma , melanoma brain metastases , and metastatic breast cancer.19 , 27 , 28 , 29 we previously showed proof of concept for increased therapeutic efficacy in mouse models of metastatic nb , using tumor - tropic nscs expressing a modified rabbit carboxylesterase ( rce ) to convert the prodrug irinotecan to sn-38.10 , 19 , 30 we now present data using a well - characterized , clonal human nsc line ( hb1.f3.cd clone 21 ) that has demonstrated clinical safety and proof of concept for brain tumor localized , nsc - expressed enzyme - mediated conversion of a prodrug ( 5-fluocytosine ) to its active chemotherapeutic ( 5-fluorouracil ) ( investigational new drug [ ind ] application 14041 ; nct01172964 ) . we transduced this nsc line with replication - deficient adenovirus designed to allow high - level , transient expression and secretion of a modified human ce1 ( hce1m6 ) . the hce1m6-expression vector was generated from the human liver ce hce1 , specifically to allow for efficient conversion of irinotecan to sn-38 , and has demonstrated functional equivalence to rce , both in vitro and in vivo . this nsc - secreted form of ce accumulates at tumor foci ( because of nsc tropism to tumor sites ) , where it can convert administered irinotecan to sn-38 and can generate a greater therapeutic radius of tumor kill around each nsc ( the bystander effect ) , as compared with irinotecan alone.34 , 35 toward clinical translation , our goal was to maximize therapeutic efficacy by determining the optimal clinically relevant dose and schedule of hce1m6-nscs + irinotecan in a mouse model of metastatic nb . repeat treatment of mice bearing nb with intravenously administered hce1m6-nscs and irinotecan resulted in both a significant decrease in tumor burden ( 1.4-fold , p = 0.0093 ) and increased long - term survival versus treatment with irinotecan alone . these studies support further development of nsc - mediated gene therapy for improved clinical outcome in nb patients . to determine whether hce1m6 expressed by nscs could enhance the cell - killing effects of irinotecan on nb cells , we measured the half - maximal inhibitory drug concentration ( ic50 ) for the drug in the presence and absence of nsc - secreted hce1m6 . the cytotoxicity of irinotecan and sn-38 was determined for a panel of human - derived nb cell lines ( sms - kcnr , chla-255 , chla-136 , and sk - n - as ) ( figure 1 ; table 1 ) . in brief , nb cells were incubated for 4 hr in cell culture medium containing irinotecan ( 0.0011,000 m ) or sn-38 ( 1100 nm ) , or in conditioned media ( cm ) harvested from nscs expressing hce1m6 to which irinotecan ( 1100 m ) was added . all nb cell lines were significantly more sensitive to irinotecan in the presence of hce1m6 as compared with irinotecan alone ( figures 1a1d ) ( table 1 ) , because of conversion of irinotecan to sn-38 , as demonstrated before . for the most sensitive cell line , sms - kcnr , the cytotoxicity of irinotecan ( ic50 = 7.95 m ) was increased up to 900-fold by addition of hce1m6-nsc cm ( ic50 = 0.009 m ) . these data suggest that hce1m6 produced by nscs can effectively convert irinotecan to the active metabolite sn-38 , reaching therapeutic ic50 levels similar to those observed for sn-38 alone . before translating use of hce1m6-nscs to a therapeutic approach , it will be necessary to ensure that these cells do not localize to non - tumor - harboring tissues and organs . this allows for assessment of clinically relevant human doses of irinotecan in these mice . one hour after intravenous administration into non - tumor - bearing es1/scid mice , nscs were detected in the lungs , liver , spleen , blood , and brain ( figure 2a ) . statistically significant differences were found when numbers of nscs in lung were compared with blood ( p = 0.02 ) , liver ( p = 0.003 ) , spleen ( p = 0.003 ) , and brain ( p = 0.007 ) , because it is likely that the majority of stem cells are initially trapped in the lungs . we did not see any cells that were positive for prussian blue staining in any of the sample tissue sections examined ( figure 2b ) , indicating the retention of exogenous hce1m6-nscs in normal tissues is a rare event and , given the assays used , detectable only by qpcr . to confirm that hce1m6-nscs could localize to nb tumors , we developed subcutaneous xenografts of all four human nb cell lines in es1/scid mice . this was similar to previous observations for which nsc distribution was heterogeneous in other solid tumor models.27 , 30 following establishment of subcutaneous chla-255 or chla-136 nb xenografts in es1/scid mice , we intravenously injected tumor - bearing mice with 2 10 hce1m6-nscs on day 0 and with irinotecan ( 15 mg / kg ) on day 2 . sn-38 levels in both chla-255 and chla-136 nb tumors in mice that received hce1m6-nscs were highest when irinotecan was given 2 days after nsc administration ( figure 4 ) . we saw an approximately 2-fold increase in sn-38 concentrations in tumors from mice that received hce1m6-nscs on day 2 after nsc injection versus treatment with irinotecan alone ( chla-255 : 445.1 69.0 versus 217.7 60.7 ng / ml [ 2.0-fold difference , p = 0.0001 ] ; chla-136 : 766.7 154.1 versus 460.9 87.1 ng / ml [ 1.7-fold difference , p = 0.0069 ) . to assess the efficacy of treatment of hce1m6 nscs and irinotecan , we intravenously injected 2 10 chla-136 nb cells expressing firefly luciferase ffluc into es1/scid mice on day 0 , and treatment was initiated on day 10 . two days after intravenous administration of hce1m6-nscs , mice were treated with three rounds of irinotecan using an established clinical dosing regimen . mice that received hce1m6-nscs in combination with irinotecan ( 15 mg / kg , approximately equivalent to human dose of 50 mg / m ) , demonstrated a significant delay in tumor growth as compared with those that received irinotecan alone ( figure 5a ) , resulting in a 1.7-fold log scale decrease in luciferase signal intensity at day 88 ( p = 0.0001 ) . the observed delay in tumor growth was associated with a significant extension of median survival in mice treated with hce1m6-nscs and irinotecan ( 15 mg / kg ) ( 137 days ; 95% confidence interval [ ci ] , 126139 days ) versus drug alone ( 117 days ; 95% ci , 112126 days ; p = 0.034 ) ( figure 5b ) . similar studies using the chla-255 tumor line also demonstrated a significant delay in tumor growth in mice treated with hce1m6-nscs and irinotecan ( 15 mg / kg ) treatment versus drug alone ( 0.74-fold decrease in tumor growth at day 42 , p = 0.0080 ) . to further analyze the effect of this nsc - mediated gene therapy , we histologically examined whether metastatic foci were present in the liver , lungs , and kidneys ( figure 6 ) . mice treated with hce1m6-nscs and irinotecan ( 15 mg / kg ) in group e showed a significant reduction in tumor burden in the liver , with only two of eight animals demonstrating tumors in this organ as compared with all ( 8/8 ) control mice ( no treatment , tumor only ; p = 0.0070 ) ( figure 6 ; table 2 ) . we also saw fewer tumor foci in the irinotecan alone ( 15 mg / kg ) treatment group ( 5/7 ) ; however , this result was not significant ( p = 0.31 ; table 2 ) . nb lesions were infrequently seen in the lungs of untreated mice ( 3/8 ) , and none was observed in mice that underwent high - dose ( 15 mg / kg ) irinotecan alone therapy . the data presented here support further efforts toward clinical translation studies of this ce - irinotecan enzyme - prodrug gene therapy approach for high - risk nb patients using an nsc line that has demonstrated clinical safety in brain tumor patients ( nct01172964 ) . these cells have been transduced ex vivo with an adenovirus designed to express a modified human ce ( hce1m6 ) , an enzyme highly proficient at irinotecan activation . ex vivo transduction of nscs with replication - deficient adenovirus offers several advantages , including : ( 1 ) high , transient secretion of hce1m6 , providing an expanded radius of action via the enzyme - prodrug bystander effect34 , 35 ; ( 2 ) non - integration into the dna of the parent nsc line , avoiding the possibility of insertional mutagenesis ; and ( 3 ) the potential to increase the effectiveness of a clinically approved chemotherapeutic agent by significantly increasing intratumoral concentrations of the active drug . treatment of several human - derived nb lines with media harvested from nscs secreting hce1m6 and irinotecan demonstrated 139- to 883-fold lower ic50 values when compared with treatment with drug alone . this is consistent with the enhanced hydrolysis of irinotecan in the media , resulting in significantly higher concentrations of sn-38 , leading to increased cytotoxicity . in vivo studies in es1/scid mice demonstrated that hce1m6-nscs are cleared from normal organs within 24 hr of intravenous administration . this is an advantage over mesenchymal stem cells ( mscs ) , which are larger and can get trapped in the lungs . this finding is consistent with other results showing heterogeneous nsc distribution within metastatic tumor foci in other metastatic solid tumor models.27 , 28 , 39 additional studies that may enhance nsc - tumor tropism would allow for further optimization , but this would require a detailed understanding of the mechanism by which these cells target tumors in vivo . however , it is possible that administering the nscs in multiple doses ( such as two to four times in one day ) rather than as a single bolus dose may improve biodistribution within and among tumor foci . no significant increase in sn-38 levels were observed at days 3 and 4 ( as compared with non - nsc - injected animals ) , and the exact reasons for this are unclear . however , based upon these results , therapeutic efficacy studies were conducted in nb metastatic models , using clinically relevant concentrations of irinotecan given 2 days post - nsc administration . this resulted in significantly delayed progression of tumor growth in mice that received hce1m6-nscs in combination with the drug ( 15 mg / kg ) . in addition , there was a significant delay in tumor growth in these mice as compared with animals that received irinotecan alone . although this establishes proof of concept for this approach , further studies are warranted to optimize the conditions for maximal therapeutic efficacy . for example , cell dosing strategies that lead to an increase in both the number and the coverage of nscs in tumors would be expected to result in even greater intratumoral conversion of the irinotecan to sn-38 and may further prolong survival . finally , mice treated with hce1m6-nscs and irinotecan ( 15 mg / kg ) demonstrated significantly less tumor burden in the liver as compared with control animals ( 6/8 had no tumor ) . minimizing liver metastases interestingly , mice with liver tumors in the irinotecan - treated groups ( either with or without hce1m6-nscs ) had fewer large , consolidated tumors , as compared with higher numbers of small tumors distributed throughout this organ in control animals . additional studies are currently ongoing in other mouse models of liver metastases to determine whether there is an organ - site - specific effect of the nscs , and if so , what the mechanism of this effect might be . collectively , the studies described herein demonstrate that hce1m6-nscs can increase sn-38 levels at tumor sites as compared with irinotecan treatment alone and have a marked effect on nb progression . in the future , the addition of temozolomide may further enhance the activity of treatment with hce1m6-nscs plus irinotecan by creating topoisomerase 1-dna complexes that may augment the activity of irinotecan . furthermore , patients with other cancers that are often treated with irinotecan , such as metastatic colon cancer that involves the liver , may also benefit from this therapy . as a consequence , further studies that seek to develop this nsc - mediated gene therapy strategy are in progress for facile translation to the clinic . chla-255 , chla-136 , and sms - kcnr human nb lines were transduced with the firefly luciferase ( fluc ) gene using a lentivirus vector . chla-255-fluc and chla-136-fluc were cultured in iscove s modified dulbecco s medium ( imdm ) supplemented with 10% fetal bovine serum ( fbs ) , 1% l - glutamine ( 100 , 25030 - 164 ; gibco ) and 1% penicillin - streptomycin ( catalog no . 25030 - 164 ; gibco ) and 1% penicillin - streptomycin for cytotoxicity assays , nb cells were seeded into each well of 96-well white opaque culture plates ( catalog no . nb cells were then exposed for 4 hr to : ( 1 ) sn-38 ( 0.0011 nm ) in cell culture media , ( 2 ) irinotecan ( 0.0011,000 m ) in cell culture media , or 3 ) irinotecan ( 0.0011000 nm ) in conditioned media from hce1m6-nscs . e1605 ; promega ) was then added to each well , and cells were incubated for 5 min in the dark , after which cell viability was measured as ffluc luminescence using a glomax multi - detection system ( model e8032 ; promega ) . nscs were then transduced overnight with a clinical - grade recombinant replication - deficient adenovirus encoding hce1m6 ( adv.hce1m6 ) , resulting in transient secretion hce1m6 . non - tumor - bearing naive es1/scid mice , both male and female , 812 weeks old , were intravenously injected with hce1m6-nscs ( 2 10 cells/200 l ) , pre - labeled with feraheme ( fe ) as previously described . all mice were pre - treated with rat anti - mouse cd122 antibody ( 200 g / mouse , injected intraperitoneally ) and total body irradiation at 200 cgy before nb cell injection . once tumors were detectable ( generally 23 weeks after nb cell injection ) , all mice were intravenously injected with hce1m6-nscs ( 2 10 cells/200 l pbs ) . two days after administration of hce1m6-nscs ( 2 10 nscs were intravenously administered to mice on day 10 after nb cell injection ) , mice were treated with three rounds of irinotecan using an established clinical dosing regimen .

nearly all of the 45% of nb patients diagnosed with high - risk tumors present with metastases , including those diagnosed at any age with amplification of the mycn oncogene and those older than 18 months with non - mycn - amplified tumors.2 , 3 , 4 , 5 although patient outcomes have steadily improved over the past 20 years , 3-year event - free survival rate is only 40% for patients who have high - risk , stage 4 metastatic disease . the current standard of care for these patients consists of : ( 1 ) intensive induction chemotherapy , ( 2 ) myeloablative consolidation chemoradiotherapy and autologous hematopoietic stem cell transplantation , and ( 3 ) 13-cis retinoic acid with immunotherapy that targets disialoganglioside ( gd2).6 , 7 , 8 , 9 treatment failures occur at both primary and metastatic sites , and particularly in metastases to the bone and bone marrow , suggesting that minimal residual disease is an important cause of recurrence . current regimens of dose - intensive chemotherapy and irradiation are likely at the limit of both anti - tumor efficacy and patient tolerance , and post - consolidation therapy does not eradicate minimal residual disease ( mrd ) in many patients.6 , 7 , 8 , 9 therefore , there is a critical need for improved , less toxic therapeutic approaches for tumor cyto - reduction ( induction and consolidation phases ) and eradication of mrd ( post - consolidation phase ) to improve clinical outcome in children with this disease . irinotecan is currently being used in front - line treatment for nb and tested in combination with other drugs in a phase i clinical trial for this disease , as well as colon cancer.12 , 13 clinical trials with this agent are also under way in variety of other solid malignancies ( e.g. , sarcomas and non - small - cell lung cancer).14 , 15 , 16 , 17 , 18 neural stem cells ( nscs ) are inherently tumor tropic and selectively localize to solid tumor foci in multiple organs after intravenous administration in several metastatic tumor models , including breast cancer,19 , 20 , 21 ovarian cancer , lung cancer , and nb.24 , 25 , 26 nsc - mediated enzyme and prodrug therapy has been shown to be effective in several tumor models including glioma , medulloblastoma , melanoma brain metastases , and metastatic breast cancer.19 , 27 , 28 , 29 we previously showed proof of concept for increased therapeutic efficacy in mouse models of metastatic nb , using tumor - tropic nscs expressing a modified rabbit carboxylesterase ( rce ) to convert the prodrug irinotecan to sn-38.10 , 19 , 30 we now present data using a well - characterized , clonal human nsc line ( hb1.f3.cd clone 21 ) that has demonstrated clinical safety and proof of concept for brain tumor localized , nsc - expressed enzyme - mediated conversion of a prodrug ( 5-fluocytosine ) to its active chemotherapeutic ( 5-fluorouracil ) ( investigational new drug [ ind ] application 14041 ; nct01172964 ) . the hce1m6-expression vector was generated from the human liver ce hce1 , specifically to allow for efficient conversion of irinotecan to sn-38 , and has demonstrated functional equivalence to rce , both in vitro and in vivo . this nsc - secreted form of ce accumulates at tumor foci ( because of nsc tropism to tumor sites ) , where it can convert administered irinotecan to sn-38 and can generate a greater therapeutic radius of tumor kill around each nsc ( the bystander effect ) , as compared with irinotecan alone.34 , 35 toward clinical translation , our goal was to maximize therapeutic efficacy by determining the optimal clinically relevant dose and schedule of hce1m6-nscs + irinotecan in a mouse model of metastatic nb . repeat treatment of mice bearing nb with intravenously administered hce1m6-nscs and irinotecan resulted in both a significant decrease in tumor burden ( 1.4-fold , p = 0.0093 ) and increased long - term survival versus treatment with irinotecan alone . to determine whether hce1m6 expressed by nscs could enhance the cell - killing effects of irinotecan on nb cells , we measured the half - maximal inhibitory drug concentration ( ic50 ) for the drug in the presence and absence of nsc - secreted hce1m6 . the cytotoxicity of irinotecan and sn-38 was determined for a panel of human - derived nb cell lines ( sms - kcnr , chla-255 , chla-136 , and sk - n - as ) ( figure 1 ; table 1 ) . in brief , nb cells were incubated for 4 hr in cell culture medium containing irinotecan ( 0.0011,000 m ) or sn-38 ( 1100 nm ) , or in conditioned media ( cm ) harvested from nscs expressing hce1m6 to which irinotecan ( 1100 m ) was added . all nb cell lines were significantly more sensitive to irinotecan in the presence of hce1m6 as compared with irinotecan alone ( figures 1a1d ) ( table 1 ) , because of conversion of irinotecan to sn-38 , as demonstrated before . for the most sensitive cell line , sms - kcnr , the cytotoxicity of irinotecan ( ic50 = 7.95 m ) was increased up to 900-fold by addition of hce1m6-nsc cm ( ic50 = 0.009 m ) . although the change in sensitivity was not as dramatic in the other cell lines , addition of hce1m6-nsc cm led to an increase of at least 139-fold ( table 1 ) . these data suggest that hce1m6 produced by nscs can effectively convert irinotecan to the active metabolite sn-38 , reaching therapeutic ic50 levels similar to those observed for sn-38 alone . because the hce1m6-nscs have a single copy of the v - myc gene per cell , we used qpcr ( taqman qpcr ) to quantify the relative amounts of nscs in the circulation and major organs . one hour after intravenous administration into non - tumor - bearing es1/scid mice , nscs were detected in the lungs , liver , spleen , blood , and brain ( figure 2a ) . however , hce1m6-nscs were undetectable by pcr at 24 hr , 48 hr , and 5 days post - injection ( figure 2a , 1 and 24 hr time points are shown ) . statistically significant differences were found when numbers of nscs in lung were compared with blood ( p = 0.02 ) , liver ( p = 0.003 ) , spleen ( p = 0.003 ) , and brain ( p = 0.007 ) , because it is likely that the majority of stem cells are initially trapped in the lungs . brain , liver , lung , and kidneys harvested at 1 and 24 hr after nsc administration were further analyzed by prussian blue staining and histological examination to detect iron - labeled hce1m6-nscs ( positive control for prussian blue staining of molday iron - labeled hce1m6-nscs was subcutaneous sknas tumor sections with nscs ; data not shown ) . we did not see any cells that were positive for prussian blue staining in any of the sample tissue sections examined ( figure 2b ) , indicating the retention of exogenous hce1m6-nscs in normal tissues is a rare event and , given the assays used , detectable only by qpcr . to confirm that hce1m6-nscs could localize to nb tumors , we developed subcutaneous xenografts of all four human nb cell lines in es1/scid mice . the average nsc tumor distributions for chla-136 , sms - kcnr , chla-255 , and sk - n - as nb xenografts were 269 340 , 153 312 , 124 223 , and 38 27 nscs per 100,000 tumor cells , respectively . the levels of v - myc among the samples , both within the same tumor samples or among mouse tumors , were highly variable , and averages were not significantly different ( as assessed by analysis of variance ) . this was similar to previous observations for which nsc distribution was heterogeneous in other solid tumor models.27 , 30 following establishment of subcutaneous chla-255 or chla-136 nb xenografts in es1/scid mice , we intravenously injected tumor - bearing mice with 2 10 hce1m6-nscs on day 0 and with irinotecan ( 15 mg / kg ) on day 2 . one hour after drug administration , mice were sacrificed and nb tumors were excised and analyzed for drug ( irinotecan and sn-38 ) levels by high - performance liquid chromatography - ms / ms . we saw an approximately 2-fold increase in sn-38 concentrations in tumors from mice that received hce1m6-nscs on day 2 after nsc injection versus treatment with irinotecan alone ( chla-255 : 445.1 69.0 versus 217.7 60.7 ng / ml [ 2.0-fold difference , p = 0.0001 ] ; chla-136 : 766.7 154.1 versus 460.9 87.1 ng / ml [ 1.7-fold difference , p = 0.0069 ) . to assess the efficacy of treatment of hce1m6 nscs and irinotecan , we intravenously injected 2 10 chla-136 nb cells expressing firefly luciferase ffluc into es1/scid mice on day 0 , and treatment was initiated on day 10 . treatment groups included : group a , tumor only ; group b , irinotecan only ( 7.5 mg / kg , three times a week ) ; group c , hce1m6-nscs + irinotecan ( 7.5 mg / kg , three times a week ) ; group d , irinotecan only ( 15 mg / kg , three times a week ) ; and group e , hce1m6-nscs + irinotecan at ( 15 mg / kg , three times a week ) . mice that received hce1m6-nscs in combination with irinotecan ( 15 mg / kg , approximately equivalent to human dose of 50 mg / m ) , demonstrated a significant delay in tumor growth as compared with those that received irinotecan alone ( figure 5a ) , resulting in a 1.7-fold log scale decrease in luciferase signal intensity at day 88 ( p = 0.0001 ) . the observed delay in tumor growth was associated with a significant extension of median survival in mice treated with hce1m6-nscs and irinotecan ( 15 mg / kg ) ( 137 days ; 95% confidence interval [ ci ] , 126139 days ) versus drug alone ( 117 days ; 95% ci , 112126 days ; p = 0.034 ) ( figure 5b ) . similar studies using the chla-255 tumor line also demonstrated a significant delay in tumor growth in mice treated with hce1m6-nscs and irinotecan ( 15 mg / kg ) treatment versus drug alone ( 0.74-fold decrease in tumor growth at day 42 , p = 0.0080 ) . to further analyze the effect of this nsc - mediated gene therapy , we histologically examined whether metastatic foci were present in the liver , lungs , and kidneys ( figure 6 ) . mice treated with hce1m6-nscs and irinotecan ( 15 mg / kg ) in group e showed a significant reduction in tumor burden in the liver , with only two of eight animals demonstrating tumors in this organ as compared with all ( 8/8 ) control mice ( no treatment , tumor only ; p = 0.0070 ) ( figure 6 ; table 2 ) . we also saw fewer tumor foci in the irinotecan alone ( 15 mg / kg ) treatment group ( 5/7 ) ; however , this result was not significant ( p = 0.31 ; table 2 ) . nb lesions were infrequently seen in the lungs of untreated mice ( 3/8 ) , and none was observed in mice that underwent high - dose ( 15 mg / kg ) irinotecan alone therapy . the data presented here support further efforts toward clinical translation studies of this ce - irinotecan enzyme - prodrug gene therapy approach for high - risk nb patients using an nsc line that has demonstrated clinical safety in brain tumor patients ( nct01172964 ) . ex vivo transduction of nscs with replication - deficient adenovirus offers several advantages , including : ( 1 ) high , transient secretion of hce1m6 , providing an expanded radius of action via the enzyme - prodrug bystander effect34 , 35 ; ( 2 ) non - integration into the dna of the parent nsc line , avoiding the possibility of insertional mutagenesis ; and ( 3 ) the potential to increase the effectiveness of a clinically approved chemotherapeutic agent by significantly increasing intratumoral concentrations of the active drug . treatment of several human - derived nb lines with media harvested from nscs secreting hce1m6 and irinotecan demonstrated 139- to 883-fold lower ic50 values when compared with treatment with drug alone . this is consistent with the enhanced hydrolysis of irinotecan in the media , resulting in significantly higher concentrations of sn-38 , leading to increased cytotoxicity . because clearance of nscs from normal tissues will be important for limiting off - target effects , based on these results , we decided to wait at least 2 days after nsc administration before treating with irinotecan in in vivo studies . we also confirmed ( by qpcr for v - myc ) that hce1m6-nscs selectively localize to nb tumors , although v - myc expression among the samples , both within the same tumor and between tumors , was highly variable . this finding is consistent with other results showing heterogeneous nsc distribution within metastatic tumor foci in other metastatic solid tumor models.27 , 28 , 39 additional studies that may enhance nsc - tumor tropism would allow for further optimization , but this would require a detailed understanding of the mechanism by which these cells target tumors in vivo . no significant increase in sn-38 levels were observed at days 3 and 4 ( as compared with non - nsc - injected animals ) , and the exact reasons for this are unclear . it may result from reduced expression of hce1m6 from the nscs in vivo , diffusion of the secreted ce away from the tumor xenografts , and/or enhanced proteolysis of the secreted enzyme . however , based upon these results , therapeutic efficacy studies were conducted in nb metastatic models , using clinically relevant concentrations of irinotecan given 2 days post - nsc administration . this resulted in significantly delayed progression of tumor growth in mice that received hce1m6-nscs in combination with the drug ( 15 mg / kg ) . for example , cell dosing strategies that lead to an increase in both the number and the coverage of nscs in tumors would be expected to result in even greater intratumoral conversion of the irinotecan to sn-38 and may further prolong survival . finally , mice treated with hce1m6-nscs and irinotecan ( 15 mg / kg ) demonstrated significantly less tumor burden in the liver as compared with control animals ( 6/8 had no tumor ) . minimizing liver metastases interestingly , mice with liver tumors in the irinotecan - treated groups ( either with or without hce1m6-nscs ) had fewer large , consolidated tumors , as compared with higher numbers of small tumors distributed throughout this organ in control animals . additional studies are currently ongoing in other mouse models of liver metastases to determine whether there is an organ - site - specific effect of the nscs , and if so , what the mechanism of this effect might be . collectively , the studies described herein demonstrate that hce1m6-nscs can increase sn-38 levels at tumor sites as compared with irinotecan treatment alone and have a marked effect on nb progression . in the future , the addition of temozolomide may further enhance the activity of treatment with hce1m6-nscs plus irinotecan by creating topoisomerase 1-dna complexes that may augment the activity of irinotecan . nb cells were then exposed for 4 hr to : ( 1 ) sn-38 ( 0.0011 nm ) in cell culture media , ( 2 ) irinotecan ( 0.0011,000 m ) in cell culture media , or 3 ) irinotecan ( 0.0011000 nm ) in conditioned media from hce1m6-nscs . non - tumor - bearing naive es1/scid mice , both male and female , 812 weeks old , were intravenously injected with hce1m6-nscs ( 2 10 cells/200 l ) , pre - labeled with feraheme ( fe ) as previously described . subcutaneous xenograft models of chla-255 and chla-136 ffluc - labeled human nb tumors ( 5 10 cells ) were established in 6- to 12-week - old male and female es1/scid mice ( n = 5 per nb type ) to determine the biodistribution of intravenously administered hce1m6-nscs at the tumor site . two days after administration of hce1m6-nscs ( 2 10 nscs were intravenously administered to mice on day 10 after nb cell injection ) , mice were treated with three rounds of irinotecan using an established clinical dosing regimen . treatment groups included : group a , tumor only ; group b , irinotecan only ( 7.5 mg / kg , three times a week ) ; group c , hce1m6-nscs + irinotecan ( 7.5 mg / kg , three times a week ) ; group d , irinotecan only ( 15 mg / kg , three times a week ) ; and group e , hce1m6-nscs + irinotecan ( 15 mg / kg , three times a week ) .

malignant gliomas have remained a malignancy with an extremely unfortunate prognosis [ 1 , 2 ] . recent improvements of standard therapies including , when feasible , surgical resection followed by radio - chemotherapy have only extended the 50% survival from 12 months to 16 months [ 35 ] . long - term survival is rare , only 5% of patients are alive after 5 years . as a consequence , one new strategy is the use of replication competent oncolytic viruses that specifically target and destroy tumor cells while leaving normal cells intact . a number of candidate oncolytic viruses for glioma therapy are currently under investigation including genetically modified herpesviruses , adenoviruses , or poliovirus , and wildtype viruses such as reovirus , vesicular - stomatitis virus , and measles virus . we previously reported the efficient killing of glioma cells of human and rat origin by parvovirus h-1 ( h-1pv ) , a single stranded nonenveloped dna virus of 5.1 kb . h-1pv induced lytic infection of glioma cells even when the cells were resistant to agents inducing apoptosis [ 12 , 13 ] . in animal experiments , h-1pv infection of rats bearing large intracranial gliomas led to tumor regression and prolonged survival ( geletneky et al . , accepted , 2010 ) . the natural host of h-1pv is the rat ; however , the virus can efficiently infect cells of other species including humans . in contrast to some other wildtype viruses under investigation for glioma - treatment , h-1pv does not cause any pathology in rodents or humans . radiation therapy prolongs survival in patients with malignant gliomas and is used as standard treatment of primary high - grade glial tumors . however , as high grade gliomas are resistant to radiation therapy and a clear dose - limitation exists due to cytotoxic effects on the surrounding brain tissue , this treatment modality is not curative and strategies to improve radiation efficiency are under investigation . the combination of radiation therapy with the oral alkylating agent temozolomide has already proved to be superior to either therapy alone and has become the standard of care for the majority of patients with newly diagnosed glioblastoma multiforme [ 3 , 5 ] . recent studies of the role of radiation therapy for recurrent gliomas that were already irradiated as part of the primary treatment demonstrated some effect when radiation was applied as a boost to smaller tumor regions . therefore , depending on the individual situation of the patient , radiation therapy can also be an option for recurrent tumors , but methods to augment the limited therapeutic effect would clearly be beneficial . clinical trials with different oncolytic viruses were able to demonstrate the safety of this novel therapeutic approach ; however , the positive therapeutic effects were restricted to individual patients . therefore , the combination of oncolytic viruses with standard therapeutics has become one important focus to improve viral cytotoxicity . radiation therapy is a part of therapeutic protocols for the majority of malignancies , and an enhanced effect of the oncolytic activity of viruses by radiation therapy could be observed for tumor cells of various histology . this is of particular interest for gliomas , as both treatments , radiation therapy and virotherapy , are primarily designed as regional therapies . for glioma cells , the oncolytic effect of herpesvirus , adenoviruses , reovirus , and measles virus the aim of this study was to assess the influence of ir on the oncolytic activity of h-1pv in glioma cells . the possible interaction of ir with h-1pv oncolytic virotherapy could be twofold : ( i ) as the use of an oncolytic virus in glioma patients would preferably include the treatment of recurrent tumors originating from previously irradiated remaining tumor cells , it has to be shown whether previous radiation therapy would interfere with viral oncolysis or replication in pretreated gliomas and ( ii ) administration of radiation therapy together with h-1pv oncolytic virotherapy could lead to improved efficacy of either treatment alone . we therefore investigated the treatment of early - passage glioma cells with ir before or after infection with h-1pv and assessed cytotoxicity , viral replication , and treatment - induced changes of the cell cycle . these findings are important to define patient populations for a clinical trial of oncolytic virotherapy of malignant gliomas and to possibly use h-1pv to increase radiation efficacy in this dismal tumor entity . human short term cultures derived from histologically confirmed glioblastomas ( nch-82 , nch-89 , nch-307 ) and a gliosarcoma ( nch-37 ) were established and characterized at the department of neurosurgery , heidelberg , germany as described previously . nch-307 is a recurrent glioblastoma cell line that had been irradiated in vivo prior to resection of the recurrent tumor . nch-37 , nch-89 , and nch-307 were tested at low - passage numbers < 30 ; nch-82 was tested at a passage number of 100 . the ethylnitrosourea - induced rat glioma cell line rg2 was previously shown to be highly susceptibly for h-1pv and was used for virus - titration experiments . all cells were grown in deme ( sigma - aldrich , steinheim , germany ) supplemented with 10% fcs ( biochromkg , berlin , germany ) and 1% antibiotics ( penicillin / streptomycin ; gibca , invitrogen corporation , karlsruhe , germany ) in a 5% co2 humidified atmosphere at 37c . radiation of cell cultures was performed at room temperature in a linear accelerator ( siemens mevatron kd2 , 6-mv photons ) at doses of 5 gy,10 gy , or 20 gy as indicated for the respective experiment ( dose rate : 3 gy / min ; distance from source to flask : 95 cm ) . control cells were transported to the accelerator but not exposed to ir ( 0 gy ) . nch-307 monolayers of all glioma cell cultures were infected under standard conditions : cells were inoculated with h-1pv diluted in pbs at a multiplicity of infection ( moi ) of 5 plaque forming units ( pfu ) per cell or 100 pfu / cell as indicated for the respective experiment . after 60 minutes , virus suspensions were removed , cells were washed with medium , and cultures were allowed to grow . the corresponding mock - infected cultures were subjected to the same procedure using virus - free pbs instead of virus suspensions . to asses cell viability , glioma cells ( nch-37 , nch-82 , nch-89 ) were seeded at 3 10 cells / well into 12-well dishes and irradiated after 24 hours with 0 gy , 5 gy , 10 gy , or 20 gy . cells were infected ( moi = 5 pfu / cell or mock - infection ) at 2 different time points : 24 hours post - ir ( early infection ) or 9 days post - ir ( late infection ) . in vivo irradiated nch-307 cells were seeded at 3 10 cells / well into 12-well dishes and infected ( moi = 5 pfu / cell , 100 pfu / cell or mock - infection ) 24 hours postseeding . the moi of h-1pv was calculated based on counts of living cells immediately prior to infection . cells were harvested 3 days after h-1pv infection / mock - infection and counted with an electronic counter ( casy , schaerfe system , reutlingen , germany ) in triplicate and results were confirmed with the typan blue exclusion test as described previously . the numbers of living cells were estimated as means and standard deviations of three independent assays . cell viability ( in % + / standard error ) was defined as the number of living treated cells over the number of living untreated cells multiplied by 100 ; standard error was calculated using the gaussian law of error propagation . statistical analysis was performed using a two - way anova with virotherapy and ir as independent factors . comparative analyses between groups were performed using post - hoc analysis . the spss software package ( spss inc . , chicago , il ) was used to perform statistical analysis . for rt - pcr analysis of viral rna , cells ( nch-37 , nch-82 , nch-89 ) were plated at a density of 3 10 cells in 17 cm culture flasks , irradiated ( 10 gy ) after 24 hours and infected ( moi = 5 pfu / cell or mock - infection ) 24 hours post - ir ; nch-307 cells were infected ( moi = 5 pfu / cell or mock - infection ) 24 hours postseeding . rna was purified using the high pure rna isolation kit ( roche diagnostics , mannheim , germany ) according to the manufacturer 's instructions . polymerase one - step rt - pcr - system ( roche diagnostics , mannheim , germany ) according to the manufacturer 's instructions . to detect h-1pv ns - transcripts , the following specific primers were used : sense primer ( position nt 19962016 ) 5-tca atg cgc tca cca tct ctg-3 ; antisense primer ( position nt 25102490 ) 5-tcg tag gct tcg tcg tgt tct-3. cells ( nch-37 , nch-82 , nch-89 ) were plated at a density of 3 10 cells in 17 cm culture flasks , irradiated ( 0 gy , 10 gy ) after 24 hours and infected ( moi = 5 pfu / cell or mock - infection ) 24 hours post - ir ( early infection ) . for late infection , cells were infected 9 days post - ir . recurrent glioblastoma nch-307 cells were infected ( moi = 5 pfu / cell or mock - infection ) 24 hours postseeding . cells were fixed with 4% paraformaldehyde and 100% methanol and permeabilized with 0.1% triton - x-100 ( sigma - aldrich , taufkirchen , germany ) . to identify ns-1 protein , probes were blocked with fetal calf serum and incubated on ice for 30 minutes with a polyclonal rabbit - anti - ns-1 antibody ( sp8 , courtesy of n. salome , dkfz , heidelberg , germany ) in a concentration of 1 : 25 . the fitc - conjugated secondary goat - anti - rabbit - antibody ( jackson immunoresearch , suffolk , uk ) was incubated in a 1:250 dilution for 20 minutes on ice . probes were analyzed for intracellular ns-1 content by measuring of fluorescence intensity , using a cytometer ( facscan flow cytometer , becton dickinson , heidelberg , germany ) at an excitation wavelength of 525 nm . the data were analyzed with the aid of a software program ( flowjo , tree star , olten , switzerland ) with dead cells gated out using pulse processing . a cell was determined as ns-1-positive when its fluorescence intensity ( fl1-h ) was greater than a certain threshold value of 5% of false positive mock infected cells . cells ( nch-37 , nch-82 , nch-89 ) were seeded at a density of 1.5 10 cells / well in 6-well dishes , irradiated 24 hours postseeding ( 10 gy ) , and infected ( moi = 5 pfu / cell or mock - infection ) 24 hours post - ir . nch-307 was infected ( moi = 5 pfu / cell or mock - infection ) 24 hours postseeding . the quantity of infectious viral particles in the supernatant of glioma cells was determined 1 hour and 3 days p.i . cells ( nch-37 , nch-82 , nch-89 ) were plated at a density of 3 10 cells in 17 cm culture flasks and irradiated ( 0 cells were harvested 24 hours and 48 hours post - ir and fixed in 80% ice - cold ethanol at 4c overnight . after fixation , cells were incubated in 2 mg / ml rnase ( sigma - aldrich , taufkirchen , germany ) , and 0,1 mg / ml pi ( sigma - aldrich , taufkirchen , germany ) for 30 minutes in the dark . samples of 10,000 cells were analyzed for dna content by flow cytometry ( facscan flow cytometer , becton dickinson , heidelberg , germany ) , and cell cycle phase distributions were analyzed with flowjo software using the dean - jett - fox model . as previous radiation therapy of tumor cells induces genetic alterations that could interfere with the susceptibility and efficiency of h-1pv infection , we infected glioma cells with a delay of 9 days after ir ( late infection ) when the cells had reentered the cell cycle . the goal of this experiment was to mimic and evaluate the possibility of h-1pv virotherapy of recurrent tumors after completion of radiation therapy as part of the initial standard treatment . in addition to testing tumor cells from primary gliomas ( nch-37 , nch-82 , nch-89 ) , we also included nch-307 cells that were established from a recurrent glioblastoma that had been irradiated several months prior to secondary surgical resection . cells ( nch-37 , nch-82 , and nch-89 ) were treated with ir of 10 gy and had resumed to proliferate 9 days after radiation therapy . however , the growth rate was clearly reduced compared with untreated controls indicating persisting long - term effects of the treatment . in comparison , cell cultures of nch-37 cells were less affected by radiation - induced growth retardation than nch-82 , and nch-89 cells . upon late infection with h-1pv with an moi of 5 pfu / cell , all cells ( nch-37 , nch-82 , and nch-89 ) showed a significant ( p < .001 ) reduction of surviving cells 3 days p.i .. cell viability of infected cells was 65.04 ( + /11.5 ) % in nch-37 cultures , 67.00 ( + /11.9 ) % in nch-82 cells , and 61.04 ( + /13.8 ) % in nch-89 cell cultures indicating intact susceptibility to h-1pv induced cell killing ( figure 1 ) . nch-307 recurrent glioma cells were infected with a low ( 5 pfu / cell ) and a high ( 100 pfu / cell ) moi ( figure 1 ) . cell viability of nch-307 cells was significantly ( p < .001 ) reduced to 55.39 ( + / 6.6 ) % ( low moi ) and 25.97 ( + / 8.8 ) % ( high moi ) indicating dose - dependent cytotoxicity of h-1pv also in recurrent glioma cells . in initial experiments , the effect of radiation therapy or h-1pv infection alone was examined prior to testing combination treatment . at radiation - doses of 5 gy , growth rates in all cell lines ( nch-37 , nch-82 , nch-89 ) were only slightly affected : cell viability was 70 ( + /9.9 ) % in nch-37 , 76 ( + /4.5 ) % in nch-82 , and 91 ( + /7.0 ) % in nch-89 . ir with 10 gy had a strong effect on nch-82 and nch-89 cells with a cell viability of 25.64 ( + /1.8 ) % ( nch-82 ) and 22.81 ( + /4.7 ) % ( nch-89 ) . nch-37 cells were much less sensitive , the cell viability was reduced to 54.25 ( + /7.2 ) % . a dose of 20 gy had a slightly stronger effect in all cell cultures : nch-82 21.53 ( + /3.8 ) % and nch-89 15.93 ( + /5.6 ) % cell viability , however in nch-37 cultures 45.19 ( + /5.6 ) % of cells were still alive ( figure 2 ) . the infection of glioma cells with h-1pv at an moi of 5 pfu / cell had a strong cytopathic effect in nch-82 and nch-89 cell cultures , with only 22.19 ( + / 3.0 ) % ( nch-82 ) and 9.73 ( + / 2.1 ) % ( nch-89 ) cell viability . nch-37 cells were less sensitive to h-1pv ; cell viability was 45.94 ( + / 6.0 ) % ( figure 2 ) . to assess whether the combination of radiation therapy and h-1pv virotherapy increases the therapeutic efficacy of either treatment alone and to evaluate the influence of the order of treatments on cytotoxicity , we performed two separate experiments : ( i ) glioma cells were irradiated with 3 different doses and infected 1 day after ir ( figure 2 , combination treatment : ir h-1 ) and ( ii ) glioma cells were infected first and subsequently irradiated with a dose of 10 gy 24 hours p.i . ( figure 2 , combination treatment : h-1 ir , far right column ) . two - way - anova showed that in all cell lines tested ( nch-37 , nch-82 , nch-89 ) , both independent factors ( ir and h-1pv infection ) had a significant influence on the number of surviving cells . there were significant interactions between ir and h-1pv infection for all of the data presented in figure 2 . comparative analyses between groups revealed the following : in all glioma cell cultures ( nch-37 , nch-82 , nch-89 ) , combination of h-1pv infection 1 day after ir was significantly ( p < .05 ) more effective than ir alone ( figure 2 ) . compared with h-1pv infection alone , combination treatment was significantly ( p < .05 ) more effective after previous ir with 5 gy , 10 gy , or 20 gy in nch-37 cells and after previous ir with 20 gy in nch-82 cells . when the order of treatments was reversed and h-1pv infection was performed 24 hours prior to ir , combination treatment only led to significantly ( p < .05 ) improved cell killing in nch-37 when compared to ir alone , but not when compared to h-1pv infection alone or in the other cell lines tested . even though high - dose radiation of nch-37 , nch-82 , and nch-89 cells with 20 gy or infection with h-1pv was highly cytotoxic , approximately 2 weeks after single treatment with ir or h-1pv alone , all cell lines resumed to proliferate from surviving clones , albeit at a much reduced rate ( table 1 ) . thus , neither ir nor h-1pv infection alone was able to eradicate all tumor cells . in contrast , when glioma cell cultures were treated with the combination of ir ( 20 gy ) and h-1pv infection ( moi = 5 pfu / cell ) 24 hours after ir , no surviving tumor cells could be observed on day 21 p.i . or at later time points after treatment in any of the tested cell cultures ( nch-37 , nch-82 , nch-89 ) indicating long - term efficiency of combination treatment ( table 1 and figure 3 ) . replication of h-1pv in infected glioma cells was tested ( i ) by the presence of viral ns-1-specific rna by rt - pcr , ( ii ) by the detection of the viral protein ns-1 as the main effector of parvoviral cytotoxicity by facs analysis , and ( iii ) by the release of infectious viral particles into the supernatant of cell cultures . ( i ) detection of viral rna : cells ( nch-37 , nch-82 , and nch-89 ) were irradiated with 10 gy and infected with h-1pv at an moi of 5 pfu / cell 24 hours later . rt - pcr was positive for the presence of viral rna indicating transcription from viral dna ( figure 4(e ) ) . ( ii ) expression of ns-1 protein : irradiated ( 10 gy ) or untreated control cells were either h-1pv infected ( moi = 5 pfu / cell ) or mock - infected 24 hours post - ir ( early infection ) or 9 days post - ir ( late infection ) . recurrent glioblastoma cells ( nch-307 ) were infected with an moi of 5 pfu / cell without additional ir . 24 hours after ( mock- ) infection , intracellular ns-1 was marked with a fitc - conjugated antibody as described above . ns-1 protein expression of unirradiated glioma cells ranged from 50% of ns-1-positive nch-37 cells to 43% in nch-82 cells and 42% in nch-89 cells 24 hours p.i . ( figure 4(a)4(c ) ) . the recurrent glioblastoma cell line nch-307 showed ns-1 expression in 44% of cells 24 hours p.i . ( figure 4(d ) ) . when cells were irradiated with 10 gy and infected with h-1pv 24 hours ( early infection ) or 9 days ( late infection ) after ir , ratios of ns-1 expression 24 hours p.i . were as follows : nch-37 cells showed an increase of ns-1-positive cells to 78% after early infection and to 67% after late infection . in nch-82 cells , ns-1 expression increased to 51% after early infection and dropped to 21% after late infection . the ns-1 expression level in nch-89 remained nearly unchanged with 42% of ns-1-positive cells after early infection and 39% after late infection . ( iii ) production of infectious h-1 virus particles : in order to assess whether cytopathic h-1pv infection of irradiated glioma cells resulted in the production of infectious progeny particles , virus yields were determined by titration on highly susceptibly rg2 cells . as demonstrated in table 2 , a 10 log - fold higher virus titer could be detected compared with input virus within 3 days after infection irrespective if cells were irradiated ( 10 gy ) or not ( 0 gy ) . results were similar in all cell lines tested ( nch-37 , nch-82 , nch-89 ) , demonstrating persisting assembly of progeny virus after ir . one possible mechanism for an improved cytotoxicity of h-1pv infection after ir could be associated to changes of the cell cycle as h-1pv replication is restricted to cells in s - phase . we therefore analyzed the effect of ir on the cell cycle of glioma cell lines ( nch-37 , nch-82 , nch-89 ) . ir of glioma cell cultures with 10 gy resulted in an increase of cells in s - phase 24 hours post - ir ( figure 5 ) . in nch-37 the increase was + 67% , in nch-82 + 114% , and in nch-89 + 72% ( figure 5(b ) ) . the increase was transient as 48 hours post - ir ; the amount of cells in the s - phase decreased to + 12% in nch-37 , to control level in nch-82 , and to + 10% in nch-89 . in parallel , at this time , the overall cell cycle distribution of nch-37 and nch-89 cells had almost returned to control levels , whereas nch-82 cells were blocked in g2/m ( figure 5(a ) ) and reached control levels 4 days post - ir ( data not shown ) . in comparison , upon h-1pv infection , cell cultures showed less homogeneous results . while nch-82 and nch-89 cells that were most sensitive to h-1 infection showed increased ratios of cells in s - phase , the amount of less sensitive nch-37 cells in s - phase was unchanged . the cell cycle changes after combination of radiation therapy and h-1pv infection were identical to h-1pv infection alone ( data not shown ) . oncolytic virotherapy is a promising new approach for the treatment of a variety of malignancies including malignant brain tumors . in early phase clinical trials , intracerebral infection of patients with oncolytic viruses of different genera however , only in a few patients tumor regression and a prolongation of progression - free survival could be demonstrated . this led to numerous attempts not only to further improve the oncolytic activity of viruses but also to investigate the combination treatment of viral infection of tumor cells with established conventional therapies . recurrences occur in 70 to 80% of cases within 2 cm of the primary tumor site and will therefore develop from cells that were already hit by a radiation dose of up to 60 gy . as radiation therapy is known to induce long - term changes in cellular genomes , this could potentially lead to an altered effect of virus infection compared with unirradiated primary glioma cells . in addition to other studies , we therefore specifically investigated the oncolytic potential of h-1pv in glioma cells that grew from irradiated clones . a cell culture established from a recurrent glioma that was irradiated with 56 gy during the initial treatment of the patient , and that had its origin in the radiation field , was fully permissive to h-1pv infection and cell killing was dose - dependent . this finding was confirmed in primary glioma cultures that were irradiated in vitro with a sublethal dose , allowed to regrow , and infected with h-1pv in a time interval of several days . the intact susceptibility of glioma cells to h-1pv infection after ir is of clinical significance as patients with recurrent gliomas who face an even worse prognosis with oftentimes less therapeutic options are prime candidates for experimental therapies . as a consequence , the group of patients with recurrent gliomas is usually the main patient population of early clinical trials of oncolytic virotherapy . however , to our knowledge the response of previously irradiated glioma cells to the oncolytic infection has never been specifically addressed for other oncolytic viruses . when h-1pv infection was performed early after ir , our data show improved killing of glioma cells , most pronounced in the most radioresistant cell line tested . these findings are in line with studies conducted with other oncolytic viruses that is , herpesvirus , adenovirus , reovirus , and measles virus that also showed an enhanced efficacy of viral oncolysis in combination with radiation therapy . whether this effect can also be demonstrated in vivo was beyond this proof of concept study and when radiation treatment was performed one day before h-1pv infection , combination treatment was significantly better in all cell lines than single treatment . virus infection followed by ir was less efficient in all cell cultures and had a reduced cytotoxic effect . one possible reason for the improved cytotoxicity of h-1pv after ir is the increased level of glioma cells positive for ns-1 expression 24 hours after early infection . previous studies revealed that ns-1 is the key - mediator of parvoviral cytotoxicity and its expression is strongly s - phase dependent [ 14 , 25 ] . cell cycle analyses revealed that in all primary glioma cell cultures tested , the rate of cells in s - phase was increased 24 hours post - ir . even though improved viral cytotoxicity may depend on several factors , this altered cell cycle distribution supports the finding of increased viral transcription and increased cell killing . as a consequence , when cells were infected when the cell cycle had returned to control levels ( late infection ) , ns-1 expression decreased . the glioma cell cultures in our study , like other glioma cell lines , were relatively radio - resistant and even after treatment with 20 gy remaining cell clones continued to grow . recent data suggests that stem - like cells exist within high - grade gliomas which are radioresistant and capable of initiating tumour regrowth . this is considered to be due to upregulated dna damage checkpoint pathways . in our system , neither radiation therapy nor h-1pv infection was able to kill all tumor cells . however , combined treatment with a high - radiation dose resulted in complete cytotoxicity in all cell cultures , indicating improved efficacy also in relatively resistant clones . these results may warrant to test whether the combination of radiation and h-1pv infection could also overcome the resistance of glioma cells expressing stem cell markers thereby offering a new treatment opportunity in these therapy refractory cells . in conclusion , irradiated glioma - cells show intact susceptibility for h-1pv infection with even improved cell killing by combining ir with h-1pv , most pronounced in radioresistant glioma cells . these results further support the ongoing development of a phase - i clinical trial for the use of h-1pv in malignant gliomas , allowing for the inclusion of pretreated patients into the study population . the authors do not have a financial interest / arrangement or affiliation with one or more organisations that could be perceived as a real or apparent conflict of interest in the context of the subject of this article .

purpose . to elucidate the influence of ionizing radiation ( ir ) on the oncolytic activity of parvovirus h-1 ( h-1pv ) in human high - grade glioma cells . methods . short term cultures of human high - grade gliomas were irradiated at different doses and infected with h-1pv . cell viability was assessed by determining relative numbers of surviving cells . replication of h-1pv was measured by rt - pcr of viral rna , fluorescence - activated cell sorter ( facs ) analysis and the synthesis of infectious virus particles . to identify a possible mechanism for radiation induced change in the oncolytic activity of h-1pv we performed cell cycle analyses . results . previous irradiation rendered glioma cells fully permissive to h-1pv infection . irradiation 24 hours prior to h-1pv infection led to increased cell killing most notably in radioresistant glioma cells . intracellular levels of ns-1 , the main effector of h-1pv induced cytotoxicity , were elevated after irradiation . s - phase levels were increased one day after irradiation improving s - phase dependent viral replication and cytotoxicity . conclusion . this study demonstrates intact susceptibility of previously irradiated glioma - cells for h-1pv induced oncolysis . the combination of ionizing radiation followed by h-1pv infection increased viral cytotoxicity , especially in radioresistant gliomas . these findings support the ongoing development of a clinical trial of h-1pv in patients with recurrent glioblastomas .

1. Introduction 2. Methods and Materials 3. Results 4. Discussion Conflicts of Interest Notification

we previously reported the efficient killing of glioma cells of human and rat origin by parvovirus h-1 ( h-1pv ) , a single stranded nonenveloped dna virus of 5.1 kb . h-1pv induced lytic infection of glioma cells even when the cells were resistant to agents inducing apoptosis [ 12 , 13 ] . in animal experiments , h-1pv infection of rats bearing large intracranial gliomas led to tumor regression and prolonged survival ( geletneky et al . the natural host of h-1pv is the rat ; however , the virus can efficiently infect cells of other species including humans . radiation therapy prolongs survival in patients with malignant gliomas and is used as standard treatment of primary high - grade glial tumors . however , as high grade gliomas are resistant to radiation therapy and a clear dose - limitation exists due to cytotoxic effects on the surrounding brain tissue , this treatment modality is not curative and strategies to improve radiation efficiency are under investigation . the combination of radiation therapy with the oral alkylating agent temozolomide has already proved to be superior to either therapy alone and has become the standard of care for the majority of patients with newly diagnosed glioblastoma multiforme [ 3 , 5 ] . therefore , the combination of oncolytic viruses with standard therapeutics has become one important focus to improve viral cytotoxicity . radiation therapy is a part of therapeutic protocols for the majority of malignancies , and an enhanced effect of the oncolytic activity of viruses by radiation therapy could be observed for tumor cells of various histology . for glioma cells , the oncolytic effect of herpesvirus , adenoviruses , reovirus , and measles virus the aim of this study was to assess the influence of ir on the oncolytic activity of h-1pv in glioma cells . the possible interaction of ir with h-1pv oncolytic virotherapy could be twofold : ( i ) as the use of an oncolytic virus in glioma patients would preferably include the treatment of recurrent tumors originating from previously irradiated remaining tumor cells , it has to be shown whether previous radiation therapy would interfere with viral oncolysis or replication in pretreated gliomas and ( ii ) administration of radiation therapy together with h-1pv oncolytic virotherapy could lead to improved efficacy of either treatment alone . we therefore investigated the treatment of early - passage glioma cells with ir before or after infection with h-1pv and assessed cytotoxicity , viral replication , and treatment - induced changes of the cell cycle . these findings are important to define patient populations for a clinical trial of oncolytic virotherapy of malignant gliomas and to possibly use h-1pv to increase radiation efficacy in this dismal tumor entity . human short term cultures derived from histologically confirmed glioblastomas ( nch-82 , nch-89 , nch-307 ) and a gliosarcoma ( nch-37 ) were established and characterized at the department of neurosurgery , heidelberg , germany as described previously . nch-307 is a recurrent glioblastoma cell line that had been irradiated in vivo prior to resection of the recurrent tumor . to asses cell viability , glioma cells ( nch-37 , nch-82 , nch-89 ) were seeded at 3 10 cells / well into 12-well dishes and irradiated after 24 hours with 0 gy , 5 gy , 10 gy , or 20 gy . cells were infected ( moi = 5 pfu / cell or mock - infection ) at 2 different time points : 24 hours post - ir ( early infection ) or 9 days post - ir ( late infection ) . in vivo irradiated nch-307 cells were seeded at 3 10 cells / well into 12-well dishes and infected ( moi = 5 pfu / cell , 100 pfu / cell or mock - infection ) 24 hours postseeding . the moi of h-1pv was calculated based on counts of living cells immediately prior to infection . cells were harvested 3 days after h-1pv infection / mock - infection and counted with an electronic counter ( casy , schaerfe system , reutlingen , germany ) in triplicate and results were confirmed with the typan blue exclusion test as described previously . for rt - pcr analysis of viral rna , cells ( nch-37 , nch-82 , nch-89 ) were plated at a density of 3 10 cells in 17 cm culture flasks , irradiated ( 10 gy ) after 24 hours and infected ( moi = 5 pfu / cell or mock - infection ) 24 hours post - ir ; nch-307 cells were infected ( moi = 5 pfu / cell or mock - infection ) 24 hours postseeding . polymerase one - step rt - pcr - system ( roche diagnostics , mannheim , germany ) according to the manufacturer 's instructions . cells ( nch-37 , nch-82 , nch-89 ) were plated at a density of 3 10 cells in 17 cm culture flasks , irradiated ( 0 gy , 10 gy ) after 24 hours and infected ( moi = 5 pfu / cell or mock - infection ) 24 hours post - ir ( early infection ) . to identify ns-1 protein , probes were blocked with fetal calf serum and incubated on ice for 30 minutes with a polyclonal rabbit - anti - ns-1 antibody ( sp8 , courtesy of n. salome , dkfz , heidelberg , germany ) in a concentration of 1 : 25 . cells ( nch-37 , nch-82 , nch-89 ) were seeded at a density of 1.5 10 cells / well in 6-well dishes , irradiated 24 hours postseeding ( 10 gy ) , and infected ( moi = 5 pfu / cell or mock - infection ) 24 hours post - ir . the quantity of infectious viral particles in the supernatant of glioma cells was determined 1 hour and 3 days p.i . as previous radiation therapy of tumor cells induces genetic alterations that could interfere with the susceptibility and efficiency of h-1pv infection , we infected glioma cells with a delay of 9 days after ir ( late infection ) when the cells had reentered the cell cycle . the goal of this experiment was to mimic and evaluate the possibility of h-1pv virotherapy of recurrent tumors after completion of radiation therapy as part of the initial standard treatment . upon late infection with h-1pv with an moi of 5 pfu / cell , all cells ( nch-37 , nch-82 , and nch-89 ) showed a significant ( p < .001 ) reduction of surviving cells 3 days p.i .. cell viability of infected cells was 65.04 ( + /11.5 ) % in nch-37 cultures , 67.00 ( + /11.9 ) % in nch-82 cells , and 61.04 ( + /13.8 ) % in nch-89 cell cultures indicating intact susceptibility to h-1pv induced cell killing ( figure 1 ) . nch-307 recurrent glioma cells were infected with a low ( 5 pfu / cell ) and a high ( 100 pfu / cell ) moi ( figure 1 ) . cell viability of nch-307 cells was significantly ( p < .001 ) reduced to 55.39 ( + / 6.6 ) % ( low moi ) and 25.97 ( + / 8.8 ) % ( high moi ) indicating dose - dependent cytotoxicity of h-1pv also in recurrent glioma cells . in initial experiments , the effect of radiation therapy or h-1pv infection alone was examined prior to testing combination treatment . at radiation - doses of 5 gy , growth rates in all cell lines ( nch-37 , nch-82 , nch-89 ) were only slightly affected : cell viability was 70 ( + /9.9 ) % in nch-37 , 76 ( + /4.5 ) % in nch-82 , and 91 ( + /7.0 ) % in nch-89 . nch-37 cells were much less sensitive , the cell viability was reduced to 54.25 ( + /7.2 ) % . a dose of 20 gy had a slightly stronger effect in all cell cultures : nch-82 21.53 ( + /3.8 ) % and nch-89 15.93 ( + /5.6 ) % cell viability , however in nch-37 cultures 45.19 ( + /5.6 ) % of cells were still alive ( figure 2 ) . the infection of glioma cells with h-1pv at an moi of 5 pfu / cell had a strong cytopathic effect in nch-82 and nch-89 cell cultures , with only 22.19 ( + / 3.0 ) % ( nch-82 ) and 9.73 ( + / 2.1 ) % ( nch-89 ) cell viability . nch-37 cells were less sensitive to h-1pv ; cell viability was 45.94 ( + / 6.0 ) % ( figure 2 ) . to assess whether the combination of radiation therapy and h-1pv virotherapy increases the therapeutic efficacy of either treatment alone and to evaluate the influence of the order of treatments on cytotoxicity , we performed two separate experiments : ( i ) glioma cells were irradiated with 3 different doses and infected 1 day after ir ( figure 2 , combination treatment : ir h-1 ) and ( ii ) glioma cells were infected first and subsequently irradiated with a dose of 10 gy 24 hours p.i . two - way - anova showed that in all cell lines tested ( nch-37 , nch-82 , nch-89 ) , both independent factors ( ir and h-1pv infection ) had a significant influence on the number of surviving cells . there were significant interactions between ir and h-1pv infection for all of the data presented in figure 2 . comparative analyses between groups revealed the following : in all glioma cell cultures ( nch-37 , nch-82 , nch-89 ) , combination of h-1pv infection 1 day after ir was significantly ( p < .05 ) more effective than ir alone ( figure 2 ) . compared with h-1pv infection alone , combination treatment was significantly ( p < .05 ) more effective after previous ir with 5 gy , 10 gy , or 20 gy in nch-37 cells and after previous ir with 20 gy in nch-82 cells . when the order of treatments was reversed and h-1pv infection was performed 24 hours prior to ir , combination treatment only led to significantly ( p < .05 ) improved cell killing in nch-37 when compared to ir alone , but not when compared to h-1pv infection alone or in the other cell lines tested . even though high - dose radiation of nch-37 , nch-82 , and nch-89 cells with 20 gy or infection with h-1pv was highly cytotoxic , approximately 2 weeks after single treatment with ir or h-1pv alone , all cell lines resumed to proliferate from surviving clones , albeit at a much reduced rate ( table 1 ) . in contrast , when glioma cell cultures were treated with the combination of ir ( 20 gy ) and h-1pv infection ( moi = 5 pfu / cell ) 24 hours after ir , no surviving tumor cells could be observed on day 21 p.i . replication of h-1pv in infected glioma cells was tested ( i ) by the presence of viral ns-1-specific rna by rt - pcr , ( ii ) by the detection of the viral protein ns-1 as the main effector of parvoviral cytotoxicity by facs analysis , and ( iii ) by the release of infectious viral particles into the supernatant of cell cultures . ( i ) detection of viral rna : cells ( nch-37 , nch-82 , and nch-89 ) were irradiated with 10 gy and infected with h-1pv at an moi of 5 pfu / cell 24 hours later . rt - pcr was positive for the presence of viral rna indicating transcription from viral dna ( figure 4(e ) ) . ( ii ) expression of ns-1 protein : irradiated ( 10 gy ) or untreated control cells were either h-1pv infected ( moi = 5 pfu / cell ) or mock - infected 24 hours post - ir ( early infection ) or 9 days post - ir ( late infection ) . recurrent glioblastoma cells ( nch-307 ) were infected with an moi of 5 pfu / cell without additional ir . ns-1 protein expression of unirradiated glioma cells ranged from 50% of ns-1-positive nch-37 cells to 43% in nch-82 cells and 42% in nch-89 cells 24 hours p.i . the recurrent glioblastoma cell line nch-307 showed ns-1 expression in 44% of cells 24 hours p.i . when cells were irradiated with 10 gy and infected with h-1pv 24 hours ( early infection ) or 9 days ( late infection ) after ir , ratios of ns-1 expression 24 hours p.i . ( iii ) production of infectious h-1 virus particles : in order to assess whether cytopathic h-1pv infection of irradiated glioma cells resulted in the production of infectious progeny particles , virus yields were determined by titration on highly susceptibly rg2 cells . one possible mechanism for an improved cytotoxicity of h-1pv infection after ir could be associated to changes of the cell cycle as h-1pv replication is restricted to cells in s - phase . we therefore analyzed the effect of ir on the cell cycle of glioma cell lines ( nch-37 , nch-82 , nch-89 ) . ir of glioma cell cultures with 10 gy resulted in an increase of cells in s - phase 24 hours post - ir ( figure 5 ) . the increase was transient as 48 hours post - ir ; the amount of cells in the s - phase decreased to + 12% in nch-37 , to control level in nch-82 , and to + 10% in nch-89 . in parallel , at this time , the overall cell cycle distribution of nch-37 and nch-89 cells had almost returned to control levels , whereas nch-82 cells were blocked in g2/m ( figure 5(a ) ) and reached control levels 4 days post - ir ( data not shown ) . in comparison , upon h-1pv infection , cell cultures showed less homogeneous results . while nch-82 and nch-89 cells that were most sensitive to h-1 infection showed increased ratios of cells in s - phase , the amount of less sensitive nch-37 cells in s - phase was unchanged . the cell cycle changes after combination of radiation therapy and h-1pv infection were identical to h-1pv infection alone ( data not shown ) . this led to numerous attempts not only to further improve the oncolytic activity of viruses but also to investigate the combination treatment of viral infection of tumor cells with established conventional therapies . as radiation therapy is known to induce long - term changes in cellular genomes , this could potentially lead to an altered effect of virus infection compared with unirradiated primary glioma cells . in addition to other studies , we therefore specifically investigated the oncolytic potential of h-1pv in glioma cells that grew from irradiated clones . a cell culture established from a recurrent glioma that was irradiated with 56 gy during the initial treatment of the patient , and that had its origin in the radiation field , was fully permissive to h-1pv infection and cell killing was dose - dependent . this finding was confirmed in primary glioma cultures that were irradiated in vitro with a sublethal dose , allowed to regrow , and infected with h-1pv in a time interval of several days . the intact susceptibility of glioma cells to h-1pv infection after ir is of clinical significance as patients with recurrent gliomas who face an even worse prognosis with oftentimes less therapeutic options are prime candidates for experimental therapies . as a consequence , the group of patients with recurrent gliomas is usually the main patient population of early clinical trials of oncolytic virotherapy . however , to our knowledge the response of previously irradiated glioma cells to the oncolytic infection has never been specifically addressed for other oncolytic viruses . when h-1pv infection was performed early after ir , our data show improved killing of glioma cells , most pronounced in the most radioresistant cell line tested . these findings are in line with studies conducted with other oncolytic viruses that is , herpesvirus , adenovirus , reovirus , and measles virus that also showed an enhanced efficacy of viral oncolysis in combination with radiation therapy . whether this effect can also be demonstrated in vivo was beyond this proof of concept study and when radiation treatment was performed one day before h-1pv infection , combination treatment was significantly better in all cell lines than single treatment . one possible reason for the improved cytotoxicity of h-1pv after ir is the increased level of glioma cells positive for ns-1 expression 24 hours after early infection . previous studies revealed that ns-1 is the key - mediator of parvoviral cytotoxicity and its expression is strongly s - phase dependent [ 14 , 25 ] . cell cycle analyses revealed that in all primary glioma cell cultures tested , the rate of cells in s - phase was increased 24 hours post - ir . even though improved viral cytotoxicity may depend on several factors , this altered cell cycle distribution supports the finding of increased viral transcription and increased cell killing . as a consequence , when cells were infected when the cell cycle had returned to control levels ( late infection ) , ns-1 expression decreased . recent data suggests that stem - like cells exist within high - grade gliomas which are radioresistant and capable of initiating tumour regrowth . these results may warrant to test whether the combination of radiation and h-1pv infection could also overcome the resistance of glioma cells expressing stem cell markers thereby offering a new treatment opportunity in these therapy refractory cells . in conclusion , irradiated glioma - cells show intact susceptibility for h-1pv infection with even improved cell killing by combining ir with h-1pv , most pronounced in radioresistant glioma cells . these results further support the ongoing development of a phase - i clinical trial for the use of h-1pv in malignant gliomas , allowing for the inclusion of pretreated patients into the study population .

as a consequence , one new strategy is the use of replication competent oncolytic viruses that specifically target and destroy tumor cells while leaving normal cells intact . a number of candidate oncolytic viruses for glioma therapy are currently under investigation including genetically modified herpesviruses , adenoviruses , or poliovirus , and wildtype viruses such as reovirus , vesicular - stomatitis virus , and measles virus . we previously reported the efficient killing of glioma cells of human and rat origin by parvovirus h-1 ( h-1pv ) , a single stranded nonenveloped dna virus of 5.1 kb . h-1pv induced lytic infection of glioma cells even when the cells were resistant to agents inducing apoptosis [ 12 , 13 ] . the natural host of h-1pv is the rat ; however , the virus can efficiently infect cells of other species including humans . radiation therapy prolongs survival in patients with malignant gliomas and is used as standard treatment of primary high - grade glial tumors . however , as high grade gliomas are resistant to radiation therapy and a clear dose - limitation exists due to cytotoxic effects on the surrounding brain tissue , this treatment modality is not curative and strategies to improve radiation efficiency are under investigation . the combination of radiation therapy with the oral alkylating agent temozolomide has already proved to be superior to either therapy alone and has become the standard of care for the majority of patients with newly diagnosed glioblastoma multiforme [ 3 , 5 ] . recent studies of the role of radiation therapy for recurrent gliomas that were already irradiated as part of the primary treatment demonstrated some effect when radiation was applied as a boost to smaller tumor regions . therefore , depending on the individual situation of the patient , radiation therapy can also be an option for recurrent tumors , but methods to augment the limited therapeutic effect would clearly be beneficial . clinical trials with different oncolytic viruses were able to demonstrate the safety of this novel therapeutic approach ; however , the positive therapeutic effects were restricted to individual patients . therefore , the combination of oncolytic viruses with standard therapeutics has become one important focus to improve viral cytotoxicity . radiation therapy is a part of therapeutic protocols for the majority of malignancies , and an enhanced effect of the oncolytic activity of viruses by radiation therapy could be observed for tumor cells of various histology . for glioma cells , the oncolytic effect of herpesvirus , adenoviruses , reovirus , and measles virus the aim of this study was to assess the influence of ir on the oncolytic activity of h-1pv in glioma cells . the possible interaction of ir with h-1pv oncolytic virotherapy could be twofold : ( i ) as the use of an oncolytic virus in glioma patients would preferably include the treatment of recurrent tumors originating from previously irradiated remaining tumor cells , it has to be shown whether previous radiation therapy would interfere with viral oncolysis or replication in pretreated gliomas and ( ii ) administration of radiation therapy together with h-1pv oncolytic virotherapy could lead to improved efficacy of either treatment alone . we therefore investigated the treatment of early - passage glioma cells with ir before or after infection with h-1pv and assessed cytotoxicity , viral replication , and treatment - induced changes of the cell cycle . these findings are important to define patient populations for a clinical trial of oncolytic virotherapy of malignant gliomas and to possibly use h-1pv to increase radiation efficacy in this dismal tumor entity . human short term cultures derived from histologically confirmed glioblastomas ( nch-82 , nch-89 , nch-307 ) and a gliosarcoma ( nch-37 ) were established and characterized at the department of neurosurgery , heidelberg , germany as described previously . radiation of cell cultures was performed at room temperature in a linear accelerator ( siemens mevatron kd2 , 6-mv photons ) at doses of 5 gy,10 gy , or 20 gy as indicated for the respective experiment ( dose rate : 3 gy / min ; distance from source to flask : 95 cm ) . nch-307 monolayers of all glioma cell cultures were infected under standard conditions : cells were inoculated with h-1pv diluted in pbs at a multiplicity of infection ( moi ) of 5 plaque forming units ( pfu ) per cell or 100 pfu / cell as indicated for the respective experiment . to asses cell viability , glioma cells ( nch-37 , nch-82 , nch-89 ) were seeded at 3 10 cells / well into 12-well dishes and irradiated after 24 hours with 0 gy , 5 gy , 10 gy , or 20 gy . cells were infected ( moi = 5 pfu / cell or mock - infection ) at 2 different time points : 24 hours post - ir ( early infection ) or 9 days post - ir ( late infection ) . in vivo irradiated nch-307 cells were seeded at 3 10 cells / well into 12-well dishes and infected ( moi = 5 pfu / cell , 100 pfu / cell or mock - infection ) 24 hours postseeding . cell viability ( in % + / standard error ) was defined as the number of living treated cells over the number of living untreated cells multiplied by 100 ; standard error was calculated using the gaussian law of error propagation . for rt - pcr analysis of viral rna , cells ( nch-37 , nch-82 , nch-89 ) were plated at a density of 3 10 cells in 17 cm culture flasks , irradiated ( 10 gy ) after 24 hours and infected ( moi = 5 pfu / cell or mock - infection ) 24 hours post - ir ; nch-307 cells were infected ( moi = 5 pfu / cell or mock - infection ) 24 hours postseeding . cells ( nch-37 , nch-82 , nch-89 ) were plated at a density of 3 10 cells in 17 cm culture flasks , irradiated ( 0 gy , 10 gy ) after 24 hours and infected ( moi = 5 pfu / cell or mock - infection ) 24 hours post - ir ( early infection ) . cells ( nch-37 , nch-82 , nch-89 ) were seeded at a density of 1.5 10 cells / well in 6-well dishes , irradiated 24 hours postseeding ( 10 gy ) , and infected ( moi = 5 pfu / cell or mock - infection ) 24 hours post - ir . cells ( nch-37 , nch-82 , nch-89 ) were plated at a density of 3 10 cells in 17 cm culture flasks and irradiated ( 0 cells were harvested 24 hours and 48 hours post - ir and fixed in 80% ice - cold ethanol at 4c overnight . after fixation , cells were incubated in 2 mg / ml rnase ( sigma - aldrich , taufkirchen , germany ) , and 0,1 mg / ml pi ( sigma - aldrich , taufkirchen , germany ) for 30 minutes in the dark . as previous radiation therapy of tumor cells induces genetic alterations that could interfere with the susceptibility and efficiency of h-1pv infection , we infected glioma cells with a delay of 9 days after ir ( late infection ) when the cells had reentered the cell cycle . the goal of this experiment was to mimic and evaluate the possibility of h-1pv virotherapy of recurrent tumors after completion of radiation therapy as part of the initial standard treatment . in addition to testing tumor cells from primary gliomas ( nch-37 , nch-82 , nch-89 ) , we also included nch-307 cells that were established from a recurrent glioblastoma that had been irradiated several months prior to secondary surgical resection . however , the growth rate was clearly reduced compared with untreated controls indicating persisting long - term effects of the treatment . upon late infection with h-1pv with an moi of 5 pfu / cell , all cells ( nch-37 , nch-82 , and nch-89 ) showed a significant ( p < .001 ) reduction of surviving cells 3 days p.i .. cell viability of infected cells was 65.04 ( + /11.5 ) % in nch-37 cultures , 67.00 ( + /11.9 ) % in nch-82 cells , and 61.04 ( + /13.8 ) % in nch-89 cell cultures indicating intact susceptibility to h-1pv induced cell killing ( figure 1 ) . nch-307 recurrent glioma cells were infected with a low ( 5 pfu / cell ) and a high ( 100 pfu / cell ) moi ( figure 1 ) . cell viability of nch-307 cells was significantly ( p < .001 ) reduced to 55.39 ( + / 6.6 ) % ( low moi ) and 25.97 ( + / 8.8 ) % ( high moi ) indicating dose - dependent cytotoxicity of h-1pv also in recurrent glioma cells . at radiation - doses of 5 gy , growth rates in all cell lines ( nch-37 , nch-82 , nch-89 ) were only slightly affected : cell viability was 70 ( + /9.9 ) % in nch-37 , 76 ( + /4.5 ) % in nch-82 , and 91 ( + /7.0 ) % in nch-89 . ir with 10 gy had a strong effect on nch-82 and nch-89 cells with a cell viability of 25.64 ( + /1.8 ) % ( nch-82 ) and 22.81 ( + /4.7 ) % ( nch-89 ) . a dose of 20 gy had a slightly stronger effect in all cell cultures : nch-82 21.53 ( + /3.8 ) % and nch-89 15.93 ( + /5.6 ) % cell viability , however in nch-37 cultures 45.19 ( + /5.6 ) % of cells were still alive ( figure 2 ) . the infection of glioma cells with h-1pv at an moi of 5 pfu / cell had a strong cytopathic effect in nch-82 and nch-89 cell cultures , with only 22.19 ( + / 3.0 ) % ( nch-82 ) and 9.73 ( + / 2.1 ) % ( nch-89 ) cell viability . to assess whether the combination of radiation therapy and h-1pv virotherapy increases the therapeutic efficacy of either treatment alone and to evaluate the influence of the order of treatments on cytotoxicity , we performed two separate experiments : ( i ) glioma cells were irradiated with 3 different doses and infected 1 day after ir ( figure 2 , combination treatment : ir h-1 ) and ( ii ) glioma cells were infected first and subsequently irradiated with a dose of 10 gy 24 hours p.i . two - way - anova showed that in all cell lines tested ( nch-37 , nch-82 , nch-89 ) , both independent factors ( ir and h-1pv infection ) had a significant influence on the number of surviving cells . comparative analyses between groups revealed the following : in all glioma cell cultures ( nch-37 , nch-82 , nch-89 ) , combination of h-1pv infection 1 day after ir was significantly ( p < .05 ) more effective than ir alone ( figure 2 ) . compared with h-1pv infection alone , combination treatment was significantly ( p < .05 ) more effective after previous ir with 5 gy , 10 gy , or 20 gy in nch-37 cells and after previous ir with 20 gy in nch-82 cells . when the order of treatments was reversed and h-1pv infection was performed 24 hours prior to ir , combination treatment only led to significantly ( p < .05 ) improved cell killing in nch-37 when compared to ir alone , but not when compared to h-1pv infection alone or in the other cell lines tested . even though high - dose radiation of nch-37 , nch-82 , and nch-89 cells with 20 gy or infection with h-1pv was highly cytotoxic , approximately 2 weeks after single treatment with ir or h-1pv alone , all cell lines resumed to proliferate from surviving clones , albeit at a much reduced rate ( table 1 ) . in contrast , when glioma cell cultures were treated with the combination of ir ( 20 gy ) and h-1pv infection ( moi = 5 pfu / cell ) 24 hours after ir , no surviving tumor cells could be observed on day 21 p.i . or at later time points after treatment in any of the tested cell cultures ( nch-37 , nch-82 , nch-89 ) indicating long - term efficiency of combination treatment ( table 1 and figure 3 ) . replication of h-1pv in infected glioma cells was tested ( i ) by the presence of viral ns-1-specific rna by rt - pcr , ( ii ) by the detection of the viral protein ns-1 as the main effector of parvoviral cytotoxicity by facs analysis , and ( iii ) by the release of infectious viral particles into the supernatant of cell cultures . ( i ) detection of viral rna : cells ( nch-37 , nch-82 , and nch-89 ) were irradiated with 10 gy and infected with h-1pv at an moi of 5 pfu / cell 24 hours later . ( ii ) expression of ns-1 protein : irradiated ( 10 gy ) or untreated control cells were either h-1pv infected ( moi = 5 pfu / cell ) or mock - infected 24 hours post - ir ( early infection ) or 9 days post - ir ( late infection ) . ns-1 protein expression of unirradiated glioma cells ranged from 50% of ns-1-positive nch-37 cells to 43% in nch-82 cells and 42% in nch-89 cells 24 hours p.i . when cells were irradiated with 10 gy and infected with h-1pv 24 hours ( early infection ) or 9 days ( late infection ) after ir , ratios of ns-1 expression 24 hours p.i . ( iii ) production of infectious h-1 virus particles : in order to assess whether cytopathic h-1pv infection of irradiated glioma cells resulted in the production of infectious progeny particles , virus yields were determined by titration on highly susceptibly rg2 cells . as demonstrated in table 2 , a 10 log - fold higher virus titer could be detected compared with input virus within 3 days after infection irrespective if cells were irradiated ( 10 gy ) or not ( 0 gy ) . results were similar in all cell lines tested ( nch-37 , nch-82 , nch-89 ) , demonstrating persisting assembly of progeny virus after ir . one possible mechanism for an improved cytotoxicity of h-1pv infection after ir could be associated to changes of the cell cycle as h-1pv replication is restricted to cells in s - phase . we therefore analyzed the effect of ir on the cell cycle of glioma cell lines ( nch-37 , nch-82 , nch-89 ) . ir of glioma cell cultures with 10 gy resulted in an increase of cells in s - phase 24 hours post - ir ( figure 5 ) . the increase was transient as 48 hours post - ir ; the amount of cells in the s - phase decreased to + 12% in nch-37 , to control level in nch-82 , and to + 10% in nch-89 . in parallel , at this time , the overall cell cycle distribution of nch-37 and nch-89 cells had almost returned to control levels , whereas nch-82 cells were blocked in g2/m ( figure 5(a ) ) and reached control levels 4 days post - ir ( data not shown ) . while nch-82 and nch-89 cells that were most sensitive to h-1 infection showed increased ratios of cells in s - phase , the amount of less sensitive nch-37 cells in s - phase was unchanged . in early phase clinical trials , intracerebral infection of patients with oncolytic viruses of different genera however , only in a few patients tumor regression and a prolongation of progression - free survival could be demonstrated . this led to numerous attempts not only to further improve the oncolytic activity of viruses but also to investigate the combination treatment of viral infection of tumor cells with established conventional therapies . in addition to other studies , we therefore specifically investigated the oncolytic potential of h-1pv in glioma cells that grew from irradiated clones . a cell culture established from a recurrent glioma that was irradiated with 56 gy during the initial treatment of the patient , and that had its origin in the radiation field , was fully permissive to h-1pv infection and cell killing was dose - dependent . the intact susceptibility of glioma cells to h-1pv infection after ir is of clinical significance as patients with recurrent gliomas who face an even worse prognosis with oftentimes less therapeutic options are prime candidates for experimental therapies . as a consequence , the group of patients with recurrent gliomas is usually the main patient population of early clinical trials of oncolytic virotherapy . however , to our knowledge the response of previously irradiated glioma cells to the oncolytic infection has never been specifically addressed for other oncolytic viruses . these findings are in line with studies conducted with other oncolytic viruses that is , herpesvirus , adenovirus , reovirus , and measles virus that also showed an enhanced efficacy of viral oncolysis in combination with radiation therapy . whether this effect can also be demonstrated in vivo was beyond this proof of concept study and when radiation treatment was performed one day before h-1pv infection , combination treatment was significantly better in all cell lines than single treatment . cell cycle analyses revealed that in all primary glioma cell cultures tested , the rate of cells in s - phase was increased 24 hours post - ir . these results may warrant to test whether the combination of radiation and h-1pv infection could also overcome the resistance of glioma cells expressing stem cell markers thereby offering a new treatment opportunity in these therapy refractory cells . in conclusion , irradiated glioma - cells show intact susceptibility for h-1pv infection with even improved cell killing by combining ir with h-1pv , most pronounced in radioresistant glioma cells . these results further support the ongoing development of a phase - i clinical trial for the use of h-1pv in malignant gliomas , allowing for the inclusion of pretreated patients into the study population .

animal use was approved by the institutional animal care and use committees at the university of oklahoma health sciences center , oklahoma city , oklahoma , and the university of houston and conformed to the arvo statement for the use of animals in ophthalmic and vision research . the cngb1 line , the rds line ( originally obtained from neeraj agarwal currently at the national eye institute , bethesda , md , usa ) , and the rho line ( originally obtained from janice lem , tufts university , boston , ma , usa ) have been described previously . cngb1 mice on the rds , rds , rho , and rho backgrounds were generated in our facility . wild - type ( i.e. , cngb1/ rds / rho ) , rds , rds , rho , and rho littermates were used from our colony as controls . all animals were maintained in cyclic light ( 12 hours light , 12 hours dark , 30 lux ) and fed standard lab chow . experiments were repeated three to seven times , and densitometry was done using the image lab software ( bio - rad , temecula , ca , usa ) . eyes for immunofluorescence were harvested , fixed , sectioned , and immunolabeled as previously described . primary antibodies are described in the table , and anti - mouse , anti - rabbit , or anti - goat alexafluor conjugated secondary antibodies ( life technologies , grand island , ny , usa ) were used . images were captured on an olympus bx-62 microscope ( center valley , pa , usa ) equipped with a spinning disc confocal unit and analyzed as described previously . antibodies used for immunofluorescence and western blotting tissue collection , processing , and plastic embedding and transmission electron microscopy ( tem ) have been described previously . light microscopy images were captured at 40 using a zeiss ( peabody , ma , usa ) universal microscope . to evaluate outer nuclear layer ( onl ) and os layer thickness , images were captured from central superior and inferior regions containing the optic nerve head , and at least three retinal sections from two to four eyes / genotype were used . assessment of rod photoreceptor function ( scotopic erg ) was performed on dark - adapted animals with a strobe flash stimulus of 157 cd - s / m intensity while photopic responses were recorded using a similar stimulus from light - adapted animals . differences between genotypes were assessed by 1-way or 2-way anova with bonferroni 's post hoc pairwise comparisons . p < 0.05 was considered significant ( p < 0.05 , p < 0.01 , and p < 0.001 ) . animal use was approved by the institutional animal care and use committees at the university of oklahoma health sciences center , oklahoma city , oklahoma , and the university of houston and conformed to the arvo statement for the use of animals in ophthalmic and vision research . the cngb1 line , the rds line ( originally obtained from neeraj agarwal currently at the national eye institute , bethesda , md , usa ) , and the rho line ( originally obtained from janice lem , tufts university , boston , ma , usa ) have been described previously . cngb1 mice on the rds , rds , rho , and rho backgrounds were generated in our facility . wild - type ( i.e. , cngb1/ rds / rho ) , rds , rds , rho , and rho littermates were used from our colony as controls . all animals were maintained in cyclic light ( 12 hours light , 12 hours dark , 30 lux ) and fed standard lab chow . experiments were repeated three to seven times , and densitometry was done using the image lab software ( bio - rad , temecula , ca , usa ) . eyes for immunofluorescence were harvested , fixed , sectioned , and immunolabeled as previously described . primary antibodies are described in the table , and anti - mouse , anti - rabbit , or anti - goat alexafluor conjugated secondary antibodies ( life technologies , grand island , ny , usa ) were used . images were captured on an olympus bx-62 microscope ( center valley , pa , usa ) equipped with a spinning disc confocal unit and analyzed as described previously . tissue collection , processing , and plastic embedding and transmission electron microscopy ( tem ) have been described previously . light microscopy images were captured at 40 using a zeiss ( peabody , ma , usa ) universal microscope . to evaluate outer nuclear layer ( onl ) and os layer thickness , images were captured from central superior and inferior regions containing the optic nerve head , and at least three retinal sections from two to four eyes / genotype were used . layer thickness was measured using adobe photoshop cs5 ( adobe systems , inc . , san jose , ca , usa ) . assessment of rod photoreceptor function ( scotopic erg ) was performed on dark - adapted animals with a strobe flash stimulus of 157 cd - s / m intensity while photopic responses were recorded using a similar stimulus from light - adapted animals . differences between genotypes were assessed by 1-way or 2-way anova with bonferroni 's post hoc pairwise comparisons . p < 0.05 was considered significant ( p < 0.05 , p < 0.01 , and p < 0.001 ) . previously we evaluated the interplay between rds and rhodopsin in os morphogenesis , and here we wanted to understand the effects of eliminating cngb1 at the same time as either rhodopsin or rds . we crossed the cngb1 line , which lacks all cngb1 gene products including cngb1a and garp1/2 , onto the rds , rds , rho , and rho backgrounds ( which were all used as control lines for the purpose of comparison throughout the study ) . at postnatal day ( p ) 30 we assessed levels of rhodopsin , rds , and rom-1 by reducing sds - page / western blot . data are presented as percentage of wt after normalization to actin ( a loading control ) ( figs . c ) and are plotted as means sem ( p < 0.05 , p < 0.01 , and p < 0.001 by 1-way anova with bonferroni 's post hoc comparison ) . though rhodopsin levels were virtually normal in the cngb1 and the rds ( fig . 1a ) , they were reduced in cngb1/rds to 56% of levels in the rds . levels of rhodopsin in the rds were reduced by 84% and were not further reduced by ablating cngb1 . retinal extracts were isolated from p30 eyes of the indicated genotypes and were analyzed by reducing sds - page / western blot . the blots were probed with ( a ) mab 1d4 against rhodopsin , ( b ) anti - rds - ct , and ( c ) anti - rom-1-ct . protein was quantified densitometrically , normalized to actin , and plotted as a percentage of wt ( shown are means sem , * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 by 1-way anova with bonferroni 's post hoc comparison ) . nonreducing sucrose gradient velocity sedimentation ( d , e ) was performed on p30 retinal extracts from wt and cngb1 . fractions from gradients ( 112 ) were further separated by sds - page / reducing western blot . resulting blots were probed with anti - rds - ct ( d ) and anti - rom-1-ct ( e ) . the relative percent of total rds or rom-1 in each fraction was assessed densitometrically and plotted as mean sem from 3 or 4 independent experiments . this more pronounced effect of cngb1 ablation in the rds compared to the rho continued when we examined rds levels ( fig . in cngb1/rds retinas , rds levels were reduced to 54% of those in the rds while rds levels in cngb1/rho remained at 87% of those in the rho . rod outer segment membrane protein-1 levels were not altered in either the cngb1/rds versus the rds or the cngb1/rho versus the rho ( fig . we also observed that rds and rom-1 are severely reduced by the coablation of cngb1 and rhodopsin ( cngb1/rho had rds / rom-1 levels that were 8.8% and 7.9% those of rho , respectively ) . cyclic nucleotide gated channel b1a is known to bind to rds in the os , and proper binding and complex assembly of rds / rom-1 are necessary for os formation , so we asked whether eliminating cngb1 altered rds / rom-1 oligomerization . retinal extracts from wt and cngb1 animals were fractionated on 5% to 20% nonreducing sucrose gradients and then separated on reducing sds - page . in the wt retina , rds is present as tetramers ( fractions 68 ) , octamers ( fractions 45 ) , and higher - order oligomers ( fractions 13 ) while rom-1 is detected only in tetrameric and octameric fractions . 1e ) complex formation in cngb1 retinas , suggesting that rds / rom-1 oligomerization is not tied to cngb1 . previously we observed that cngb1a and garp2 protein levels are reduced by 50% in the rds and are undetectable in the rds . when combined with our data here showing that rds levels are decreased in the cngb1/rds compared to the rds cyclic nucleotide gated channel b1a and rds binding occurs during the os targeting process prior to arrival in the os , so we next used immunofluorescence labeling to ask whether the targeting of rds , rom-1 , rhodopsin , or cngb1a / garp1/2 is affected by co - elimination of cngb1 , rhodopsin , or rds . retinal degeneration slow and rom-1 labeling was properly restricted to the os layer in the wt , cngb1 , rds , cngb1/rho , rho , and cngb1/rho retinas ( fig . 2a , green ) . similarly , rhodopsin localization is not affected in most of these genotypes ; however , some accumulation of rhodopsin in the onl is observed in the cngb1/rho ( fig . large amounts of rhodopsin accumulate in the inner segment and onl , and this is recapitulated in the cngb1/rds ( fig . in contrast , in the rho and cngb1/rho retinas , the majority of rds is not found in the inner segment , but is properly restricted to the distal tips of the photoreceptors , consistent with our previous observation showing that rds and rom-1 are found in the small nascent oss of the rho . however , in the rho and cngb1/rho retinas , some mild mislocalization of rds ( but not rom-1 ) to the inner segment , onl , and outer plexiform layer is seen ( arrows , fig . combined , these data suggest that while rhodopsin is more susceptible to mislocalization than rds , cngb1 ablation has little to no effect on the os targeting of rds , rom-1 , or rhodopsin . eliminating cngb1 does not affect targeting of rds or rhodopsin . paraffin - embedded retinal sections from p30 animals of the indicated genotypes were labeled ( a ) with antibodies against rds ( rds - ct , green ) , rhodopsin ( mab-1d4 , red ) , or rom-1 ( rom1-ct , green ) ; ( b ) mab 1d4 ( red ) or rom1-ct ( green ) ; ( c ) rds - ct or rom1-ct ( green ) , or ( d ) cngb1 gene products ( garp-4b1 , red ) . in all cases nuclei os , outer segment ; onl , outer nuclear layer ; is , inner segment ; opl , outer plexiform layer . conversely , to see whether reduction / elimination of rds or rhodopsin affected cngb1a / garp1/2 targeting , we labeled with garp-4b1 , which recognizes all cngb1 gene products ( fig . 2d , red ) . in wt , rds , and rho retinas , cngb1a / garp1/2 properly localized to the os layer . this observation was replicated in the rho retina ; cngb1a / garp1/2 properly localized to the os layer , even though the rho does not form fully elaborated oss . in striking contrast , and consistent with our western blot data , in the absence of rds ( rds ) , cngb1a and garp1/2 are virtually undetectable in the retina . this decrease in cngb1 gene products in the absence of rds is clearly not due to a requirement for proper os formation , since the oss in the rho retina , while more formed than in the rds , are still just tiny sacs of vesicle - containing membrane at the tip of the os , yet the rho model still expresses cngb1a / garp1/2 . rather it suggests that the stability or transport of cngb1a and/or garp 1/2 are dependent directly on rds or on the initiation / formation of oss by rds ( which can be seen in nascent form even in the rho ) . however , though the differences do not attain statistical significance , mean onl thickness in the cngb1 , cngb1/rds , and cngb1/rho was decreased by 10% to 15% compared to their respective controls ( wt , rds , and rho ) , suggesting that degeneration in the cngb1 starts earlier than that in the rds and rho . there is no difference in onl thickness in rho versus cngb1/rho or rds versus cngb1/rds , suggesting that the photoreceptor degeneration in the rds and rho is not additive with that in cngb1 . representative light micrographs ( lm ) were captured from plastic - embedded retinal sections harvested at p30 from the indicated genotypes . rpe , retinal pigment epithelium ; os , outer segment ; is , inner segment ; onl , outer nuclear layer . ( b , c ) outer nuclear layer and os layer thicknesses were measured in sections captured from the central superior and central inferior retina from 2 to 4 eyes per genotype . plotted are means sem , * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 by 2-way anova with bonferroni 's post hoc comparison . it was previously shown that oss are shorter and more disorganized in the cngb1 and rds retinas compared to wt , and our data confirm this ( fig . 3c ) . interestingly , this effect was additive : in the cngb1/rds , mean os length was 41% of wt , compared to 52% and 73% of wt for rds and cngb1 , respectively ( fig . as expected , at p30 , oss in the rho are similar to those in wt , but we do see os shortening in the cngb1/rho due to the absence of cngb1a ; oss in the rho are 89% of wt , while those in the cngb1/rho are 59% of wt . again , the effects of eliminating cngb1a from the rds retina are more severe than in the rho retina ; for example , oss in the cngb1/rds are decreased to 56% of those in cngb1 ( p < 0.05 ) , while the oss of the cngb1/rho are 81% of cngb1 ( not significant ) . figure 4a shows representative waveforms , and maximum saturating a- and b - wave amplitudes are plotted in figure 4b ( mean sem , p < 0.001 by 1-way anova with bonferroni 's post hoc comparison ) . our results for control animals ( cngb1 , rds , and rho ) are consistent with previous publications ( fig . 4b ) . again , elimination of cngb1 had a much more severe effect in the rds background than it did in the rho background . scotopic a - wave values in the cngb1/rho were not significantly reduced compared to the cngb1 , suggesting that all of the functional decrease in the cngb1/rho is due to the absence of cngb1 . in contrast , in the cngb1/rds , scotopic erg responses were severely reduced compared to both cngb1 and rds ( fig . this effect was seen in both the scotopic a- and b - waves , and scotopic a - waves in the cngb1/rds were virtually undetectable . scotopic erg function is undetectable in the rds and rho retina at p30 , so we did not perform ergs on animals in those backgrounds . the additive erg defect in the cngb1/rds could be due to reductions in cnga1 ; however , levels of cnga1 in cngb1 are virtually undetectable , and this finding is recapitulated in cngb1/rds ( supplementary fig . thus while it is possible that further reduction in cnga1 levels contributes to the additive defects seen in the cngb1/rds , we can not conclusively assess the possibility given that the levels of cnga1 are below the limit of detection . ( a ) representative waveforms ; ( b ) plots of mean amplitude sem from 4 to 7 mice per genotype ( * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 by 1-way anova with bonferroni 's post hoc comparison ) . ( c ) scale bars : 10 m for low - magnification em images and 500 nm for higher - magnification em images . rpe , retinal pigment epithelium ; os , outer segment ; cc , connecting cilium . we conducted tem to determine whether the severe effects of simultaneously eliminating cngb1 and reducing rhodopsin / rds led to defects in os ultrastructure ( fig . the os shortening we quantified on the light microscopy level in the cngb1 is also evident by low - magnification em ( 3000 , top , fig . 4c ) , we find that the disc diameter and often the disc alignment are abnormal in the cngb1 retina . in some cases , new discs grew abnormally parallel to the plasma membrane ( arrow , fig . outer segments in the rds were characterized by abnormal disc size and the formation of highly dysmorphic whorl structures , but interestingly , this phenotype was not worsened in the cngb1/rds retina . outer segment structure in the cngb1/rho was worse than in the rho ( which is largely normal ) . the phenotype in the cngb1/rho was of the same type seen in the cngb1 , namely , abnormalities in disc size and alignment but not whorl formation ; however , the defects appeared to be more severe in the cngb1/rho compared to the cngb1 . the rds retina forms no os at the tip of the connecting cilia ( fig . 5 , cc marks connecting cilia ) , and the cngb1/rds is not different from the rds . in contrast , the rho forms well - characterized tiny nascent oss ( arrows , fig . similar structures at the distal tip of the connecting cilium were seen in the cngb1/rho ( arrows , fig . outer segment ultrastructure in the rds and rho is not affected by the ablation of cngb1 . scale bars : 10 m for low - magnification em images ( top ) and 500 nm for higher - magnification em images ( bottom ) . though cngb1 is expressed only in rods , secondary cone loss can occur after rod defects . figure 6a shows representative photopic erg waveforms while figure 6b plots mean photopic b - wave amplitudes ( mean sem , p < 0.01 by 1-way anova with bonferroni 's post hoc comparison ) . consistent with previous reports , at this time point significant decreases in cone function are not detected in the rds or cngb1 . likewise , no significant reductions in cone function were noticed in cngb1/rho versus rho or wt . however , a striking 75% reduction in cone function was observed in cngb1/rds compared to wt , rds , and cngb1 . to identify whether this defect was associated with loss of cone cells , m - opsin - positive cones were counted in a region in the superior central retina while s - opsin - positive cones were counted in the inferior central portion of the retina ( representative images in fig . 6c ; fig . consistent with the dramatic decrease in photopic erg response in the cngb1/rds compared to the cngb1 or the rds , we observed a 50% reduction in s - cones in the cngb1/rds compared to cngb1 , rds , or wt controls ( p < 0.001 by 1-way anova ) . photopic ergs were recorded from wt , cngb1 , rds , cngb1/rds , rho , and cngb1/rho at p30 . ( a ) representative waveforms ; ( b ) plots of the mean maximum photopic b - wave amplitude . n = 4 to 7 mice per genotype ( * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 by 1-way anova with bonferroni 's post hoc comparison ) . ( c ) paraffin - embedded retinal sections from p30 animals were labeled with anti s - opsin ( green , top ) and anti - m - opsin ( green , bottom ) and were all counterstained with dapi ( blue ) . os , outer segment ; onl , outer nuclear layer ; opl , outer plexiform layer . ( d ) cells positive for cone opsins were counted in a 300-m region of the central retina either inferior ( s - opsin - positive cells ) or superior ( m - opsin - positive cells ) of the optic nerve . plotted previously we evaluated the interplay between rds and rhodopsin in os morphogenesis , and here we wanted to understand the effects of eliminating cngb1 at the same time as either rhodopsin or rds . we crossed the cngb1 line , which lacks all cngb1 gene products including cngb1a and garp1/2 , onto the rds , rds , rho , and rho backgrounds ( which were all used as control lines for the purpose of comparison throughout the study ) . at postnatal day ( p ) 30 we assessed levels of rhodopsin , rds , and rom-1 by reducing sds - page / western blot . data are presented as percentage of wt after normalization to actin ( a loading control ) ( figs . c ) and are plotted as means sem ( p < 0.05 , p < 0.01 , and p < 0.001 by 1-way anova with bonferroni 's post hoc comparison ) . though rhodopsin levels were virtually normal in the cngb1 and the rds ( fig . 1a ) , they were reduced in cngb1/rds to 56% of levels in the rds . levels of rhodopsin in the rds were reduced by 84% and were not further reduced by ablating cngb1 . retinal extracts were isolated from p30 eyes of the indicated genotypes and were analyzed by reducing sds - page / western blot . the blots were probed with ( a ) mab 1d4 against rhodopsin , ( b ) anti - rds - ct , and ( c ) anti - rom-1-ct . protein was quantified densitometrically , normalized to actin , and plotted as a percentage of wt ( shown are means sem , * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 by 1-way anova with bonferroni 's post hoc comparison ) . nonreducing sucrose gradient velocity sedimentation ( d , e ) was performed on p30 retinal extracts from wt and cngb1 . fractions from gradients ( 112 ) were further separated by sds - page / reducing western blot . resulting blots were probed with anti - rds - ct ( d ) and anti - rom-1-ct ( e ) . the relative percent of total rds or rom-1 in each fraction was assessed densitometrically and plotted as mean sem from 3 or 4 independent experiments . this more pronounced effect of cngb1 ablation in the rds compared to the rho continued when we examined rds levels ( fig . in cngb1/rds retinas , rds levels were reduced to 54% of those in the rds while rds levels in cngb1/rho remained at 87% of those in the rho . rod outer segment membrane protein-1 levels were not altered in either the cngb1/rds versus the rds or the cngb1/rho versus the rho ( fig . we also observed that rds and rom-1 are severely reduced by the coablation of cngb1 and rhodopsin ( cngb1/rho had rds / rom-1 levels that were 8.8% and 7.9% those of rho , respectively ) . cyclic nucleotide gated channel b1a is known to bind to rds in the os , and proper binding and complex assembly of rds / rom-1 are necessary for os formation , so we asked whether eliminating cngb1 altered rds / rom-1 oligomerization . retinal extracts from wt and cngb1 animals were fractionated on 5% to 20% nonreducing sucrose gradients and then separated on reducing sds - page . in the wt retina , rds is present as tetramers ( fractions 68 ) , octamers ( fractions 45 ) , and higher - order oligomers ( fractions 13 ) while rom-1 is detected only in tetrameric and octameric fractions . 1e ) complex formation in cngb1 retinas , suggesting that rds / rom-1 oligomerization is not tied to cngb1 . previously we observed that cngb1a and garp2 protein levels are reduced by 50% in the rds and are undetectable in the rds . when combined with our data here showing that rds levels are decreased in the cngb1/rds compared to the rds cyclic nucleotide gated channel b1a and rds binding occurs during the os targeting process prior to arrival in the os , so we next used immunofluorescence labeling to ask whether the targeting of rds , rom-1 , rhodopsin , or cngb1a / garp1/2 is affected by co - elimination of cngb1 , rhodopsin , or rds . retinal degeneration slow and rom-1 labeling was properly restricted to the os layer in the wt , cngb1 , rds , cngb1/rho , rho , and cngb1/rho retinas ( fig . similarly , rhodopsin localization is not affected in most of these genotypes ; however , some accumulation of rhodopsin in the onl is observed in the cngb1/rho ( fig . large amounts of rhodopsin accumulate in the inner segment and onl , and this is recapitulated in the cngb1/rds ( fig . in contrast , in the rho and cngb1/rho retinas , the majority of rds is not found in the inner segment , but is properly restricted to the distal tips of the photoreceptors , consistent with our previous observation showing that rds and rom-1 are found in the small nascent oss of the rho . however , in the rho and cngb1/rho retinas , some mild mislocalization of rds ( but not rom-1 ) to the inner segment , onl , and outer plexiform layer is seen ( arrows , fig . combined , these data suggest that while rhodopsin is more susceptible to mislocalization than rds , cngb1 ablation has little to no effect on the os targeting of rds , rom-1 , or rhodopsin . eliminating cngb1 does not affect targeting of rds or rhodopsin . paraffin - embedded retinal sections from p30 animals of the indicated genotypes were labeled ( a ) with antibodies against rds ( rds - ct , green ) , rhodopsin ( mab-1d4 , red ) , or rom-1 ( rom1-ct , green ) ; ( b ) mab 1d4 ( red ) or rom1-ct ( green ) ; ( c ) rds - ct or rom1-ct ( green ) , or ( d ) cngb1 gene products ( garp-4b1 , red ) . in all cases nuclei os , outer segment ; onl , outer nuclear layer ; is , inner segment ; opl , outer plexiform layer . conversely , to see whether reduction / elimination of rds or rhodopsin affected cngb1a / garp1/2 targeting , we labeled with garp-4b1 , which recognizes all cngb1 gene products ( fig . 2d , red ) . in wt , rds , and rho retinas , this observation was replicated in the rho retina ; cngb1a / garp1/2 properly localized to the os layer , even though the rho does not form fully elaborated oss . in striking contrast , and consistent with our western blot data , in the absence of rds ( rds ) , cngb1a and garp1/2 are virtually undetectable in the retina . this decrease in cngb1 gene products in the absence of rds is clearly not due to a requirement for proper os formation , since the oss in the rho retina , while more formed than in the rds , are still just tiny sacs of vesicle - containing membrane at the tip of the os , yet the rho model still expresses cngb1a / garp1/2 . rather it suggests that the stability or transport of cngb1a and/or garp 1/2 are dependent directly on rds or on the initiation / formation of oss by rds ( which can be seen in nascent form even in the rho ) . 3a ) . as expected at early time points , onl thickness is not changed in rds or rho versus wt ( figs . however , though the differences do not attain statistical significance , mean onl thickness in the cngb1 , cngb1/rds , and cngb1/rho was decreased by 10% to 15% compared to their respective controls ( wt , rds , and rho ) , suggesting that degeneration in the cngb1 starts earlier than that in the rds and rho . there is no difference in onl thickness in rho versus cngb1/rho or rds versus cngb1/rds , suggesting that the photoreceptor degeneration in the rds and rho is not additive with that in cngb1 . representative light micrographs ( lm ) were captured from plastic - embedded retinal sections harvested at p30 from the indicated genotypes . rpe , retinal pigment epithelium ; os , outer segment ; is , inner segment ; onl , outer nuclear layer . ( b , c ) outer nuclear layer and os layer thicknesses were measured in sections captured from the central superior and central inferior retina from 2 to 4 eyes per genotype . plotted are means sem , * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 by 2-way anova with bonferroni 's post hoc comparison . it was previously shown that oss are shorter and more disorganized in the cngb1 and rds retinas compared to wt , and our data confirm this ( fig . 3c ) . interestingly , this effect was additive : in the cngb1/rds , mean os length was 41% of wt , compared to 52% and 73% of wt for rds and cngb1 , respectively ( fig . as expected , at p30 , oss in the rho are similar to those in wt , but we do see os shortening in the cngb1/rho due to the absence of cngb1a ; oss in the rho are 89% of wt , while those in the cngb1/rho are 59% of wt . again , the effects of eliminating cngb1a from the rds retina are more severe than in the rho retina ; for example , oss in the cngb1/rds are decreased to 56% of those in cngb1 ( p < 0.05 ) , while the oss of the cngb1/rho are 81% of cngb1 ( not significant ) . figure 4a shows representative waveforms , and maximum saturating a- and b - wave amplitudes are plotted in figure 4b ( mean sem , p < 0.001 by 1-way anova with bonferroni 's post hoc comparison ) . our results for control animals ( cngb1 , rds , and rho ) are consistent with previous publications ( fig . , elimination of cngb1 had a much more severe effect in the rds background than it did in the rho background . scotopic a - wave values in the cngb1/rho were not significantly reduced compared to the cngb1 , suggesting that all of the functional decrease in the cngb1/rho is due to the absence of cngb1 . in contrast , in the cngb1/rds , scotopic erg responses were severely reduced compared to both cngb1 and rds ( fig . this effect was seen in both the scotopic a- and b - waves , and scotopic a - waves in the cngb1/rds were virtually undetectable . scotopic erg function is undetectable in the rds and rho retina at p30 , so we did not perform ergs on animals in those backgrounds . the additive erg defect in the cngb1/rds could be due to reductions in cnga1 ; however , levels of cnga1 in cngb1 are virtually undetectable , and this finding is recapitulated in cngb1/rds ( supplementary fig . thus while it is possible that further reduction in cnga1 levels contributes to the additive defects seen in the cngb1/rds , we can not conclusively assess the possibility given that the levels of cnga1 are below the limit of detection . ( a ) representative waveforms ; ( b ) plots of mean amplitude sem from 4 to 7 mice per genotype ( * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 by 1-way anova with bonferroni 's post hoc comparison ) . scale bars : 10 m for low - magnification em images and 500 nm for higher - magnification em images . rpe , retinal pigment epithelium ; os , outer segment ; cc , connecting cilium . we conducted tem to determine whether the severe effects of simultaneously eliminating cngb1 and reducing rhodopsin / rds led to defects in os ultrastructure ( fig . the os shortening we quantified on the light microscopy level in the cngb1 is also evident by low - magnification em ( 3000 , top , fig . 4c ) , we find that the disc diameter and often the disc alignment are abnormal in the cngb1 retina . in some cases , outer segments in the rds were characterized by abnormal disc size and the formation of highly dysmorphic whorl structures , but interestingly , this phenotype was not worsened in the cngb1/rds retina . outer segment structure in the cngb1/rho was worse than in the rho ( which is largely normal ) . the phenotype in the cngb1/rho was of the same type seen in the cngb1 , namely , abnormalities in disc size and alignment but not whorl formation ; however , the defects appeared to be more severe in the cngb1/rho compared to the cngb1 . the rds retina forms no os at the tip of the connecting cilia ( fig . 5 , cc marks connecting cilia ) , and the cngb1/rds is not different from the rds . in contrast , the rho forms well - characterized tiny nascent oss ( arrows , fig . similar structures at the distal tip of the connecting cilium were seen in the cngb1/rho ( arrows , fig . outer segment ultrastructure in the rds and rho is not affected by the ablation of cngb1 . scale bars : 10 m for low - magnification em images ( top ) and 500 nm for higher - magnification em images ( bottom ) . though cngb1 is expressed only in rods , secondary cone loss can occur after rod defects . figure 6a shows representative photopic erg waveforms while figure 6b plots mean photopic b - wave amplitudes ( mean sem , p < 0.01 by 1-way anova with bonferroni 's post hoc comparison ) . consistent with previous reports , at this time point significant decreases in cone function are not detected in the rds or cngb1 . likewise , no significant reductions in cone function were noticed in cngb1/rho versus rho or wt . however , a striking 75% reduction in cone function was observed in cngb1/rds compared to wt , rds , and cngb1 . to identify whether this defect was associated with loss of cone cells , m - opsin - positive cones were counted in a region in the superior central retina while s - opsin - positive cones were counted in the inferior central portion of the retina ( representative images in fig . consistent with the dramatic decrease in photopic erg response in the cngb1/rds compared to the cngb1 or the rds , we observed a 50% reduction in s - cones in the cngb1/rds compared to cngb1 , rds , or wt controls ( p < 0.001 by 1-way anova ) . cone function is significantly reduced in the cngb1/rds retina . photopic ergs were recorded from wt , cngb1 , rds , cngb1/rds , rho , and cngb1/rho at p30 . ( a ) representative waveforms ; ( b ) plots of the mean maximum photopic b - wave amplitude . n = 4 to 7 mice per genotype ( * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 by 1-way anova with bonferroni 's post hoc comparison ) . ( c ) paraffin - embedded retinal sections from p30 animals were labeled with anti s - opsin ( green , top ) and anti - m - opsin ( green , bottom ) and were all counterstained with dapi ( blue ) . os , outer segment ; onl , outer nuclear layer ; opl , outer plexiform layer . ( d ) cells positive for cone opsins were counted in a 300-m region of the central retina either inferior ( s - opsin - positive cells ) or superior ( m - opsin - positive cells ) of the optic nerve . plotted in this study , our goal was to understand how rds ( a rim protein ) , rhodopsin ( primarily a disc protein ) , and cngb1a ( a plasma membrane protein ) are interrelated in their ability to support os structure and function . recent work showed that rds can bind rhodopsin and suggested that a complex composed of cngb1a , rds , and rhodopsin may play a structural role in anchoring the rim to the plasma membrane ( via rds - cngb1a interactions ) , and a functional role ensuring that proteins are properly localized for optimal phototransduction . when combined with other work showing that cngb1a / garp interactions with phosphodiesterase 6 may provide a role for garp in cgmp turnover , these observations suggest that rds may organize the rim region into a hotspot area or environment for highly regulated phototransduction . this hypothesis is consistent with our results here showing that cngb1a and rds can act additively , exacerbating rds haploinsufficiency , and resulting in ( 1 ) severely decreased rod function in cngb1/rds versus cngb1 or rds and ( 2 ) modest additional reductions in the thickness of the os layer and the onl in the cngb1/rds versus the rds and cngb1 . critically , however , these additive effects occur without any difference in rod ultrastructure in cngb1/rds versus rds . this suggests that defects in rod function and overall retinal structure can not be completely attributed to a role for cngb1/rds interactions in maintaining rim structure , but rather that these interactions also organize a functional environment critical for proper signal transduction . this role for rds as an interactor / organizer of many proteins ( i.e. , not just rom-1 ) is consistent with the observation that rds also interacts with rhodopsin as well as the known broader role of tetraspanins , which often act as organizers of membrane domains containing a wide variety of different proteins . an interesting outcome from our work cngb1 is expressed only in rods , and cone function in cngb1 animals is normal at p30 and starts decreasing only later , likely a secondary phenomenon due to rod loss . similarly , while rds is expressed in rods and cones , cones can better tolerate reduced rds levels than rods , so cone function in the rds is also preserved until approximately 4 months of age . yet here we see cone degeneration and a dramatic reduction in cone function in cngb1/rds as early as p30 . this likely represents a rapid acceleration of rds haploinsufficiency in cones ; however , why elimination of cngb1 in rods should accelerate rds - associated cone degeneration is not clear . this cone phenotype is not completely due to overt rod loss since onl thickness is only mildly affected in the cngb1/rds versus the rds . there may be several underlying causes , including overall alterations in retinal homeostasis , altered secretion of signaling factors from rods needed for cone health , or alterations in other cell types such as horizontal cells and retinal glia that interact with both rods and cones . further exploration of the mechanisms underlying this cone defect are of great interest given the proliferation of retinal disease wherein secondary cone loss following rod - specific defects leads to worsened patient visual outcomes . the trafficking pathways for rhodopsin have been extensively studied , and rds and rhodopsin are thought to traffic separately . recent work has shown that in contrast to rhodopsin , the majority of rds does not traffic through the trans - golgi , but uses an unconventional secretory pathway during os targeting . little is known about the regulation of cngb1a / garp1/2 trafficking ; however , recent work from goldberg 's group shows that rds / cngb1a interactions first occur in the inner segment , raising the intriguing possibility that cngb1a may traffic with rds . however , whether the cngb1a would traffic with conventionally or unconventionally processed rds remains to be further explored . here we find that in general , trafficking of rds , rom-1 , and rhodopsin was not affected by cngb1 ablation . the presence of a clear region of cngb1a / garp1/2 immunoreactivity in the distal inner segment region , likely inside the small abnormally formed os nubs , suggests that overall , cngb1a / garp1/2 targeting is not affected by elimination of rhodopsin ; however , we can not assess the role of rds in cngb1a / garp1/2 trafficking because cngb1a / garp1/2 are not detectable in rds mice . thus , it is not possible to differentiate whether cngb1a / garp1/2 trafficking to the os requires rds , whether cngb1a protein stability requires rds , or whether cngb1a trafficking / stability require the rds - mediated assembly of oss . interestingly , in some cases eliminating the cngb1 channel can be beneficial in preventing or retarding retinal degeneration . for example , the removal of cngb1 significantly rescued the rapid degenerative phenotypes in the rd1 mouse . degeneration in the rd1 was hypothesized to be tied to observed elevated cgmp levels . however , the removal of cng channels led to rescue of rod photoreceptors without ameliorating cgmp levels , suggesting that removing cgmp - mediated signaling was beneficial rather than removing cgmp per se . as a parallel , eliminating rds in the rd1 retina ( rds / pde6b ) also delayed retinal degeneration regardless of the massive accumulation of cgmp . our observation that cngb1a levels are drastically reduced in the rds suggests that attenuation of rd1-associated retinal degeneration in the rd1 rds double knockout is likely due to an elimination of the cng channel / cngb1a rather than to the loss of rds per se . in conclusion , we here present data showing that elimination of cngb1 exacerbates rds - associated functional but not structural haploinsufficiency . these observations suggest that rds / cngb1 interactions have a role in os function in addition to structure and are consistent with the hypothesis that rds functions as a component of a multiprotein plasma membrane - rim - disc complex critical for oss .

purposerod photoreceptor outer segment ( os ) morphogenesis , structural integrity , and proper signal transduction rely on critical proteins found in the different os membrane domains ( e.g. , plasma , disc , and disc rim membrane ) . among these key elements are retinal degeneration slow ( rds , also known as peripherin-2 ) , rhodopsin , and the beta subunit of the cyclic nucleotide gated channel ( cngb1a ) , which have been found to interact in a complex . the purpose of this study was to evaluate the potential interplay between these three proteins by examining retinal disease phenotypes in animal models expressing varying amounts of cngb1a , rhodopsin , and rds.methodsouter segment trafficking , retinal function , and photoreceptor structure were evaluated using knockout mouse lines.resultseliminating cngb1 and reducing rds leads to additive defects in rds expression levels and rod electroretinogram ( erg ) function , ( e.g. , cngb1//rds+/ versus rds+/ or cngb1/ ) but not to additive defects in rod ultrastructure . these additive effects also manifested in cone function : photopic erg responses were significantly lower in the cngb1//rds+/ versus rds+/ or cngb1/ , suggesting that eliminating cngb1 can accelerate the cone degeneration that usually presents later in the rds+/. this was not the case with rhodopsin ; reducing rhodopsin levels in concert with eliminating cngb1a did not lead to phenotypes more severe than those observed in the cngb1 knockout alone.conclusionsthese data support a role for rds as the core component of a multiprotein plasma membrane - rim - disc complex that has both a structural role in photoreceptor os formation and maintenance and a functional role in orienting proteins for optimal signal transduction .

Materials and Methods Ethics Statement and Animal Care and Use Immunofluorescence Labeling and Protein Chemistry Light and Transmission Electron Microscopy Electroretinography Statistical Analysis Results Levels of Rhodopsin, RDS, and ROM-1 Are Altered by Co-elimination of CNGB1 and RDS/Rhodopsin Trafficking of OS Protein Is Largely Unaffected by Removing Elimination of CNGB1 Exacerbates OS Shortening When RDS Is Also Reduced Eliminating Elimination of Discussion Supplementary Material

the cngb1 line , the rds line ( originally obtained from neeraj agarwal currently at the national eye institute , bethesda , md , usa ) , and the rho line ( originally obtained from janice lem , tufts university , boston , ma , usa ) have been described previously . , cngb1/ rds / rho ) , rds , rds , rho , and rho littermates were used from our colony as controls . the cngb1 line , the rds line ( originally obtained from neeraj agarwal currently at the national eye institute , bethesda , md , usa ) , and the rho line ( originally obtained from janice lem , tufts university , boston , ma , usa ) have been described previously . previously we evaluated the interplay between rds and rhodopsin in os morphogenesis , and here we wanted to understand the effects of eliminating cngb1 at the same time as either rhodopsin or rds . we crossed the cngb1 line , which lacks all cngb1 gene products including cngb1a and garp1/2 , onto the rds , rds , rho , and rho backgrounds ( which were all used as control lines for the purpose of comparison throughout the study ) . at postnatal day ( p ) 30 we assessed levels of rhodopsin , rds , and rom-1 by reducing sds - page / western blot . though rhodopsin levels were virtually normal in the cngb1 and the rds ( fig . 1a ) , they were reduced in cngb1/rds to 56% of levels in the rds . the blots were probed with ( a ) mab 1d4 against rhodopsin , ( b ) anti - rds - ct , and ( c ) anti - rom-1-ct . cyclic nucleotide gated channel b1a is known to bind to rds in the os , and proper binding and complex assembly of rds / rom-1 are necessary for os formation , so we asked whether eliminating cngb1 altered rds / rom-1 oligomerization . when combined with our data here showing that rds levels are decreased in the cngb1/rds compared to the rds cyclic nucleotide gated channel b1a and rds binding occurs during the os targeting process prior to arrival in the os , so we next used immunofluorescence labeling to ask whether the targeting of rds , rom-1 , rhodopsin , or cngb1a / garp1/2 is affected by co - elimination of cngb1 , rhodopsin , or rds . retinal degeneration slow and rom-1 labeling was properly restricted to the os layer in the wt , cngb1 , rds , cngb1/rho , rho , and cngb1/rho retinas ( fig . similarly , rhodopsin localization is not affected in most of these genotypes ; however , some accumulation of rhodopsin in the onl is observed in the cngb1/rho ( fig . large amounts of rhodopsin accumulate in the inner segment and onl , and this is recapitulated in the cngb1/rds ( fig . however , in the rho and cngb1/rho retinas , some mild mislocalization of rds ( but not rom-1 ) to the inner segment , onl , and outer plexiform layer is seen ( arrows , fig . paraffin - embedded retinal sections from p30 animals of the indicated genotypes were labeled ( a ) with antibodies against rds ( rds - ct , green ) , rhodopsin ( mab-1d4 , red ) , or rom-1 ( rom1-ct , green ) ; ( b ) mab 1d4 ( red ) or rom1-ct ( green ) ; ( c ) rds - ct or rom1-ct ( green ) , or ( d ) cngb1 gene products ( garp-4b1 , red ) . in striking contrast , and consistent with our western blot data , in the absence of rds ( rds ) , cngb1a and garp1/2 are virtually undetectable in the retina . this decrease in cngb1 gene products in the absence of rds is clearly not due to a requirement for proper os formation , since the oss in the rho retina , while more formed than in the rds , are still just tiny sacs of vesicle - containing membrane at the tip of the os , yet the rho model still expresses cngb1a / garp1/2 . however , though the differences do not attain statistical significance , mean onl thickness in the cngb1 , cngb1/rds , and cngb1/rho was decreased by 10% to 15% compared to their respective controls ( wt , rds , and rho ) , suggesting that degeneration in the cngb1 starts earlier than that in the rds and rho . it was previously shown that oss are shorter and more disorganized in the cngb1 and rds retinas compared to wt , and our data confirm this ( fig . again , the effects of eliminating cngb1a from the rds retina are more severe than in the rho retina ; for example , oss in the cngb1/rds are decreased to 56% of those in cngb1 ( p < 0.05 ) , while the oss of the cngb1/rho are 81% of cngb1 ( not significant ) . scotopic a - wave values in the cngb1/rho were not significantly reduced compared to the cngb1 , suggesting that all of the functional decrease in the cngb1/rho is due to the absence of cngb1 . in contrast , in the cngb1/rds , scotopic erg responses were severely reduced compared to both cngb1 and rds ( fig . we conducted tem to determine whether the severe effects of simultaneously eliminating cngb1 and reducing rhodopsin / rds led to defects in os ultrastructure ( fig . the phenotype in the cngb1/rho was of the same type seen in the cngb1 , namely , abnormalities in disc size and alignment but not whorl formation ; however , the defects appeared to be more severe in the cngb1/rho compared to the cngb1 . 5 , cc marks connecting cilia ) , and the cngb1/rds is not different from the rds . however , a striking 75% reduction in cone function was observed in cngb1/rds compared to wt , rds , and cngb1 . to identify whether this defect was associated with loss of cone cells , m - opsin - positive cones were counted in a region in the superior central retina while s - opsin - positive cones were counted in the inferior central portion of the retina ( representative images in fig . consistent with the dramatic decrease in photopic erg response in the cngb1/rds compared to the cngb1 or the rds , we observed a 50% reduction in s - cones in the cngb1/rds compared to cngb1 , rds , or wt controls ( p < 0.001 by 1-way anova ) . plotted previously we evaluated the interplay between rds and rhodopsin in os morphogenesis , and here we wanted to understand the effects of eliminating cngb1 at the same time as either rhodopsin or rds . we crossed the cngb1 line , which lacks all cngb1 gene products including cngb1a and garp1/2 , onto the rds , rds , rho , and rho backgrounds ( which were all used as control lines for the purpose of comparison throughout the study ) . at postnatal day ( p ) 30 we assessed levels of rhodopsin , rds , and rom-1 by reducing sds - page / western blot . though rhodopsin levels were virtually normal in the cngb1 and the rds ( fig . 1a ) , they were reduced in cngb1/rds to 56% of levels in the rds . cyclic nucleotide gated channel b1a is known to bind to rds in the os , and proper binding and complex assembly of rds / rom-1 are necessary for os formation , so we asked whether eliminating cngb1 altered rds / rom-1 oligomerization . in the wt retina , rds is present as tetramers ( fractions 68 ) , octamers ( fractions 45 ) , and higher - order oligomers ( fractions 13 ) while rom-1 is detected only in tetrameric and octameric fractions . when combined with our data here showing that rds levels are decreased in the cngb1/rds compared to the rds cyclic nucleotide gated channel b1a and rds binding occurs during the os targeting process prior to arrival in the os , so we next used immunofluorescence labeling to ask whether the targeting of rds , rom-1 , rhodopsin , or cngb1a / garp1/2 is affected by co - elimination of cngb1 , rhodopsin , or rds . retinal degeneration slow and rom-1 labeling was properly restricted to the os layer in the wt , cngb1 , rds , cngb1/rho , rho , and cngb1/rho retinas ( fig . similarly , rhodopsin localization is not affected in most of these genotypes ; however , some accumulation of rhodopsin in the onl is observed in the cngb1/rho ( fig . large amounts of rhodopsin accumulate in the inner segment and onl , and this is recapitulated in the cngb1/rds ( fig . in contrast , in the rho and cngb1/rho retinas , the majority of rds is not found in the inner segment , but is properly restricted to the distal tips of the photoreceptors , consistent with our previous observation showing that rds and rom-1 are found in the small nascent oss of the rho . however , in the rho and cngb1/rho retinas , some mild mislocalization of rds ( but not rom-1 ) to the inner segment , onl , and outer plexiform layer is seen ( arrows , fig . paraffin - embedded retinal sections from p30 animals of the indicated genotypes were labeled ( a ) with antibodies against rds ( rds - ct , green ) , rhodopsin ( mab-1d4 , red ) , or rom-1 ( rom1-ct , green ) ; ( b ) mab 1d4 ( red ) or rom1-ct ( green ) ; ( c ) rds - ct or rom1-ct ( green ) , or ( d ) cngb1 gene products ( garp-4b1 , red ) . in wt , rds , and rho retinas , this observation was replicated in the rho retina ; cngb1a / garp1/2 properly localized to the os layer , even though the rho does not form fully elaborated oss . in striking contrast , and consistent with our western blot data , in the absence of rds ( rds ) , cngb1a and garp1/2 are virtually undetectable in the retina . this decrease in cngb1 gene products in the absence of rds is clearly not due to a requirement for proper os formation , since the oss in the rho retina , while more formed than in the rds , are still just tiny sacs of vesicle - containing membrane at the tip of the os , yet the rho model still expresses cngb1a / garp1/2 . however , though the differences do not attain statistical significance , mean onl thickness in the cngb1 , cngb1/rds , and cngb1/rho was decreased by 10% to 15% compared to their respective controls ( wt , rds , and rho ) , suggesting that degeneration in the cngb1 starts earlier than that in the rds and rho . there is no difference in onl thickness in rho versus cngb1/rho or rds versus cngb1/rds , suggesting that the photoreceptor degeneration in the rds and rho is not additive with that in cngb1 . it was previously shown that oss are shorter and more disorganized in the cngb1 and rds retinas compared to wt , and our data confirm this ( fig . again , the effects of eliminating cngb1a from the rds retina are more severe than in the rho retina ; for example , oss in the cngb1/rds are decreased to 56% of those in cngb1 ( p < 0.05 ) , while the oss of the cngb1/rho are 81% of cngb1 ( not significant ) . scotopic a - wave values in the cngb1/rho were not significantly reduced compared to the cngb1 , suggesting that all of the functional decrease in the cngb1/rho is due to the absence of cngb1 . in contrast , in the cngb1/rds , scotopic erg responses were severely reduced compared to both cngb1 and rds ( fig . we conducted tem to determine whether the severe effects of simultaneously eliminating cngb1 and reducing rhodopsin / rds led to defects in os ultrastructure ( fig . in some cases , outer segments in the rds were characterized by abnormal disc size and the formation of highly dysmorphic whorl structures , but interestingly , this phenotype was not worsened in the cngb1/rds retina . the phenotype in the cngb1/rho was of the same type seen in the cngb1 , namely , abnormalities in disc size and alignment but not whorl formation ; however , the defects appeared to be more severe in the cngb1/rho compared to the cngb1 . 5 , cc marks connecting cilia ) , and the cngb1/rds is not different from the rds . consistent with previous reports , at this time point significant decreases in cone function are not detected in the rds or cngb1 . however , a striking 75% reduction in cone function was observed in cngb1/rds compared to wt , rds , and cngb1 . to identify whether this defect was associated with loss of cone cells , m - opsin - positive cones were counted in a region in the superior central retina while s - opsin - positive cones were counted in the inferior central portion of the retina ( representative images in fig . consistent with the dramatic decrease in photopic erg response in the cngb1/rds compared to the cngb1 or the rds , we observed a 50% reduction in s - cones in the cngb1/rds compared to cngb1 , rds , or wt controls ( p < 0.001 by 1-way anova ) . plotted in this study , our goal was to understand how rds ( a rim protein ) , rhodopsin ( primarily a disc protein ) , and cngb1a ( a plasma membrane protein ) are interrelated in their ability to support os structure and function . recent work showed that rds can bind rhodopsin and suggested that a complex composed of cngb1a , rds , and rhodopsin may play a structural role in anchoring the rim to the plasma membrane ( via rds - cngb1a interactions ) , and a functional role ensuring that proteins are properly localized for optimal phototransduction . this hypothesis is consistent with our results here showing that cngb1a and rds can act additively , exacerbating rds haploinsufficiency , and resulting in ( 1 ) severely decreased rod function in cngb1/rds versus cngb1 or rds and ( 2 ) modest additional reductions in the thickness of the os layer and the onl in the cngb1/rds versus the rds and cngb1 . critically , however , these additive effects occur without any difference in rod ultrastructure in cngb1/rds versus rds . this suggests that defects in rod function and overall retinal structure can not be completely attributed to a role for cngb1/rds interactions in maintaining rim structure , but rather that these interactions also organize a functional environment critical for proper signal transduction . this role for rds as an interactor / organizer of many proteins ( i.e. , not just rom-1 ) is consistent with the observation that rds also interacts with rhodopsin as well as the known broader role of tetraspanins , which often act as organizers of membrane domains containing a wide variety of different proteins . yet here we see cone degeneration and a dramatic reduction in cone function in cngb1/rds as early as p30 . further exploration of the mechanisms underlying this cone defect are of great interest given the proliferation of retinal disease wherein secondary cone loss following rod - specific defects leads to worsened patient visual outcomes . the trafficking pathways for rhodopsin have been extensively studied , and rds and rhodopsin are thought to traffic separately . here we find that in general , trafficking of rds , rom-1 , and rhodopsin was not affected by cngb1 ablation . the presence of a clear region of cngb1a / garp1/2 immunoreactivity in the distal inner segment region , likely inside the small abnormally formed os nubs , suggests that overall , cngb1a / garp1/2 targeting is not affected by elimination of rhodopsin ; however , we can not assess the role of rds in cngb1a / garp1/2 trafficking because cngb1a / garp1/2 are not detectable in rds mice . as a parallel , eliminating rds in the rd1 retina ( rds / pde6b ) also delayed retinal degeneration regardless of the massive accumulation of cgmp . our observation that cngb1a levels are drastically reduced in the rds suggests that attenuation of rd1-associated retinal degeneration in the rd1 rds double knockout is likely due to an elimination of the cng channel / cngb1a rather than to the loss of rds per se . these observations suggest that rds / cngb1 interactions have a role in os function in addition to structure and are consistent with the hypothesis that rds functions as a component of a multiprotein plasma membrane - rim - disc complex critical for oss .

previously we evaluated the interplay between rds and rhodopsin in os morphogenesis , and here we wanted to understand the effects of eliminating cngb1 at the same time as either rhodopsin or rds . we crossed the cngb1 line , which lacks all cngb1 gene products including cngb1a and garp1/2 , onto the rds , rds , rho , and rho backgrounds ( which were all used as control lines for the purpose of comparison throughout the study ) . we also observed that rds and rom-1 are severely reduced by the coablation of cngb1 and rhodopsin ( cngb1/rho had rds / rom-1 levels that were 8.8% and 7.9% those of rho , respectively ) . cyclic nucleotide gated channel b1a is known to bind to rds in the os , and proper binding and complex assembly of rds / rom-1 are necessary for os formation , so we asked whether eliminating cngb1 altered rds / rom-1 oligomerization . in the wt retina , rds is present as tetramers ( fractions 68 ) , octamers ( fractions 45 ) , and higher - order oligomers ( fractions 13 ) while rom-1 is detected only in tetrameric and octameric fractions . when combined with our data here showing that rds levels are decreased in the cngb1/rds compared to the rds cyclic nucleotide gated channel b1a and rds binding occurs during the os targeting process prior to arrival in the os , so we next used immunofluorescence labeling to ask whether the targeting of rds , rom-1 , rhodopsin , or cngb1a / garp1/2 is affected by co - elimination of cngb1 , rhodopsin , or rds . retinal degeneration slow and rom-1 labeling was properly restricted to the os layer in the wt , cngb1 , rds , cngb1/rho , rho , and cngb1/rho retinas ( fig . in contrast , in the rho and cngb1/rho retinas , the majority of rds is not found in the inner segment , but is properly restricted to the distal tips of the photoreceptors , consistent with our previous observation showing that rds and rom-1 are found in the small nascent oss of the rho . however , in the rho and cngb1/rho retinas , some mild mislocalization of rds ( but not rom-1 ) to the inner segment , onl , and outer plexiform layer is seen ( arrows , fig . paraffin - embedded retinal sections from p30 animals of the indicated genotypes were labeled ( a ) with antibodies against rds ( rds - ct , green ) , rhodopsin ( mab-1d4 , red ) , or rom-1 ( rom1-ct , green ) ; ( b ) mab 1d4 ( red ) or rom1-ct ( green ) ; ( c ) rds - ct or rom1-ct ( green ) , or ( d ) cngb1 gene products ( garp-4b1 , red ) . this decrease in cngb1 gene products in the absence of rds is clearly not due to a requirement for proper os formation , since the oss in the rho retina , while more formed than in the rds , are still just tiny sacs of vesicle - containing membrane at the tip of the os , yet the rho model still expresses cngb1a / garp1/2 . rather it suggests that the stability or transport of cngb1a and/or garp 1/2 are dependent directly on rds or on the initiation / formation of oss by rds ( which can be seen in nascent form even in the rho ) . however , though the differences do not attain statistical significance , mean onl thickness in the cngb1 , cngb1/rds , and cngb1/rho was decreased by 10% to 15% compared to their respective controls ( wt , rds , and rho ) , suggesting that degeneration in the cngb1 starts earlier than that in the rds and rho . there is no difference in onl thickness in rho versus cngb1/rho or rds versus cngb1/rds , suggesting that the photoreceptor degeneration in the rds and rho is not additive with that in cngb1 . interestingly , this effect was additive : in the cngb1/rds , mean os length was 41% of wt , compared to 52% and 73% of wt for rds and cngb1 , respectively ( fig . as expected , at p30 , oss in the rho are similar to those in wt , but we do see os shortening in the cngb1/rho due to the absence of cngb1a ; oss in the rho are 89% of wt , while those in the cngb1/rho are 59% of wt . again , the effects of eliminating cngb1a from the rds retina are more severe than in the rho retina ; for example , oss in the cngb1/rds are decreased to 56% of those in cngb1 ( p < 0.05 ) , while the oss of the cngb1/rho are 81% of cngb1 ( not significant ) . scotopic a - wave values in the cngb1/rho were not significantly reduced compared to the cngb1 , suggesting that all of the functional decrease in the cngb1/rho is due to the absence of cngb1 . the additive erg defect in the cngb1/rds could be due to reductions in cnga1 ; however , levels of cnga1 in cngb1 are virtually undetectable , and this finding is recapitulated in cngb1/rds ( supplementary fig . thus while it is possible that further reduction in cnga1 levels contributes to the additive defects seen in the cngb1/rds , we can not conclusively assess the possibility given that the levels of cnga1 are below the limit of detection . outer segments in the rds were characterized by abnormal disc size and the formation of highly dysmorphic whorl structures , but interestingly , this phenotype was not worsened in the cngb1/rds retina . the phenotype in the cngb1/rho was of the same type seen in the cngb1 , namely , abnormalities in disc size and alignment but not whorl formation ; however , the defects appeared to be more severe in the cngb1/rho compared to the cngb1 . to identify whether this defect was associated with loss of cone cells , m - opsin - positive cones were counted in a region in the superior central retina while s - opsin - positive cones were counted in the inferior central portion of the retina ( representative images in fig . consistent with the dramatic decrease in photopic erg response in the cngb1/rds compared to the cngb1 or the rds , we observed a 50% reduction in s - cones in the cngb1/rds compared to cngb1 , rds , or wt controls ( p < 0.001 by 1-way anova ) . plotted previously we evaluated the interplay between rds and rhodopsin in os morphogenesis , and here we wanted to understand the effects of eliminating cngb1 at the same time as either rhodopsin or rds . we crossed the cngb1 line , which lacks all cngb1 gene products including cngb1a and garp1/2 , onto the rds , rds , rho , and rho backgrounds ( which were all used as control lines for the purpose of comparison throughout the study ) . we also observed that rds and rom-1 are severely reduced by the coablation of cngb1 and rhodopsin ( cngb1/rho had rds / rom-1 levels that were 8.8% and 7.9% those of rho , respectively ) . cyclic nucleotide gated channel b1a is known to bind to rds in the os , and proper binding and complex assembly of rds / rom-1 are necessary for os formation , so we asked whether eliminating cngb1 altered rds / rom-1 oligomerization . in the wt retina , rds is present as tetramers ( fractions 68 ) , octamers ( fractions 45 ) , and higher - order oligomers ( fractions 13 ) while rom-1 is detected only in tetrameric and octameric fractions . when combined with our data here showing that rds levels are decreased in the cngb1/rds compared to the rds cyclic nucleotide gated channel b1a and rds binding occurs during the os targeting process prior to arrival in the os , so we next used immunofluorescence labeling to ask whether the targeting of rds , rom-1 , rhodopsin , or cngb1a / garp1/2 is affected by co - elimination of cngb1 , rhodopsin , or rds . retinal degeneration slow and rom-1 labeling was properly restricted to the os layer in the wt , cngb1 , rds , cngb1/rho , rho , and cngb1/rho retinas ( fig . in contrast , in the rho and cngb1/rho retinas , the majority of rds is not found in the inner segment , but is properly restricted to the distal tips of the photoreceptors , consistent with our previous observation showing that rds and rom-1 are found in the small nascent oss of the rho . however , in the rho and cngb1/rho retinas , some mild mislocalization of rds ( but not rom-1 ) to the inner segment , onl , and outer plexiform layer is seen ( arrows , fig . paraffin - embedded retinal sections from p30 animals of the indicated genotypes were labeled ( a ) with antibodies against rds ( rds - ct , green ) , rhodopsin ( mab-1d4 , red ) , or rom-1 ( rom1-ct , green ) ; ( b ) mab 1d4 ( red ) or rom1-ct ( green ) ; ( c ) rds - ct or rom1-ct ( green ) , or ( d ) cngb1 gene products ( garp-4b1 , red ) . in wt , rds , and rho retinas , this observation was replicated in the rho retina ; cngb1a / garp1/2 properly localized to the os layer , even though the rho does not form fully elaborated oss . this decrease in cngb1 gene products in the absence of rds is clearly not due to a requirement for proper os formation , since the oss in the rho retina , while more formed than in the rds , are still just tiny sacs of vesicle - containing membrane at the tip of the os , yet the rho model still expresses cngb1a / garp1/2 . rather it suggests that the stability or transport of cngb1a and/or garp 1/2 are dependent directly on rds or on the initiation / formation of oss by rds ( which can be seen in nascent form even in the rho ) . however , though the differences do not attain statistical significance , mean onl thickness in the cngb1 , cngb1/rds , and cngb1/rho was decreased by 10% to 15% compared to their respective controls ( wt , rds , and rho ) , suggesting that degeneration in the cngb1 starts earlier than that in the rds and rho . there is no difference in onl thickness in rho versus cngb1/rho or rds versus cngb1/rds , suggesting that the photoreceptor degeneration in the rds and rho is not additive with that in cngb1 . interestingly , this effect was additive : in the cngb1/rds , mean os length was 41% of wt , compared to 52% and 73% of wt for rds and cngb1 , respectively ( fig . as expected , at p30 , oss in the rho are similar to those in wt , but we do see os shortening in the cngb1/rho due to the absence of cngb1a ; oss in the rho are 89% of wt , while those in the cngb1/rho are 59% of wt . again , the effects of eliminating cngb1a from the rds retina are more severe than in the rho retina ; for example , oss in the cngb1/rds are decreased to 56% of those in cngb1 ( p < 0.05 ) , while the oss of the cngb1/rho are 81% of cngb1 ( not significant ) . scotopic a - wave values in the cngb1/rho were not significantly reduced compared to the cngb1 , suggesting that all of the functional decrease in the cngb1/rho is due to the absence of cngb1 . the additive erg defect in the cngb1/rds could be due to reductions in cnga1 ; however , levels of cnga1 in cngb1 are virtually undetectable , and this finding is recapitulated in cngb1/rds ( supplementary fig . thus while it is possible that further reduction in cnga1 levels contributes to the additive defects seen in the cngb1/rds , we can not conclusively assess the possibility given that the levels of cnga1 are below the limit of detection . in some cases , outer segments in the rds were characterized by abnormal disc size and the formation of highly dysmorphic whorl structures , but interestingly , this phenotype was not worsened in the cngb1/rds retina . the phenotype in the cngb1/rho was of the same type seen in the cngb1 , namely , abnormalities in disc size and alignment but not whorl formation ; however , the defects appeared to be more severe in the cngb1/rho compared to the cngb1 . to identify whether this defect was associated with loss of cone cells , m - opsin - positive cones were counted in a region in the superior central retina while s - opsin - positive cones were counted in the inferior central portion of the retina ( representative images in fig . consistent with the dramatic decrease in photopic erg response in the cngb1/rds compared to the cngb1 or the rds , we observed a 50% reduction in s - cones in the cngb1/rds compared to cngb1 , rds , or wt controls ( p < 0.001 by 1-way anova ) . plotted in this study , our goal was to understand how rds ( a rim protein ) , rhodopsin ( primarily a disc protein ) , and cngb1a ( a plasma membrane protein ) are interrelated in their ability to support os structure and function . recent work showed that rds can bind rhodopsin and suggested that a complex composed of cngb1a , rds , and rhodopsin may play a structural role in anchoring the rim to the plasma membrane ( via rds - cngb1a interactions ) , and a functional role ensuring that proteins are properly localized for optimal phototransduction . when combined with other work showing that cngb1a / garp interactions with phosphodiesterase 6 may provide a role for garp in cgmp turnover , these observations suggest that rds may organize the rim region into a hotspot area or environment for highly regulated phototransduction . this hypothesis is consistent with our results here showing that cngb1a and rds can act additively , exacerbating rds haploinsufficiency , and resulting in ( 1 ) severely decreased rod function in cngb1/rds versus cngb1 or rds and ( 2 ) modest additional reductions in the thickness of the os layer and the onl in the cngb1/rds versus the rds and cngb1 . this suggests that defects in rod function and overall retinal structure can not be completely attributed to a role for cngb1/rds interactions in maintaining rim structure , but rather that these interactions also organize a functional environment critical for proper signal transduction . there may be several underlying causes , including overall alterations in retinal homeostasis , altered secretion of signaling factors from rods needed for cone health , or alterations in other cell types such as horizontal cells and retinal glia that interact with both rods and cones . little is known about the regulation of cngb1a / garp1/2 trafficking ; however , recent work from goldberg 's group shows that rds / cngb1a interactions first occur in the inner segment , raising the intriguing possibility that cngb1a may traffic with rds . the presence of a clear region of cngb1a / garp1/2 immunoreactivity in the distal inner segment region , likely inside the small abnormally formed os nubs , suggests that overall , cngb1a / garp1/2 targeting is not affected by elimination of rhodopsin ; however , we can not assess the role of rds in cngb1a / garp1/2 trafficking because cngb1a / garp1/2 are not detectable in rds mice . thus , it is not possible to differentiate whether cngb1a / garp1/2 trafficking to the os requires rds , whether cngb1a protein stability requires rds , or whether cngb1a trafficking / stability require the rds - mediated assembly of oss . however , the removal of cng channels led to rescue of rod photoreceptors without ameliorating cgmp levels , suggesting that removing cgmp - mediated signaling was beneficial rather than removing cgmp per se . our observation that cngb1a levels are drastically reduced in the rds suggests that attenuation of rd1-associated retinal degeneration in the rd1 rds double knockout is likely due to an elimination of the cng channel / cngb1a rather than to the loss of rds per se . these observations suggest that rds / cngb1 interactions have a role in os function in addition to structure and are consistent with the hypothesis that rds functions as a component of a multiprotein plasma membrane - rim - disc complex critical for oss .

the structure and property relationship ( spr ) in polymer nanocarriers are normally hard to be defined clearly , due to the polydispersity of polymer and the limited capability for the site - specific functionalization . in contrast , the biomacromolecule systems , e.g. peptide , protein , and the oligonucleotides have a precise structure ( sequence)property relationship in regulating the activities of the biologics . it is clear that the well - defined structures of materials are essential for the quantitative analysis of the spr in functional materials and allow for the application of the theoretical and computational approaches to rationally design and predict the properties of the nanocarriers . these approaches would significantly accelerate the development of the efficient nanocarriers for drug delivery , compared with the empirical approaches in nanocarrier development . anticancer drugs encapsulated in nanoparticles , e.g. liposomes , polymer micelles , and polymer nanoparticles , are normally administered via intravenous injections that require the nanocarriers to be hemocompatible and have stealth property in order to avoid fast clearance by the reticuloendothelial system ( res ) . the formation of the nanocarriers is mostly driven by the hydrophobic aggregation or ionic interactions of the amphiphilic materials , which have a tendency to interact with cell membranes via hydrophobic or charge interactions . such plasma membrane lytic activity may raise safety issues for in vivo applications of nanocarriers . synthetic amphiphilic polymers have been synthesized to form micelle nanocarriers for drug delivery . to elucidate the spr in membrane activity is crucial in optimizing the nanocarrier for safe and efficient in vivo drug delivery . we have developed a biohybrid system , i.e. poly(ethylene glycol ) ( peg)-b - dendritic oligocholic acid ( ca ) ( named as telodendrimer ) via peptide chemistry . it has a well - defined structure with the capability of the precise structural design and functionalization . telodendrimers self - assemble into stable micelles for efficient paclitaxel ( ptx ) encapsulation and a few other anticancer drugs ( see citations in supporting information [ si ] ) . while the paclitaxel - loaded micelles formed by such telodendrimers exhibited a safe profile in in vivo administration for anticancer treatment in mouse models and companion dogs , the empty telodendrimer micelles showed moderate hemolytic properties at relative high concentrations ( 1 mg / ml ) in vitro . the former can be explained by enhanced hydrophobic interactions within the nanotherapeutics by loading highly hydrophobic ptx . however , the loading of some other drug molecules may not be able to stabilize the micelle and may result in hemolysis when given intravenously . the cross - linking approaches are able to stabilize micelles and reduce hemolytic properties . however , the additional reagents and treatments for micelle cross - linking usually require extensive purification before in vivo administration , which may lead to the loss of drug molecules and will be a significant burden for clinical application in terms of quality control and regulations . therefore , there is a need to study the structure and hemolytic activity relationship of nanocarrier to eliminate the hemolytic potential , which could be studied via a series of structural modifications and the property characterization . cholic acid , the key building block in telodendrimers , is known to have strong membrane activity as a small - molecule surfactant . the stacking between the hydrophilic surfaces of cholic acid units via hydrogen bonding is essential to shelter the polar groups allowing for the insertion of a whole complex into the hydrophobic phospholipids bilayer membrane . we hypothesize that introducing bulky hydrophilic groups onto hydroxyl groups of ca may disrupt the compact interactions between the polar surfaces of cas and increase the polar surface area ( psa ) of the whole complex , therefore reducing the membrane activity of telodendrimers . in order to modify the telodendrimer with a well - defined structure , we chose the bottom - up approach to modify cholic acid before being conjugated onto the telodendrimer . glycerol or amino glycerol groups are introduced onto the cholic acid via ether bond formation with hydroxyl groups at the 3- and 7- or 12-positions . the core - forming subunits of the telodendrimers have been studied via molecular dynamics to study the conformations and the corresponding polar surface area ( psa ) to predict the membrane activities of the nanocarriers . further , the telodendrimer subunits with different glycerol substitution were synthesized directly and the membrane activities were tested to validate the theoretical predictions . the anticancer drugs , paclitaxel ( ptx ) and doxorubicin ( dox ) , were successfully loaded into the modified telodendrimer micelles . the drug loading capacity , stability , tumor targeting , and anticancer effects were systemically studied in vitro and in vivo . monomethylterminated poly(ethylene glycol ) monoamine hydrochloride(meo - peg - nh2hcl , mw : 5000 da ) was purchased from jenkem technology ( dallas , tx ) . paclitaxel ( ptx ) was purchased from ak scientific ( union city , ca ) . nirf dye did ( 1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine perchlorate , d-307 ) , was purchased from invitrogen . tetrazolium compound [ 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium , mts ] and phenazine methosulfate ( pms ) were purchased from promega ( madison , wi ) . cholic acid ( ca ) , epichlorohydrin , diisopropyl carbodimide ( dic ) , n - hydroxybenzotriazole ( hobt ) , and all other chemicals were purchased from sigma - aldrich ( st . louis , mo ) . molecular dynamics simulations were carried out using the freely distributed gromacs 4.5.5 software package.h nmr spectra of the small molecules and polymers were recorded using bruker avance 600 mhz spectrometer in cdcl3 or dmso - d6 . particle sizes of micelle were measured using a dynamic lighter scattering ( dls ) particle sizer ( zetatrac , microtrac inc . ) and the area - based mean particle sizes were presented . the tem images of micelles were recorded on a transmission electron microscope ( jeol jem-2100 ) and samples were stained with uranyl aceate . vis nanodrop spectrometer and a simadzu hplc ( lc20at ) equipped with a c18 column and 55% mecn as mobile phase were used for detection of drug loading efficiency and drug release studies . fortessa ( becton dickinson , san jose , ca ) flow cytometer was used for cell uptake study , and ivis-200 small animal imager was used for in vivo tumor imaging . the nomenclature of the telodendrimers and the dimer molecules followed the system used in the previous studies : for example , telodendrimer pegca8 indicates that the molecular weight of peg is 5 kda and there are eight cholic acids conjugated on the periphery of dendritic polylysine . the additional functional groups on ca were described as ca-4oh , ca-5oh , ca-3oh - nh2 , representing one glycerol , two glycerols and one animoglycerol introduced onto cholic acid , respectively . the corresponding telodendrimers were named as peg(ca-4oh)8 , peg(ca-5oh)8 , peg(ca-3oh - nh2)8 , the dimer molecules were synthesized from ca and ca derivatives via a lysine ( k in abbreviation ) bridge and named as kca2 , k(ca-4oh)2 , k(ca-5oh)2 . the preparations of cholic acid derivatives and the dimer molecules are described in si . as reported in our previous procedure , telodendrimers have been prepared from meopeg - nh2 with 5000 da molecular weight via stepwise solution phase fmoc - peptide chemistry . briefly , fmoc peptide chemistry was used to couple ( fmoc)lys(fmoc)-oh onto the n - terminal of peg ( mw = 5 or 10 kda ) using diisopropyl carbodimide ( dic , 3 equiv ) and n - hydroxybenzotriazole ( hobt , 3 equiv ) as coupling reagents in dmf overnight . upon confirmation of negative results from the kaiser test for the reaction , chilled ether was added to the reaction solution to precipitate out the polymer , which was further washed twice with chilled ether . fmoc protecting groups were removed by treatment with 20% piperidine in dmf for 30 min , and polymer was precipitated and washed with cold ether . after three steps of repeated coupling of ( fmoc)lysine(fmoc)-oh and de - fmoc reactions , a dendritic polylysine was synthesized at one end of peg . then , the active nhs esters of ca derivatives ( shown in si ) reacted with the free amino groups at the periphery of polylysine in the presence of triethylamine . the dry polymer product was dissolved in water and purified via dialysis ( mwco : 3500 da ) against pure water . ptx was loaded into telodendrimer micelles via a dry - down ( evaporation ) method as described previously . briefly , 20 mg of telodendrimer along with 4 mg of paclitaxel were first dissolved in chcl3 and evaporated on rotavapor to obtain a homogeneous dry polymer film . the film was reconstituted in 1 ml phosphate - buffered solution ( pbs ) , followed by sonication for 30 min to allow micelle dispersion . no precipitation was observed with the monodispersed particle size from 15 to 30 nm during dls measurement , which indicated the complete drug loading . to track the biodistribution of nanoparticles , hydrophobic nirf dye did was coencapsulated with ptx into the micelles using the same method as described above . the particle sizes of the micelle solution were measured via a dls particle sizer . finally , the micelle formulation was filtered with a 0.22 m filter to sterilize the sample . the encapsulation of dox into the polymeric micelles was carried out by following the procedure described previously . briefly , 1 mg of dox - hcl was stirred with 3 mol equiv of triethylamine ( tea ) in chloroform ( chcl3)/methanol ( meoh ) ( 1:1 , v / v ) overnight to neutralize hcl in dox - hcl . ten mg of peg - ca8 or peg-(ca-4oh)8 telodendrimer was added into above dox solution in chloroform / methanol and mixed , evaporated on rotavapor to obtain a homogeneous dry polymer film , and further dried under high vacuum to further remove tea and solvents . the film was reconstituted in 1 ml phosphate - buffered solution ( pbs ) , followed by sonication for 30 min , allowing the sample film to disperse into micelle solution . the particle sizes of the micelle solution were measured via a dls particle sizer . finally , the micelle formulation was filtered with a 0.22 m filter to sterilize the sample . two ml of blood was added into 10 ml of pbs , and then red blood cells ( rbcs ) were separated from plasma by centrifugation at 1000 g for 10 min . the rbcs were washed three times with 10 ml of pbs solution , and resuspended in 20 ml pbs . two hundred l of diluted rbc suspension was mixed with polymers at serial concentrations ( 10 , 100 , 500 , and 1000 ug / ml ) by gentle vortex and incubated at 37 c . after 0.5 h , 4 h , and overnight , the mixtures were centrifuged at 1000 g for 5 min . the supernatant free of hemoglobin was measured by the absorbance at 540 nm using a uv vis spectrometer . rbcs incubation with triton-100 ( 2% ) and pbs were used as the positive and negative controls , respectively . the percent hemolysis of rbcs was calculated using the following formula : ptx loaded nanoformulations ( 500 ul ) were placed in the dialysis bags with the mwco of 3.5 kda and dialyzed against 4 five ul of drug solutions were sampled at the designed time intervals and pbs solution were refreshed at each measurement . the ptx concentrations were analyzed via hplc ( simadzu lc20 t ) equipped with a c-18 column , and mobile phase was 55% mecn / water . the releases of dox from the nanoformulations were also studied using the samiliar dialysis method . however , the dox concentration was monitored at 480 nm using uv vis absorbance ( nanodrop ) . the quantitative cellular uptake of various dox formulations by raji tumor cells was analyzed by flow cytometry . briefly , 3 10 raji cells were incubated with free dox , dox - peg - ca8 , or dox - peg - ca4 at different dox concentrations ( 1 , 3 , and 9 m ) for 30 min or 2 h at 37 c , respectively . then , the cells were washed with pbs three times and resuspended in pbs for the flow cytometry analysis using the fortessa ( becton dickinson , san jose , ca ) . cell - associated dox was excited with an argon laser ( 488 nm ) , and fluorescence was detected at 560 nm ; 10,000 events were collected for each sample . ovarian cancer cell line skov-3 , colon cancer cell line ht-29 , breast cancer cell line mda - mb-231 and lymphoma raji cell line were purchased from american type culture collection ( atcc ; manassas , va , u.s.a . ) . ovarian cancer cell line skov-3 , colon cancer cell line ht-29 , and lymphoma raji cell line were purchased from american type culture collection ( atcc ; manassas , va , u.s.a . ) . all these cells were cultured in mccoy s 5a ovarian cancer cell line skov-3 , colon cancer cell line ht-29 , breast cancer cell line mda - mb-231 and lymphoma raji cell line were purchased from american type culture collection ( atcc ; manassas , va , u.s.a . ) . skov-3 and ht-29 cells were cultured in mccov s 5a medium and mda - mb-231 and raji cells were cultured with rpmi 1640 medium supplemented with 10% fetal bovine serum ( fbs ) , 100 u / ml penicillin g , and 100 g / ml streptomycin at 37 c using a humidified 5% co2 incubator . skov-3 cells were seeded in 96-well plates at a density of 10,000 cells / well 24 h prior to the treatment . empty micelles and various formulations of ptx with different dilutions were added to the plate and then incubated in a humidified 37 c , 5% co2 incubator . after 72 h incubation , celltiter 96 aqueous cell proliferation reagent , which is composed of mts and an electron coupling reagent pms was added to each well according to the manufacturer s instructions . the cell viability was determined by measuring the absorbance at 490 nm using a microplate reader ( biotek synergy 2 microplate reader ) . results are shown as the average cell viability [ ( odtreat odblank)/(odcontrol odblank ) 100% ] of triplicate wells . female athymic nude mice ( nu / nu strain ) , 68 weeks age , were purchased from harlan . all animals were kept under pathogen - free conditions according to aaalac ( association for assessment and accreditation of laboratory animal care ) guidelines and were allowed to acclimatize for at least 4 days prior to any experiments . all animal experiments were performed in compliance with institutional guidelines and according to protocol approved by the committee for the humane use of animals of the state university of new york upstate medical university . a subcutaneous colon cancer xenograft mouse model was established by injecting 1 10 ht-29 or mda - mb-231 cancer cells in a 100 l of mixture of pbs and matrigel ( 1:1 v / v ) subcutaneously at the right flank in female nude mice . nude mice with subcutaneous tumors of an approximate 810 mm in diameter were subjected to in vivo nirf optical imaging . at different time points postinjection of did- and ptx - coloaded micelles formed by telodendrimer ii ( the mass ratio of did and ptx within nanocarrier was 0.25:1:10 ) , mice were scanned using a ivis-200 small animal imager at cy5.5 excitation and emission channels . the mice were anaesthetized by isofluorane gas before and during each imaging . after in vivo imaging , animals were euthanized by co2 overdose at 48 or 72 h after injection . tumors and major organs were excised and imaged with the ivis-200 small animal imager . advanced molecular dynamics simulations were performed to identify the most stable structures of ca dimers in polar ( water ) and nonpolar ( decane ) solvents . the initial structures of ca dimers were generated in the chembio 3d ultra 13.0.0.3015 software followed by molecular dynamics simulations in gromacs molecular dynamics package 4.5 . gromos96 45a3 force field was used to model ca dimers and decane , while water was modeled using the spc / e potential . each of the dimers was subjected to energy minimization in vacuum using the steepest decent algorithm ; however , to explore the potential energy landscape and to identify a global minimum energy geometry , simulated annealing ( sa ) method was employed . for each of the ca dimers , a set of 1000 independent annealing simulations were performed from randomly selected starting structures generated by a canonical nvt ensemble simulation run at t = 1000 k. the annealing was then performed by gradually reducing the temperature from 1000 to 5 k over 1 ns . each of the 1000 independent annealing profiles were analyzed to identify the one that led to the most stable structure . the minimum energy structure was then solvated in water and decane and equilibrated using isothermal isobaric npt ensemble at t = 300 k. the production molecular dynamics runs were performed at 300 k for 100 ns to calculate the average psa and representative equilibrium structure geometries . cholic acid , the key building block in telodendrimers , is known to have strong membrane activity as a small - molecule surfactant . the membrane activity of the telodendrimer micelles is believed due to the facial amphiphilic structure of ca subunit , which self - assembles through hydrogen bonding with the hydrophobic surfaces exposed to the lipid bilayer . it has been reported that the oligomer of cholic acid ( so - called molecular umbrella ) can effectively deliver hydrophilic biomacromolecules through the phospholipid bilayer membrane . also , the insertion of the assembled dimers or tetramers of ca into the phospholipid s bilayer membrane have been reported to form artificial ion channels , where hydrophilic hydroxyl groups are sheltered from the hydrophobic lipids . therefore , the disruption of the packing between the hydrophilic surfaces of oligo - cholic acid is expected to reduce the plasma membrane lytic activity of these types of molecules and the peg - block - oligoca telodendrimers . in this study , glycerol and amino - glycerol have been introduced into the 3- and 7-oh or 12-oh groups of ca via ether bond formation to increase the steric hindrance while maintaining the facial amphiphilicity of ca ( scheme 1 ) . reagents and conditions : ( i ) tfaa , anhydrous thf , 05 c , 1.5 h ; ( ii ) t - buoh , below 5 c , then at rt for 7 h ; ( iii ) nh3h2o ( 2030% ) , 05 c for 12 h , then rt for 4 h ; ( iv ) epichlorohydrin , 50% naoh , ( n - bu)4noh30 h2o , ch2cl2 , rt , 16 h ; ( v ) nh3 in meoh ( 7 m ) , licl ( 1.2 equiv ) in a sealed container , rt , 24 h ; ( vi ) fmoc - osu ( 1.2 equiv ) , diea ( 1.5 equiv ) , ch2cl2 , 12 h ; ( vii ) 50% tfa in ch2cl2 , 0 c , 30 min ; ( iii ) lioh water solution ( 10 equiv ) , rt , 1836 h ; ( ix ) hosu ( 1.2 equiv ) , dcc ( 1.2 equiv ) , ch2cl2 , rt , 12 h. first , the carboxylic acid of ca was protected via an acid labile tert - butyl ester via an intermediate of the hybrid anhydride with trifluoroacetic acid ( tfa ) . a white powder of ca tert - butyl ester 1 was obtained with a yield over 97% . next , epichlorohydrin was conjugated onto hydroxyl groups of ca in the presence of aqueous sodium hydroxide . through flash column , the monosubstituted 2 and diepoxy substituted compound 3 it should be mentioned that the diepoxy - substituted compound 3 may be a mixture of the compounds of 3,5-disubstituted and 3,7-disubstituted species due to the similar reactivity of 5-oh and 7-oh groups . it was evidenced via the partial decrease of the proton signals on both 5 and 7 carbons ( 3.82 and 3.93 ppm ) , which corresponds to the c h with the unsubstituted hydroxyl groups ( figure s1 in si ) . in addition , two new peaks appeared at 3.3 and 3.4 ppm , indicating the graft of glycerol onto both 5 and 7 carbons via ether groups . in this study , we did not attempt to separate these two isomers , since they may have very similar physical properties in telodendrimers . the tert - butyl ester of mono- and diepoxy - substituted compounds 2 and 3 were cleaved via a treatment with 50% of tfa in dcm at 0 c . in the next step , the epoxy groups were opened via the treatment with lithium hydroxide solution to yield diol derivatives 4 and 5 at yields of 84.4% and 36.5% , respectively . in addition , monoepoxy - derived compound 2 was treated with an nh3 solution in methanol in the presence of licl as a catalyst to introduce amino groups on the polar surface of ca ( compound 6 ) . furthermore , free amine in compound 6 was protected via fmoc - osu , then tert - butyl ester in compound 7 was removed via the treatment of 50% tfa in dcm to yield fmoc - protected amino cholic acid 8 with an 88.4% yield after flash column purification . all the ca derivatives with free carboxylic acids 4 , 5 , and 8 were activated via coupling with hosu using dcc as a condensation reagent to yield active esters 9 , 10 , and 11 , respectively , for telodendrimer conjugation . all the nmr spectra ( figure s1 in si ) and mass spectra ( figure s2 in si ) indicated the correct chemical structures of these intermediates of ca derivatives . following our previous procedure , we further synthesized the telodendrimers i , ii , iii , and iv ( scheme 2 ) via liquid peptide chemistry using ca and ca derivatives as building blocks to cap the periphery of dendritic polylysine . the proton signal of 3-oh on ca disappeared in telodendrimers ii and iv , and new hydroxyl groups on glycerol and amino - glycerol appeared in telodendrimers ii and iv , indicating the expected 3-position glycerol substitution . five hydroxyl groups on diglycerol - substituted compound 5 were observed in the proton nmr spectrum ( figure s1 in si ) , which was also shown in the nmr spectrum of telodendrimer iii . the complete disappearance of the 3-hydroxyl group and the partially decreased proton signals of both 7- and 12- hydroxyl groups on ca in telodendrimer iii were observed in the nmr spectrum ( figure 1 ) . in addition , the substitution on 12-oh caused the proton signal of c-18 methyl groups shifted partially toward the low field from 0.57 to 0.60 ppm . similar phenomena have been reported in the literature . the above nmr results indicated that ca-5oh derivative may have two different isomers , e.g. 3,7- and 3,12-disubstitutions . the number of cholic acid and its derivatives coupled onto the telodendrimer were calculated to be 7.47.6 ( theoretical number of 8) via the integration of peg proton signal and 18-methyl group on cholic acid . as expected , the engineered telodendrimer ii peg(ca-4oh)8 micelles exhibited negligible hemolytic properties compared with typical telodendrimer i pegca8 ( figure 2 ) after incubation with red blood cells for both 4 and 20 h at 37 c . after being loaded with ptx , peg(ca-4oh)8 micelles were observed to be nonhemolytic up to 1 mg / ml after 20 h incubation . however , 100% and 50% hemolysis were caused by the original telodendrimer i pegca8 before and after ptx loading , respectively , at the same 1 mg / ml concentration . telodendrimer iv with amino - derived cholic acid as building block shows almost 100% hemolysis even at a concentration as low as 0.1 mg / ml for a 4 h incubation period ( figure 2 ) . it is due to the positive charges on this telodendrimer iv increasing the interaction with negatively charged cell membrane . very surprisingly , much more hydrophilic telodendrimer iii with five hydroxyl groups on each cholic acid scaffold also showed significant hemolysis at high concentrations of about 0.5 to 1 mg / ml after 4 h incubation , which was even higher than the parent telodendrimer pegca8 ( figure 2 ) . it has been reported that the bulky hydrophilic block in an amphiphilic polymer may disturb the liquid crystalline packing of lipid bilayer in addition to the effect brought by the hydrophobic block alone . in particular , block copolymers having branched polyglycerol as hydrophilic blocks show a far more pronounced effect on membrane structure as compared to copolymers with linear peo blocks . however , it is still unclear why the plasma membrane lytic activity of telodendrimers varied with the number of glycerols introduced on ca . proton nmr spectra of the telodendrimers with ca and ca derivatives as building blocks . hemolytic properties of telodendrimers with different glycerol modifications and with / without ptx loading after incubation with human red blood cells for 4 h ( a ) and 20 h ( b ) . the triplicated data are presented as mean sd ( * : p < 0.05 ; * * : p < 0.005 ) . although , it is not easy to study the interactions between micelles and plasma membrane directly via molecular simulation at the atom level , we could apply these approaches to study the conformations of the hydrophobic subunits of the telodendrimers , which directly interact with plasma membrane . it is believed that the plasma membrane lytic activity of a polymer micelle solution are caused by a small portion of the free amphiphilic polymers existing in solution rather than the intact micelles which are covered by a layer of inert hydrophilic polymer . polar surface area ( psa ) is commonly used to characterize the permeability of a drug molecule . a small molecule with a psa less than 1.40 nm is likely to be able to penetrate through the cell membrane . therefore , the psa of the polymer subunits can be used to compare their membrane activities . the density of the polar functional groups , i.e. hydroxyl groups in cholic acid , contributes to the psa of telodendrimers . a dimer of cholic acids bridged via a lysine , kca2 , is a subunit of telodendrimer i , which was calculated to have 3.15 nm of psa solely based on the functional group contribution . however , the varying conformation of a molecule may change its topological polarity significantly . therefore , the stable conformation of the kca2 was studied via simulated annealing molecular dynamics simulations . the psa of the most stable conformations were calculated on the basis of the topology of the molecules . energy - minimized folded conformations of the ca dimers ( in vacuum ) with varying glycerol substitutions displayed in two modes and orientations . as shown in figure 3 , a hand - in - hand like hairpin conformation of kca2 with the efficient shelter of their hydroxyl groups at the interface of the complex formed via hydrogen bonding . a majority of kca2 hydrophobic surface is exposed , which favors membrane insertion and may directly contribute to the observed hemolytic properties . a considerably reduced psa of kca2 with the minimized energy was calculated in vacuum to be 1.08 nm ( table 1 ) , which was much smaller than the psa solely based on the functional group contributions ( 3.15 nm ) . in a biological system , the interaction of polymers with the plasma membrane occurs in an aqueous condition . therefore , a comparison of the psa of ca dimers in the presence of water molecules may be more representative for the membrane activity of the free amphiphilic molecules in water . on the other hand , the disintegrated polymer micelle may merge with the cell membrane from the exposed hydrophobic core , especially when nanoparticles are chemically or physically anchored onto the cell membrane via targeting ligands or nonspecific interactions . in this case , the core - forming ca dimers aggregate in the hydrophobic core of the micelles . therefore , the conformations of the ca dimers were equilibrated in hydrophobic decane to probe these scenarios . it is noticed that the molecules have small psa after being simulated in vacuum . after equilibration with the solvent molecules ( water or decane ) at 300 k for 100 ns , solvent molecules could insert into the folded structures . the hydroxyl groups were exposed more , and the psa increased , especially in aqueous conditions ( table 1 ) . however , the dimer molecules still remained in the folded conformations restricted by the hydrogen bonding . the stable structures in vacuum were first obtained by the simulated annealing process and then solvated in water or decane at 300 k and equilibrated . the production md simulation runs were performed at 300 k for 100 ns to calculate the average psa and the equilibrium structure geometries . according to our hypothesis , we expect to see the increased psa and the disrupted folding of the ca dimers after introducing bulky hydrophilic groups to ca . the molecular dynamics simulations revealed that the single - glycerol modified ca dimer , k(ca-4oh)2 , also forms a hairpin - like folded conformation via hydrogen bonding ( figure 3 ) . compared to kca2 the k(ca-4oh)2 dimer resulted in a lower - energy structure ( figure s3 in si ) using a simulated annealing approach that is attributed to the enhanced hydrogen bonding . however , as shown in figure 3 , the extra hydroxyl groups are exposed to the solvent molecules at the bottom of the molecular clips . as expected , the k(ca-4oh)2 dimer possesses a higher psa in vacuum ( 1.19 nm ) and after equilibration in water ( 1.65 nm ) or decane ( 1.34 nm ) , which may prohibit the insertion of the telodendrimer into the hydrophobic lipid bilayer membrane . as shown in figure 3 , the ca dimer with diglycerol modification , possessing five hydroxyl groups on each cholic acid , k(ca-5oh)2 , forms a very stable folded conformation via an extensive hydrogen - bonding network . the stable conformations of folded k(ca-5oh)2 lead to the highest energy stabilization ( figure s3 in si ) during the annealing and folding process compared to other ca modifications . most polar groups have been sheltered within the folded conformation , and the psa was calculated to be the smallest at 1.04 nm for k(ca-5oh_7)2 in vacuum , which is even smaller than that for kca2 in the original telodendrimer i. the possible isomers of the disubstituted ca dimers with the second glycerol on the 7 or 12 position ( e.g. , k(ca-5oh_7)2 ; k(ca-5oh_12)2 and k(ca-5oh_7/12)2 ( figure s4 in si ) ) have the similar small psa values and low binding energies during the simulated annealing process ( figure s3 in si ) . the smaller psa and stable conformation both contribute to the hemolytic activity of the telodendrimer iii . as shown in figure 3 , the subunit of telodendrimer iv , k(ca-3oh - nh2)2 , also formed a folded conformation via hydrogen bonding to partially shelter the polar groups after equilibration through molecular dynamics simulations . the psa of the amino - derived ca dimer with an energy - minimized folded conformation was calculated to be higher , 1.21 nm in vacuum , which is further increased to 1.43 nm upon protonation . however , the positively charged amino groups are located at the bottom of the resulting ca dimers , which are believed to induce stronger electrostatic interactions with phospholipid bilayer cell membranes . accordingly , a 100% hemolysis was observed for telodendrimer iv even at 0.1 mg / ml after a 4 h incubation ( figure 2 ) , which is the combination of charge and hydrophobic effects . structure of the cholic acid dimers with different glycerol substitution prepared via solution - phase organic synthesis ( a ) ; hemolytic properties of small - molecule dimers of cholic acid with different glycerol modifications after incubation with human red blood cells for 30 min ( b ) and 4 h ( c ) . the triplicate data are presented as mean sd ( * : p < 0.01 ; * * : p < 0.001 ) . the above molecular simulation studies have clearly revealed the potential spr between ca modification and the hemolytic activity of telodendrimers . however , the direct validation of the computational simulation might be essential for applying these approaches to study the spr of nanocarriers . therefore , cholic acid dimers studied in molecular simulation were synthesized and evaluated . cholic acid dimers with the different glycerol substitution were prepared via solution - phase peptide chemistry ( figure 4a ) . as shown in the si , k(ca)2 , k(ca-4oh)2 , and k(ca-5oh)2 have been characterized with proton nmr ( figure s5 in si ) and maldi - tof ms ( figure s6 in si ) , demonstrating the right structures . the hemolysis properties of these three dimers were tested directly via red blood cell incubation . it is expected that these small molecules showed higher hemolysis compared with telodendrimers , due to the lack of peg sheltering . as predicated in the md simulation , the k(ca-4oh)2 exhibited a significantly lower membrane activity in hemolysis assays than both k(ca-5oh)2 and k(ca)2 for 30 min and 4 h incubation ( figure 4b , c ) . it was also observed that unmodified k(ca)2 showed a significantly higher membrane activity than k(ca-5oh)2 , especially at relatively low concentration of 0.1 mm . however , telodendrimer i pegca8 showed significantly lower hemolysis than telodendrimer iii peg(ca-5oh)8 after 4 h incubation ( figure 2a ) . this may due to the better integrity of pegca8 micelles with the relative low cmc ( as discussed below ) compared with peg(ca-5oh)8 micelles , which are more hydrophilic with higher cmc . the dynamic nature of peg(ca-5oh)8 micelles makes them readily interact with the plasma membrane . ( a ) dls particle sizes of telodendrimer i and ii and the micelles loaded with ptx and dox . the mean particle sizes are presented based on area - distribution ; ( b ) tem images of telodendrimer ii after being loaded with ptx and dox , respectively , with uranyl acetate staining ( scale bar : 100 nm ) . the above chemical modifications of ca on the hydrophilic surface maintained the facial amphiphilic property of ca within the telodendrimer , which ensured the formation of micellar nanoparticles for drug loading . however , such modification on ca increased the critical micellization concentrations ( cmcs ) of the telodendrimers . the cmcs were measured via the classic pyrene 1:3 method to be 17.4 g / ml , 24.2 g / ml , 95.9 g / ml and 49.3 g / ml for telodendrimers i , ii , iii , and iv , respectively ( figure s-7 in si ) . the significantly increased cmcs for telodendrimers iii and iv indicated the higher free polymer concentration in micelle solutions , which may further contribute to the enhanced hemolytic properties . the telodendrimer ii peg(ca-4oh)8 showed similar particle size and stability before and after ptx loading at 3/20 drug / polymer ratio , compared with the original telodendrimer i pegca8 , due to the similar structures and cmcs . in addition , telodendrimers i and ii both exhibited good loading capacity and stability for another anticancer drug , doxorubicin ( dox ) , at 1/10 drug / polymer w / w ratio . the particle sizes have been detected via dls to be 34 and 19.5 nm with narrow distribution , respectively ( figure 5a ) . at these ptx and dox loading ratios , clear micelle solutions were obtained without any drug precipitation observed both by eye and by dls analysis , which indicated the complete drug loading efficiency . further , no drug precipitation was observed from these ptx and dox nanoformulations after storages for weeks at 4 c . the tem studies have revealed the spherical micelles formed by telodendrimer i before and after ptx and dox encapsulation . in this study , we also utilized tem to study the morphology of the nanoparticles formed by peg(ca-4oh)8 . narrow dispersed , homogeneous spherical nanoparticles were observed ( figure 5b ) , which is similar to the dls analysis ( figure 5a ) . on the contrary , telodendrimer iii can only assemble into smaller particles with heterogonous distributions , e.g. 1 nm and 5 nm sizes . it is due to the overwhelming hydroxyl groups introduced to the ca polar surface , which broke the balance of the amphiphilicity of telodendrimers . after ptx loading ( 2:10 , w / w , ptx / iii ) , the sizes of the micelles increased to 62 nm with a small portion of larger sizes at 295 nm ( figure s8 in si ) . telodendrimer iv was observed to aggregate into micelles about 343 nm in size in pure water solution , which were reduced to about 11 nm upon adjusting ph to 5.5 due to the increased polarity and charge repulsion of positively charged amino groups . such ptx - loaded nanoparticles ( 2:10 w / w ptx / iii ) exhibited heterogeneous size distribution of 51 and 341 nm in pbs ( figure s9 in si ) , which is not preferred for in vivo applications . the drug release of both ptx and dox from the telodendrimer ii were observed to be slightly slower than those from telodendrimer i ( figure 6a , b ) , which may due to the stronger hydrogen bonding . ptx released slowly from both nanocarriers with 50% drug release at about 18 h ; in contrast , burst releases were observed for dox and 50% of drug released at 8 h , due to the better water solubility of drug molecules . accumulative drug release profile of ptx and dox loaded in the nanocarrier formed with telodendrimer i ( a ) and monoglycerol derived telodendrimer ii ( b ) ; quantitative flow cytometry analysis of the cellular uptake of various dox formulations by raji cells after 30 min ( c ) and 2 h ( d ) incubation at three drug concentrations . the cell viability assays to evaluate the cytotoxicity of telodendrimer ii and the anticancer effects of the micelles loaded with ptx ( e ) and dox ( f ) on the skov-3 ovarian cell line and raji lymphoma cell line , respectively . given the lowered membrane activity for telodendrimer ii , one may ask whether it will decrease the cell uptake and lower the efficacy of the encapsulated chemodrugs ? to address this issue , we performed the quantitative flow cytometry study to analyze the cell uptake of the chemodrugs . doxorubicin is a fluorescent anticancer drug molecule , which was loaded in nanocarriers for cell uptake study in flow cytometry experiments . as shown in figure 6c , d , the quantitative flow cytometric analysis demonstrated the similar cell uptake for free dox and dox loaded in telodendrimer micelles in raji cells after 30 min and 2 h incubation at different drug concentrations . the cellular uptakes of all dox formulations were in concentration- and time - dependent patterns . furthermore , the in vitro cytotoxicity studies ( figure 6f ) revealed the similar anticancer effects on the raji lymphoma cells for the free dox and dox loaded in the nanocarriers i and ii after 72 h incubation . additionally the ptx - loaded telodendrimer ii ( ic50 : 1.6 ng / ml ) and ptx - loaded telodendrimer i ( ic50 : 3.4 ng / ml ) exhibited slightly higher anticancer effects than free ptx ( ic 50:14.5 ng / ml ) formulated in a mixture of cremophore el and ethanol ( 1:1 v / v ) in an ovarian cancer cell line , skov-3 ( figure 6e ) . this may be due to the partial precipitation of ptx from the cremophore formulation after dilution with cell culture medium . overall , the telodendrimer ii was nontoxic up to 0.5 mg / ml concentration for both cell lines . ex vivo biodistribution of telodendrimer nanocarrier ii loaded with ptx and nirf dye did in colon cancer - bearing nude mice after 48 h postinjection via tail vein ( a ) ; the comparison of the average fluorescence intensity in various organs and ht-29 tumors between the animals given did - ptx - np ii or free dye did ( b ) ; ex vivo biodistribution of telodendrimer nanocarrier ii loaded with ptx and nirf dye did in breast cancer bearing nude mice after 72 h postinjection via tail vein ( c ) ; quantitative analysis of the average fluorescence intensity in various organs and mda - mb-231 tumors between the animals given did - ptx - np ii or free dye did ( d ) . as reported in our previous studies , telodendrimer i nanocarrier was able to target various solid tumors efficiently in animal models . therefore , we would like to further evaluate the biodistribution and tumor targeting properties of the optimized telodendrimers in solid tumor xenograft models , such as colon and breast cancers after intravenous injection of telodendrimer ii micelles coloaded with nirf dye did and ptx . did is a hydrophobic near - infrared fluorescence cyanine dye , which could be stably entrapped within nanocarrier with very slow release profile ( figure s-10 in si ) and could be used as a surrogate to probe the distribution of the nanocarriers . as shown in figure 7 , the ex vivo images revealed the highest uptake of fluorescent signal in tumors compared to other organs in both tumor types . the same targeting effects for telodendrimer i were also observed previously in animal models bearing solid tumors . the free did administration led to a relatively low accumulation in tumors and significant high deposition in lung , liver , and spleen , which were significantly reduced in animals treated with the nanoformulations . as shown in figure 7b and figure 6d , nanocarriers might be able to deliver much higher concentrations of hydrophobic drug molecules to tumor sites ( 24 fold ) , compared with the free did injections . with the enhanced in vivo anticancer effects for the ptx formulated in telodendrimer i nanocarriers , we anticipate these nonhemolytic nanotherapeutics formed by telodendrimer ii will further improve the anticancer effects via efficient tumor - targeted drug delivery . the further pk / pd studies and the in vivo efficacy of this optimized nanocarrier in delivering different chemodrugs in animal models will be tested and reported in a separate account . the spr of a telodendrimer system in interacting with plasma membranes has been clearly revealed via computational and experimental approaches . to minimize the hemolytic properties of telodendrimer nanocarriers , we took a rational approach to modify telodendrimer structures by introducing hydrophilic glycerol or amino - glycerol groups onto the polar surface of cholic acid . with the aid of molecular dynamics simulations , we had a better understanding of the relationship between plasma membrane lytic activity and the structural conformations of the amphiphiles . the introduction of hydrophilic monoglycerol onto the polar surface of cholic acid was predicated via molecular dynamics to be optimal in disrupting the molecular folding of the subunit of telodendrimer and increasing the polar surface area of the complex . on the contrary , introduction of two glycerols or an amino - glycerol on each cholic acid led to the increased hemolytic properties of the resulting telodendrimers , which were correlated with the decreased psa and the exposed positive charges in the molecular dynamics simulations , respectively . further , the computation results have been verified in the experiments via the synthesis of the telodendrimer subunits , which were tested to be consistent with the theoretical prediction for the membrane activities . such spatial modification of ca via monoglycerol grafting sustained the balance of the facial amphiphilicity of ca , therefore maintaining the ptx and dox loading capacity , stability , and anticancer effects of the nanotherapeutics . given the nonhemolytic property , efficient cancer cell uptake , and the excellent in vivo tumor - targeted properties , the monoglycerol derivatized telodendrimer ii has been demonstrated as a promising nanocarrier for efficient anticancer drug delivery .

a series of telodendrimer ( a linear polyethyelene glycol - block - dendritic oligo - cholic acid ) have been synthesized via a bottom - up approach to optimize the hemocompatibility of the nanocarrier . numbers of hydrophilic glycerol groups were introduced onto the polar surface of cholic acid to reduce the plasma membrane lytic activity of telodendrimers . an interesting result was observed : only an optimum number of glycerol introduced could reduce the hemolytic properties of the nanocarrier ; on the contrary , more glycerols or the amino - glycerol substitution onto cholic acid significantly increased the hemolytic properties of the nanocarriers . to further elucidate the structure property relationship , the molecular dynamic approach was used to simulate the conformation of the subunits of telodendrimers with different glycerol substitution , and the binding energies and the polar surface areas of the hairpin conformations were calculated to explain the membrane activities of nanocarriers . in addition , these telodendrimer subunits were synthesized and their membrane activities were tested directly , which validated the computational prediction and correlated with the observed hemolytic activity of nanocarriers . the glycerol substitution sustained the facial amphiphilicity of cholic acid , maintaining the superior drug loading capacity ( paclitaxel and doxorubicin ) , stability , cell uptake , and anticancer efficacy of payloads . the in vivo optical imaging study indicated that the optimized nanocarriers can specifically deliver drug molecules to the tumor sites more efficiently than free drug administration , which is essential for the enhanced cancer treatment .

Introduction Experimental Section Results and Discussion Conclusions

the structure and property relationship ( spr ) in polymer nanocarriers are normally hard to be defined clearly , due to the polydispersity of polymer and the limited capability for the site - specific functionalization . peptide , protein , and the oligonucleotides have a precise structure ( sequence)property relationship in regulating the activities of the biologics . it is clear that the well - defined structures of materials are essential for the quantitative analysis of the spr in functional materials and allow for the application of the theoretical and computational approaches to rationally design and predict the properties of the nanocarriers . liposomes , polymer micelles , and polymer nanoparticles , are normally administered via intravenous injections that require the nanocarriers to be hemocompatible and have stealth property in order to avoid fast clearance by the reticuloendothelial system ( res ) . such plasma membrane lytic activity may raise safety issues for in vivo applications of nanocarriers . to elucidate the spr in membrane activity is crucial in optimizing the nanocarrier for safe and efficient in vivo drug delivery . while the paclitaxel - loaded micelles formed by such telodendrimers exhibited a safe profile in in vivo administration for anticancer treatment in mouse models and companion dogs , the empty telodendrimer micelles showed moderate hemolytic properties at relative high concentrations ( 1 mg / ml ) in vitro . however , the additional reagents and treatments for micelle cross - linking usually require extensive purification before in vivo administration , which may lead to the loss of drug molecules and will be a significant burden for clinical application in terms of quality control and regulations . therefore , there is a need to study the structure and hemolytic activity relationship of nanocarrier to eliminate the hemolytic potential , which could be studied via a series of structural modifications and the property characterization . the stacking between the hydrophilic surfaces of cholic acid units via hydrogen bonding is essential to shelter the polar groups allowing for the insertion of a whole complex into the hydrophobic phospholipids bilayer membrane . we hypothesize that introducing bulky hydrophilic groups onto hydroxyl groups of ca may disrupt the compact interactions between the polar surfaces of cas and increase the polar surface area ( psa ) of the whole complex , therefore reducing the membrane activity of telodendrimers . in order to modify the telodendrimer with a well - defined structure , we chose the bottom - up approach to modify cholic acid before being conjugated onto the telodendrimer . glycerol or amino glycerol groups are introduced onto the cholic acid via ether bond formation with hydroxyl groups at the 3- and 7- or 12-positions . the core - forming subunits of the telodendrimers have been studied via molecular dynamics to study the conformations and the corresponding polar surface area ( psa ) to predict the membrane activities of the nanocarriers . further , the telodendrimer subunits with different glycerol substitution were synthesized directly and the membrane activities were tested to validate the theoretical predictions . the drug loading capacity , stability , tumor targeting , and anticancer effects were systemically studied in vitro and in vivo . fortessa ( becton dickinson , san jose , ca ) flow cytometer was used for cell uptake study , and ivis-200 small animal imager was used for in vivo tumor imaging . the nomenclature of the telodendrimers and the dimer molecules followed the system used in the previous studies : for example , telodendrimer pegca8 indicates that the molecular weight of peg is 5 kda and there are eight cholic acids conjugated on the periphery of dendritic polylysine . the additional functional groups on ca were described as ca-4oh , ca-5oh , ca-3oh - nh2 , representing one glycerol , two glycerols and one animoglycerol introduced onto cholic acid , respectively . the preparations of cholic acid derivatives and the dimer molecules are described in si . no precipitation was observed with the monodispersed particle size from 15 to 30 nm during dls measurement , which indicated the complete drug loading . cholic acid , the key building block in telodendrimers , is known to have strong membrane activity as a small - molecule surfactant . the membrane activity of the telodendrimer micelles is believed due to the facial amphiphilic structure of ca subunit , which self - assembles through hydrogen bonding with the hydrophobic surfaces exposed to the lipid bilayer . therefore , the disruption of the packing between the hydrophilic surfaces of oligo - cholic acid is expected to reduce the plasma membrane lytic activity of these types of molecules and the peg - block - oligoca telodendrimers . in this study , glycerol and amino - glycerol have been introduced into the 3- and 7-oh or 12-oh groups of ca via ether bond formation to increase the steric hindrance while maintaining the facial amphiphilicity of ca ( scheme 1 ) . through flash column , the monosubstituted 2 and diepoxy substituted compound 3 it should be mentioned that the diepoxy - substituted compound 3 may be a mixture of the compounds of 3,5-disubstituted and 3,7-disubstituted species due to the similar reactivity of 5-oh and 7-oh groups . it was evidenced via the partial decrease of the proton signals on both 5 and 7 carbons ( 3.82 and 3.93 ppm ) , which corresponds to the c h with the unsubstituted hydroxyl groups ( figure s1 in si ) . in addition , monoepoxy - derived compound 2 was treated with an nh3 solution in methanol in the presence of licl as a catalyst to introduce amino groups on the polar surface of ca ( compound 6 ) . the proton signal of 3-oh on ca disappeared in telodendrimers ii and iv , and new hydroxyl groups on glycerol and amino - glycerol appeared in telodendrimers ii and iv , indicating the expected 3-position glycerol substitution . in addition , the substitution on 12-oh caused the proton signal of c-18 methyl groups shifted partially toward the low field from 0.57 to 0.60 ppm . the number of cholic acid and its derivatives coupled onto the telodendrimer were calculated to be 7.47.6 ( theoretical number of 8) via the integration of peg proton signal and 18-methyl group on cholic acid . however , it is still unclear why the plasma membrane lytic activity of telodendrimers varied with the number of glycerols introduced on ca . hemolytic properties of telodendrimers with different glycerol modifications and with / without ptx loading after incubation with human red blood cells for 4 h ( a ) and 20 h ( b ) . although , it is not easy to study the interactions between micelles and plasma membrane directly via molecular simulation at the atom level , we could apply these approaches to study the conformations of the hydrophobic subunits of the telodendrimers , which directly interact with plasma membrane . it is believed that the plasma membrane lytic activity of a polymer micelle solution are caused by a small portion of the free amphiphilic polymers existing in solution rather than the intact micelles which are covered by a layer of inert hydrophilic polymer . therefore , the psa of the polymer subunits can be used to compare their membrane activities . hydroxyl groups in cholic acid , contributes to the psa of telodendrimers . a dimer of cholic acids bridged via a lysine , kca2 , is a subunit of telodendrimer i , which was calculated to have 3.15 nm of psa solely based on the functional group contribution . therefore , the stable conformation of the kca2 was studied via simulated annealing molecular dynamics simulations . the psa of the most stable conformations were calculated on the basis of the topology of the molecules . as shown in figure 3 , a hand - in - hand like hairpin conformation of kca2 with the efficient shelter of their hydroxyl groups at the interface of the complex formed via hydrogen bonding . a majority of kca2 hydrophobic surface is exposed , which favors membrane insertion and may directly contribute to the observed hemolytic properties . a considerably reduced psa of kca2 with the minimized energy was calculated in vacuum to be 1.08 nm ( table 1 ) , which was much smaller than the psa solely based on the functional group contributions ( 3.15 nm ) . in a biological system , the interaction of polymers with the plasma membrane occurs in an aqueous condition . therefore , a comparison of the psa of ca dimers in the presence of water molecules may be more representative for the membrane activity of the free amphiphilic molecules in water . on the other hand , the disintegrated polymer micelle may merge with the cell membrane from the exposed hydrophobic core , especially when nanoparticles are chemically or physically anchored onto the cell membrane via targeting ligands or nonspecific interactions . the hydroxyl groups were exposed more , and the psa increased , especially in aqueous conditions ( table 1 ) . the molecular dynamics simulations revealed that the single - glycerol modified ca dimer , k(ca-4oh)2 , also forms a hairpin - like folded conformation via hydrogen bonding ( figure 3 ) . however , as shown in figure 3 , the extra hydroxyl groups are exposed to the solvent molecules at the bottom of the molecular clips . as expected , the k(ca-4oh)2 dimer possesses a higher psa in vacuum ( 1.19 nm ) and after equilibration in water ( 1.65 nm ) or decane ( 1.34 nm ) , which may prohibit the insertion of the telodendrimer into the hydrophobic lipid bilayer membrane . most polar groups have been sheltered within the folded conformation , and the psa was calculated to be the smallest at 1.04 nm for k(ca-5oh_7)2 in vacuum , which is even smaller than that for kca2 in the original telodendrimer i. the possible isomers of the disubstituted ca dimers with the second glycerol on the 7 or 12 position ( e.g. the smaller psa and stable conformation both contribute to the hemolytic activity of the telodendrimer iii . the psa of the amino - derived ca dimer with an energy - minimized folded conformation was calculated to be higher , 1.21 nm in vacuum , which is further increased to 1.43 nm upon protonation . accordingly , a 100% hemolysis was observed for telodendrimer iv even at 0.1 mg / ml after a 4 h incubation ( figure 2 ) , which is the combination of charge and hydrophobic effects . structure of the cholic acid dimers with different glycerol substitution prepared via solution - phase organic synthesis ( a ) ; hemolytic properties of small - molecule dimers of cholic acid with different glycerol modifications after incubation with human red blood cells for 30 min ( b ) and 4 h ( c ) . the above molecular simulation studies have clearly revealed the potential spr between ca modification and the hemolytic activity of telodendrimers . however , the direct validation of the computational simulation might be essential for applying these approaches to study the spr of nanocarriers . cholic acid dimers with the different glycerol substitution were prepared via solution - phase peptide chemistry ( figure 4a ) . as shown in the si , k(ca)2 , k(ca-4oh)2 , and k(ca-5oh)2 have been characterized with proton nmr ( figure s5 in si ) and maldi - tof ms ( figure s6 in si ) , demonstrating the right structures . the hemolysis properties of these three dimers were tested directly via red blood cell incubation . ( a ) dls particle sizes of telodendrimer i and ii and the micelles loaded with ptx and dox . the above chemical modifications of ca on the hydrophilic surface maintained the facial amphiphilic property of ca within the telodendrimer , which ensured the formation of micellar nanoparticles for drug loading . the significantly increased cmcs for telodendrimers iii and iv indicated the higher free polymer concentration in micelle solutions , which may further contribute to the enhanced hemolytic properties . in addition , telodendrimers i and ii both exhibited good loading capacity and stability for another anticancer drug , doxorubicin ( dox ) , at 1/10 drug / polymer w / w ratio . narrow dispersed , homogeneous spherical nanoparticles were observed ( figure 5b ) , which is similar to the dls analysis ( figure 5a ) . it is due to the overwhelming hydroxyl groups introduced to the ca polar surface , which broke the balance of the amphiphilicity of telodendrimers . telodendrimer iv was observed to aggregate into micelles about 343 nm in size in pure water solution , which were reduced to about 11 nm upon adjusting ph to 5.5 due to the increased polarity and charge repulsion of positively charged amino groups . such ptx - loaded nanoparticles ( 2:10 w / w ptx / iii ) exhibited heterogeneous size distribution of 51 and 341 nm in pbs ( figure s9 in si ) , which is not preferred for in vivo applications . accumulative drug release profile of ptx and dox loaded in the nanocarrier formed with telodendrimer i ( a ) and monoglycerol derived telodendrimer ii ( b ) ; quantitative flow cytometry analysis of the cellular uptake of various dox formulations by raji cells after 30 min ( c ) and 2 h ( d ) incubation at three drug concentrations . the cell viability assays to evaluate the cytotoxicity of telodendrimer ii and the anticancer effects of the micelles loaded with ptx ( e ) and dox ( f ) on the skov-3 ovarian cell line and raji lymphoma cell line , respectively . furthermore , the in vitro cytotoxicity studies ( figure 6f ) revealed the similar anticancer effects on the raji lymphoma cells for the free dox and dox loaded in the nanocarriers i and ii after 72 h incubation . therefore , we would like to further evaluate the biodistribution and tumor targeting properties of the optimized telodendrimers in solid tumor xenograft models , such as colon and breast cancers after intravenous injection of telodendrimer ii micelles coloaded with nirf dye did and ptx . did is a hydrophobic near - infrared fluorescence cyanine dye , which could be stably entrapped within nanocarrier with very slow release profile ( figure s-10 in si ) and could be used as a surrogate to probe the distribution of the nanocarriers . as shown in figure 7b and figure 6d , nanocarriers might be able to deliver much higher concentrations of hydrophobic drug molecules to tumor sites ( 24 fold ) , compared with the free did injections . with the enhanced in vivo anticancer effects for the ptx formulated in telodendrimer i nanocarriers , we anticipate these nonhemolytic nanotherapeutics formed by telodendrimer ii will further improve the anticancer effects via efficient tumor - targeted drug delivery . the further pk / pd studies and the in vivo efficacy of this optimized nanocarrier in delivering different chemodrugs in animal models will be tested and reported in a separate account . to minimize the hemolytic properties of telodendrimer nanocarriers , we took a rational approach to modify telodendrimer structures by introducing hydrophilic glycerol or amino - glycerol groups onto the polar surface of cholic acid . with the aid of molecular dynamics simulations , we had a better understanding of the relationship between plasma membrane lytic activity and the structural conformations of the amphiphiles . the introduction of hydrophilic monoglycerol onto the polar surface of cholic acid was predicated via molecular dynamics to be optimal in disrupting the molecular folding of the subunit of telodendrimer and increasing the polar surface area of the complex . on the contrary , introduction of two glycerols or an amino - glycerol on each cholic acid led to the increased hemolytic properties of the resulting telodendrimers , which were correlated with the decreased psa and the exposed positive charges in the molecular dynamics simulations , respectively . further , the computation results have been verified in the experiments via the synthesis of the telodendrimer subunits , which were tested to be consistent with the theoretical prediction for the membrane activities . such spatial modification of ca via monoglycerol grafting sustained the balance of the facial amphiphilicity of ca , therefore maintaining the ptx and dox loading capacity , stability , and anticancer effects of the nanotherapeutics . given the nonhemolytic property , efficient cancer cell uptake , and the excellent in vivo tumor - targeted properties , the monoglycerol derivatized telodendrimer ii has been demonstrated as a promising nanocarrier for efficient anticancer drug delivery .

it is clear that the well - defined structures of materials are essential for the quantitative analysis of the spr in functional materials and allow for the application of the theoretical and computational approaches to rationally design and predict the properties of the nanocarriers . liposomes , polymer micelles , and polymer nanoparticles , are normally administered via intravenous injections that require the nanocarriers to be hemocompatible and have stealth property in order to avoid fast clearance by the reticuloendothelial system ( res ) . the formation of the nanocarriers is mostly driven by the hydrophobic aggregation or ionic interactions of the amphiphilic materials , which have a tendency to interact with cell membranes via hydrophobic or charge interactions . while the paclitaxel - loaded micelles formed by such telodendrimers exhibited a safe profile in in vivo administration for anticancer treatment in mouse models and companion dogs , the empty telodendrimer micelles showed moderate hemolytic properties at relative high concentrations ( 1 mg / ml ) in vitro . however , the additional reagents and treatments for micelle cross - linking usually require extensive purification before in vivo administration , which may lead to the loss of drug molecules and will be a significant burden for clinical application in terms of quality control and regulations . therefore , there is a need to study the structure and hemolytic activity relationship of nanocarrier to eliminate the hemolytic potential , which could be studied via a series of structural modifications and the property characterization . we hypothesize that introducing bulky hydrophilic groups onto hydroxyl groups of ca may disrupt the compact interactions between the polar surfaces of cas and increase the polar surface area ( psa ) of the whole complex , therefore reducing the membrane activity of telodendrimers . the core - forming subunits of the telodendrimers have been studied via molecular dynamics to study the conformations and the corresponding polar surface area ( psa ) to predict the membrane activities of the nanocarriers . the nomenclature of the telodendrimers and the dimer molecules followed the system used in the previous studies : for example , telodendrimer pegca8 indicates that the molecular weight of peg is 5 kda and there are eight cholic acids conjugated on the periphery of dendritic polylysine . briefly , fmoc peptide chemistry was used to couple ( fmoc)lys(fmoc)-oh onto the n - terminal of peg ( mw = 5 or 10 kda ) using diisopropyl carbodimide ( dic , 3 equiv ) and n - hydroxybenzotriazole ( hobt , 3 equiv ) as coupling reagents in dmf overnight . the percent hemolysis of rbcs was calculated using the following formula : ptx loaded nanoformulations ( 500 ul ) were placed in the dialysis bags with the mwco of 3.5 kda and dialyzed against 4 five ul of drug solutions were sampled at the designed time intervals and pbs solution were refreshed at each measurement . ovarian cancer cell line skov-3 , colon cancer cell line ht-29 , breast cancer cell line mda - mb-231 and lymphoma raji cell line were purchased from american type culture collection ( atcc ; manassas , va , u.s.a . ) all these cells were cultured in mccoy s 5a ovarian cancer cell line skov-3 , colon cancer cell line ht-29 , breast cancer cell line mda - mb-231 and lymphoma raji cell line were purchased from american type culture collection ( atcc ; manassas , va , u.s.a . ) skov-3 and ht-29 cells were cultured in mccov s 5a medium and mda - mb-231 and raji cells were cultured with rpmi 1640 medium supplemented with 10% fetal bovine serum ( fbs ) , 100 u / ml penicillin g , and 100 g / ml streptomycin at 37 c using a humidified 5% co2 incubator . a subcutaneous colon cancer xenograft mouse model was established by injecting 1 10 ht-29 or mda - mb-231 cancer cells in a 100 l of mixture of pbs and matrigel ( 1:1 v / v ) subcutaneously at the right flank in female nude mice . at different time points postinjection of did- and ptx - coloaded micelles formed by telodendrimer ii ( the mass ratio of did and ptx within nanocarrier was 0.25:1:10 ) , mice were scanned using a ivis-200 small animal imager at cy5.5 excitation and emission channels . for each of the ca dimers , a set of 1000 independent annealing simulations were performed from randomly selected starting structures generated by a canonical nvt ensemble simulation run at t = 1000 k. the annealing was then performed by gradually reducing the temperature from 1000 to 5 k over 1 ns . the membrane activity of the telodendrimer micelles is believed due to the facial amphiphilic structure of ca subunit , which self - assembles through hydrogen bonding with the hydrophobic surfaces exposed to the lipid bilayer . also , the insertion of the assembled dimers or tetramers of ca into the phospholipid s bilayer membrane have been reported to form artificial ion channels , where hydrophilic hydroxyl groups are sheltered from the hydrophobic lipids . therefore , the disruption of the packing between the hydrophilic surfaces of oligo - cholic acid is expected to reduce the plasma membrane lytic activity of these types of molecules and the peg - block - oligoca telodendrimers . in this study , glycerol and amino - glycerol have been introduced into the 3- and 7-oh or 12-oh groups of ca via ether bond formation to increase the steric hindrance while maintaining the facial amphiphilicity of ca ( scheme 1 ) . reagents and conditions : ( i ) tfaa , anhydrous thf , 05 c , 1.5 h ; ( ii ) t - buoh , below 5 c , then at rt for 7 h ; ( iii ) nh3h2o ( 2030% ) , 05 c for 12 h , then rt for 4 h ; ( iv ) epichlorohydrin , 50% naoh , ( n - bu)4noh30 h2o , ch2cl2 , rt , 16 h ; ( v ) nh3 in meoh ( 7 m ) , licl ( 1.2 equiv ) in a sealed container , rt , 24 h ; ( vi ) fmoc - osu ( 1.2 equiv ) , diea ( 1.5 equiv ) , ch2cl2 , 12 h ; ( vii ) 50% tfa in ch2cl2 , 0 c , 30 min ; ( iii ) lioh water solution ( 10 equiv ) , rt , 1836 h ; ( ix ) hosu ( 1.2 equiv ) , dcc ( 1.2 equiv ) , ch2cl2 , rt , 12 h. first , the carboxylic acid of ca was protected via an acid labile tert - butyl ester via an intermediate of the hybrid anhydride with trifluoroacetic acid ( tfa ) . through flash column , the monosubstituted 2 and diepoxy substituted compound 3 it should be mentioned that the diepoxy - substituted compound 3 may be a mixture of the compounds of 3,5-disubstituted and 3,7-disubstituted species due to the similar reactivity of 5-oh and 7-oh groups . it was evidenced via the partial decrease of the proton signals on both 5 and 7 carbons ( 3.82 and 3.93 ppm ) , which corresponds to the c h with the unsubstituted hydroxyl groups ( figure s1 in si ) . furthermore , free amine in compound 6 was protected via fmoc - osu , then tert - butyl ester in compound 7 was removed via the treatment of 50% tfa in dcm to yield fmoc - protected amino cholic acid 8 with an 88.4% yield after flash column purification . the proton signal of 3-oh on ca disappeared in telodendrimers ii and iv , and new hydroxyl groups on glycerol and amino - glycerol appeared in telodendrimers ii and iv , indicating the expected 3-position glycerol substitution . the complete disappearance of the 3-hydroxyl group and the partially decreased proton signals of both 7- and 12- hydroxyl groups on ca in telodendrimer iii were observed in the nmr spectrum ( figure 1 ) . as expected , the engineered telodendrimer ii peg(ca-4oh)8 micelles exhibited negligible hemolytic properties compared with typical telodendrimer i pegca8 ( figure 2 ) after incubation with red blood cells for both 4 and 20 h at 37 c . very surprisingly , much more hydrophilic telodendrimer iii with five hydroxyl groups on each cholic acid scaffold also showed significant hemolysis at high concentrations of about 0.5 to 1 mg / ml after 4 h incubation , which was even higher than the parent telodendrimer pegca8 ( figure 2 ) . although , it is not easy to study the interactions between micelles and plasma membrane directly via molecular simulation at the atom level , we could apply these approaches to study the conformations of the hydrophobic subunits of the telodendrimers , which directly interact with plasma membrane . it is believed that the plasma membrane lytic activity of a polymer micelle solution are caused by a small portion of the free amphiphilic polymers existing in solution rather than the intact micelles which are covered by a layer of inert hydrophilic polymer . as shown in figure 3 , a hand - in - hand like hairpin conformation of kca2 with the efficient shelter of their hydroxyl groups at the interface of the complex formed via hydrogen bonding . on the other hand , the disintegrated polymer micelle may merge with the cell membrane from the exposed hydrophobic core , especially when nanoparticles are chemically or physically anchored onto the cell membrane via targeting ligands or nonspecific interactions . the molecular dynamics simulations revealed that the single - glycerol modified ca dimer , k(ca-4oh)2 , also forms a hairpin - like folded conformation via hydrogen bonding ( figure 3 ) . most polar groups have been sheltered within the folded conformation , and the psa was calculated to be the smallest at 1.04 nm for k(ca-5oh_7)2 in vacuum , which is even smaller than that for kca2 in the original telodendrimer i. the possible isomers of the disubstituted ca dimers with the second glycerol on the 7 or 12 position ( e.g. structure of the cholic acid dimers with different glycerol substitution prepared via solution - phase organic synthesis ( a ) ; hemolytic properties of small - molecule dimers of cholic acid with different glycerol modifications after incubation with human red blood cells for 30 min ( b ) and 4 h ( c ) . as predicated in the md simulation , the k(ca-4oh)2 exhibited a significantly lower membrane activity in hemolysis assays than both k(ca-5oh)2 and k(ca)2 for 30 min and 4 h incubation ( figure 4b , c ) . the above chemical modifications of ca on the hydrophilic surface maintained the facial amphiphilic property of ca within the telodendrimer , which ensured the formation of micellar nanoparticles for drug loading . the cmcs were measured via the classic pyrene 1:3 method to be 17.4 g / ml , 24.2 g / ml , 95.9 g / ml and 49.3 g / ml for telodendrimers i , ii , iii , and iv , respectively ( figure s-7 in si ) . the telodendrimer ii peg(ca-4oh)8 showed similar particle size and stability before and after ptx loading at 3/20 drug / polymer ratio , compared with the original telodendrimer i pegca8 , due to the similar structures and cmcs . such ptx - loaded nanoparticles ( 2:10 w / w ptx / iii ) exhibited heterogeneous size distribution of 51 and 341 nm in pbs ( figure s9 in si ) , which is not preferred for in vivo applications . accumulative drug release profile of ptx and dox loaded in the nanocarrier formed with telodendrimer i ( a ) and monoglycerol derived telodendrimer ii ( b ) ; quantitative flow cytometry analysis of the cellular uptake of various dox formulations by raji cells after 30 min ( c ) and 2 h ( d ) incubation at three drug concentrations . the cell viability assays to evaluate the cytotoxicity of telodendrimer ii and the anticancer effects of the micelles loaded with ptx ( e ) and dox ( f ) on the skov-3 ovarian cell line and raji lymphoma cell line , respectively . as shown in figure 6c , d , the quantitative flow cytometric analysis demonstrated the similar cell uptake for free dox and dox loaded in telodendrimer micelles in raji cells after 30 min and 2 h incubation at different drug concentrations . furthermore , the in vitro cytotoxicity studies ( figure 6f ) revealed the similar anticancer effects on the raji lymphoma cells for the free dox and dox loaded in the nanocarriers i and ii after 72 h incubation . additionally the ptx - loaded telodendrimer ii ( ic50 : 1.6 ng / ml ) and ptx - loaded telodendrimer i ( ic50 : 3.4 ng / ml ) exhibited slightly higher anticancer effects than free ptx ( ic 50:14.5 ng / ml ) formulated in a mixture of cremophore el and ethanol ( 1:1 v / v ) in an ovarian cancer cell line , skov-3 ( figure 6e ) . ex vivo biodistribution of telodendrimer nanocarrier ii loaded with ptx and nirf dye did in colon cancer - bearing nude mice after 48 h postinjection via tail vein ( a ) ; the comparison of the average fluorescence intensity in various organs and ht-29 tumors between the animals given did - ptx - np ii or free dye did ( b ) ; ex vivo biodistribution of telodendrimer nanocarrier ii loaded with ptx and nirf dye did in breast cancer bearing nude mice after 72 h postinjection via tail vein ( c ) ; quantitative analysis of the average fluorescence intensity in various organs and mda - mb-231 tumors between the animals given did - ptx - np ii or free dye did ( d ) . therefore , we would like to further evaluate the biodistribution and tumor targeting properties of the optimized telodendrimers in solid tumor xenograft models , such as colon and breast cancers after intravenous injection of telodendrimer ii micelles coloaded with nirf dye did and ptx . with the enhanced in vivo anticancer effects for the ptx formulated in telodendrimer i nanocarriers , we anticipate these nonhemolytic nanotherapeutics formed by telodendrimer ii will further improve the anticancer effects via efficient tumor - targeted drug delivery . the further pk / pd studies and the in vivo efficacy of this optimized nanocarrier in delivering different chemodrugs in animal models will be tested and reported in a separate account . to minimize the hemolytic properties of telodendrimer nanocarriers , we took a rational approach to modify telodendrimer structures by introducing hydrophilic glycerol or amino - glycerol groups onto the polar surface of cholic acid . with the aid of molecular dynamics simulations , we had a better understanding of the relationship between plasma membrane lytic activity and the structural conformations of the amphiphiles . the introduction of hydrophilic monoglycerol onto the polar surface of cholic acid was predicated via molecular dynamics to be optimal in disrupting the molecular folding of the subunit of telodendrimer and increasing the polar surface area of the complex . on the contrary , introduction of two glycerols or an amino - glycerol on each cholic acid led to the increased hemolytic properties of the resulting telodendrimers , which were correlated with the decreased psa and the exposed positive charges in the molecular dynamics simulations , respectively . further , the computation results have been verified in the experiments via the synthesis of the telodendrimer subunits , which were tested to be consistent with the theoretical prediction for the membrane activities . such spatial modification of ca via monoglycerol grafting sustained the balance of the facial amphiphilicity of ca , therefore maintaining the ptx and dox loading capacity , stability , and anticancer effects of the nanotherapeutics . given the nonhemolytic property , efficient cancer cell uptake , and the excellent in vivo tumor - targeted properties , the monoglycerol derivatized telodendrimer ii has been demonstrated as a promising nanocarrier for efficient anticancer drug delivery .

mesoporous semiconductor oxide electrodes are used in different applications including electrochemical sensing , electrochromic devices and photoelectrochemical generation of fuels or electrical energy . all these applications rely on the external manipulation or tracking of the charge transfer between an optically and/or chemically active layer and an external contact . the macroscopic rate of charge transfer between the mesoporous film and a conductive substrate is the result of a sequence of intermingled microscopic processes . these processes , which are associated with carrier generation , recombination , transport and transfer , take place on different time scales and compete kinetically with each other . due to a high concentration of trap states electron transport in mesoporous semiconductor oxide films is orders of magnitude slower than in single crystals . in photoelectrocatalytic and photovoltaic applications electron collection at the external contact competes with charge recombination in the bulk of the semiconductor and at the interfaces within the porous film , limiting solar conversion efficiency . the use of electrochemical doping ( charge transfer reductive doping ) , where electron / proton or electron / li pairs incorporated within the oxide passivate the electron traps , is a very efficient way to temporarily improve photoelectrochemical activity , and as such , constitutes an elegant way to improve charge separation within these materials . although many efforts are being pursued in this direction , a comprehensive study of these electronic defects is still missing . to reach such a level of understanding , one needs to carefully identify the nature of the recombination centers and transport - limiting traps in mesoporous semiconductor electrodes , which is a significant challenge for both experimentalists and theoreticians . it is well established that the fundamental processes associated with the transport , transfer , and recombination of photogenerated charge carriers in mesoporous semiconductor electrodes are determined by both the distribution of band gap states and their population , i.e. , the position of the fermi level within the film . electrochemical methods such as cyclic voltammetry and electrochemical impedance spectroscopy have proven useful in characterizing electronic states in nanostructured semiconductor oxide electrodes , though the chemical nature of the traps remains controversial . specifically , charge / discharge measurements provide information on the distribution of electrochemically active states in mesoporous electrodes , where electron accumulation is compensated by the adsorption of ions at the oxide surface . for tio2 electrodes in an aqueous acidic electrolyte the generation of ti centers is compensated by proton uptake ( eq 1)1importantly , in the case of small cations ( such as h or li ) , charge injection and compensation not only take place at the semiconductor / electrolyte interface , but can become a three - dimensional process via insertion of ions into subsurface regions of the nanocrystals . the reversibility of this charge accumulation opens up the possibility of driving fast reduction reactions at the semiconductor / electrolyte interface . on the other hand , electrochemical doping was found to modify , at least temporarily , the electrode performance in different applications ranging from dye - sensitized solar cells to photocatalysis and supercapacitors . the doping of mesoporous tio2 electrodes with li ( which is isovalent with h ) has recently been demonstrated to enhance electron transport and improve efficiency in perovskite solar cells . whereas different studies discuss an increase of the electrode performance upon electrochemical doping phenomenologically by accelerated charge transport and reduced recombination , the underlying microscopic details remain to be elucidated . for tio2 , theoretical studies have recently addressed at the single particle level the geometry and energetics of electron trap states in the bulk and at the semiconductor / electrolyte interface . deep electron traps located at the grain boundary are found to slow down charge transport unless high current densities ensure a high average occupation of transport - limiting traps . such trap filling effects have recently been highlighted for deep traps in oriented tio2 nanotube arrays by dynamic photocurrent measurements . in addition , these states may act as recombination sites exerting a further deleterious impact on the photocurrent . the high structural and electronic complexity of mesoporous semiconductor oxide electrodes makes an investigation of the nature , concentration , and location of electronic trap states and the elucidation of their impact on charge recombination and transport very challenging . designing appropriate model systems to understand the action of these states is difficult . indeed , their complexity must be high enough to realistically mimic processes in technologically relevant materials , but low enough to result in clear structure activity relationships that can be supported by both experiments and theoretical modeling . in this work , we combine first - principles theoretical calculations with the electrochemical characterization of nanostructured rutile tio2 films to demonstrate that particle / particle interfaces introduce deep traps . these interfaces represent favorable locations for proton segregation , which can be induced by the electrochemical doping of the porous electrodes . a long lasting ( hours to days ) , but reversible accumulation of electrons and protons ( i.e. , e / h or h doping ) at the interface is tracked by cyclic voltammetry via the shift toward more positive potentials of a pair of capacitive peaks , which is associated with trap states at the particle / particle interface . the passivation of recombination centers by e / h doping leads to a transient photocurrent enhancement due to improved electron / hole separation for those electrodes , which are characterized by a high concentration of particle / particle interfaces ( i.e. , random particle networks ) . for electrodes lacking a high density of particle / particle interfaces ( i.e. , arrays of oriented nanocolumns ) this study highlights the importance of particle / particle interfaces in mesoporous films and provides strategies to actively manipulate the density of electronic states and their population by electrochemical methods . the resulting long - lasting ( > 15 h ) improvement of photoelectrode performance after electrochemical doping is explained by using theoretical calculations that are in qualitative agreement with experiments . slurries of rutile tio2 nanoparticles ( sachtleben , nano rutile ) were prepared by grinding 1 g of tio2 powder with 3.2 ml of h2o , 60 l of acetylacetone ( 99+% , aldrich ) , and 60 l of triton x ( aldrich ) and were spread with a glass rod onto fluorine - doped tin oxide ( fto ) conducting glass ( pilkington , tec 15 , resistance 15 / ) using scotch tape as a spacer . alternatively , ti foils ( goodfellow , 99.6+% , 250 m ) were used to investigate a possible impact of the substrate type on the electrochemical and photoelectrochemical properties . however , the nanoparticle ( np ) films were annealed and sintered for 1 h at 450 c in air . after sintering a film thickness of 3.5 2.0 m was determined by scanning electron microscopy ( sem ) . the films are formed by a random network of elongated particles with a length of 50 nm and a width of 20 nm ( figure s1a , b and ref ( 33 ) ) and are of pure rutile phase ( figure s2 ) . as shown in a previous study , furthermore , it was estimated that a high fraction of the exposed surface is formed by ( 110 ) facets . electrodes formed by a rutile tio2 nanocolumn ( nc ) array were prepared using a hydrothermal synthesis . concretely , 21.6 ml of a 6 m hcl solution were mixed with 360 l of tetra - n - butylorthotitanat ( 98% , merck millipore ) . the solution was placed in a teflon - lined steel autoclave ( 45 ml , parr instruments ) containing fto substrates and was heated to 150 c for 15 h. after synthesis , the electrodes were thoroughly rinsed with water . these films consist of rutile tio2 nanocolumns with a rectangular cross section and with a width of 80180 nm and a length of 1.5 m ( figures s1c , d and s2 ) . as highlighted previously , individual nanocolumns are porous and consist of a bundle of oriented and single crystalline nanowires with a diameter of 1020 nm . the nanowires are elongated along the direction and are expected to expose ( 110 ) facets at the surface . the endings of the nanowires can be observed at higher magnifications at the top parts of the nanocolumns ( inset in figure s1c ) . for both types of electrode the contact area and the uncovered parts of the substrate were finally sealed by epoxy resin . spin polarized density functional theory ( dft ) calculations are performed using the projector augmented wave formalism as implemented in the vienna ab initio simulation package . the 3d and 4s electrons of ti , and the 2s and 2p electrons of o are treated as valence electrons and expanded in a plane wave basis with energies up to 500 ev . ernzerhof exchange correlation functional and correct for the self - interaction error ( sie ) for electrons by employing a dft+u approach . the hubbard u parameter for the ti 3d - states is taken from previous work which fitted to spectroscopic properties of surface oxygen vacancies ( uti = 4.2 ev ) . we also employ a hubbard u term to correct the sie on o 2p - states ( uo = 7.5 ev ) in order to make the results transferable to future calculations which will consider electrons and holes . however , we note that the addition of a hubbard u term on o does not affect the calculated trapping energies reported in the present work . for the conventional cell of rutile a 6 6 9 monkhorst pack k - point grid is used and structural optimization is performed until forces are less than 0.01 ev / . using the perdew burke ernzerhof exchange correlation functional we obtain lattice parameters within 2% of experiment ( a = 4.67 and c = 3.03 ) . to investigate the interaction of electrons with the grain boundary defect , we attempt to localize an electron polaron at all inequivalent ti sites within the grain boundary supercell . to achieve this , we create a precursor potential well for electron trapping by displacing nearest neighbor anions away from a particular ti site by 0.1 followed by full self - consistent optimization of the structure . in cases where this displacement procedure alone is insufficient to direct the self - consistent optimization into the desired charge localized metastable state , we manually set the orbital occupancy using a modification to the vasp code developed by allen and watson . however , we stress that in all cases the resulting metastable states are fully and self - consistently optimized . for calculations involving charged defects ( such as electron polarons ) , overall neutrality to identify prospective proton incorporation sites in the grain boundary supercell , we make use of the fact that protons will form a bond with lattice oxygen ions at a distance of approximately 1.0 . we computationally identify the set of positions within 1.0 0.1 of each lattice oxygen ion . we further reduce the number of possible proton positions by identifying the proton positions around each oxygen ion that has the lowest electrostatic potential ( thereby representing the most favorable position for the proton on electrostatic grounds ) . in this way , we can readily obtain a large number of prospective proton positions , which provide the initial coordinates for full geometry relaxations . using this procedure , inequivalent proton sites with the this procedure is straightforward to implement and may , with suitable modification , be applicable to modeling protons in low symmetry structures ( such as nanoparticles or surfaces ) in a wider range of oxide materials . rutile tio2 nanoparticle ( np ) electrodes ( consisting of a random network of tio2 particles , figure s1a , b ) and nanocolumn ( nc ) electrodes ( consisting of an array of oriented nanocolumns , figure s1c , d ) have been used in the present study as model systems for investigating the effect of nanocrystal organization and interconnection on electrochemical and photoelectrochemical properties . the voltammetric response of a rutile tio2 np electrode is characterized , in the absence of significant faradaic currents ( i.e. , in 1 m methanol/0.1 m hclo4 aqueous solution purged from o2 ) , by a charge accumulation region at low potentials ( figure 1a ) . the photocurrent onset potential , which yields an estimate of the conduction band edge position in the semiconductor , lies for this electrode at eag / agcl 0.5 v ( figure s3a ) . cvs for rutile tio2 np ( a ) and nc ( b ) electrodes before and after an electrochemical doping at eag / agcl = 0.6 v for 3 h. for the np electrode the effect of subsequent polarization for 15 h at 0.8 v ( dedoping ) is also shown . previous analyses of the density of electrochemically active band gap states in mesoporous tio2 films by cyclic voltammetry and electrochemical impedance spectroscopy have demonstrated the presence of a broad exponential distribution of states below the conduction band edge in the accumulation region in the case of anatase electrodes , which is absent in rutile tio2 films . currents in the accumulation region ( eag / agcl < 0.25 v , figure 1 ) of rutile tio2 electrodes have been attributed to the population / depopulation of electronic states in the conduction band compensated by proton adsorption at the oxide surface . however , for both tio2 modifications , a narrow distribution of deep trap states is typically present and gives rise to a pair of capacitive peaks in the cyclic voltammograms ( cvs ) , which is also observed on rutile tio2 np electrodes ( figure 1a ) . 0.13 v and lie thus 0.30.5 v below the conduction band edge of the semiconductor . in the following , we will focus in detail on the intensity of these signals and on the energetics of the associated trap states in mesoporous films featuring different morphology ( i.e. , np versus nc films ) and will follow their modification upon electrochemical doping . the corresponding signal is much less pronounced for the rutile tio2 nc electrode ( figure 1b ) as compared to the np electrode ( figure 1a ) . previous studies reported that for both ordered one - dimensional nanostructures and single crystal electrodes , the peaks are virtually absent , whereas they show a high intensity for thin film electrodes consisting of random nanoparticle networks . in line with previous interpretations , we assign the couple of capacitive peaks observed for the np electrode to the contribution of electron traps at particle / particle interfaces . interestingly , the contribution is asymmetric , the anodic peak being much broader than the cathodic peak ( figures 1a and s4a ) . slow kinetics for h extraction ( compare eq 1 ) or a change in the electrode conductivity may contribute to this effect . the cvs in figure 1 were obtained by applying to the electrode a linear potential profile with a scan rate of 20 mvs . importantly , at fast scan rates , it is possible that not all of the deep traps in the mesoporous film are equilibrated with the fermi level of the conducting substrate . this is the reason why even large perturbation techniques such as cyclic voltammetry may yield for deep trap states only apparent chemical capacitances . therefore , we performed charging and discharging measurements using extremely long lasting perturbations in the potential range featuring the capacitive peaks ( figure s5 ) . we measured the capacitive currents upon stepping the electrode potential in potential steps eag / agcl = 0.02 v first from 0.2 to 0.2 v ( charge accumulation ) and then from 0.2 v back to 0.2 v ( charge extraction ) . after every step , the potential was kept constant for 60 s and the accumulated / extracted charge density associated with each potential step ( left axis in figure s4b ) was determined by integration of the resulting current transient ( figure s5 ) . to obtain the chemical capacitance associated with interface traps ( right axis in figure s4b ) the charge was then referred to eag / agcl . such an analysis yields a much higher symmetry of charging and discharging branches , nevertheless , there is still an imbalance of positive and negative charge pointing to a partial irreversibility of charge accumulation . the chemical capacitance extracted from these measurements ( right axes in figure s4a , b ) has thus to be considered an apparent capacitance . from the total charge accumulated upon stepping the potential from 0.2 to 0.2 v ( 35 ccm , figure s4b ) , we estimate ( using the average values of film thickness and particle size and assuming a film porosity of 0.5 ) the number of extracted charges to correspond to 25 electrons per tio2 nanoparticle . as previously reported , tio2 electrodes can be electrochemically doped by cathodic polarization . following polarization at eag / agcl = 0.6 v significant changes are observed in the cv of a rutile tio2 np electrode ( figures 1a and s4a ) : the peak corresponding to deep traps is displaced by 0.08 v toward more positive potentials , while a slight increase of the peak intensity is observed upon doping . the same observations are made in the absence of methanol ( figure s6 ) . qualitatively the same conclusions can be drawn from the large perturbation charging / discharging experiment ( figure s4b ) . electrochemical doping induces only minor changes at eag / agcl < 0.2 v , although a slight increase of the capacitive current is observed at 0.45 v < eag / agcl < 0.25 v. importantly , we do not observe a shift of the photocurrent onset potential upon electrochemical doping ( figure s3b ) indicating that the band edges are not displaced significantly . all changes in the cvs are reversible with respect to prolonged polarization at 0.8 v ( t > 15 h , figure 1a ) . these observations point to a dynamic and transient change of the density of electrochemically active states upon electrochemical charge accumulation in np electrodes . importantly , no significant change of the cv is observed upon doping of a nc electrode ( figure 1b ) . electrochemical doping has a beneficial effect on the photoelectrochemical performance of rutile tio2 np electrodes as deduced from photocurrent transients ( figures 2a and s7 ) and cvs ( figure s8a ) . concretely , the photocurrent generated by the electrode in a 0.1 m hclo4 aqueous solution containing 1 m methanol as a hole scavenger depends significantly on the electrochemical pretreatment of the film as shown in the following . first the photocurrent of a pristine electrode was recorded at eag / agcl = 0.8 v. then the electrode was polarized at progressively more negative potentials in the accumulation region corresponding to the electrochemical doping of the film . after every doping step the photocurrent was again recorded to sample the impact of doping on the photoelectrocatalytic activity of the electrode . two doping parameters were systematically changed doping potential ( figure s7 ) and doping time ( figure 2 ) . whereas electrode polarization for 20 min at edop = 0.5 v induces only minor changes of the photoelectrocatalytic activity , we observe an up to 3-fold photocurrent increase when doping at edop = 0.6 v ( figure s7 ) . the photocurrent enhancement by electrochemical doping is a very slow process . only after 4 h of polarization at 0.6 v no further changes after such a long doping time the photocurrent has experienced an increase by a factor of 7 ( photocurrent enhancement factor , pcef = 7 , figure 2a ) . photocurrent transients recorded upon uv exposure of rutile tio2 np ( a ) and nc ( b ) electrodes before and after electrochemical doping at eag / agcl = 0.6 v for different doping times ( t0.6vdop ) . importantly , the photocurrent increase is reversible with respect to prolonged polarization at positive potentials ( figure s8 ) . however , even after 15 h of charge extraction ( by electrode polarization at 0.8 v ) the photocurrent still exceeds its initial value by 30% . these results highlight that the beneficial effect of electrochemical doping is transient , but long lasting . the relative photocurrent enhancement upon doping is much less pronounced for rutile tio2 nc electrodes ( pcef = 2 , figure 2b ) . also in this case the beneficial effect is reversible with respect to polarization at 0.8 v ( not shown ) . a comparison of the photocurrent evolution following the progressive electrochemical doping of np and nc electrodes is shown in the chronoamperometric profiles in figure s9 . importantly , these results confirm that the increased current measured upon uv exposure of doped electrodes corresponds indeed to a faradaic photocurrent and does not simply result from a light - induced extraction of charges accumulated in the doping step . the additional charge transferred ( after doping ) from the tio2 film to the conducting substrate upon uv exposure exceeds by far the charge injected from the conducting substrate into the tio2 film upon electrochemical doping ( figure s9a ) . from the electrochemical characterization of np and nc films we have gained the following pieces of information about the impact of electrode morphology and electrochemical doping on the density of electrochemically active states and on the photoelectrocatalytic activity : ( i ) for porous films consisting of a random particle network ( np electrodes ) a high density of deep electron traps gives rise to a couple of capacitive peaks in the cv . this signal is virtually absent in films consisting of oriented nanocolumn arrays ( nc electrodes ) . ( ii ) prolonged polarization of np electrodes at eag / agcl = 0.6 v ( electrochemical or charge transfer reductive doping ) induces a displacement of these capacitive peaks by 0.08 v toward more positive potentials and a minor increase in the chemical capacitance at 0.45 v < eag / agcl < 0.25 v. these changes are reversible with respect to charge extraction ( dedoping ) upon prolonged electrode polarization at eag / agcl = 0.8 v. both processes ( doping and dedoping ) are extremely slow ( hours to days ) . ( iii ) electrochemical doping increases the photoelectrocatalytic activity of np electrodes toward methanol oxidation as sampled by a 7-fold increase of the photocurrent ( pcef = 7 ) . the activity enhancement is transient and the photocurrent relaxes slowly back to its initial value ( t > 15 h ) . the beneficial effect of electrochemical doping is much less pronounced for rutile tio2 nc electrodes ( pcef = 2 ) . to help interpret the experimental results discussed above and provide deeper atomistic insight into the effect of protons on electron trapping we perform first - principles theoretical calculations for a model interface in nanocrystalline tio2 . in particular , we consider the ( 210 ) rutile tio2 grain boundary , the structure of which has been investigated previously both experimentally and theoretically ( figure 3a ) . while this interface possesses a high degree of symmetry it has atomistic features which are expected to be representative of more general interfaces in nanocrystalline tio2 , namely , reduced ion coordination and local strain at the interface . in a recent theoretical study , it was demonstrated that this grain boundary is associated with interfacial ti ions which can trap electrons more strongly than bulk ti lattice sites . this effect is due to local variations in the electrostatic potential near the grain boundary and changes in ion coordination and bond strain with similar effects found at tio2 surfaces.figure 4 shows the distribution of electron trapping energies ( defined with respect to the energy of an electron trapped on a bulk ti site ) for ti ions within 6 of the grain boundary plane . we note that at finite temperature electrons may hop between ti sites at the interface . the activation energy for electron hopping between adjacent sites was calculated previously to be about 0.3 ev in the bulk and up to 50% higher at the interface . owing to their increased stability the equilibrium occupation of interfacial traps will remain higher than that in the bulk . therefore , the presence of deep traps at this interface provides a semiquantitative model for the voltammetric feature of electron trapping states ( pair of capacitive peaks ) observed in the pristine tio2 np electrodes by cyclic voltammetry ( figure 1a ) . as such it is a useful reference system on which to explore the interaction of protons with interfaces and the subsequent effect they have on electron trapping . for the following discussion , it is important to keep in mind that the energy scale and the electrochemical potential scale have opposite signs , i.e. , a trap state becoming more stable ( i.e. , deeper ) will be characterized by a more negative trapping energy and a more positive electrochemical potential . ( a ) optimized structure of the pristine ( 210 ) rutile tio2 grain boundary showing the electron spin density associated with an electron in the most stable site ( isosurface shown in purple ) . ( c ) ( h)(e ) decorated ( 210 ) rutile tio2 grain boundary . ( d ) ( h)(e ) decorated ( 210 ) rutile tio2 grain boundary with an additional electron trapped in the most stable site . ti sites , o sites , and h ions are represented by blue , red , and green spheres , respectively . distribution of electron trapping energies ( et ) within 6 of the pristine and ( h)(e)-doped grain boundary . et is defined with respect to the energy of an electron trapped on a bulk ti site ( horizontal dashed red line ) . all levels in the shaded region correspond to interfacial sites which are available to trap electrons more strongly than the bulk crystal ( hereafter referred to as interface traps ) . the side panels show the spatial distribution of interface traps ( highlighted by orange spheres ) . the ti site which already has a trapped electron in the ( h)(e)-doped interface is unavailable to trap additional electrons ( indicated by the light blue sphere on the right side panel ) . on ( h)(e)-doping , the number of available interface traps is reduced by 50% ( from 1.89 10 to 0.95 10 cm ) . electrochemical doping of tio2 by prolonged polarization is likely to be associated with the incorporation of h ions from the aqueous solution to compensate the negative electron charge trapped at interfaces . to assess this possibility we first investigate the interaction of protons with the ( 210 ) rutile tio2 grain boundary . on introduction into the tio2 lattice protons form bonds with lattice o ions resulting in oh species . while previous theoretical studies have identified the most stable structure of the oh species in bulk rutile tio2 , it is not straightforward to deduce the likely proton configurations in the lower symmetry grain boundary region . to address this problem we identify prospective positions for h incorporation based on analysis of the three - dimensional electrostatic potential and screen 80 different configurations to identify the most stable structure ( see experimental section ) . for these calculations we consider one h ion in a supercell of dimensions 9.1046 10.439 corresponding to a density of 1.05 10 cm . the most stable h incorporation site is found at the grain boundary and is 0.6 ev more stable than in the bulk ( figure 3b ) . the presence of h induces a transformation in structure near the grain boundary as compared to the pristine structure . in particular , one of the ti ions near the grain boundary relaxes toward the oh ion . we next investigate the interaction of electrons with the h decorated grain boundary structure identified above . by making suitable initial atomic distortions around each ti site in the supercell followed by full optimization of the total energy with respect to relaxation of all ion coordinates we obtain a series of metastable configurations corresponding to electrons trapped on different ti ions ( see experimental section ) . the most stable electron trapping ti site is located at the grain boundary directly adjacent to the oh species ( figure 3c ) . this defect can be considered as a h atom with the proton and electron dissociated onto neighboring sites . very similar defect centers are found in nanocrystalline mgo where they have been characterized in detail by electron spin resonance and theoretical calculations . hereafter , we will refer to these proton plus electron defects produced by h doping as ( h)(e ) centers , following the nomenclature of previous studies . the ( h)(e ) center should provide a reasonable model for the electron traps in the np electrodes following electrochemical doping and polarized for sufficiently short times at a positive potential . we have also computed the fermi contact hyperfine coupling parameter for ( h)(e ) in the most stable position segregated at grain boundary 7.5 mhz . this is significantly reduced compared to that calculated for the isolated h atom 1402.6 mhz ( close to the experimental value of 1422 mhz ) . this could provide an experimental signature of ( h)(e ) centers at the grain boundary . if each proton at the grain boundary has already trapped an electron forming a ( h)(e ) center ( as shown in figure 3c ) one may ask how additional electrons added to the system would interact with the interface ( for example as realized experimentally by cv measurements on electrochemically doped electrodes ) . to address this question we obtain fully optimized metastable configurations corresponding to the localization of a second electron on all ti sites in the supercell . an electron trapped on a bulk - like ti site has a very similar local geometry and spin density to the bulk - like polaron in the pristine grain boundary . this provides a reference with which to assess the trapping energies of sites in the vicinity of the grain boundary . we find a distribution of trapping energies for ti ions within 6 of the grain boundary plane spanning a similar range to that found for the pristine interface and the most stable electron trap is again located close to the grain boundary ( figure 3d ) . the distribution of electron trapping energies associated with the pristine and ( h)(e)-doped grain boundaries are compared in figure 4 . the pristine grain boundary presents 18 ti sites per supercell that can trap electrons more strongly than in the bulk ( i.e. , traps with et < 0 , hereafter referred to as interface traps ) . this corresponds to an interface trap concentration of 1.89 10 cm with an average trapping energy of 0.14 ev . following ( h)(e)-doping the number of interface traps are found to trap electrons corresponding to an interface trap concentration of 0.95 10 cm . analysis of atomic structures indicates that a number of effects are responsible for the modification of interfacial traps on ( h)(e)-doping . as noted above , the presence of h induces a localized deformation , which changes the structural and electrostatic environment of ti sites near the grain boundary . we find a strong correlation between the electron trapping energy of a given ti site and its corresponding electrostatic potential , as discussed previously for the pristine case . in particular , the presence of the ( h)(e ) center modifies the electrostatic potential on ti sites near the grain boundary destabilizing a number of traps . at the same time one of the ti sites adjacent to h that was not a trap in the pristine case becomes a trap after doping . the net result is that the number of ti sites available to trap electrons is reduced by 50% . the optimized atomic structures for the pristine and doped grain boundaries are provided in the supporting information . in addition to the reduced concentration of interface traps there is also a reduction in the average trapping energy ( i.e. , from 0.14 to 0.26 ev ) . the shift in average trapping energy of about 0.12 ev is of the same order as that observed experimentally for doped electrodes by cv ( 0.08 ev , figure 1a ) . although the average depth of grain boundary traps is increased , the significant decrease in the density of interface traps provides an explanation for the improved photoelectrocatalytic activity of electrochemically doped electrodes observed experimentally . due to the high specific surface area of mesoporous semiconductor electrodes , the main contribution to the density of electronic band gap states as sampled by electrochemical methods such as cyclic voltammetry results from processes at the semiconductor / electrolyte interface . indeed these electrodes typically show a reversible charging / discharging behavior on short time scales . for tio2 electrodes the corresponding accumulated charge was shown to scale linearly with the internal area of the semiconductor / electrolyte interface . nevertheless , processes with different kinetics contribute with different relative intensities to the overall signal . consequently , when extracting a chemical capacitance from the measured capacitive current , those electronic states getting populated by very slow charging processes will be underrepresented . this is true for electronic states in subsurface regions of the semiconductor , such as the electron traps at particle / particle interfaces giving rise to the pair of capacitive peaks in the cvs of rutile tio2 np electrodes ( figure 1a ) . importantly , whereas electrochemical methods based on charge / discharge measurements provide information on the distribution of electrochemically active states , it must not be ignored that a persistent charge accumulation in the mesoporous film may modify both the fermi level and the density of states itself . in this context , it is well established nowadays that long lasting reductive treatments of mesoporous films may result in a long lasting accumulation of charges ( electrochemical or charge transfer reductive doping ) , thereby significantly influencing the macroscopic electrode behavior in different applications . whereas the technological implications of such an electrochemical manipulation of the electrode properties are clear , the underlying reasons and microscopic details of this phenomenon are unknown . our observations from electrochemical measurements and results from first principle theoretical calculations give a consistent picture of electron and proton trapping in nanocrystalline tio2 films of different morphology . calculations indicate that ( h)(e ) decoration modifies the distribution of electron traps at particle / particle interfaces ( figure 5i ) . an increase of the depth of interface traps goes along with a 50% decrease in their density . consistent changes of the density of electrochemically active states are tracked by voltammetry ( figure 5ii ) . upon electrochemical doping of rutile tio2 np electrodes , i.e. , upon a long lasting accumulation of electron / proton pairs in the film , we observe a reversible shift toward more positive potentials of capacitive peaks associated with trap states at particle / particle interfaces . in addition an increase of the chemical capacitance is observed at more negative potentials , i.e. , at 0.45 v < eag / agcl < 0.25 v. such a modification was previously related to the population of subsurface states upon a light - induced insertion of protons and electrons affording faster charge transport in dye - sensitized tio2 films . here we show that the partial removal of interface traps upon ( h)(e ) decoration of particle / particle interfaces may contribute to such a capacitance change . the main contribution to currents in the accumulation region of both pristine and doped electrodes ( contributions at eag / agcl < 0.25 v highlighted in red in figure 5ii ) , however , are associated with the population / depopulation of electronic states in the rutile tio2 conduction band compensated by proton adsorption at the oxide surface ( ecb / hads states , figure 5c , d ) as discussed in detail in a previous study . scheme highlighting the effect of electrochemical doping on the density of electrochemically active states and on light induced charge separation . electrochemical charge accumulation in np electrodes is reversible on the time scale of a cv measurement , i.e. when recording the electrode s voltammetric response between 0.8 and 0.6 v ( and vice versa ) at a scan rate of 20 mvs ( figure 5a c ) . in this case charge accumulation takes place mainly at the particle surface and at those grain boundary states located near the oxide / electrolyte interface . prolonged polarization of np electrodes at eag / agcl = 0.6 v ( t0.6vdop = 4 h ) induces electrochemical doping and thus a population of trap states deep within the particle / particle interfaces ( figure 5 d ) . this charge accumulation is not reversible on the time scale of a cv measurement ( figure 5d rather there is a long lasting ( though reversible , vide infra ) modification of the density of electrochemically active states . in the voltammetric experiment ( figure 5d f ) charge accumulation and charge extraction take now place on a film featuring particle / particle interfaces , which are partially decorated by ( h)(e ) . consequently , band gap states are associated with modified trapping energies resulting in a modified density of electrochemically active states ( figure 5i and ii . the concerted uptake of e / h pairs in particle / particle interface regions upon cathodic polarization ( i.e. , electron injection from the conducting substrate and insertion of protons from the electrolyte into the oxide ) and the reverse process taking place upon anodic polarization ( i.e. , electron transfer to the substrate and proton diffusion through the solid phase into the electrolyte ) are expected to proceed very slowly . the increased intensity of the pair of capacitive peaks associated with traps at the particle / particle interface can be explained by the enhanced conductivity in the doped film which allows populating and depopulating electron traps faster and deeper within the particle / particle contact area . the persistence of electrochemical doping is associated with the slow kinetics of h diffusion from the gb core to the oxide / electrolyte interface . the dedoping of the film can thus only be achieved upon prolonged polarization at eag / agcl > 0.2 v ( e.g. , t0.8vdedop = 15 h , figure 5a , f ) . the increased photoelectrocatalytic activity of doped electrodes can be attributed to the deactivation of a major fraction of interface traps and recombination sites by the decoration of particle / particle interfaces with ( h)(e ) ( figure 5a , f ) . indeed , calculations point to a 50% decrease in density of interface traps following ( h)(e ) doping ( figure 4 ) , which is expected to affect charge separation at the particle / particle interface in two ways : by accelerating electron transfer across the grain boundary and by reducing electron / hole recombination . on the other hand , kamat and co - workers reported on the deactivation of recombination centers due to trap filling and the generation of ti / h centers in tio2 electrodes . enhancement of the transport properties of mesoporous tio2 electrodes for perovskite solar cells via li doping has also been demonstrated and is proposed to involve a similar mechanism . while not investigated theoretically in this work , hole trapping may also be modified in a favorable way upon ( h)(e ) doping . beneficial effects of electrochemical doping have been reported not only for tio2 , but also for zno , wo3 , and bivo4 films . we believe that our findings will contribute to a better understanding of interfacial processes at play in different metal oxide - based materials . we have tracked the long lasting accumulation of electron / proton pairs in rutile tio2 films consisting of a random nanoparticle network ( i ) via a reversible shift of a capacitive peak in the cv , which we associate with trap states located at particle / particle interfaces , and ( ii ) via the transient enhancement of the photoelectrocatalytic activity toward methanol photooxidation . theoretical calculations indicate that interfaces between crystals in tio2 represent favorable locations for the segregation of proton defects , being up to 0.6 ev more stable than in the bulk crystal . importantly , ( h)(e ) doping of grain boundaries significantly modifies the electronic properties of the particle / particle interface . for ti ions within 6 of the interface a shift in the average trapping energy of deep traps of 0.12 ev . a 50% reduction of the overall number of deep electron traps at the grain boundary is considered to be the main reason for the beneficial effect of electrochemical doping of rutile tio2 np electrodes on their photoelectrocatalytic activity . the qualitative agreement between our experimental results and theoretical calculations strongly supports our detailed description of these complex interfacial systems .

particle / particle interfaces play a crucial role in the functionality and performance of nanocrystalline materials such as mesoporous metal oxide electrodes . defects at these interfaces are known to impede charge separation via slow - down of transport and increase of charge recombination , but can be passivated via electrochemical doping ( i.e. , incorporation of electron / proton pairs ) , leading to transient but large enhancement of photoelectrode performance . although this process is technologically very relevant , it is still poorly understood . here we report on the electrochemical characterization and the theoretical modeling of electron traps in nanocrystalline rutile tio2 films . significant changes in the electrochemical response of porous films consisting of a random network of tio2 particles are observed upon the electrochemical accumulation of electron / proton pairs . the reversible shift of a capacitive peak in the voltammetric profile of the electrode is assigned to an energetic modification of trap states at particle / particle interfaces . this hypothesis is supported by first - principles theoretical calculations on a tio2 grain boundary , providing a simple model for particle / particle interfaces . in particular , it is shown how protons readily segregate to the grain boundary ( being up to 0.6 ev more stable than in the tio2 bulk ) , modifying its structure and electron - trapping properties . the presence of hydrogen at the grain boundary increases the average depth of traps while at the same time reducing their number compared to the undoped situation . this provides an explanation for the transient enhancement of the photoelectrocatalytic activity toward methanol photooxidation which is observed following electrochemical hydrogen doping of rutile tio2 nanoparticle electrodes .

Introduction Experimental Section Results and Discussion General Discussion Conclusions

in photoelectrocatalytic and photovoltaic applications electron collection at the external contact competes with charge recombination in the bulk of the semiconductor and at the interfaces within the porous film , limiting solar conversion efficiency . the use of electrochemical doping ( charge transfer reductive doping ) , where electron / proton or electron / li pairs incorporated within the oxide passivate the electron traps , is a very efficient way to temporarily improve photoelectrochemical activity , and as such , constitutes an elegant way to improve charge separation within these materials . for tio2 electrodes in an aqueous acidic electrolyte the generation of ti centers is compensated by proton uptake ( eq 1)1importantly , in the case of small cations ( such as h or li ) , charge injection and compensation not only take place at the semiconductor / electrolyte interface , but can become a three - dimensional process via insertion of ions into subsurface regions of the nanocrystals . whereas different studies discuss an increase of the electrode performance upon electrochemical doping phenomenologically by accelerated charge transport and reduced recombination , the underlying microscopic details remain to be elucidated . for tio2 , theoretical studies have recently addressed at the single particle level the geometry and energetics of electron trap states in the bulk and at the semiconductor / electrolyte interface . deep electron traps located at the grain boundary are found to slow down charge transport unless high current densities ensure a high average occupation of transport - limiting traps . the high structural and electronic complexity of mesoporous semiconductor oxide electrodes makes an investigation of the nature , concentration , and location of electronic trap states and the elucidation of their impact on charge recombination and transport very challenging . indeed , their complexity must be high enough to realistically mimic processes in technologically relevant materials , but low enough to result in clear structure activity relationships that can be supported by both experiments and theoretical modeling . in this work , we combine first - principles theoretical calculations with the electrochemical characterization of nanostructured rutile tio2 films to demonstrate that particle / particle interfaces introduce deep traps . these interfaces represent favorable locations for proton segregation , which can be induced by the electrochemical doping of the porous electrodes . a long lasting ( hours to days ) , but reversible accumulation of electrons and protons ( i.e. , e / h or h doping ) at the interface is tracked by cyclic voltammetry via the shift toward more positive potentials of a pair of capacitive peaks , which is associated with trap states at the particle / particle interface . the passivation of recombination centers by e / h doping leads to a transient photocurrent enhancement due to improved electron / hole separation for those electrodes , which are characterized by a high concentration of particle / particle interfaces ( i.e. for electrodes lacking a high density of particle / particle interfaces ( i.e. the resulting long - lasting ( > 15 h ) improvement of photoelectrode performance after electrochemical doping is explained by using theoretical calculations that are in qualitative agreement with experiments . slurries of rutile tio2 nanoparticles ( sachtleben , nano rutile ) were prepared by grinding 1 g of tio2 powder with 3.2 ml of h2o , 60 l of acetylacetone ( 99+% , aldrich ) , and 60 l of triton x ( aldrich ) and were spread with a glass rod onto fluorine - doped tin oxide ( fto ) conducting glass ( pilkington , tec 15 , resistance 15 / ) using scotch tape as a spacer . for calculations involving charged defects ( such as electron polarons ) , overall neutrality to identify prospective proton incorporation sites in the grain boundary supercell , we make use of the fact that protons will form a bond with lattice oxygen ions at a distance of approximately 1.0 . rutile tio2 nanoparticle ( np ) electrodes ( consisting of a random network of tio2 particles , figure s1a , b ) and nanocolumn ( nc ) electrodes ( consisting of an array of oriented nanocolumns , figure s1c , d ) have been used in the present study as model systems for investigating the effect of nanocrystal organization and interconnection on electrochemical and photoelectrochemical properties . the voltammetric response of a rutile tio2 np electrode is characterized , in the absence of significant faradaic currents ( i.e. previous analyses of the density of electrochemically active band gap states in mesoporous tio2 films by cyclic voltammetry and electrochemical impedance spectroscopy have demonstrated the presence of a broad exponential distribution of states below the conduction band edge in the accumulation region in the case of anatase electrodes , which is absent in rutile tio2 films . currents in the accumulation region ( eag / agcl < 0.25 v , figure 1 ) of rutile tio2 electrodes have been attributed to the population / depopulation of electronic states in the conduction band compensated by proton adsorption at the oxide surface . however , for both tio2 modifications , a narrow distribution of deep trap states is typically present and gives rise to a pair of capacitive peaks in the cyclic voltammograms ( cvs ) , which is also observed on rutile tio2 np electrodes ( figure 1a ) . in the following , we will focus in detail on the intensity of these signals and on the energetics of the associated trap states in mesoporous films featuring different morphology ( i.e. the corresponding signal is much less pronounced for the rutile tio2 nc electrode ( figure 1b ) as compared to the np electrode ( figure 1a ) . in line with previous interpretations , we assign the couple of capacitive peaks observed for the np electrode to the contribution of electron traps at particle / particle interfaces . importantly , at fast scan rates , it is possible that not all of the deep traps in the mesoporous film are equilibrated with the fermi level of the conducting substrate . from the total charge accumulated upon stepping the potential from 0.2 to 0.2 v ( 35 ccm , figure s4b ) , we estimate ( using the average values of film thickness and particle size and assuming a film porosity of 0.5 ) the number of extracted charges to correspond to 25 electrons per tio2 nanoparticle . following polarization at eag / agcl = 0.6 v significant changes are observed in the cv of a rutile tio2 np electrode ( figures 1a and s4a ) : the peak corresponding to deep traps is displaced by 0.08 v toward more positive potentials , while a slight increase of the peak intensity is observed upon doping . electrochemical doping induces only minor changes at eag / agcl < 0.2 v , although a slight increase of the capacitive current is observed at 0.45 v < eag / agcl < 0.25 v. importantly , we do not observe a shift of the photocurrent onset potential upon electrochemical doping ( figure s3b ) indicating that the band edges are not displaced significantly . importantly , no significant change of the cv is observed upon doping of a nc electrode ( figure 1b ) . electrochemical doping has a beneficial effect on the photoelectrochemical performance of rutile tio2 np electrodes as deduced from photocurrent transients ( figures 2a and s7 ) and cvs ( figure s8a ) . concretely , the photocurrent generated by the electrode in a 0.1 m hclo4 aqueous solution containing 1 m methanol as a hole scavenger depends significantly on the electrochemical pretreatment of the film as shown in the following . first the photocurrent of a pristine electrode was recorded at eag / agcl = 0.8 v. then the electrode was polarized at progressively more negative potentials in the accumulation region corresponding to the electrochemical doping of the film . after every doping step the photocurrent was again recorded to sample the impact of doping on the photoelectrocatalytic activity of the electrode . whereas electrode polarization for 20 min at edop = 0.5 v induces only minor changes of the photoelectrocatalytic activity , we observe an up to 3-fold photocurrent increase when doping at edop = 0.6 v ( figure s7 ) . photocurrent transients recorded upon uv exposure of rutile tio2 np ( a ) and nc ( b ) electrodes before and after electrochemical doping at eag / agcl = 0.6 v for different doping times ( t0.6vdop ) . a comparison of the photocurrent evolution following the progressive electrochemical doping of np and nc electrodes is shown in the chronoamperometric profiles in figure s9 . the additional charge transferred ( after doping ) from the tio2 film to the conducting substrate upon uv exposure exceeds by far the charge injected from the conducting substrate into the tio2 film upon electrochemical doping ( figure s9a ) . from the electrochemical characterization of np and nc films we have gained the following pieces of information about the impact of electrode morphology and electrochemical doping on the density of electrochemically active states and on the photoelectrocatalytic activity : ( i ) for porous films consisting of a random particle network ( np electrodes ) a high density of deep electron traps gives rise to a couple of capacitive peaks in the cv . ( iii ) electrochemical doping increases the photoelectrocatalytic activity of np electrodes toward methanol oxidation as sampled by a 7-fold increase of the photocurrent ( pcef = 7 ) . to help interpret the experimental results discussed above and provide deeper atomistic insight into the effect of protons on electron trapping we perform first - principles theoretical calculations for a model interface in nanocrystalline tio2 . in particular , we consider the ( 210 ) rutile tio2 grain boundary , the structure of which has been investigated previously both experimentally and theoretically ( figure 3a ) . this effect is due to local variations in the electrostatic potential near the grain boundary and changes in ion coordination and bond strain with similar effects found at tio2 surfaces.figure 4 shows the distribution of electron trapping energies ( defined with respect to the energy of an electron trapped on a bulk ti site ) for ti ions within 6 of the grain boundary plane . therefore , the presence of deep traps at this interface provides a semiquantitative model for the voltammetric feature of electron trapping states ( pair of capacitive peaks ) observed in the pristine tio2 np electrodes by cyclic voltammetry ( figure 1a ) . for the following discussion , it is important to keep in mind that the energy scale and the electrochemical potential scale have opposite signs , i.e. ( a ) optimized structure of the pristine ( 210 ) rutile tio2 grain boundary showing the electron spin density associated with an electron in the most stable site ( isosurface shown in purple ) . ( d ) ( h)(e ) decorated ( 210 ) rutile tio2 grain boundary with an additional electron trapped in the most stable site . while previous theoretical studies have identified the most stable structure of the oh species in bulk rutile tio2 , it is not straightforward to deduce the likely proton configurations in the lower symmetry grain boundary region . the most stable h incorporation site is found at the grain boundary and is 0.6 ev more stable than in the bulk ( figure 3b ) . the presence of h induces a transformation in structure near the grain boundary as compared to the pristine structure . in particular , one of the ti ions near the grain boundary relaxes toward the oh ion . the ( h)(e ) center should provide a reasonable model for the electron traps in the np electrodes following electrochemical doping and polarized for sufficiently short times at a positive potential . this provides a reference with which to assess the trapping energies of sites in the vicinity of the grain boundary . we find a distribution of trapping energies for ti ions within 6 of the grain boundary plane spanning a similar range to that found for the pristine interface and the most stable electron trap is again located close to the grain boundary ( figure 3d ) . the pristine grain boundary presents 18 ti sites per supercell that can trap electrons more strongly than in the bulk ( i.e. as noted above , the presence of h induces a localized deformation , which changes the structural and electrostatic environment of ti sites near the grain boundary . in particular , the presence of the ( h)(e ) center modifies the electrostatic potential on ti sites near the grain boundary destabilizing a number of traps . at the same time one of the ti sites adjacent to h that was not a trap in the pristine case becomes a trap after doping . in addition to the reduced concentration of interface traps there is also a reduction in the average trapping energy ( i.e. although the average depth of grain boundary traps is increased , the significant decrease in the density of interface traps provides an explanation for the improved photoelectrocatalytic activity of electrochemically doped electrodes observed experimentally . this is true for electronic states in subsurface regions of the semiconductor , such as the electron traps at particle / particle interfaces giving rise to the pair of capacitive peaks in the cvs of rutile tio2 np electrodes ( figure 1a ) . in this context , it is well established nowadays that long lasting reductive treatments of mesoporous films may result in a long lasting accumulation of charges ( electrochemical or charge transfer reductive doping ) , thereby significantly influencing the macroscopic electrode behavior in different applications . our observations from electrochemical measurements and results from first principle theoretical calculations give a consistent picture of electron and proton trapping in nanocrystalline tio2 films of different morphology . calculations indicate that ( h)(e ) decoration modifies the distribution of electron traps at particle / particle interfaces ( figure 5i ) . upon electrochemical doping of rutile tio2 np electrodes , i.e. , upon a long lasting accumulation of electron / proton pairs in the film , we observe a reversible shift toward more positive potentials of capacitive peaks associated with trap states at particle / particle interfaces . in addition an increase of the chemical capacitance is observed at more negative potentials , i.e. here we show that the partial removal of interface traps upon ( h)(e ) decoration of particle / particle interfaces may contribute to such a capacitance change . the main contribution to currents in the accumulation region of both pristine and doped electrodes ( contributions at eag / agcl < 0.25 v highlighted in red in figure 5ii ) , however , are associated with the population / depopulation of electronic states in the rutile tio2 conduction band compensated by proton adsorption at the oxide surface ( ecb / hads states , figure 5c , d ) as discussed in detail in a previous study . prolonged polarization of np electrodes at eag / agcl = 0.6 v ( t0.6vdop = 4 h ) induces electrochemical doping and thus a population of trap states deep within the particle / particle interfaces ( figure 5 d ) . this charge accumulation is not reversible on the time scale of a cv measurement ( figure 5d rather there is a long lasting ( though reversible , vide infra ) modification of the density of electrochemically active states . in the voltammetric experiment ( figure 5d f ) charge accumulation and charge extraction take now place on a film featuring particle / particle interfaces , which are partially decorated by ( h)(e ) . the increased intensity of the pair of capacitive peaks associated with traps at the particle / particle interface can be explained by the enhanced conductivity in the doped film which allows populating and depopulating electron traps faster and deeper within the particle / particle contact area . the increased photoelectrocatalytic activity of doped electrodes can be attributed to the deactivation of a major fraction of interface traps and recombination sites by the decoration of particle / particle interfaces with ( h)(e ) ( figure 5a , f ) . indeed , calculations point to a 50% decrease in density of interface traps following ( h)(e ) doping ( figure 4 ) , which is expected to affect charge separation at the particle / particle interface in two ways : by accelerating electron transfer across the grain boundary and by reducing electron / hole recombination . we have tracked the long lasting accumulation of electron / proton pairs in rutile tio2 films consisting of a random nanoparticle network ( i ) via a reversible shift of a capacitive peak in the cv , which we associate with trap states located at particle / particle interfaces , and ( ii ) via the transient enhancement of the photoelectrocatalytic activity toward methanol photooxidation . theoretical calculations indicate that interfaces between crystals in tio2 represent favorable locations for the segregation of proton defects , being up to 0.6 ev more stable than in the bulk crystal . importantly , ( h)(e ) doping of grain boundaries significantly modifies the electronic properties of the particle / particle interface . a 50% reduction of the overall number of deep electron traps at the grain boundary is considered to be the main reason for the beneficial effect of electrochemical doping of rutile tio2 np electrodes on their photoelectrocatalytic activity .

the macroscopic rate of charge transfer between the mesoporous film and a conductive substrate is the result of a sequence of intermingled microscopic processes . in photoelectrocatalytic and photovoltaic applications electron collection at the external contact competes with charge recombination in the bulk of the semiconductor and at the interfaces within the porous film , limiting solar conversion efficiency . the use of electrochemical doping ( charge transfer reductive doping ) , where electron / proton or electron / li pairs incorporated within the oxide passivate the electron traps , is a very efficient way to temporarily improve photoelectrochemical activity , and as such , constitutes an elegant way to improve charge separation within these materials . to reach such a level of understanding , one needs to carefully identify the nature of the recombination centers and transport - limiting traps in mesoporous semiconductor electrodes , which is a significant challenge for both experimentalists and theoreticians . it is well established that the fundamental processes associated with the transport , transfer , and recombination of photogenerated charge carriers in mesoporous semiconductor electrodes are determined by both the distribution of band gap states and their population , i.e. electrochemical methods such as cyclic voltammetry and electrochemical impedance spectroscopy have proven useful in characterizing electronic states in nanostructured semiconductor oxide electrodes , though the chemical nature of the traps remains controversial . specifically , charge / discharge measurements provide information on the distribution of electrochemically active states in mesoporous electrodes , where electron accumulation is compensated by the adsorption of ions at the oxide surface . for tio2 electrodes in an aqueous acidic electrolyte the generation of ti centers is compensated by proton uptake ( eq 1)1importantly , in the case of small cations ( such as h or li ) , charge injection and compensation not only take place at the semiconductor / electrolyte interface , but can become a three - dimensional process via insertion of ions into subsurface regions of the nanocrystals . on the other hand , electrochemical doping was found to modify , at least temporarily , the electrode performance in different applications ranging from dye - sensitized solar cells to photocatalysis and supercapacitors . for tio2 , theoretical studies have recently addressed at the single particle level the geometry and energetics of electron trap states in the bulk and at the semiconductor / electrolyte interface . the high structural and electronic complexity of mesoporous semiconductor oxide electrodes makes an investigation of the nature , concentration , and location of electronic trap states and the elucidation of their impact on charge recombination and transport very challenging . in this work , we combine first - principles theoretical calculations with the electrochemical characterization of nanostructured rutile tio2 films to demonstrate that particle / particle interfaces introduce deep traps . , e / h or h doping ) at the interface is tracked by cyclic voltammetry via the shift toward more positive potentials of a pair of capacitive peaks , which is associated with trap states at the particle / particle interface . the passivation of recombination centers by e / h doping leads to a transient photocurrent enhancement due to improved electron / hole separation for those electrodes , which are characterized by a high concentration of particle / particle interfaces ( i.e. , arrays of oriented nanocolumns ) this study highlights the importance of particle / particle interfaces in mesoporous films and provides strategies to actively manipulate the density of electronic states and their population by electrochemical methods . slurries of rutile tio2 nanoparticles ( sachtleben , nano rutile ) were prepared by grinding 1 g of tio2 powder with 3.2 ml of h2o , 60 l of acetylacetone ( 99+% , aldrich ) , and 60 l of triton x ( aldrich ) and were spread with a glass rod onto fluorine - doped tin oxide ( fto ) conducting glass ( pilkington , tec 15 , resistance 15 / ) using scotch tape as a spacer . we also employ a hubbard u term to correct the sie on o 2p - states ( uo = 7.5 ev ) in order to make the results transferable to future calculations which will consider electrons and holes . to investigate the interaction of electrons with the grain boundary defect , we attempt to localize an electron polaron at all inequivalent ti sites within the grain boundary supercell . in cases where this displacement procedure alone is insufficient to direct the self - consistent optimization into the desired charge localized metastable state , we manually set the orbital occupancy using a modification to the vasp code developed by allen and watson . for calculations involving charged defects ( such as electron polarons ) , overall neutrality to identify prospective proton incorporation sites in the grain boundary supercell , we make use of the fact that protons will form a bond with lattice oxygen ions at a distance of approximately 1.0 . using this procedure , inequivalent proton sites with the this procedure is straightforward to implement and may , with suitable modification , be applicable to modeling protons in low symmetry structures ( such as nanoparticles or surfaces ) in a wider range of oxide materials . rutile tio2 nanoparticle ( np ) electrodes ( consisting of a random network of tio2 particles , figure s1a , b ) and nanocolumn ( nc ) electrodes ( consisting of an array of oriented nanocolumns , figure s1c , d ) have been used in the present study as model systems for investigating the effect of nanocrystal organization and interconnection on electrochemical and photoelectrochemical properties . the photocurrent onset potential , which yields an estimate of the conduction band edge position in the semiconductor , lies for this electrode at eag / agcl 0.5 v ( figure s3a ) . cvs for rutile tio2 np ( a ) and nc ( b ) electrodes before and after an electrochemical doping at eag / agcl = 0.6 v for 3 h. for the np electrode the effect of subsequent polarization for 15 h at 0.8 v ( dedoping ) is also shown . previous analyses of the density of electrochemically active band gap states in mesoporous tio2 films by cyclic voltammetry and electrochemical impedance spectroscopy have demonstrated the presence of a broad exponential distribution of states below the conduction band edge in the accumulation region in the case of anatase electrodes , which is absent in rutile tio2 films . currents in the accumulation region ( eag / agcl < 0.25 v , figure 1 ) of rutile tio2 electrodes have been attributed to the population / depopulation of electronic states in the conduction band compensated by proton adsorption at the oxide surface . however , for both tio2 modifications , a narrow distribution of deep trap states is typically present and gives rise to a pair of capacitive peaks in the cyclic voltammograms ( cvs ) , which is also observed on rutile tio2 np electrodes ( figure 1a ) . in the following , we will focus in detail on the intensity of these signals and on the energetics of the associated trap states in mesoporous films featuring different morphology ( i.e. we measured the capacitive currents upon stepping the electrode potential in potential steps eag / agcl = 0.02 v first from 0.2 to 0.2 v ( charge accumulation ) and then from 0.2 v back to 0.2 v ( charge extraction ) . from the total charge accumulated upon stepping the potential from 0.2 to 0.2 v ( 35 ccm , figure s4b ) , we estimate ( using the average values of film thickness and particle size and assuming a film porosity of 0.5 ) the number of extracted charges to correspond to 25 electrons per tio2 nanoparticle . following polarization at eag / agcl = 0.6 v significant changes are observed in the cv of a rutile tio2 np electrode ( figures 1a and s4a ) : the peak corresponding to deep traps is displaced by 0.08 v toward more positive potentials , while a slight increase of the peak intensity is observed upon doping . electrochemical doping induces only minor changes at eag / agcl < 0.2 v , although a slight increase of the capacitive current is observed at 0.45 v < eag / agcl < 0.25 v. importantly , we do not observe a shift of the photocurrent onset potential upon electrochemical doping ( figure s3b ) indicating that the band edges are not displaced significantly . electrochemical doping has a beneficial effect on the photoelectrochemical performance of rutile tio2 np electrodes as deduced from photocurrent transients ( figures 2a and s7 ) and cvs ( figure s8a ) . concretely , the photocurrent generated by the electrode in a 0.1 m hclo4 aqueous solution containing 1 m methanol as a hole scavenger depends significantly on the electrochemical pretreatment of the film as shown in the following . first the photocurrent of a pristine electrode was recorded at eag / agcl = 0.8 v. then the electrode was polarized at progressively more negative potentials in the accumulation region corresponding to the electrochemical doping of the film . photocurrent transients recorded upon uv exposure of rutile tio2 np ( a ) and nc ( b ) electrodes before and after electrochemical doping at eag / agcl = 0.6 v for different doping times ( t0.6vdop ) . importantly , these results confirm that the increased current measured upon uv exposure of doped electrodes corresponds indeed to a faradaic photocurrent and does not simply result from a light - induced extraction of charges accumulated in the doping step . the additional charge transferred ( after doping ) from the tio2 film to the conducting substrate upon uv exposure exceeds by far the charge injected from the conducting substrate into the tio2 film upon electrochemical doping ( figure s9a ) . from the electrochemical characterization of np and nc films we have gained the following pieces of information about the impact of electrode morphology and electrochemical doping on the density of electrochemically active states and on the photoelectrocatalytic activity : ( i ) for porous films consisting of a random particle network ( np electrodes ) a high density of deep electron traps gives rise to a couple of capacitive peaks in the cv . ( ii ) prolonged polarization of np electrodes at eag / agcl = 0.6 v ( electrochemical or charge transfer reductive doping ) induces a displacement of these capacitive peaks by 0.08 v toward more positive potentials and a minor increase in the chemical capacitance at 0.45 v < eag / agcl < 0.25 v. these changes are reversible with respect to charge extraction ( dedoping ) upon prolonged electrode polarization at eag / agcl = 0.8 v. both processes ( doping and dedoping ) are extremely slow ( hours to days ) . to help interpret the experimental results discussed above and provide deeper atomistic insight into the effect of protons on electron trapping we perform first - principles theoretical calculations for a model interface in nanocrystalline tio2 . in particular , we consider the ( 210 ) rutile tio2 grain boundary , the structure of which has been investigated previously both experimentally and theoretically ( figure 3a ) . this effect is due to local variations in the electrostatic potential near the grain boundary and changes in ion coordination and bond strain with similar effects found at tio2 surfaces.figure 4 shows the distribution of electron trapping energies ( defined with respect to the energy of an electron trapped on a bulk ti site ) for ti ions within 6 of the grain boundary plane . therefore , the presence of deep traps at this interface provides a semiquantitative model for the voltammetric feature of electron trapping states ( pair of capacitive peaks ) observed in the pristine tio2 np electrodes by cyclic voltammetry ( figure 1a ) . ( a ) optimized structure of the pristine ( 210 ) rutile tio2 grain boundary showing the electron spin density associated with an electron in the most stable site ( isosurface shown in purple ) . electrochemical doping of tio2 by prolonged polarization is likely to be associated with the incorporation of h ions from the aqueous solution to compensate the negative electron charge trapped at interfaces . while previous theoretical studies have identified the most stable structure of the oh species in bulk rutile tio2 , it is not straightforward to deduce the likely proton configurations in the lower symmetry grain boundary region . to address this problem we identify prospective positions for h incorporation based on analysis of the three - dimensional electrostatic potential and screen 80 different configurations to identify the most stable structure ( see experimental section ) . by making suitable initial atomic distortions around each ti site in the supercell followed by full optimization of the total energy with respect to relaxation of all ion coordinates we obtain a series of metastable configurations corresponding to electrons trapped on different ti ions ( see experimental section ) . we find a distribution of trapping energies for ti ions within 6 of the grain boundary plane spanning a similar range to that found for the pristine interface and the most stable electron trap is again located close to the grain boundary ( figure 3d ) . although the average depth of grain boundary traps is increased , the significant decrease in the density of interface traps provides an explanation for the improved photoelectrocatalytic activity of electrochemically doped electrodes observed experimentally . due to the high specific surface area of mesoporous semiconductor electrodes , the main contribution to the density of electronic band gap states as sampled by electrochemical methods such as cyclic voltammetry results from processes at the semiconductor / electrolyte interface . this is true for electronic states in subsurface regions of the semiconductor , such as the electron traps at particle / particle interfaces giving rise to the pair of capacitive peaks in the cvs of rutile tio2 np electrodes ( figure 1a ) . importantly , whereas electrochemical methods based on charge / discharge measurements provide information on the distribution of electrochemically active states , it must not be ignored that a persistent charge accumulation in the mesoporous film may modify both the fermi level and the density of states itself . in this context , it is well established nowadays that long lasting reductive treatments of mesoporous films may result in a long lasting accumulation of charges ( electrochemical or charge transfer reductive doping ) , thereby significantly influencing the macroscopic electrode behavior in different applications . , upon a long lasting accumulation of electron / proton pairs in the film , we observe a reversible shift toward more positive potentials of capacitive peaks associated with trap states at particle / particle interfaces . , at 0.45 v < eag / agcl < 0.25 v. such a modification was previously related to the population of subsurface states upon a light - induced insertion of protons and electrons affording faster charge transport in dye - sensitized tio2 films . here we show that the partial removal of interface traps upon ( h)(e ) decoration of particle / particle interfaces may contribute to such a capacitance change . the main contribution to currents in the accumulation region of both pristine and doped electrodes ( contributions at eag / agcl < 0.25 v highlighted in red in figure 5ii ) , however , are associated with the population / depopulation of electronic states in the rutile tio2 conduction band compensated by proton adsorption at the oxide surface ( ecb / hads states , figure 5c , d ) as discussed in detail in a previous study . the increased intensity of the pair of capacitive peaks associated with traps at the particle / particle interface can be explained by the enhanced conductivity in the doped film which allows populating and depopulating electron traps faster and deeper within the particle / particle contact area . the persistence of electrochemical doping is associated with the slow kinetics of h diffusion from the gb core to the oxide / electrolyte interface . the increased photoelectrocatalytic activity of doped electrodes can be attributed to the deactivation of a major fraction of interface traps and recombination sites by the decoration of particle / particle interfaces with ( h)(e ) ( figure 5a , f ) . indeed , calculations point to a 50% decrease in density of interface traps following ( h)(e ) doping ( figure 4 ) , which is expected to affect charge separation at the particle / particle interface in two ways : by accelerating electron transfer across the grain boundary and by reducing electron / hole recombination . we have tracked the long lasting accumulation of electron / proton pairs in rutile tio2 films consisting of a random nanoparticle network ( i ) via a reversible shift of a capacitive peak in the cv , which we associate with trap states located at particle / particle interfaces , and ( ii ) via the transient enhancement of the photoelectrocatalytic activity toward methanol photooxidation . theoretical calculations indicate that interfaces between crystals in tio2 represent favorable locations for the segregation of proton defects , being up to 0.6 ev more stable than in the bulk crystal . a 50% reduction of the overall number of deep electron traps at the grain boundary is considered to be the main reason for the beneficial effect of electrochemical doping of rutile tio2 np electrodes on their photoelectrocatalytic activity .

the development of efficient catalytic methods for the formation of bonds between main group elements is of considerable interest for the continued development of main group chemistry . such processes enable new discoveries to be made in the promising application areas that main group species are now occupying , such as high performance polymers , emissive materials , etch resists for lithography , and precursors to ceramic thin films or devices . however , the development of this field lags substantially behind the advances made in catalytic c c and c x bond formation , for which there are now a myriad of efficient ways to promote such unions that are important for the construction of new molecules . catalytic dehydrocoupling of amine and phosphine boranes is one method that has emerged for the formation of b p bonds , and development in the area has been spurred on by the potential for ammonia in addition , polymeric materials that can arise from dehydropolymerization of primary analogues are also of significant interest as they are valence isoelectronic with technologically ubiquitous polyolefins . although the metal catalyzed formation of polyaminoboranes has attracted recent attention , catalytic routes to polyphosphinoboranes have also been known since 1999 . perhaps the best example is that of the [ rh(cod)2][otf ] catalyzed dehydrocoupling of secondary , h3bpr2h , and primary , h3bprh2 , phosphine boranes to give oligomeric and polymeric materials ( scheme 1 ) . although initial reports demonstrated that catalysis using [ rh(cod)2][otf ] was a homogeneous process ( i.e. , not colloidal ) , there has been only sporadic further work on elucidating the mechanistic details . progress has no doubt been slowed due to the fact that the reaction conditions reported for phosphine borane dehydrocoupling often require melt conditions , thus making interrogation of the catalytic cycle problematic . recently , we have reported that the rh(i ) complexes [ rh(pbu2h)2(-fc6h5)][bar4 ] , and [ rh(l)(-fc6h5)][bar4 ] , [ l = ph2p(ch2)3pph2 ] are particularly well - suited to the study of the dehydrocoupling mechanism of secondary phosphine boranes in solvents such as fluorobenzene ; and on the basis of the observation of intermediates , kinetic studies , and h / d exchange experiments we have proposed a catalytic cycle for the dehydrocoupling of h3bpr2h ( r= ph , bu ; scheme 2 ) . for this cycle , intermediate species were isolated , but their structures could not be confirmed by x - ray crystallography . in particular for r = ph , a -b - agostic complex b , and the product of dehydrocoupling f , that is proposed to sit off cycle , could be isolated and spectroscopically characterized . under stoichiometric conditions the observation that b transforms into f on gentle heating allowed for kinetic parameters to be determined that suggested that the rate - determining step(s ) for dehydrocoupling were located within the transformations b to d. in solution phase the turnover limiting step for catalysis is proposed to be the displacement of the linear diboraphosphine product ( i.e. , f to a ) , although under the melt conditions used for efficient catalysis this may well be different . further insight comes from the observations that for r = bu the barrier to dehydrocoupling is higher ( 70 c versus 25 c for reaction ) , p h activation appears also to be a higher energy process , different intermediates ( a and e ) are observed , and the turnover limiting process in catalysis is now suggested to be the p h activation / dehydrocoupling steps . prior work has demonstrated a similar difference in relative rates of dehydrocoupling of secondary h3bpr2h [ r = p-(cf3)c6h4 , ph , bu , bu ] and primary h3bprh2 [ r = ph , bu , bu ] phosphine boranes using the [ rh(cod)2][otf ] catalyst , and this was suggested to be due to a combination of steric and electronic ( relative p h bond strengths ) factors , although the mechanism of dehydrocoupling of phosphine boranes using this catalyst is currently not known . h bond is such that spontaneous dehydrocoupling occurs in the absence of catalyst , with electron - withdrawing aryl groups [ p-(cf3)c6h4 ] undergoing faster reaction than electron - donating [ p-(ome)c6h4 ] . very recent work has shown that paramagnetic ti(iii ) centers might also be involved in dehydrocoupling of phosphine and amine boranes when using cp2ti - based catalysts , while oligomerization of base - stabilized phosphino likely decomposition routes in rh - systems for phosphine borane dehydrocoupling to form bis(phosphine)boronium salts have also recently been discussed . , we report an extension of our investigations into the mechanism of phosphine borane dehydrocoupling using the { rh(ph2p(ch2)3pph2 ) } fragment , by varying the electronic and steric profile of the secondary phosphine boranes h3bpr2h [ r = 3,5-(cf3)2c6h3 ; p-(cf3)c6h4 ; p-(ome)c6h4 ; adamantyl ] , as well as investigations with the primary phosphine borane h3bpcyh2 . dehydrocoupling forms the corresponding metal bound linear diboraphosphines h3bpr2bh2pr2h and h3bprhbh2prh2 , respectively these studies provide insight into the determining role of the electronics and sterics of the phosphine borane in the dehydrocoupling process , as well as providing as yet unreported examples of the solid - state structures of the intermediates related to the catalytic cycle . we also report for the first time the partial control of diastereoselectivity in dehydrocoupling of primary phosphine boranes , that can additionally be biased by use of a chiral chelating phosphine on the rhodium center . a range of secondary phosphine boranes with differing electronic and steric properties have been used in this study ( 1 , 2 , 3 , and 4 , figure 1 ) , which also provide comparison with the previously reported ph , 6 , and bu , 7 , analogues . the primary phosphine compounds 2(33 ) and 3(38 ) are known adducts and offer electron - withdrawing and donating aryl groups , respectively . the synthesis of adamantyl - substituted phosphine , 4 , an analogue of 7 , has been reported in the patent literature . compared with the butyl group , adamantyl has a greater steric bulk due to its larger volume and rigid structure . the new linear diboraphosphines , 1013 , have also been synthesized to aid in the identification of final dehydrocoupling products . complexes 1012 are synthesized by a rh - catalyzed process from the corresponding phosphine boranes , while primary phosphine containing 13 has been synthesized in good isolated yield ( 85% ) by addition of [ nbu4][bh4 ] to the bis(phopshine)boronium [ ( cyh2p)2bh2]br . addition of 2 equiv of 1 to [ rh(l)(-fc6h5)][bar4 ] [ l = ph2p(ch2)3pph2 ] in 1,2-f2c6h4 solution at 25 c rapidly ( on time of mixing ) resulted in the formation of [ rh(l)(h)(,-pr2bh3)(-h3bpr2h)][bar4 ] , 14 [ r = 3,5-(cf3)2(c6h3 ) , scheme 3 ] , which was characterized by nmr spectroscopy , esi - ms ( electrospray ionization mass spectrometry ) , and single crystal x - ray diffraction . likewise , the use of 2 equiv of phosphine borane 2 or 3 results in the formation of the analogous complexes 15 [ r = p-(cf3)c6h4 ] and 16 [ r = p-(ome)c6h4 ] , respectively , which were fully characterized using solution techniques . all these complexes proceed to dehydrocouple ( vide infra ) , and only for 14 was an analytically pure crystalline solid obtained . even so , dissolution of crystalline material of 14 resulted in the observation of small amounts ( approximately 510% ) of the associated dehydrocoupling product in the solution nmr spectra after short periods of time . complexes 15 and 16 could only be isolated as oils , but their characterization by nmr spectroscopy and esi - ms was fully consistent with their formulation . [ bar4 ] anions are not shown . the solution nmr spectra for 14 , 15 , and 16 are very similar to those previously reported for [ rh(l)(h)(,-pph2bh3)(-h3bpph2h)][bar4 ] ( i.e. , b , scheme 2(31 ) ) , and data for 14 is discussed in detail . two of the resonances are broadened significantly compared to the other two , suggesting these phosphorus atoms are bound to a quadrupolar boron center . one of these shows both a large trans pp coupling [ j(pp ) 244 hz ] and coupling to rh [ j(rhp ) 75 hz ] , while the other is a broad singlet . the other two signals are sharper and are assigned to the two p environments of the ph2p(ch2)3pph2 ligand . one of these sharper resonances [ 29.5 , ddd , j(rhp ) 130 , j(pp ) 35 , j(pp ) 21 hz ] is assigned to the phosphorus atom trans to the weakly bound -b - agostic interaction on the basis of the larger rh coupling constant , while the other signal [ 11.3 , ddd , j(rhp ) 103 , j(pp ) 244 , j(pp ) 35 hz ] is assigned to the phosphorus atom trans to the coordinated phosphido ligand . in the h nmr spectrum of 14 one broad , relative integral 3h , signal is observed at 0.78 , indicative of a rhh3b interaction in which the b h bonds are undergoing rapid site exchange on the nmr spectroscopic time scale between terminal and bridging sites . a broad , relative integral 1h , resonance at 6.12 cooling of the solution to 0 c led to the resolution of this signal as doublet [ j(ph ) = 65 hz ] , fully consistent with its trans disposition to a phosphine . the remaining bh(terminal ) signals are not observed , and it is likely they are coincident with the { ch2}3 signals . a sharper signal at 16.21 , relative integral 1h , is assigned to a metal hydride resonance , in which the coupling to both rh and p is clearly small and unresolved . the ph group is observed at 5.81 that collapses into a singlet in the h{p } nmr spectrum . the b nmr spectrum shows a broad signal centered at 39.8 , which is not shifted significantly from that of free phosphine this is assigned to a coincidence of the -b hrh agostic and rhh3b signals , as has been noted previously . complexes 15 and 16 have similar h , b , and p nmr spectra , and thus we assign very similar structures . crystals of complex 14 of suitable quality for analysis by x - ray diffraction were obtained by layering of a 1,2-f2c6h4 solution with pentane at 26 c . the structure of 14 in the solid - state ( figure 2 ) is fully consistent with the structure deduced from the solution nmr spectroscopic data . the formally rh(iii ) center adopts a pseudo - octahedral geometry , with the chelating phosphine ligand and the hydride located on one of the faces of the octahedron . h activation , and is bound to the metal via a phosphido bond [ rh1p3 , 2.3045(10 ) ] and a -b - agostic bond [ rh1b1 , 2.515(4 ) ] . the other phosphine borane unit occupies the last coordination site via a -rhh all the hydrides ( b h and rh h ) were located in the final difference map . the structure is in full accord with the solution nmr spectroscopic data , confirming the spectroscopic assignments that have been made previously . -b - agostic interactions are known , and we have recently reported [ rh(,-pph2bh2pph3)(pph3)2][bar4 ] in which a base - stabilized phosphine borane adopts a -b - agostic interaction with the rh - center . phosphine boranes are also known , and bimetallic complexes showing both b - agostic and borane coordination modes have been reported . compared to a rh(i ) complex that shows a bidentate -coordination mode for the borane , [ rh(pbu2h)2(-h3bpbu2h)][bar4 ] , the rhb distance for the -interaction in 14 is considerably longer [ 2.188(3 ) versus 2.740(4 ) , respectively ] , consistent with this different binding motif . similar changes in mb distance have been noted on moving between and coordination modes in chelating phosphine boranes . some hydrogen atoms are omitted for clarity . selected bond lengths ( ) and angles ( deg ) : rh1p1 , 2.778(10 ) ; rh1p2 , 2.3163(9 ) ; rh1p3 , 2.3045(10 ) ; p3b1 , 1.913(4 ) ; p4b2 , 1.918(4 ) ; rh1b1 , 2.515(4 ) ; rh1b2 , 2.740(4 ) ; rh1p3b1 , 72.54(14 ) ; rh1b2p4 , 121.3(2 ) . complexes 1416 undergo spontaneous dehydrocoupling ( 25 c ) to form products of the general formula [ rh(l)h(,-pr2bh2pr2bh3)][bar4 ] : 17 , r = 3,5-(cf3)2c6h3 ; 18 , r = p-(cf3)c6h4 ; 19 , r = p-(ome)c6h4 ( scheme 4 ) . this process also results in the liberation of h2 ( observed , h nmr spectroscopy ) . for 17 and 18 there are additional products formed , assigned as [ rh(l)(pr2h)2][bar4 ] , 21 and 22 , respectively , on the basis of nmr spectroscopic data . these complexes are formed in parallel to 17 and 18 , as preformed 17 ( vide infra ) does not proceed to form 21 . complex 21 has been independently prepared by addition of two equivalents of hp((cf3)2c6h3)2 to [ rh(l)(-fc6h5)][bar4 ] . time = 6 h 17/21 , 18/22 ( 25 c ) ; 8 h 16/19 ( 35 c ) . this mixture of products observed for the electron - withdrawing phosphine substituents ( i.e. , 1 and 2 ) contrasts with that found for when r = ph and p-(ome)c6h4 , which yield the dehydrocoupled ( e.g. , 19 and f , scheme 2 ) product in essentially quantitative form ( 95% by p{h } nmr spectroscopy ) . complex 17 has been synthesized cleanly from direct addition of the preformed dehydrocoupled diboraphosphine product , 10 , to [ rh(l)(-fc6h5)][bar4 ] , scheme 5 . it was from this reaction that material of 17 suitable for single crystal x - ray diffraction was obtained . figure 3 shows the solid - state structure of 17 , in which the diboraphosphine acts as a chelate to the rh(iii ) center , via a phosphido group and two b - agostic interactions : [ rh(l)h(,-pr2bh2pr2bh3)][bar4 ] [ r = 3,5-(cf3)2c6h3 ] . all the hydride ligands ( b h and rh h ) were located in the final difference map . the rh(iii ) center has pseudo - octahedral geometry , in which the oligomeric phosphine borane is bound tridentate to the metal through -bh2rh [ b2rh1 , 2.280(5 ) ] and phosphido [ p3rh1 , 2.3925(10 ) ] interactions . the rhb distance is considerably shorter than those observed in 14 , consistent with the -bidentate binding mode of the borane . selected bond lengths ( ) and angles ( deg ) : rh1p1 , 2.3241(11 ) ; rh1p2 , 2.2650(11 ) ; rh1p3 , 2.3925(10 ) ; rh1b2 , 2.280(5 ) ; rh1p3b1 , 110.88(15 ) ; b1p4b2 , 107.5(2 ) . the nmr spectroscopic data for 17 are fully consistent with the solid - state structure being retained in solution and are also very similar to that reported for the analogous complex formed from the deydrocoupling of 6 ( r = ph ) . two of these signals are well - resolved and show coupling to rh , 46.6 [ j(rhp ) 111 hz ] and 12.8 [ j(rhp ) 91 hz ] , and are attributed to the chelating phosphine ligand . one of these signals ( 12.8 ) also shows large p p coupling [ j(pp ) 260 hz ] suggesting a trans position relative to the phosphido center . the other two environments are broad , typical of those observed when coupling to a quadrupolar boron center . for one of these trans the h nmr spectrum shows three different broad , relative integral 1h , environments assigned to the bh3 moiety [ 4.54 , 1.20 , and 4.37 ] . this indicates that the bh3 unit is not undergoing exchange on the nmr spectroscopic time scale , as noted previously for similar -mh3b systems . the rh h signal is observed at 13.98 as a sharper signal , although this also shows unresolved coupling . the b nmr spectrum shows two different environments [ 27.1 and 0.21 ] for the two boron atoms present in the diboraphosphine , with the latter assigned to the -h3b unit on the basis of the large downfield shift from free ligand ( = + 36.8 ) . spectroscopic data for complexes 18 and 19 , that are produced by the direct dehydrocoupling route are similar , although for 18 this is also formed as a mixture with 22 . the dehydrocoupling reaction ( i.e. , 14 to 17 ) shows a dependence on the substituents on the phosphine . for electron - withdrawing aryl groups ( e.g. , p - cf3 ) , it is faster when compared with electron rich groups ( i.e. , p - ome ) . following these processes in situ using nmr spectroscopy demonstrated that these dehydrocoupling reactions follow a first order rate profile for the consumption of the starting material over at least three half - lives ( see supporting information ) : 1 3 h ( 25 c ) ; 2 3 h ( 25 c ) ; 6 14 h ( 25 c);3 8 h ( 35 c ) , 120 h ( 25 c ) . that the parallel products 21 and 22 are formed in approximately equal ratio to the dehydrocoupled product ( 17 , 18 , respectively ) ) suggests that k1 k2 ( scheme 4 ) . in addition to this parallel process , direct comparison of the rate constants is further complicated by the fact that 16 19 required heating to 35 c to make the reaction run over a convenient time scale for analysis by nmr spectroscopy . nevertheless these relative rates reflect previous observations on the rate of catalytic dehydrocoupling when the electronics of a system are changed , in as much as electron - withdrawing groups promote the reaction . interestingly , for all the aryl complexes initial p h activation to form a phosphido hydride complex ( i.e. , 14 ) is very rapid , occurring on time of mixing . h activation that is rate - determining for the dehydrocoupling event , as we have commented on for r = ph . in this study we suggested that b h activation / reorganization in intermediates such as b ( scheme 2 ) prior to p h bond , and calculations on analogous h3bl ( l = lewis base ) systems show that the b h bond is considerably weaker when there are electron - withdrawing groups on the lewis base . however , we can not rule out that the relative p h bond strengths in intermediates such as 14 also might play a role , or that there is a change in the rate determining step on changing the phosphine borane ligand , as the intimate details of the mechanism leading to p the observation that for an electron - withdrawing phosphine there is a significant proportion of parallel product formed that results from p b bond cleavage has been noted previously in phosphine borane complexes to give either simple adducts or further reaction to yield bis(phosphine)boronium salts . prior to the formation of the parallel product 21 ( r = 3,5-(cf3)2c6h3 ) an intermediate is observed that has been characterized by h and p{h } nmr spectroscopy as [ rh(l)h(,-pr2bh3)(pr2h)][bar4 ] 20 , i.e. , a complex that sits directly between 14 and 21 by loss of one bh3 fragment ( scheme 6 ) . b bond cleavage , formally of the -h3bpr2h ligand , to afford a complex with a -b - agostic interaction from a phosphide borane ligand ( as for 14 ) and a simple pr2h ligand trans to a hydride . complex 20 was not isolated in pure form , being observed alongside 14 and the final products 17/21 . however , after 2 h reaction a significant proportion of 20 is present ( 20% by p nmr spectroscopy ) , allowing for its identification aided by comparison with the nmr spectroscopic data for 14 ( supporting information ) . in particular four environments are observed in the p nmr spectrum , with only one of these broadened significantly by coupling to quadrupolar boron . this signal also shows a large , mutual , trans j(pp ) coupling with another phosphine environment . in the high - field region of the h nmr spectrum a broad doublet is observed at 7.06 [ j(hp ) = 76 hz ] which is assigned to the -b - agostic interaction , while there is a relatively sharper one at 9.61 [ j(hp ) = 165 hz ] assigned to rh h , and again rh coupling is not resolved . these assignments were confirmed by h{p } , h{b } , and h / p correlation experiments . borane h3bp(adamantyl)2h , 4 , to [ rh(l)(-fc6h5)][bar4 ] in 1,2-f2c6h4 solution at 25 c rapidly results in a color change from orange to purple and the formation of the new bound rh(i ) phosphine borane complex [ rh(l)(-h3bp(adamantyl)2h)][bar4 ] , 23 , which was characterized in situ by nmr spectroscopy . this complex could not be isolated as it undergoes further reaction , by p b bond cleavage at room temperature , to form 24 ( scheme 7 ) . addition of 1 equiv of 4 resulted in a final mixture of 24 and [ rh(l)(-fc6h5)][bar4 ] . the h nmr spectrum of complex 23 immediately after preparation shows a broad , relative integral 3h , signal at 1.36 characteristic of a -bound phosphine borane that is undergoing site exchange between the coordinated and uncoordinated b h environments . two signals are observed in the p{h } nmr spectrum , in a 2:1 ratio at 35.1 [ j(rhp ) 167 hz ] and 30.1 ( br ) . over time ( 1 h ) , complex 23 disappears to be replaced by a new complex that has been characterized by nmr spectroscopy and a solid - state x - ray diffraction experiment as [ rh(l)(phr2)(h3bphr2)][bar4 ] ( 24 , r = adamantyl ) . figure 4 shows the structure of the cation present in 24 in the solid - state . a rh(i ) center is in a pseudo - square - planar geometry with a chelating ligand , and the other two coordination sites are occupied by p(adamantyl)2h and a -h3bp(adamantyl)2h [ rhb , 2.457(7 ) ] ligands , respectively . the solution nmr spectroscopic data for 24 are fully consistent with the solid - state structure , and in particular the trans disposition of p1 and p3 , and the -h3bpr2h ligand . some hydrogen atoms are omitted for clarity . selected bond lengths ( ) : rh1p1 , 2.2262(16 ) ; rh1p2 , 2.2861(16 ) ; rh1p3 , 2.3568(15 ) ; rh1b1 , 2.457(7 ) ; b1p4 , 1.936(7 ) . a significant amount of p b bond cleavage product is thus observed for both electron poor aryl phosphine boranes ( e.g. , 14 ) and very bulky electron rich phosphine boranes ( e.g. , 24 ) , but not the electron rich aryl phosphine 3 or h3bpph2h ( 6 ) . interestingly we have recently reported that for h3bpbu2h p b bond cleavage is also observed during dehydrocoupling catalysis being accompanied by a further dehydrocoupling step , through which bis(phosphine)boronium salts are ultimately formed . similar complexes can be prepared on rhodium using h3bpph2h and pph3 under stoichiometeric conditions . one suggested mechanism for this process is the reaction of a short - lived phosphino borane ( or its masked equivalent ) with coordinated phosphine , not dissimilar to the mechanism suggested for the formation of diaminoboranes from amine boranes and amines catalyzed by alkaline earth catalysts . complexes 20 and 24 serve as models for intermediates in this process [ rh(iii ) and rh(i ) , respectively ] , although we do not observe the formation of corresponding bis(phosphine)boronium salts in this case . boranes can yield polyphosphinoboranes , rather than the simple oligomers observed with secondary phosphine boranes ( scheme 1 ) . with an appreciation of the intermediate metal complexes formed with secondary phosphine boranes from this and previous work , it was of interest to explore whether the proposed dehydrocoupling mechanism for secondary phosphine boranes using [ rh(l)(-fc6h5)][bar4 ] could be applied to primary analogues . boranes is important , as these processes currently remain unresolved due to the melt conditions employed that make following intermediates or kinetics problematic . in situ investigations using stoichiometric quantities of primary phosphine boranes h3bpphh2 resulted in immediate reaction when combined with [ rh(l)(-fc6h5)][bar4 ] , but a number of products were formed which we have not been able to convincingly characterize . this mixture of species observed is in contrast with h3bpph2h where single products are formed analogous to 1416 . however , reaction of [ rh(l)(-fc6h5)][bar4 ] with a slight excess of h3bpcyh2 ( 5 ) in 1,2-f2c6h4 solution at 25 c led to the instantaneous formation of only two complexes in a 1:1 ratio , 25a and 25b , [ rh(l)h(,-pcyhbh3)(-h3bpcyh2)][bar4 ] , as a proposed diastereomeric pair ( scheme 8) . these new products are directly analogous to those formed with secondary phosphine boranes ( i.e. , 14 ) , and the nmr spectroscopic data match closely . the p{h } nmr spectrum from this reaction shows 8 resonances , in addition to a broad peak at 35.5 due to excess phosphine signals centered at 31.7 and 30.5 are assigned to one of the chelating phosphine ligand p environments in each diastereoisomer , and show characteristic j(rhp ) coupling constants consistent with a rh(iii ) center . complex overlapping multiplets at 11.8 [ 2 ddd ] represent the resonances for both diastereomers of the second chelated phosphorus center , which is trans to the phosphide position , displaying a large trans pp coupling constant [ j(pp ) 200 hz ] in addition to coupling to rh and cis - p . the remaining 4 signals are broad indicating the phosphorus centers are bound to a quadrupolar b nucleus . of these , peaks at 11.0 and 32.1 are assigned to the phosphide centers of each diastereomer trans to the chelating phosphine [ j(pp ) 200 hz ] , and resonances at 39.8 and 44.2 as assigned to phosphorus centers in the -bound phosphine borane unit . these large differences in chemical shift of the phosphido signal ( 21.2 ) might reflect significant local difference in steric pressure between 25a and 25b at this group . interestingly , a much smaller difference is observed with the dehydrocoupled products ( 26a / b , 3.5 ) in which the phosphide group is part of a chelate ring . the h nmr spectrum does not have the necessary resolution to separate out the diastereomers in the hydride region , with broad resonances observed at 2.3 ( 3 h , bh3 ) , 7.9 ( 1 h , rh h complexes 25a / b can not be isolated in pure form , and characterization by nmr spectroscopy is best performed on freshly prepared samples , as after 1 h ( 25 c ) they have undergone dehydrocoupling to give a mixture of two resolvable diastereomers 26a and 26b , with one of the diastereomers present in a significantly larger amount 6:1 ( scheme 8) , indicating that the dehydrocoupling step occurs with some stereocontrol . the decomposition product [ rh(l)(ph2cy)2 ] , analogous to 21/22 , was also observed . nmr spectroscopic and esi - ms analysis suggests that the dehydrocoupling products formed are direct analogues of 17 . this mixture of diastereomers can also be synthesized cleanly by direct reaction of [ rh(l)(-fc6h5)][bar4 ] with the preformed diaboraphosphine h3bpcyhbh2pcyh2 ( 13 ) in 1,2-f2c6h4 solution at 25 c ( figure 5 for the solid - state structure ) . immediate measurement of the p{h } nmr spectrum after mixing showed clean conversion to complexes 26a and 26b in an approximate 1:1 ratio , interestingly different from the 1:6 ratio observed from dehydrocoupling . molecular structure of 13 . displacement ellipsoids are drawn at 50% probability level . selected bond lengths ( ) and angles ( deg ) : p1b1 1.9267(18 ) , b1p2 1.9381(18 ) , p2b2 1.926(2 ) ; p1b1p2 108.34(9 ) , b1p2b2 113.32(9 ) . resonances in the p{h } nmr spectrum of 26 can , again , be assigned aided by reference to those of structurally characterized 17 . peaks centered at 37.9 and 34.5 result from the chelated phosphorus trans to the b - agostic site , while the signals for the phosphorus trans to the phosphido group overlap at 10.7 , and display characteristic j(pp ) trans coupling [ 255 hz ] . the broad resonances of the diboraphosphine are observed at 19.8 and 16.2 for the phosphido center [ j(pp ) 255 ] and 14.9 and 16.6 ppm for the remaining site . the high - field region of the h nmr spectrum of 26a/26b shows a slight downfield shift of the rh h hydride resonance to 16.1 , when compared to 25a/25b , while the -bh2rh units are observed as two broadened resonances at 2.98 ( 1h ) and 5.98 ( 1h ) . for these hydride signals the separate signals are not resolved for each diasteroisomer , although each resonance is rather asymmetric suggesting two overlapping environments . a p{h } nmr spectrum taken of this mixture after 18 h at 25 c showed a significant change in the ratios of the diastereomers 26a/26b ( scheme 9 ) . the peaks for one isomer at [ 34.5 , 16.2 , 10.7 , and 14.9 ] have reduced relative area , giving an approximate ratio of 6:1 for the two diastereoisomers . this ratio is similar to that found from direct dehydrocoupling in 25a/25b after 1 h ( vide supra ) , underscoring the stereocontrol occurring in the p leaving this solution for one week resulted in no significant change to this ratio , suggesting equilibrium had been reached . we suggest that the mechanism for equilibration involves reductive elimination of the phosphido and hydride ligands to form a rh(i ) phosphine borane complex , similar to e in scheme 2 , which then undergoes rapid oxidative addition of the other p h bond . this must be a reversible process , leading to a thermodynamic ratio of the diastereoisomers and the resulting selectivity . unfortunately we were unable to deduce the stereochemistry of the preferred isomer using roesy experiments or a solid - state structure . however , inspection of models leads us to propose that the thermodynamic product is likely to have the cyclohexyl group pointing away from the chelating phosphine ligand s phenyl groups , i.e. , 26b . that these diastereoisomers are a result of the metal activation of the prochiral terminal p h bonds in 13 is shown by addition of an excess of dppe to 26a / b . this affords [ rh(dppe)(l)][bar4 ] with the concomitant formation of free 13 ( scheme 9 ) . we have briefly explored the use of a chiral metal / ligand fragment in dehydrocoupling , [ rh(bdpp ) ] [ s , s - bdpp = ( 2s,4s)-2,4-bis(diphenylphosphino)pentane ] . this chiral ligand was chosen as electronically and sterically ( i.e. , bite angle ) it is similar to ph2p(ch2)3pph2 . addition of h3bpcyh2 , 5 , to [ rh(bdpp)(-fc6h5)][bar4 ] results in the immediate formation of two diastereoisomers of [ rh(bdpp)h(,-pcyhbh3)(-h3bpcyh2)][bar4 ] , 27 , in a 3:1 ratio ( scheme 10 ) . although we are unable to comment on the absolute configuration of these isomers , it is interesting to note that this is biased away from the 1:1 ratio observed in the achiral system . compounds 27a / b proceed on to dehydrocouple to form diastereoisomers of [ rh(bdpp)h(,-prhbh2prhbh3)][bar4 ] , 28 , 1:5:3:0 ratio . the same mixture of diasteroisomers can be formed by direct addition of 13 to [ rh(bdpp)(-fc6h5)][bar4 ] . initially a 2:1:2:1 ratio of 4 isomers is observed , that changes to a 5:1:3:0 ratio after 18 h. we are unable to comment in more detail on the conformation of these isomers , although the observation of stereocontrol in the direct dehydrocoupling is similar to that observed for the achiral system . addition of excess dppe to this mixture forms a product identified by esi - ms as [ rh(bdpp)(dppe ) ] and free 13 ( by p and b nmr spectroscopy ) . we have not explored whether there is enantiocontrol at the central { pcyh unit } arising from this pb coupling event on release from the metal . boranes h3bpbu2h and h3bpbu2bh2pbu2h , which on addition to [ rh(l)(-fc6h5)][bar4 ] gave the corresponding rh(i ) -phosphine borane complexes with no p such selectivity for primary over secondary phosphines in p h activation at a metal center has been described previously for both phosphine and phosphine borane ligands . in particular it has been shown that addition of h3bpphh2 to pt(pet3)2h(pph2bh3 ) results in exchange of the metal bound phosphide complex to give the primary phosphido borane complex . here it was suggested that the greater thermodynamic driving force for formation of the primary phosphido p bonds to smaller primary phosphido ligands are likely to be stronger . under the standard catalytic melt conditions ( 90 c , 5 mol % ) , [ rh(l)(-fc6h5)][bar4 ] will dehydrocouple the secondary aryl phosphine boranes used in this study to form the corresponding linear diboraphosphines 1012 , although we have not explored in detail the temporal evolution of these systems due to the problems associated with directly interrogating the melt . however , trends can be observed . for electron - withdrawing groups ( 1 and 2 ) , complete consumption of starting the reaction at this temperature is not selective , and although the main product is the linear diboraphosphine , there are products that we tentatively identify as the cyclic oligomers ( bh2pr2)n ( n = 3 , 4 ) . our results are broadly in line with the previously reported catalyzed dehydrocoupling of 2 using [ rh(cod)cl]2 , which , at a slightly lower temperature ( 60 c , 16 h , melt ) , affords the linear diboraphosphine product in 69% isolated yield , while at 100 c only the cyclic oligomers are isolated . the mechanism of formation of the higher cyclic oligomers , ( bh2pr2)n , remains to be resolved . for electron - donating 3 the reaction is slower using the [ rh(l)(-fc6h5)][bar4 ] catalyst ( 8 h ) but overall is more selective . for r = ph we have previously shown that [ rh(l)(-fc6h5)][bar4 ] catalyzes dehydocoupling to give the corresponding linear diboraphosphine in greater than 95% conversion after 4 h. for secondary phosphine boranes , h3bpph2h thus offers balance between overall rate and selectivity . conditions : [ rh(l)(-fc6h5)][bar4 ] , 5 mol % , 90c , melt . given the product distributions and likely decomposition pathways in the melt it is inappropriate to comment in detail on the nature of the rate - determining steps during catalysis under these conditions . however , on the basis of the solution studies , p b bond formation , ( dehydrocoupling ) is faster with electron - withdrawing groups . the temporal differences in observed product conversion in the melt could reflect a difference in the rate of the p b bond forming event , or alternatively , they could reflect the ease at which the bound product is substituted on the metal center , i.e. , a turnover limiting step . to probe this latter scenario , reaction between 19 ( aryl - ome ) and diboraphosphine 11 ( aryl - cf3 ) to form 18 and free 12 demonstrates that an equilibrium is established slightly in favor of 18 ( scheme 11 ) . this suggests that there is not a strong inherent difference in binding strengths between the two products , with the implication being that the observed rate differences in the melt arise from the dehydrocoupling step . although this is different from what is observed in solution at room temperature , in which release of the product is likely the turnover limiting step , it is consistent with the high local concentration of h3bpr2h that being under melt conditions ( 90 c ) would promote such a substitution . [ rh(l)(-fc6h5)][bar4 ] also acts as a catalyst for the dehydrocoupling of primary phosphine boranes . under melt conditions ( 90 c , 5 mol % , 4 h ) h3bpphh2 is dehydrocoupled to give a major product which is identified by p nmr spectroscopy as being polymeric ( bh2pphh)n by comparison with previously reported data for purified material coming from the [ rh(cod)2][otf ] catalyzed process [ 49.3 , d , j(ph ) 350 hz , 1,2f2c6h4 ; lit . : 48.9 , , j(ph ) 360 hz , cdcl3 ] . there were also other species observed 55 , which could be reduced in relative concentration ( to 10% ) by precipitation into hexanes . interestingly , these proposed shorter chain oligomers are present in a greater proportion at shorter reaction times , which might suggest that polycondensation is occurring to give higher molecular weight polymer . under non - melt conditions ( toluene heated to reflux , 0.5 mol % , 16 h ) these shorter oligomers are by far the dominant species ( supporting information ) . positive mode esi - ms ( electrospray mass spectrometry ) of the melt reaction product demonstrated polymerization , showing repeat units of [ h(pphhbh2)npphh2 ] up to n = 10 ( supporting information ) . similar analyses have been reported for amine borane dehydropolymerization . that these polymers are terminated by { pphh2 } groups rather than { bh3 } this formulation also argues against cyclic oligomers being observed by esi - ms , and presumably the additional phosphine arises from p b bond cleavage . use of h3bpcyh2 under these conditions afforded significantly more complex mixtures that we were unable to resolve . the solid - state structures of the intermediates in the dehydrocoupling of secondary phosphine boranes using the { rh(ph2pch2ch2ch2pph2 ) } fragment have been determined . this demonstrates that the complex that precedes dehydrocoupling to form a linear diboraphosphine has bound and p borane ligands , while the product has a linear diboraphosphine bound to the metal center . for aryl phosphine boranes , electron - withdrawing groups ( cf3 ) promote stoichiometric dehydrocoupling faster than for more electron - donating ( ome ) groups . this increase in rate is accompanied by a significant degree of parallel and competitive p b bond cleavage to afford metal complexes with two monodentate phosphine ligands , which we suggest is due to a weakening of the p these systems also turnover catalytically under melt conditions , with the overall rate of conversion broadly following the relative dehydrocoupling rates observed in the stoichiometric studies , suggesting that the dehydrocoupling step under melt conditions might also be the turnover limiting step . boranes , to such an extent that stoichiometric dehydrocoupling is not observed . for this phosphine a significant observation is that , for primary phosphine boranes , which are precursors to polyphosphinoboranes , use of the { rh(ph2pch2ch2ch2pph2 ) } fragment results in some apparent diastereoselectivity in the dehydrocoupling step , at least in the stoichiometric reactions that produce metal - bound diboraphosphines . such selectivity could well have implications in the control of the stereochemistry of polymer that would result from further insertion events . a significant future challenge is to harness any inherent bias in each dehydrocoupling insertion step productively while also developing the necessary spectroscopic and physical characterization markers to interrogate the oligomer and polymer stereochemistry . all manipulations , unless otherwise stated , were performed under an atmosphere of argon , using standard schlenk and glovebox techniques . glassware was oven - dried at 130 c overnight and flamed under vacuum prior to use . hexane and pentane were dried using a grubbs type solvent purification system ( mbraun sps-800 ) and degassed by successive freeze pump thaw cycles . cd2cl2 , c6h5f , and 1,2-f2c6h4 were distilled under vacuum from cah2 and stored over 3 molecular sieves , 1,2-f2c6h4 was stirred over alumina for 2 h prior to drying . bis-1,3-(diphenylphosphino)propane ( dpp3 ) and ( 2s,4s)-2,4-bis(diphenylphosphino)pentane ( bdpp ) were purchased from aldrich . [ rh(nbd)cl]2 and [ rh(nbd)(dpp3)][bar4 ] were prepared as previously described . ( 4-methoxyphenyl)2hpbh3 ( 3 ) , ( adamantyl)2hpbh3 ( 4 ) , and cyh2pbh3 ( 5 ) were prepared by the same method as me3pbh3 but with the phosphine changed . ( 4-trifluoromethylphenyl)2phbh3 ( 2 ) and ( 3,5-bis(trifluoromethyl)phenyl)2pcl were prepared according to literature procedures reported by clark et al . nmr spectra were recorded on a bruker avd 500 mhz spectrometer at room temperature unless otherwise stated . in 1,2-c6h4f2 , h nmr spectra were referenced to the center of the downfield solvent multiplet ( 7.07 ) , and p and b nmr spectra were referenced against 85% h3po4 ( external ) and bf3oet2 ( external ) , respectively . the spectrometer was prelocked and preshimmed using a c6d6 ( 0.1 ml ) and 1,2-c6h4f2 ( 0.3 ml ) sample . esi - ms were recorded on a bruker microtof instrument . in all esi - ms spectra there was a good fit to both the principal molecular ion and the overall isotopic distribution . signals in the p{h } nmr spectra were integrated relative to those in similar environments ( i.e. , rh p or b p ) to obtain the relative ratios of products , and data was acquired with a pulse repetition time of 1 s. this avoids potential problems with different relaxation times for different phosphorus environments . nevertheless , the quoted relative ratios based upon this data should be treated as qualitative rather than quantitative . a solution of ( 3,5-bis(trifluoromethyl)phenyl)2pcl ( 1.48 g , 3.0 mmol ) in diethyl ether ( 5 ml ) was added dropwise to a diethyl ether ( 20 ml ) suspension of libh4 ( 0.070 g , 3.21 mmol ) cooled to 5 c with an ice bath . the diethyl ether was removed in vacuo , and the residue was dissolved in hexanes ( 30 ml ) and filtered through celite . the hexanes were reduced in vacuo to 10 ml , and the solution was placed in the freezer ( 20 c ) overnight yielding colorless crystals . excess hexanes were decanted , and crystals were dried to afford a fine white powder which was subsequently washed with 2 3 ml of cold hexanes . removal of all volatiles under reduced pressure yielded 630 mg of fine white powder ( 1 ) . h nmr ( 300 mhz , cdcl3 ) : 8.14 ( br s , 1 h , p - ar - h ) , 8.09 ( br s , 2 h , o - ar - h ) , 6.58 ( dm , jhp = 388 hz , 1 h , ph ) , 0.32.0 ( br m , 3 h , bh ) . p{h } nmr ( 121 mhz , cdcl3 ) : 4.7 ( br s , ph ) . b{h } nmr ( 160 mhz , cdcl3 ) : 41.7 ( br s , bh3 ) . f nmr ( 282 mhz , cdcl3 ) : 62.9 ( s , cf3 ) . ei - ms ( 70 ev ) m / z ( % ) : 458 ( 62 ) [ m bh3 ] . ( adamantyl)2phbh3 was prepared under the same conditions as me3pbh3 but with ( adamantyl)2ph instead of pme3 . h nmr ( 300 mhz , cdcl3 ) : 3.61 ( dm , 1 h , jhp = 379 hz , ph ) , 2.11 to 1.83 ( 30 h , adamantyl - h ) , 0.41 to 0.15 ( br m , 3 h , bh ) . p{h } nmr ( 121 mhz , cdcl3 ) : 40.1 ( br m , ph ) . b{h } nmr ( 160 mhz , cdcl3 ) : 44.8 ( br d , bh3 ) . calcd for c20h34bp : c 75.89% , h 10.84% . a youngs flask charged with 0.25 mmol of r2phbh3 ( 118 mg of 1 , 84 mg of 2 , 65 mg of 3 ) and 5 mol % of [ rh(dpp3)(c6h5f)][bar4 ] ( 18.4 mg , 0.0125 mmol ) was heated to 90 c for 4 h ( 10 and 11 ) or 8 h ( 12 ) in melt conditions . the resulting solids were washed with n - hexane and recrystallized from a mixture of diethyl ether and hexane at 18 c ( 10 30 mg , 25% ; 11 22 mg , 26% ; 12 32 mg , 49% ) . h nmr ( 300 mhz , cdcl3 ) : 8.09 to 7.89 ( 12 h , ar h ) , 7.32 ( dm , jhp = 412 hz , 1 h , ph ) , 2.45 ( br m , 2 h , bh2 ) , 1.11 ( br m , 3 h , bh3 ) . p{h } nmr ( 121 mhz , cdcl3 ) : 1.7 ( br s , phr2 ) , 14.0 ( br s , pr2 ) . b{h } nmr ( 160 mhz , cdcl3 ) : 33.2 ( br s , bh2 ) , 37.7 ( br s , bh3 ) . h nmr ( 500 mhz , cd2cl2 ) : 7.77 to 7.52 ( 16 h , ar h ) , 7.04 ( dt , jhp = 426 hz , jhh = 7.8 hz , 1 h , ph ) , 2.37 ( br m , 2 h , bh2 ) , 1.02 ( br m , 3 h , bh3 ) . p{h } nmr ( 202 mhz , cd2cl2 ) : 3.5 ( br s , phr2 ) , 15.4 ( br s , pr2 ) . b{h } nmr ( 160 mhz , cd2cl2 ) : 33.7 ( br s , bh2 ) , 37.6 ( br s , bh3 ) . h nmr ( 500 mhz , cd2cl2 ) : 7.64 to 6.77 ( 16 h , ar h ) , 6.69 ( dt , jhp = 415 hz , jhh = 6.6 hz , 1 h , ph ) , 3.84 ( s , 6 h , ch3 ) , 3.80 ( s , 6 h , ch3 ) , 2.14 ( br m , 2 h , bh2 ) , 0.96 ( br m , 3 h , bh3 ) . p{h } nmr ( 202 mhz , cd2cl2 ) : 7.6 ( br s , phr2 ) , 22.1 ( br s , pr2 ) . b{h } nmr ( 160 mhz , cd2cl2 ) : 33.3 ( br s , bh2 ) , 37.1 ( br s , bh3 ) . to a youngs flask charged with [ rh(dpp3)(c6h5f)][bar4 ] ( 50 mg , 0.034 mmol ) and 2 equiv of h3bpr2h ( 32 mg of 1 , 23 mg of 2 , 18 mg of 3 0.068 mmol ) was added 1,2-f2c6h4 ( 5 ml ) . the solution was stirred at room temperature 10 min , and a change in color from pale orange to bright yellow was observed . complexes 15 and 16 were isolated as yellow oils , and characterized in situ by nmr spectroscopy and esi - ms . complex 14 could be crystallized at 24 c in the freezer inside the glovebox ( yield 29.6 mg , 37% ) . complexes 15 and 16 could not be isolated cleanly , and attempts to do so led to intractable mixtures of 15 and 16 with 18 and 19 , respectively . slow diffusion of pentane ( 10 ml ) over a solution of 14 in 1,2-f2c6h4 at 24 c afforded yellow crystals ( one of which was employed for an x - ray diffraction study ) . h nmr ( 500 mhz , 1,2-f2c6h4 ) : 8.32 ( s , 8 h , bar4 ) , 7.69 ( s , 4 h , bar4 ) , 5.81 ( d , jhp = 435 hz , 1 h , ph ) , 3.120.81 ( 8 h , 3ch2 dpp3 + bh2 ) , 0.78 ( br , 3 h , bh3 ) , 6.12 ( br , 1 h , bh signals from the aromatics were not observed due to being overlapped by signals from 1,2-f2c6h4 . h nmr ( 500 mhz , 1,2-f2c6h4 , selected data at 0 c ) : 6.12 ( d , jhp = 65 hz , 1 h , bh rh ) . p{h } nmr ( 202 mhz , 1,2-f2c6h4 ) : 29.5 ( ddd , jprh = 130 hz , jpp(cis)= 35 hz , jpp(cis ) = 21 hz , ph2p(ch2)3pph2 ) , 11.3 ( ddd , jpp(trans ) = 244 hz , jprh = 103 hz , jpp(cis ) = 35 hz , ph2p(ch2)3pph2 ) , 0.7 ( dd , jpp(trans ) = 248 hz , jprh = 75 hz , rh - pr2bh3 ) , 2.62 ( br s , phr2bh3 ) . b{h } nmr ( 160 mhz , 1,2-f2c6h4 ) : 6.2 ( bar4 ) , 39.8 ( br , 2 bh3 ) . esi - ms ( 1,2-c6h4f2 , 60 c ) positive ion : m / z = 1431.07 ( calcd 1431.07 , m 2bh3 ) . anal . found : c 46.82% , h 2.39% . h nmr ( 500 mhz , 1,2-f2c6h4 ) : 8.32 ( s , 8 h , bar4 ) , 7.69 ( s , 4 h , bar4 ) , 4.90 ( d , jhp = 414 hz , ph ) , 3.011.10 ( 8 h , 3ch2 dpp3 + bh2 ) , 1.18 ( br , 3h , bh3 ) , 6.95 ( d , jhp = 78 hz , 1 h , bh signals from the aromatics were not observed due to being overlapped by signals from 1,2-f2c6h4 . p{h } nmr ( 202 mhz , 1,2-f2c6h4 ) : 27.3 ( ddd , jprh = 127 hz , jpp(cis ) = 37 hz , jpp(cis ) = 16 hz , ph2p(ch2)3pph2 ) , 11.8 ( ddd , jpp(trans ) = 231 hz , jprh = 100 hz , jpp(cis ) = 37 hz , ph2p(ch2)3pph2 ) , 0.3 ( dd , jpp(trans ) = 231 hz , jprh = 70 hz , rh - pr2bh3 ) , 5.96 ( br s , phr2bh3 ) . b{h } nmr ( 160 mhz , 1,2-f2c6h4 ) : 6.2 ( bar4 ) 39.9 ( br , 2 bh3 ) . esi - ms ( 1,2-c6h4f2 , 60 c ) positive ion : m / z = 1159.13 ( calcd 1159.13 , m 2bh3 ) . h nmr ( 500 mhz , 1,2-f2c6h4 ) : 8.32 ( s , 8 h , bar4 ) , 7.69 ( s , 4 h , bar4 ) , 4.93 ( d , jhp = 409 hz , 1 h , ph ) , 3.77 ( s , 6 h , och3 ) , 3.69 ( s , 6 h , och3 ) , 3.160.58 ( 8 h , 3ch2 dpp3 + bh2 ) , 1.11 ( br , 3 h , bh3 ) , 6.53 ( d , jhp = 73 hz , 1 h , bh h ) . signals from the aromatics were not observed due to being overlapped by signals from 1,2-f2c6h4 . p{h } nmr ( 202 mhz , 1,2-f2c6h4 ) : 27.3 ( ddd , jprh = 132 hz , jpp(cis ) = 37 hz , jpp(cis ) = 12 hz , ph2p(ch2)3pph2 ) , 9.5 ( ddd , jpp(trans ) = 227 hz , jprh = 98 hz , jpp(cis ) = 37 hz , ph2p(ch2)3pph2 ) , 0.7 ( br dd , jpp(trans ) = 227 hz , jprh = 65 hz , rh - pr2bh3 ) , 11.2 ( br s , phr2bh3 ) . b{h } nmr ( 160 mhz , 1,2-f2c6h4 ) : 6.2 ( bar4 ) 38.7 to 49.8 ( br , 2 bh3 ) . esi - ms ( 1,2-f2c6h4 , 60 c ) positive ion : m / z = 1021.25 ( calcd 1021.25 , m c6h4ome ) , 900.17 ( calcd 900.17 , m 2bh3 , c6h4ome ) , 775.17 charged with [ rh(dpp3)(c6h5f)][bar4 ] ( 50 mg , 0.034 mmol ) and 2 equiv of h3bpr2h ( 32 mg of 1 , 23 mg of 2 , 18 mg of 3 0.068 mmol ) was added 1,2-f2c6h4 ( 5 ml ) . the solution was stirred at room temperature for 24 h. the formation of h2 gas is also observed . complexes 17 and 18 could not be isolated cleanly ; they were observed with 22 and 23 , respectively . method b follows . to a youngs flask charged with [ rh(dpp3)(c6h5f)][bar4 ] ( 50 mg , 0.034 mmol ) and 1 equiv of 10 ( 32 mg , 0.068 mmol ) slow diffusion of pentane ( 10 ml ) over a solution of 17 in 1,2-f2c6h4 at 24 c afforded yellow crystals ( one of which was employed for an x - ray diffraction study ) . h nmr ( 500 mhz , 1,2-f2c6h4 ) : 8.32 ( s , 8h , bar4 ) , 7.69 ( s , 4h , bar4 ) , 4.40 ( vbr , 1h , bh ) 3.102.12 ( 8h , 3ch2 dpp3 + bh2 ) , 1.20 ( vbr , 1h , bh ) , 4.54 ( vbr , 1h , bh ) , 13.98 ( s , 1h , rh signals from aromatics not observed due to being overlapped by signals from 1,2-f2c6h4 . p{h } nmr ( 202 mhz , 1,2-f2c6h4 ) : 46.6 ( dd , jrh p(cis)= 36 , ph2p(ch2)3pph2 ) , 29.5 ( m , jp p(trans ) = 260 , rh - pr2bh3pr2hbh3 ) , 12.8 ( ddd , , jp p(trans ) = 260 , jrh p(cis ) = 33 , ph2p(ch2)3pph2 ) , 2.7 ( s , rh - pr2bh3pr2hbh3 ) . b{h } nmr ( 160 mhz , 1,2-f2c6h4 ) : 0.21 ( br ) , 6.2 ( s , bar4 ) , 27.1 ( br ) . esi - ms ( 1,2-f2c6h4 , 60 c ) positive ion : m / z = 1457.09 ( calcd 1457.12 , m ) . h nmr ( 500 mhz , 1,2-f2c6h4 ) : 8.32 ( s , 8 h , bar4 ) , 7.69 ( s , 4 h , bar4 ) , 4.24 ( v br , 1 h , bh ) , 2.611.72 ( 8 h , 3ch2 dpp3 + bh2 ) , 1.29 ( v br , 1 h , bh ) , 4.65 ( v br , 1 h , bh ) , 14.90 ( s , 1 h , rh p{h } nmr ( 202 mhz , 1,2-f2c6h4 ) : 42.5 ( dd , jprh = 106 hz , jpp(cis ) = 34 hz , ph2p(ch2)3pph2 ) , 28.6 ( m , jpp(trans ) = 272 hz , rh - pr2bh3pr2hbh3 ) , 15.3 ( ddd , jpp(trans ) = 272 hz , jprh = 101 hz , jpp(cis ) = 33 hz , ph2p(ch2)3pph2 ) , 6.4 ( s , rh - pr2bh3pr2hbh3 ) . b{h } nmr ( 160 mhz , 1,2-f2c6h4 ) : 0.4 ( br ) , 6.2 ( s , bar4 ) , 25.9 ( br ) . esi - ms ( 1,2-f2c6h4 ) positive ion : m / z = 1185.17 ( calcd 1185.18 , m ) . h nmr ( 500 mhz , 1,2-f2c6h4 ) : 8.32 ( s , 8 h , bar4 ) , 7.69 ( s , 4 h , bar4 ) , 3.77 ( s , 3 h , och3 ) , 3.76 ( s , 3 h , och3 ) , 3.69 ( s , 6 h , och3 ) , 2.851.72 ( 8 h , 3ch2 dpp3 + bh2 ) , 1.03 ( v br , 1 h , bh ) , 4.00 ( v br , 1 h , bh ) , 14.55 ( s , 1 h , rh h ) . signals from aromatics not observed due to being overlapped by signals from 1,2-f2c6h4 . p{h } nmr ( 202 mhz , 1,2-f2c6h4 ) : 42.7 ( ddd , jprh = 109 hz , jpp(cis ) = 35 hz , jpp(cis ) = 12 hz ph2p(ch2)3pph2 ) , 28.1 ( br m , jpp(trans ) = 279 hz , rh - pr2bh3pr2hbh3 ) , 12.5 ( ddd , jpp(trans ) = 279 hz , jprh = 88 hz , jpp(cis ) = 12 hz , ph2p(ch2)3pph2 ) , 11.7 ( br s , rh - pr2bh3pr2hbh3 ) . b{h } nmr ( 160 mhz , c6h4f2 ) : 3.42 ( br ) , 6.2 ( s , bar4 ) , 27.7 ( br ) . esi - ms ( 1,2-f2c6h4 ) positive ion : m / z = 1033.27 ( calcd 1033.27 , m ) . method a follows . to a youngs flask charged with [ rh(dpp3)(c6h5f)][bar4 ] ( 50 mg , 0.034 mmol ) and 2 equiv of h3bpr2h ( 32 mg of 1 ) the solution was stirred at room temperature for 24 h. the formation of h2 ( gas ) is also observed . method b follows . to a youngs flask charged with [ rh(dpp3)(c6h5f)][bar4 ] ( 20 mg , 0.034 mmol ) and 2 equiv of phr2 ( 31 mg , 0.068 mmol , r = 3,5-bis(trifluoromethyl)phenyl ) after stirring for 10 min the solution was evaporated to dryness and the solid washed with pentane ( 2 ml ) . complex 21 was isolated as yellow solid ( yield 17.8 mg , 57% ) . h nmr ( 500 mhz , 1,2-f2c6h4 ) : 8.33 ( s , 8 h , bar4 ) , 7.69 ( s , 4 h , bar4 ) , 6.41 ( dm , jhp = 375 hz , 2 h , ph ) , 2.62 ( br , 4 h , 2 ch2 dpp3 ) , 2.17 ( m , 2 h ch2 , dpp3 ) . . p{h } nmr ( 202 mhz , 1,2-f2c6h4 ) : 9.6 ( m , 2p , aabbm ) , 5.5 ( m , 2p , aabbm ) . esi - ms ( 1,2-f2c6h4 ) positive ion : m / z = 1431.04 ( calcd 1431.07 , m ) . h nmr ( 500 mhz , 1,2-f2c6h4 ) : 8.33 ( s , 8 h , bar4 ) , 7.69 ( s , 4 h , bar4 ) , 5.91 ( dm , jhp = 359 hz , 2 h , ph ) , 2.57 ( br , 4 h , 2 ch2 dpp3 ) , 2.06 ( m , 2 h ch2 , dpp3 ) . signals from aromatics not observed due to being overlapped by signals from 1,2-f2c6h4 . p{h } nmr ( 202 mhz , 1,2-f2c6h4 ) : 12.2 ( m , 2p , aabbm ) , 5.2 ( m , 2p , aabbm ) . to a youngs flask charged with [ rh(dpp3)(c6h5f)][bar4 ] ( 50 mg , 0.034 mmol ) and h3bpr2h ( 4 ) ( 11 mg , 0.034 mmol ) was added 1,2-f2c6h4 ( 5 ml ) . the solution was stirred at room temperature for 1 min , and a change in color from pale orange to purple was observed . h nmr ( 500 mhz , 1,2-f2c6h4 ) : 8.32 ( s , 8 h , bar4 ) , 7.68 ( s , 4 h , bar4 ) , 3.45 ( d , 1 h , jhp = 380 hz , b - ph ) , 2.521.12 ( 36 h , adamantyl - h + dpp3 ch2 ) , 1.36 ( br , 3 h , bh3 ) . signals from aromatics not observed due to being overlapped by signals from 1,2-f2c6h4 . p{h } nmr ( 202 mhz , 1,2-f2c6h4 ) : 35.1 ( d , jprh = 167 hz , dpp3 ) , 30.5 ( br , b esi - ms ( 1,2-f2c6h4 ) positive ion : m / z = 629.09 ( calcd 629.08 , [ rh(dpp3)(c6h4f2 ) ] ) . method a follows . to a youngs flask charged with [ rh(dpp3)(c6h5f)][bar4 ] ( 50 mg , 0.034 mmol ) and 2 equiv of h3bpr2h ( 4 ) ( 22 mg , 0.068 mmol ) was added 1,2-f2c6h4 ( 5 ml ) . the solution was stirred at room temperature for 24 h. complex 24 was isolated as orange solid . method b follows . to a youngs flask charged with [ rh(dpp3)(c6h5f)][bar4 ] ( 50 mg , 0.034 mmol ) and 1 equiv of phr2 ( 10 mg , 0.034 mmol ) the solution was stirred for 5 min , and then 1 equiv of h3bpr2h ( 4 ) ( 11 mg , 0.034 mmol ) was added . the solution was stirred for 5 min , and complex 24 was isolated as orange solid ( yield 48 mg , 71% ) . slow diffusion of pentane ( 10 ml ) into a solution of 24 in 1,2-f2c6h4 at 24 c afforded yellow crystals ( one of which was employed for x - ray diffraction studies ) . h nmr ( 500 mhz , 1,2-f2c6h4 ) : 8.33 ( s , 8 h , bar4 ) , 7.69 ( s , 4 h , bar4 ) , 3.30 ( d , jhp = 362 hz , 1 h , b - ph ) , 2.87 ( d , jhp = 412 hz , 1 h , ph ) , 2.451.56 ( 66 h , dpp3 ch2 and adamantyl - h ) , 0.24 ( br , 3 h , bh3 ) . p{h } nmr ( 202 mhz , 1,2-f2c6h4 ) : 60.4 ( ddd , jpp(trans ) = 266 hz , jprh = 142 hz , jpp(cis ) = 30 hz , ph2p(ch2)3pph2 ) , 30.5 ( s , phr2bh3 ) , 22.8 ( dd , jprh = 163 hz , jpp(cis ) = 30 hz , pph2(ch2)3pph2 ) , 6.6 ( ddd , jpp(trans ) = 270 hz , jprh = 144 hz , jpp(cis ) = 30 hz , rh - pr2bh3 ) . b{h } nmr ( 160 mhz , 1,2-f2c6h4 ) : 6.0 ( s , bar4 ) , 42.2 ( br , bh3 ) . esi - ms ( 1,2-f2c6h4 ) positive ion : m / z = 956.30 ( unidentified fragment ) . calcd for c99h103b2f24p4rh : c 59.50% , h 5.20% . to a youngs flask charged with [ rh(dpp3)(c6h5f)][bar4 ] ( 50 mg , 0.034 mmol ) was added 1,2-f2c6h4 ( 5 ml ) . a 2 equiv portion of h3bph2cy ( 5 ) ( 0.68 ml , 0.1 m solution in 1,2-f2c6h4 , 0.068 mmol ) was then added . the solution was stirred at room temperature for 1 min , and a change in color from orange to pale yellow was observed . complexes 25a and 25b were observed as an approximate 1:1 ratio of isomers and were characterized in situ by nmr spectroscopy . complexes 25a and 25b could not be isolated as they reacted quickly to form complexes 26a and 26b . the p{h } nmr spectrum of this reaction mixture indicates that 2 diastereomers are present ; while we were able to identify the 2 sets of 4 resonances each ( labeled and , based on coupling constants and approximate integrations ) it was not possible to determine which set of signals belonged to which diastereomer . h nmr ( 500 mhz , 1,2-f2c6h4 ) : 8.32 ( s , 8 h , bar4 ) , 7.69 ( s , 4 h , bar4 ) , 2.730.32 ( 32 h , 3 ch2 dpp3 + bh2 , cyh , ph ) , 2.29 ( v br , 3 h , bh ) , 7.92 ( br d , 1 h , bh - rh ) , 17.51 ( s , 1 h , rh p{h } nmr ( 202 mhz , 1,2-f2c6h4 ) : 31.7 ( dm , jprh = 134 hz , ph2p(ch2)3pph2 ) , 30.5 ( dm , jprh = 129 hz , ph2p(ch2)3pph2 ) , 11.8 ( overlapping ddd , jpp(trans ) = approximately 200 hz , jprh = approximately 104 hz , jpp(cis ) = approximately 25 hz , ph2p(ch2)3pph2 ) , 11.0 ( br d , jpp(trans ) = approximately 200 hz , rh - phcy - b ) , 32.1 ( br d , jpp(trans ) = approximately 200 hz , rh - phcy - b ) , 39.8 ( br s , rh h3bph2cy ) , 44.2 ( br s , rh h3bph2cy ) . esi - ms ( 1,2-f2c6h4 , 60 c ) positive ion : m / z = 747.20 ( calcd 747.21 , m 2bh3 ) . to a stirred solution of pcyh2 ( 3.40 ml , 10% wt in hexane , 2.0 mmol ) in dichloromethane ( 20 ml ) was added brh2bsme2 ( 1.0 ml , 1.0 m in ch2cl2 , 1.0 mmol ) and the solution stirred at room temperature for 2 h. the resulting colorless solution was concentrated in vacuo to approximately 3 ml , and diethyl ether ( 30 ml ) was added to precipitate a white solid . the solvent was decanted off and the solid washed with a further 10 ml of diethyl ether . the solid was redissolved in dichloromethane , filtered , and recrystallized from a mixture of dichloromethane and diethyl ether at 18 c to yield white crystals ( first crop 0.190 g , second crop 0.041 g , overall yield 71% ) . at room temperature in cd2cl2 [ ( cyh2p)2bh2]br undergoes a degenerate exchange reaction ; [ ( cyh2p)2bh2]br is in equilibrium with cyh2pbh2br + ph2cy . this exchange process does not occur at 60 c on the nmr time scale , and each of these species can be observed . in the solid - state h nmr ( 500 mhz , 60 c , cd2cl2 ) : 5.46 ( dm , jhp = 429 hz , [ ( cyh2p)2bh2 ] ) , 4.75 ( dm , jhp = 388 hz , cyh2pbh2br ) , 2.52 ( dm , jhp = 196 hz , cyh2p ) , 2.371.16 ( cyh and bh ) . p{h } nmr ( 202 mhz , 60 c , cd2cl2 ) : 35.5 ( br s , [ ( cyh2p)2bh2 ] ) , 38.2 ( br s , cyh2pbh2br ) , 107.5 ( s , cyh2p ) [ ( cyh2p)2bh2]br ( 0.150 g , 0.461 mmol ) ( prepared as above ) and [ nbu4][bh4 ] ( 0.119 g , 0.461 mmol ) were added to a schlenk tube . dichloromethane ( 10 ml ) was added , and effervescence was observed ; the solution was stirred at room temperature for 1 h. the solvent was removed in vacuo , and n - hexane ( 20 ml ) was added to the white solid . the solution was filtered to remove [ nbu4]br and concentrated to approximately 2 ml . storage of this solution for 16 h at 18 c yielded colorless crystals of 13 ( 0.102 g , 86% ) . h nmr ( 500 mhz , 25 c , cd2cl2 ) : 4.68 ( dm , jhp = 386 hz , cyh2pbh2phcybh3 ) , 3.50 ( dm , jhp = 326 hz , cyh2pbh2phcybh3 ) , 2.170.02 ( cyh and bh ) . p{h } nmr ( 202 mhz , 25 c , cd2cl2 ) : 36.6 ( br , cyh2pbh2phcybh3 ) , 45.1 ( br , cyh2pbh2phcybh3 ) . anal . found : c 55.79% , h 11.82% . method a follows . to a youngs flask charged with [ rh(dpp3)(c6h5f)][bar4 ] ( 50 mg , 0.034 mmol ) was added 1,2-f2c6h4 ( 5 ml ) followed by 2 equiv of h3bpcyh2 ( 5 ) ( 0.68 ml , 0.1 m solution in 1,2-f2c6h4 , 0.068 mmol ) . the solution was stirred at room temperature for 12 h. the formation of h2 is also observed . complexes 26a and 26b were characterized as a mixture in solution by nmr spectroscopy and esi - ms . the p{h } nmr spectrum of this reaction mixture indicates that 2 diastereomers are present ; we were able to identify the 2 sets of 4 resonances each ( labeled 26a and 26b , based on coupling constants and approximate integrations ) and tentatively assigned the individual diastereomers ( scheme s5 , supporting information ) by inspection of a model . method b follows . to a youngs flask charged with [ rh(dpp3)(c6h5f)][bar4 ] ( 50 mg , 0.034 mmol ) and 1 equiv of cyh2pbh2pcyhbh3 ( 13 ) ( 8.8 mg , 0.034 mmol ) complexes 26a and 26b were characterized as a mixture in solution by nmr spectroscopy and esi - ms . h nmr ( 500 mhz , 1,2-f2c6h4 ) : 8.32 ( s , 8 h , bar4 ) , 7.69 ( s , 4 h , bar4 ) , 4.353.01 ( ph and bh ) , 2.920.92 ( 30 h , 3ch2 dpp3 , bh2 , cyh ) , 2.98 ( br , 1 h , bh - rh ) , 5.98 ( br , 1 h , bh - rh ) , 16.08 ( s , 1 h , rh signals from aromatics not observed due to being overlapped by signals from 1,2-f2c6h4 . p{h } nmr ( 202 mhz , 1,2-f2c6h4 ) : 37.9 ( dm , jprh = 102 hz , ph2p(ch2)3pph2 ) , 34.5 ( dm , jprh = 102 hz , ph2p(ch2)3pph2 ) , 19.8 ( br d , jpp(trans ) = approximately 255 hz , rh - phcyb ) , 16.2 ( br d , jpp(trans ) = approximately 255 hz , rh - phcy - b ) , 10.7 ( overlapping dm , jpp(trans ) = 255 hz , jprh = 88 hz , jpp(cis ) = approximately 25 hz , ph2p(ch2)3pph2 ) , 14.9 ( br s , rh - phcybh2phcybh3 ) , 16.6 ( br s , rh - phcybh2phcybh3 ) . esi - ms ( 1,2-f2c6h4 , 60 c ) positive ion : m / z = 773.26 ( calcd 773.24 , m ) . [ rh(nbd)2][bar4 ] was synthesized by an adaptation of the preparation of [ rh(cod)2][bar4 ] . [ rh(nbd)2][bar4 ] ( 0.100 g , 0.0869 mmol ) was dissolved in ch2cl2 ( 10 ml ) to produce a deep red solution . ( 2s,4s)-2,4-bis(diphenylphosphino)pentane ( bdpp ) ( 0.0383 g , 0.0869 mmol ) was added , and a color change to red was observed . the solution was stirred for 1 h at room temperature before the solvent was removed in vacuo . n - pentane ( 20 ml ) was added , and the solution was sonicated to produce an orange powder . the solvent was decanted and the solid washed with pentane ( 2 20 ml ) . the product was dried in vacuo and isolated ( yield 0.102 g , 78% ) . h nmr ( 300 mhz , cd2cl2 ) : 7.72 ( s , 8 h , bar4 ) , 7.56 ( s , 4 h , bar4 ) , 7.707.36 ( m , p - ph ) , 4.86 ( m , 2 h , = c h ) , 4.30 ( m , 2 h , = c h ) , 3.90 ( m , 2 h , nbd c h ) , 2.77 ( m , 2 h , p c h ) , 1.84 ( tt , jhh = 6.45 hz , jhp = 19.95 hz , 2 h , ch2 ) , 1.58 ( m , 2 h , nbd ch2 ) , 1.13 ( m , 6 h , ch3 ) . p{h } nmr ( 121.6 mhz , cd2cl2 ) : 27.4 ( d , jprh = 149 hz ) . anal . found : c 54.32% , h 3.18% . [ rh(bdpp)(nbd)][bar4 ] prepared as above ( 0.020 g , 0.0133 mmol ) was added to a high - pressure nmr tube and dissolved in fluorobenzene ( 0.5 ml ) . h nmr ( 500 mhz , c6h5f ) : 8.32 ( s , 8 h , bar4 ) , 7.61 ( s , 4 h , bar4 ) , 5.63 ( m , 2 h , ar h ) , 2.38 ( m , 2 h , p - ch ) , 1.40 ( m , 2 h , ch2 ) , 0.78 ( m , 6 h , ch3 ) . signals from p - ph not observed due to being overlapped by signals from c6h5f . signals from norbornane ( from hydrogenation of norbornadiene ) can also be observed at 2.12 ( m , 2 h , nba c h ) , 1.40 ( m , 2 h , nba ch2 , overlapped ) , 1.11 ( m , 2 h , nba ch2 ) . p{h } nmr ( 202 mhz , c6h5f ) : 39.9 ( d , jprh = 194 hz ) . esi - ms ( c6h5f , 60 c ) positive ion : m / z = 639.13 ( calcd 639.12 , m ) . [ rh(bdpp)(c6h5f)][bar4 ] ( 26 ) was prepared in a high - pressure nmr tube as above . to this was added 2 equiv of h3bph2cy ( 5 ) ( 0.27 ml , 0.1 m solution in 1,2-f2c6h4 , 0.027 mmol ) and the solution mixed for 1 min . complexes 27a and 27b were observed as ratio of isomers and were characterized in situ by nmr spectroscopy . complexes 27a and 27b could not be isolated as they reacted quickly to form complexes of 28 ; signals for 28 can be observed in both h and p{h } nmr spectra of 27a and 27b which , along with the presence of h2 , show that the complexes rapidly undergo dehydrocoupling to form complexes of 28 . the p{h } nmr spectrum of this reaction mixture indicates that 2 diastereomers are present ; while we were able to identify the 2 sets of 4 resonances ( labeled and , based on coupling constants and approximate integrations ) , it was not possible to determine which set of signals belonged to which diastereomer . h nmr ( 500 mhz , c6h5f + 1,2-f2c6h4 ) : 8.32 ( s , 8 h , bar4 ) , 7.69 ( s , 4 h , bar4 ) , 4.19 to 0.32 ( ch3 , ch2 , ch , bdpp , bh2 , cyh , ph ) , 1.80 to 3.31 ( v br , 3 h , bh3 ) , 7.54 to 8.86 ( br d , 1 h , bh - rh ) , 17.73 ( br s , 1 h , rh signals from aromatics not observed due to being overlapped by signals from c6h5f and 1,2-f2c6h4 . h2 can be observed in the nmr spectrum as a sharp singlet at 4.52 ppm suggesting that some dehydrocoupling to complex 28 has occurred . this is further evidenced by the small rh h hydride signal at 16.07 ppm which is observed for complex 28 . p{h } nmr ( 202 mhz , c6h5f + 1,2-f2c6h4 ) : 42.5 ( dm , jprh = 129 hz , ph2p(ch2)3pph2 ) , 32.6 ( dm , jprh = 119 hz , ph2p(ch2)3pph2 ) , 28.3 ( overlapping ddd , jpp(trans ) = approximately 208 hz , jprh = approximately 98 hz , jpp(cis ) = approximately 32 hz , ph2p(ch2)3pph2 ) , 3.6 ( br d , jpp(trans ) = approximately 208 hz , rh - phcy - b ) , 12.1 ( br d , jpp(trans ) = approximately 208 hz , rh - phcy - b ) , 42.1 ( br s , rh [ rh(bdpp)(c6h5f)][bar4 ] was prepared on an nmr scale from [ rh(bdpp)(nbd)][bar4 ] ( 0.020 g , 0.0133 mmol ) as above . this solution was transferred by cannula to an nmr tube containing cyh2pbh2phcybh3 ( 13 ) ( 3.4 mg , 0.0133 mmol ) , and the solution was mixed briefly to yield a pale yellow solution . [ rh(bdpp)(c6h5f)][bar4 ] was prepared on an nmr scale from [ rh(bdpp)(nbd)][bar4 ] ( 0.020 g , 0.0133 mmol ) as above . to this was added 2 equiv of h3bph2cy ( 5 ) ( 0.27 ml , 0.1 m solution in 1,2-c6h4f2 , 0.027 mmol ) and the solution mixed for 18 h. the p{h } nmr spectrum arising from method a indicates that 4 diastereomers are present ; while we were able to identify some of the signals from the 4 sets of 4 resonances ( labeled , , $ , and & based on coupling constants and approximate integrations ) , it was not possible to determine which set of signals belonged to which of the diastereomers . see figure s7 , supporting information h nmr ( 500 mhz , c6h5f ) : 8.32 ( s , 8 h , bar4 ) , 7.69 ( s , 4 h , bar4 ) , 5.472.46 ( ph ) , 2.110.63 ( ch , ch2 , and ch3 bdpp , bh2 , cyh ) , 3.21 ( br , 1 h , bhrh ) , 5.65 to 6.85 ( br , 1 h , bhrh ) , 16.09 , 16.20 , and 17.04 ( s , 1 h , rh signals from aromatics not observed due to being overlapped by signals from c6h5f and 1,2-c6h4f2 . p{h } nmr ( 202 mhz , c6h5f ) : 58.9 ( ddd , jprh = 107 hz , jpp(cis ) = 30 hz , jpp(cis ) = 12 hz , ph2p(ch2)3pph2 ) , 58.0 ( dm , jprh = 102 hz , ph2p(ch2)3pph2 ) , 46.6 ( dm , jprh = 100 hz , ph2p ( ch2)3pph2 ) , 43.6 ( dm , jprh = 102 hz , ph2p(ch2)3pph2 ) , 28.7 ( ddd , jpp(trans ) = 254 hz , jprh = 90 hz , jpp(cis ) = 32 hz , ph2p(ch2)3pph2 ) , 27.5 ( ddd , jpp(trans ) = 250 hz , jprh = 92 hz , jpp(cis ) = 32 hz , ph2p(ch2)3pph2 ) , 24.817.7 ( overlapping m , jpp(trans ) = approximately 254 hz , jprh = approximately 87 hz , jpp(cis ) = approximately 26 hz , ph2p(ch2)3pph2 and rh - phcy - b ) , 13.5 to 20.6 ( br s of 4 isomers , rh - phcybh2phcybh3 ) . esi - ms ( 1,2-c6h4f2 , 60 c ) positive ion : m / z = 801.29 ( calcd 801.29 , m ) . in order to establish that dehydrocoupling had occurred when method b was used to form complexes 28 , excess ( 10 equiv ) of 1,2-bis(diphenylphosphino)ethane was added to the reaction mixture to release the dehydrocoupled product , cyh2pbh2phcybh3 , from the metal center . the p{h } nmr spectrum showed two broad signals at 37.9 and 43.9 which are in agreement with those found for compound 13 . in a procedure similar to that reported by manners et al . , phh2pbh3 ( 0.248 g , 2.0 mmol ) was dissolved in toluene ( 10 ml ) either in the presence of [ rh(dpp3)(c6h5f)][bar4 ] ( 14.7 mg , 0.01 mmol ) or with no catalyst present . the solution was heated to reflux for 16 h before cooling to room temperature . the solution was concentrated in vacuo and added to stirred hexane ( 100 ml ) to produce a white precipitate . the solvent was decanted and the solid washed with hexane ( 2 50 ml ) . the solid was dried in vacuo and isolated in air ( yield 0.110 g rh catalyzed , 0.101 g uncatalyzed ) . the p{h } nmr spectrum of the isolated solids produced by the different methods are very similar with several very broad peaks from 45 to 57 and very broad peaks of lower intensity from 72 to 87 . this is in agreement with the results obtained by manners et al . for uncatalyzed polymerization of phh2pbh3 . a youngs flask charged with phh2pbh3 ( 31 mg , 0.25 mmol ) and 5 mol % of [ rh(dpp3)(c6h5f)][bar4 ] ( 18.4 mg , 0.0125 mmol ) was heated to 90 c for 4 h in melt conditions . the p{h } nmr spectrum shows a peak at 49.3 ppm in agreement with that observed by manners et al . for polymeric material and a lower intensity resonance at 55.0 ppm . see supporting information figure s9 . in the p{h } nmr spectrum the peak at 49.3 ppm split into a broad doublet with a jph coupling constant of approximately 350 hz . analysis by esi - ms of the reaction mixture showed a repeating pattern corresponding to the polymeric repeat unit [phhpbh2] , see figure s10 , supporting information .

the electronic and steric effects in the stoichiometric dehydrocoupling of secondary and primary phosphine boranes h3bpr2h [ r = 3,5-(cf3)2c6h3 ; p-(cf3)c6h4 ; p-(ome)c6h4 ; adamantyl , ad ] and h3bpcyh2 to form the metal - bound linear diboraphosphines h3bpr2bh2pr2h and h3bprhbh2prh2 , respectively , are reported . reaction of [ rh(l)(6-fc6h5)][barf4 ] [ l = ph2p(ch2)3pph2 , arf = 3,5-(cf3)2c6h3 ] with 2 equiv of h3bpr2h affords [ rh(l)(h)(,-pr2bh3)(1-h3bpr2h)][barf4 ] . these complexes undergo dehydrocoupling to give the diboraphosphine complexes [ rh(l)(h)(,2-pr2bh2pr2bh3)][barf4 ] . with electron - withdrawing groups on the phosphine borane there is the parallel formation of the products of b p cleavage , [ rh(l)(pr2h)2][barf4 ] , while with electron - donating groups no parallel product is formed . for the bulky , electron rich , h3bp(ad)2h no dehydrocoupling is observed , but an intermediate rh(i ) phosphine borane complex is formed , [ rh(l){2-h3bp(ad)2h}][barf4 ] , that undergoes b p bond cleavage to give [ rh(l){1-h3bp(ad)2h}{p(ad)2h}][barf4 ] . the relative rates of dehydrocoupling of h3bpr2h ( r = aryl ) show that increasingly electron - withdrawing substituents result in faster dehydrocoupling , but also suffer from the formation of the parallel product resulting from p b bond cleavage . h3bpcyh2 undergoes a similar dehydrocoupling process , and gives a mixture of stereoisomers of the resulting metal - bound diboraphosphine that arise from activation of the prochiral p h bonds , with one stereoisomer favored . this diastereomeric mixture may also be biased by use of a chiral phosphine ligand . the selectivity and efficiencies of resulting catalytic dehydrocoupling processes are also briefly discussed .

Introduction Results and Discussion Conclusions Experimental Section

catalytic dehydrocoupling of amine and phosphine boranes is one method that has emerged for the formation of b p bonds , and development in the area has been spurred on by the potential for ammonia in addition , polymeric materials that can arise from dehydropolymerization of primary analogues are also of significant interest as they are valence isoelectronic with technologically ubiquitous polyolefins . perhaps the best example is that of the [ rh(cod)2][otf ] catalyzed dehydrocoupling of secondary , h3bpr2h , and primary , h3bprh2 , phosphine boranes to give oligomeric and polymeric materials ( scheme 1 ) . recently , we have reported that the rh(i ) complexes [ rh(pbu2h)2(-fc6h5)][bar4 ] , and [ rh(l)(-fc6h5)][bar4 ] , [ l = ph2p(ch2)3pph2 ] are particularly well - suited to the study of the dehydrocoupling mechanism of secondary phosphine boranes in solvents such as fluorobenzene ; and on the basis of the observation of intermediates , kinetic studies , and h / d exchange experiments we have proposed a catalytic cycle for the dehydrocoupling of h3bpr2h ( r= ph , bu ; scheme 2 ) . further insight comes from the observations that for r = bu the barrier to dehydrocoupling is higher ( 70 c versus 25 c for reaction ) , p h activation appears also to be a higher energy process , different intermediates ( a and e ) are observed , and the turnover limiting process in catalysis is now suggested to be the p h activation / dehydrocoupling steps . prior work has demonstrated a similar difference in relative rates of dehydrocoupling of secondary h3bpr2h [ r = p-(cf3)c6h4 , ph , bu , bu ] and primary h3bprh2 [ r = ph , bu , bu ] phosphine boranes using the [ rh(cod)2][otf ] catalyst , and this was suggested to be due to a combination of steric and electronic ( relative p h bond strengths ) factors , although the mechanism of dehydrocoupling of phosphine boranes using this catalyst is currently not known . h bond is such that spontaneous dehydrocoupling occurs in the absence of catalyst , with electron - withdrawing aryl groups [ p-(cf3)c6h4 ] undergoing faster reaction than electron - donating [ p-(ome)c6h4 ] . very recent work has shown that paramagnetic ti(iii ) centers might also be involved in dehydrocoupling of phosphine and amine boranes when using cp2ti - based catalysts , while oligomerization of base - stabilized phosphino likely decomposition routes in rh - systems for phosphine borane dehydrocoupling to form bis(phosphine)boronium salts have also recently been discussed . , we report an extension of our investigations into the mechanism of phosphine borane dehydrocoupling using the { rh(ph2p(ch2)3pph2 ) } fragment , by varying the electronic and steric profile of the secondary phosphine boranes h3bpr2h [ r = 3,5-(cf3)2c6h3 ; p-(cf3)c6h4 ; p-(ome)c6h4 ; adamantyl ] , as well as investigations with the primary phosphine borane h3bpcyh2 . dehydrocoupling forms the corresponding metal bound linear diboraphosphines h3bpr2bh2pr2h and h3bprhbh2prh2 , respectively these studies provide insight into the determining role of the electronics and sterics of the phosphine borane in the dehydrocoupling process , as well as providing as yet unreported examples of the solid - state structures of the intermediates related to the catalytic cycle . we also report for the first time the partial control of diastereoselectivity in dehydrocoupling of primary phosphine boranes , that can additionally be biased by use of a chiral chelating phosphine on the rhodium center . a range of secondary phosphine boranes with differing electronic and steric properties have been used in this study ( 1 , 2 , 3 , and 4 , figure 1 ) , which also provide comparison with the previously reported ph , 6 , and bu , 7 , analogues . complexes 1012 are synthesized by a rh - catalyzed process from the corresponding phosphine boranes , while primary phosphine containing 13 has been synthesized in good isolated yield ( 85% ) by addition of [ nbu4][bh4 ] to the bis(phopshine)boronium [ ( cyh2p)2bh2]br . addition of 2 equiv of 1 to [ rh(l)(-fc6h5)][bar4 ] [ l = ph2p(ch2)3pph2 ] in 1,2-f2c6h4 solution at 25 c rapidly ( on time of mixing ) resulted in the formation of [ rh(l)(h)(,-pr2bh3)(-h3bpr2h)][bar4 ] , 14 [ r = 3,5-(cf3)2(c6h3 ) , scheme 3 ] , which was characterized by nmr spectroscopy , esi - ms ( electrospray ionization mass spectrometry ) , and single crystal x - ray diffraction . likewise , the use of 2 equiv of phosphine borane 2 or 3 results in the formation of the analogous complexes 15 [ r = p-(cf3)c6h4 ] and 16 [ r = p-(ome)c6h4 ] , respectively , which were fully characterized using solution techniques . in the h nmr spectrum of 14 one broad , relative integral 3h , signal is observed at 0.78 , indicative of a rhh3b interaction in which the b h bonds are undergoing rapid site exchange on the nmr spectroscopic time scale between terminal and bridging sites . compared to a rh(i ) complex that shows a bidentate -coordination mode for the borane , [ rh(pbu2h)2(-h3bpbu2h)][bar4 ] , the rhb distance for the -interaction in 14 is considerably longer [ 2.188(3 ) versus 2.740(4 ) , respectively ] , consistent with this different binding motif . complexes 1416 undergo spontaneous dehydrocoupling ( 25 c ) to form products of the general formula [ rh(l)h(,-pr2bh2pr2bh3)][bar4 ] : 17 , r = 3,5-(cf3)2c6h3 ; 18 , r = p-(cf3)c6h4 ; 19 , r = p-(ome)c6h4 ( scheme 4 ) . figure 3 shows the solid - state structure of 17 , in which the diboraphosphine acts as a chelate to the rh(iii ) center , via a phosphido group and two b - agostic interactions : [ rh(l)h(,-pr2bh2pr2bh3)][bar4 ] [ r = 3,5-(cf3)2c6h3 ] . the nmr spectroscopic data for 17 are fully consistent with the solid - state structure being retained in solution and are also very similar to that reported for the analogous complex formed from the deydrocoupling of 6 ( r = ph ) . the b nmr spectrum shows two different environments [ 27.1 and 0.21 ] for the two boron atoms present in the diboraphosphine , with the latter assigned to the -h3b unit on the basis of the large downfield shift from free ligand ( = + 36.8 ) . nevertheless these relative rates reflect previous observations on the rate of catalytic dehydrocoupling when the electronics of a system are changed , in as much as electron - withdrawing groups promote the reaction . in this study we suggested that b h activation / reorganization in intermediates such as b ( scheme 2 ) prior to p h bond , and calculations on analogous h3bl ( l = lewis base ) systems show that the b h bond is considerably weaker when there are electron - withdrawing groups on the lewis base . however , we can not rule out that the relative p h bond strengths in intermediates such as 14 also might play a role , or that there is a change in the rate determining step on changing the phosphine borane ligand , as the intimate details of the mechanism leading to p the observation that for an electron - withdrawing phosphine there is a significant proportion of parallel product formed that results from p b bond cleavage has been noted previously in phosphine borane complexes to give either simple adducts or further reaction to yield bis(phosphine)boronium salts . prior to the formation of the parallel product 21 ( r = 3,5-(cf3)2c6h3 ) an intermediate is observed that has been characterized by h and p{h } nmr spectroscopy as [ rh(l)h(,-pr2bh3)(pr2h)][bar4 ] 20 , i.e. in the high - field region of the h nmr spectrum a broad doublet is observed at 7.06 [ j(hp ) = 76 hz ] which is assigned to the -b - agostic interaction , while there is a relatively sharper one at 9.61 [ j(hp ) = 165 hz ] assigned to rh h , and again rh coupling is not resolved . borane h3bp(adamantyl)2h , 4 , to [ rh(l)(-fc6h5)][bar4 ] in 1,2-f2c6h4 solution at 25 c rapidly results in a color change from orange to purple and the formation of the new bound rh(i ) phosphine borane complex [ rh(l)(-h3bp(adamantyl)2h)][bar4 ] , 23 , which was characterized in situ by nmr spectroscopy . one suggested mechanism for this process is the reaction of a short - lived phosphino borane ( or its masked equivalent ) with coordinated phosphine , not dissimilar to the mechanism suggested for the formation of diaminoboranes from amine boranes and amines catalyzed by alkaline earth catalysts . however , reaction of [ rh(l)(-fc6h5)][bar4 ] with a slight excess of h3bpcyh2 ( 5 ) in 1,2-f2c6h4 solution at 25 c led to the instantaneous formation of only two complexes in a 1:1 ratio , 25a and 25b , [ rh(l)h(,-pcyhbh3)(-h3bpcyh2)][bar4 ] , as a proposed diastereomeric pair ( scheme 8) . the h nmr spectrum does not have the necessary resolution to separate out the diastereomers in the hydride region , with broad resonances observed at 2.3 ( 3 h , bh3 ) , 7.9 ( 1 h , rh h complexes 25a / b can not be isolated in pure form , and characterization by nmr spectroscopy is best performed on freshly prepared samples , as after 1 h ( 25 c ) they have undergone dehydrocoupling to give a mixture of two resolvable diastereomers 26a and 26b , with one of the diastereomers present in a significantly larger amount 6:1 ( scheme 8) , indicating that the dehydrocoupling step occurs with some stereocontrol . this mixture of diastereomers can also be synthesized cleanly by direct reaction of [ rh(l)(-fc6h5)][bar4 ] with the preformed diaboraphosphine h3bpcyhbh2pcyh2 ( 13 ) in 1,2-f2c6h4 solution at 25 c ( figure 5 for the solid - state structure ) . we suggest that the mechanism for equilibration involves reductive elimination of the phosphido and hydride ligands to form a rh(i ) phosphine borane complex , similar to e in scheme 2 , which then undergoes rapid oxidative addition of the other p h bond . that these diastereoisomers are a result of the metal activation of the prochiral terminal p h bonds in 13 is shown by addition of an excess of dppe to 26a / b . we have briefly explored the use of a chiral metal / ligand fragment in dehydrocoupling , [ rh(bdpp ) ] [ s , s - bdpp = ( 2s,4s)-2,4-bis(diphenylphosphino)pentane ] . initially a 2:1:2:1 ratio of 4 isomers is observed , that changes to a 5:1:3:0 ratio after 18 h. we are unable to comment in more detail on the conformation of these isomers , although the observation of stereocontrol in the direct dehydrocoupling is similar to that observed for the achiral system . in particular it has been shown that addition of h3bpphh2 to pt(pet3)2h(pph2bh3 ) results in exchange of the metal bound phosphide complex to give the primary phosphido borane complex . under the standard catalytic melt conditions ( 90 c , 5 mol % ) , [ rh(l)(-fc6h5)][bar4 ] will dehydrocouple the secondary aryl phosphine boranes used in this study to form the corresponding linear diboraphosphines 1012 , although we have not explored in detail the temporal evolution of these systems due to the problems associated with directly interrogating the melt . for electron - withdrawing groups ( 1 and 2 ) , complete consumption of starting the reaction at this temperature is not selective , and although the main product is the linear diboraphosphine , there are products that we tentatively identify as the cyclic oligomers ( bh2pr2)n ( n = 3 , 4 ) . however , on the basis of the solution studies , p b bond formation , ( dehydrocoupling ) is faster with electron - withdrawing groups . the temporal differences in observed product conversion in the melt could reflect a difference in the rate of the p b bond forming event , or alternatively , they could reflect the ease at which the bound product is substituted on the metal center , i.e. this suggests that there is not a strong inherent difference in binding strengths between the two products , with the implication being that the observed rate differences in the melt arise from the dehydrocoupling step . that these polymers are terminated by { pphh2 } groups rather than { bh3 } this formulation also argues against cyclic oligomers being observed by esi - ms , and presumably the additional phosphine arises from p b bond cleavage . for aryl phosphine boranes , electron - withdrawing groups ( cf3 ) promote stoichiometric dehydrocoupling faster than for more electron - donating ( ome ) groups . this increase in rate is accompanied by a significant degree of parallel and competitive p b bond cleavage to afford metal complexes with two monodentate phosphine ligands , which we suggest is due to a weakening of the p these systems also turnover catalytically under melt conditions , with the overall rate of conversion broadly following the relative dehydrocoupling rates observed in the stoichiometric studies , suggesting that the dehydrocoupling step under melt conditions might also be the turnover limiting step . for this phosphine a significant observation is that , for primary phosphine boranes , which are precursors to polyphosphinoboranes , use of the { rh(ph2pch2ch2ch2pph2 ) } fragment results in some apparent diastereoselectivity in the dehydrocoupling step , at least in the stoichiometric reactions that produce metal - bound diboraphosphines . to a youngs flask charged with [ rh(dpp3)(c6h5f)][bar4 ] ( 50 mg , 0.034 mmol ) and 2 equiv of h3bpr2h ( 32 mg of 1 ) the solution was stirred at room temperature for 24 h. the formation of h2 ( gas ) is also observed . to this was added 2 equiv of h3bph2cy ( 5 ) ( 0.27 ml , 0.1 m solution in 1,2-c6h4f2 , 0.027 mmol ) and the solution mixed for 18 h. the p{h } nmr spectrum arising from method a indicates that 4 diastereomers are present ; while we were able to identify some of the signals from the 4 sets of 4 resonances ( labeled , , $ , and & based on coupling constants and approximate integrations ) , it was not possible to determine which set of signals belonged to which of the diastereomers .

such processes enable new discoveries to be made in the promising application areas that main group species are now occupying , such as high performance polymers , emissive materials , etch resists for lithography , and precursors to ceramic thin films or devices . however , the development of this field lags substantially behind the advances made in catalytic c c and c x bond formation , for which there are now a myriad of efficient ways to promote such unions that are important for the construction of new molecules . catalytic dehydrocoupling of amine and phosphine boranes is one method that has emerged for the formation of b p bonds , and development in the area has been spurred on by the potential for ammonia in addition , polymeric materials that can arise from dehydropolymerization of primary analogues are also of significant interest as they are valence isoelectronic with technologically ubiquitous polyolefins . recently , we have reported that the rh(i ) complexes [ rh(pbu2h)2(-fc6h5)][bar4 ] , and [ rh(l)(-fc6h5)][bar4 ] , [ l = ph2p(ch2)3pph2 ] are particularly well - suited to the study of the dehydrocoupling mechanism of secondary phosphine boranes in solvents such as fluorobenzene ; and on the basis of the observation of intermediates , kinetic studies , and h / d exchange experiments we have proposed a catalytic cycle for the dehydrocoupling of h3bpr2h ( r= ph , bu ; scheme 2 ) . under stoichiometric conditions the observation that b transforms into f on gentle heating allowed for kinetic parameters to be determined that suggested that the rate - determining step(s ) for dehydrocoupling were located within the transformations b to d. in solution phase the turnover limiting step for catalysis is proposed to be the displacement of the linear diboraphosphine product ( i.e. further insight comes from the observations that for r = bu the barrier to dehydrocoupling is higher ( 70 c versus 25 c for reaction ) , p h activation appears also to be a higher energy process , different intermediates ( a and e ) are observed , and the turnover limiting process in catalysis is now suggested to be the p h activation / dehydrocoupling steps . prior work has demonstrated a similar difference in relative rates of dehydrocoupling of secondary h3bpr2h [ r = p-(cf3)c6h4 , ph , bu , bu ] and primary h3bprh2 [ r = ph , bu , bu ] phosphine boranes using the [ rh(cod)2][otf ] catalyst , and this was suggested to be due to a combination of steric and electronic ( relative p h bond strengths ) factors , although the mechanism of dehydrocoupling of phosphine boranes using this catalyst is currently not known . very recent work has shown that paramagnetic ti(iii ) centers might also be involved in dehydrocoupling of phosphine and amine boranes when using cp2ti - based catalysts , while oligomerization of base - stabilized phosphino likely decomposition routes in rh - systems for phosphine borane dehydrocoupling to form bis(phosphine)boronium salts have also recently been discussed . , we report an extension of our investigations into the mechanism of phosphine borane dehydrocoupling using the { rh(ph2p(ch2)3pph2 ) } fragment , by varying the electronic and steric profile of the secondary phosphine boranes h3bpr2h [ r = 3,5-(cf3)2c6h3 ; p-(cf3)c6h4 ; p-(ome)c6h4 ; adamantyl ] , as well as investigations with the primary phosphine borane h3bpcyh2 . dehydrocoupling forms the corresponding metal bound linear diboraphosphines h3bpr2bh2pr2h and h3bprhbh2prh2 , respectively these studies provide insight into the determining role of the electronics and sterics of the phosphine borane in the dehydrocoupling process , as well as providing as yet unreported examples of the solid - state structures of the intermediates related to the catalytic cycle . we also report for the first time the partial control of diastereoselectivity in dehydrocoupling of primary phosphine boranes , that can additionally be biased by use of a chiral chelating phosphine on the rhodium center . a range of secondary phosphine boranes with differing electronic and steric properties have been used in this study ( 1 , 2 , 3 , and 4 , figure 1 ) , which also provide comparison with the previously reported ph , 6 , and bu , 7 , analogues . likewise , the use of 2 equiv of phosphine borane 2 or 3 results in the formation of the analogous complexes 15 [ r = p-(cf3)c6h4 ] and 16 [ r = p-(ome)c6h4 ] , respectively , which were fully characterized using solution techniques . one of these sharper resonances [ 29.5 , ddd , j(rhp ) 130 , j(pp ) 35 , j(pp ) 21 hz ] is assigned to the phosphorus atom trans to the weakly bound -b - agostic interaction on the basis of the larger rh coupling constant , while the other signal [ 11.3 , ddd , j(rhp ) 103 , j(pp ) 244 , j(pp ) 35 hz ] is assigned to the phosphorus atom trans to the coordinated phosphido ligand . compared to a rh(i ) complex that shows a bidentate -coordination mode for the borane , [ rh(pbu2h)2(-h3bpbu2h)][bar4 ] , the rhb distance for the -interaction in 14 is considerably longer [ 2.188(3 ) versus 2.740(4 ) , respectively ] , consistent with this different binding motif . figure 3 shows the solid - state structure of 17 , in which the diboraphosphine acts as a chelate to the rh(iii ) center , via a phosphido group and two b - agostic interactions : [ rh(l)h(,-pr2bh2pr2bh3)][bar4 ] [ r = 3,5-(cf3)2c6h3 ] . the rh(iii ) center has pseudo - octahedral geometry , in which the oligomeric phosphine borane is bound tridentate to the metal through -bh2rh [ b2rh1 , 2.280(5 ) ] and phosphido [ p3rh1 , 2.3925(10 ) ] interactions . following these processes in situ using nmr spectroscopy demonstrated that these dehydrocoupling reactions follow a first order rate profile for the consumption of the starting material over at least three half - lives ( see supporting information ) : 1 3 h ( 25 c ) ; 2 3 h ( 25 c ) ; 6 14 h ( 25 c);3 8 h ( 35 c ) , 120 h ( 25 c ) . in this study we suggested that b h activation / reorganization in intermediates such as b ( scheme 2 ) prior to p h bond , and calculations on analogous h3bl ( l = lewis base ) systems show that the b h bond is considerably weaker when there are electron - withdrawing groups on the lewis base . however , we can not rule out that the relative p h bond strengths in intermediates such as 14 also might play a role , or that there is a change in the rate determining step on changing the phosphine borane ligand , as the intimate details of the mechanism leading to p the observation that for an electron - withdrawing phosphine there is a significant proportion of parallel product formed that results from p b bond cleavage has been noted previously in phosphine borane complexes to give either simple adducts or further reaction to yield bis(phosphine)boronium salts . prior to the formation of the parallel product 21 ( r = 3,5-(cf3)2c6h3 ) an intermediate is observed that has been characterized by h and p{h } nmr spectroscopy as [ rh(l)h(,-pr2bh3)(pr2h)][bar4 ] 20 , i.e. b bond cleavage , formally of the -h3bpr2h ligand , to afford a complex with a -b - agostic interaction from a phosphide borane ligand ( as for 14 ) and a simple pr2h ligand trans to a hydride . in the high - field region of the h nmr spectrum a broad doublet is observed at 7.06 [ j(hp ) = 76 hz ] which is assigned to the -b - agostic interaction , while there is a relatively sharper one at 9.61 [ j(hp ) = 165 hz ] assigned to rh h , and again rh coupling is not resolved . borane h3bp(adamantyl)2h , 4 , to [ rh(l)(-fc6h5)][bar4 ] in 1,2-f2c6h4 solution at 25 c rapidly results in a color change from orange to purple and the formation of the new bound rh(i ) phosphine borane complex [ rh(l)(-h3bp(adamantyl)2h)][bar4 ] , 23 , which was characterized in situ by nmr spectroscopy . over time ( 1 h ) , complex 23 disappears to be replaced by a new complex that has been characterized by nmr spectroscopy and a solid - state x - ray diffraction experiment as [ rh(l)(phr2)(h3bphr2)][bar4 ] ( 24 , r = adamantyl ) . one suggested mechanism for this process is the reaction of a short - lived phosphino borane ( or its masked equivalent ) with coordinated phosphine , not dissimilar to the mechanism suggested for the formation of diaminoboranes from amine boranes and amines catalyzed by alkaline earth catalysts . with an appreciation of the intermediate metal complexes formed with secondary phosphine boranes from this and previous work , it was of interest to explore whether the proposed dehydrocoupling mechanism for secondary phosphine boranes using [ rh(l)(-fc6h5)][bar4 ] could be applied to primary analogues . however , reaction of [ rh(l)(-fc6h5)][bar4 ] with a slight excess of h3bpcyh2 ( 5 ) in 1,2-f2c6h4 solution at 25 c led to the instantaneous formation of only two complexes in a 1:1 ratio , 25a and 25b , [ rh(l)h(,-pcyhbh3)(-h3bpcyh2)][bar4 ] , as a proposed diastereomeric pair ( scheme 8) . the h nmr spectrum does not have the necessary resolution to separate out the diastereomers in the hydride region , with broad resonances observed at 2.3 ( 3 h , bh3 ) , 7.9 ( 1 h , rh h complexes 25a / b can not be isolated in pure form , and characterization by nmr spectroscopy is best performed on freshly prepared samples , as after 1 h ( 25 c ) they have undergone dehydrocoupling to give a mixture of two resolvable diastereomers 26a and 26b , with one of the diastereomers present in a significantly larger amount 6:1 ( scheme 8) , indicating that the dehydrocoupling step occurs with some stereocontrol . selected bond lengths ( ) and angles ( deg ) : p1b1 1.9267(18 ) , b1p2 1.9381(18 ) , p2b2 1.926(2 ) ; p1b1p2 108.34(9 ) , b1p2b2 113.32(9 ) . the high - field region of the h nmr spectrum of 26a/26b shows a slight downfield shift of the rh h hydride resonance to 16.1 , when compared to 25a/25b , while the -bh2rh units are observed as two broadened resonances at 2.98 ( 1h ) and 5.98 ( 1h ) . we suggest that the mechanism for equilibration involves reductive elimination of the phosphido and hydride ligands to form a rh(i ) phosphine borane complex , similar to e in scheme 2 , which then undergoes rapid oxidative addition of the other p h bond . we have briefly explored the use of a chiral metal / ligand fragment in dehydrocoupling , [ rh(bdpp ) ] [ s , s - bdpp = ( 2s,4s)-2,4-bis(diphenylphosphino)pentane ] . under the standard catalytic melt conditions ( 90 c , 5 mol % ) , [ rh(l)(-fc6h5)][bar4 ] will dehydrocouple the secondary aryl phosphine boranes used in this study to form the corresponding linear diboraphosphines 1012 , although we have not explored in detail the temporal evolution of these systems due to the problems associated with directly interrogating the melt . for electron - withdrawing groups ( 1 and 2 ) , complete consumption of starting the reaction at this temperature is not selective , and although the main product is the linear diboraphosphine , there are products that we tentatively identify as the cyclic oligomers ( bh2pr2)n ( n = 3 , 4 ) . our results are broadly in line with the previously reported catalyzed dehydrocoupling of 2 using [ rh(cod)cl]2 , which , at a slightly lower temperature ( 60 c , 16 h , melt ) , affords the linear diboraphosphine product in 69% isolated yield , while at 100 c only the cyclic oligomers are isolated . the temporal differences in observed product conversion in the melt could reflect a difference in the rate of the p b bond forming event , or alternatively , they could reflect the ease at which the bound product is substituted on the metal center , i.e. although this is different from what is observed in solution at room temperature , in which release of the product is likely the turnover limiting step , it is consistent with the high local concentration of h3bpr2h that being under melt conditions ( 90 c ) would promote such a substitution . under melt conditions ( 90 c , 5 mol % , 4 h ) h3bpphh2 is dehydrocoupled to give a major product which is identified by p nmr spectroscopy as being polymeric ( bh2pphh)n by comparison with previously reported data for purified material coming from the [ rh(cod)2][otf ] catalyzed process [ 49.3 , d , j(ph ) 350 hz , 1,2f2c6h4 ; lit . this increase in rate is accompanied by a significant degree of parallel and competitive p b bond cleavage to afford metal complexes with two monodentate phosphine ligands , which we suggest is due to a weakening of the p these systems also turnover catalytically under melt conditions , with the overall rate of conversion broadly following the relative dehydrocoupling rates observed in the stoichiometric studies , suggesting that the dehydrocoupling step under melt conditions might also be the turnover limiting step . for this phosphine a significant observation is that , for primary phosphine boranes , which are precursors to polyphosphinoboranes , use of the { rh(ph2pch2ch2ch2pph2 ) } fragment results in some apparent diastereoselectivity in the dehydrocoupling step , at least in the stoichiometric reactions that produce metal - bound diboraphosphines . h nmr ( 300 mhz , cd2cl2 ) : 7.72 ( s , 8 h , bar4 ) , 7.56 ( s , 4 h , bar4 ) , 7.707.36 ( m , p - ph ) , 4.86 ( m , 2 h , = c h ) , 4.30 ( m , 2 h , = c h ) , 3.90 ( m , 2 h , nbd c h ) , 2.77 ( m , 2 h , p c h ) , 1.84 ( tt , jhh = 6.45 hz , jhp = 19.95 hz , 2 h , ch2 ) , 1.58 ( m , 2 h , nbd ch2 ) , 1.13 ( m , 6 h , ch3 ) . to this was added 2 equiv of h3bph2cy ( 5 ) ( 0.27 ml , 0.1 m solution in 1,2-c6h4f2 , 0.027 mmol ) and the solution mixed for 18 h. the p{h } nmr spectrum arising from method a indicates that 4 diastereomers are present ; while we were able to identify some of the signals from the 4 sets of 4 resonances ( labeled , , $ , and & based on coupling constants and approximate integrations ) , it was not possible to determine which set of signals belonged to which of the diastereomers .

hydration mediates many processes in nature , among which are protein folding , phase partitioning of chemicals , and protein there is increasing interest in taking advantage of the water thermodynamics and structure in drug design . indeed , methods such as watermap and gist use explicit solvent simulations to score the stability of specific water sites in an enzymatic active site by the means of explicit solvent simulations . these methods have the disadvantage of being relatively slow , which opens the way for more approximate methods including grid or szmap that treat water as a fluid lacking correlations interacting with a solute via an effective solvent potential as in the case of szmap . more approximate methods based on continuum solvent models can also capture certain aspects of hydration such as the high dielectric polarization of water . they have the advantage of being orders of magnitude faster than molecular simulations , but lack the packing and h - bonding structure of water , which greatly limits the behavior of the model where the size and anisotropy of water molecules matter . an intermediate approach using liquid state integral equations , called the three - dimensional reference interaction site model ( 3d - rism ) , is very attractive for applications where the high dielectric polarization , the detailed interactions with a solute , and the multibody correlations of the solvent structure matters . the 3d - rism method produces an approximate average solvent distribution around a rigid solute . it is orders of magnitude faster than molecular simulations and does not share the sequestered - water sampling problem that molecular simulations have . compared to the independent particle approximation or continuum models , it has the advantage of treating water in a liquid state , incorporating the molecular correlations in an effective way . the 3d - rism approach becomes an interesting alternative in many applications to the more traditional methods . however , a major weakness of the 3d - rism is its poor ability to compute , with any reasonable accuracy , the hydration free energy ( hfe ) of organic solutes . this defect is related to the problem of all extended rism theories in dealing with the thermodynamics of hydrophobicity . figure 1 shows the inadequacy of the 3d - rism with the kh closure hfes for a set of 504 small organic molecules when compared to experimental results ( c.f . comparison of the 3d - rism / kh prediction of the hydration free energy with experimental results for a set of 504 small organic molecules . it is important to be able to calculate with sufficient accuracy the hfe of small solutes given that the equilibrium for ligand binding depends on both the direct interaction of the ligand as well as the desolvation penalty . explicit solvent approaches such as watermap and gist are scoring functions not designed to compute hfes since these methods do not account , for example , for the cavity free energy . the independent particle methods and continuum approaches are also missing most of the cavity free energy and usually rely on surface area ( sa ) scaling or other approximate types of methods . it is known that sa types of approaches do not produce accurate cavity terms compared to more rigorous theoretical calculations . it is therefore of great interest to find a theoretical formalism to compute the full hfe including the cavity free energy component . in this article , we test 3d - rism with the kh closure and the ng modified free energy functional against converged explicit solvent simulations . we show that only a single component of the energy functional is responsible for most of the error correction . in the next sections , we first briefly review important aspects of the 3d - rism / kh ; we then show results and present our calculations . the fundamental equations that lead to the practical framework we use can be derived using the variational principle in the density functional theory of liquids . this formally states that , given an external potential ( e.g. , the interaction potential coming from a solute ) , the true physical solvent distribution density minimizes the grand canonical potential functional . the ornstein zernike type of integral equation such as the rism approximation for molecular solvents , may be used to compute solvent density and thermodynamic functions that can be solved on a spatial grid . alone , it is difficult to use this approach to treat complicated solutes such as drug - like molecules or proteins . the 3d - rism method makes possible applications such as computing the hfe of more elaborate solutes . the idea is to fix a single solute ( implying infinite dilution of the solute ) and compute the solvent perturbation using any of a number of closures . the 3d - rism equations with a suitable closure only need the solute and solvent potential parameters as input , typically from molecular two - body additive force fields ( e.g. , amber , charmm , etc . ) , and the solvent thermodynamic conditions ( bulk solvent density , temperature , and composition ) . once solved , the 3d - rism equations yield approximate density distribution functions , direct correlation functions ( dcf ) for each h and for o in the case of pure water . the distribution function is the three - dimensional analog to the radial distribution function ( also called pair distribution function ) . it gives the spatial density of the solvent atoms in the presence of a solute . the auxiliary dcfs are essential to relating the solvent distribution functions to the solvent free energy . it is very convenient that , using thermodynamic integration , an exact equation can be derived for the hfe . the equivalent formalism applied to molecular simulations requires multiple simulations , each of which account for the variation of a transformation parameter . however , as previously shown , the approximate 3d - rism hfes lack sufficient accuracy when compared to either experiments or more rigorous simulation methods . this will be further demonstrated hereafter . in an attempt to improve the 3d - rism hfes , the theoretical corrections had success on a limited number of molecules and focus on the improvement of the closure equation with additional so - called bridge terms . universal correction ( uc ) shows that , with a two parameter fit to the partial molar volume , the 3d - rism hfe can be reasonably well corrected . the original uc is given by1where guc is the corrected hydration free energy in kcal / mol , gkh is the 3d - rism hydration free energy using the kovalenko hirata ( kh ) closure in kcal / mol , is the number density of bulk water ( 0.0333 molecule / ) , and vm is the partial molar volume in units of / molecule . the correction optimized by palmer et al . is quite interesting . here , we go beyond a fit to experimental hydration free energies . we find a correction optimized on computed quantities that ultimately only depends on fundamental atomic quantities derived from simulation and the thermodynamic state of the system . also , our proposed method stems from correcting the analytical free energy functional found in 3d - rism / kh and provides a better understanding of the nature of the hfe errors . to accomplish this , we fit with a single parameter based on a computed lennard - jones sphere hfe to obtain a demonstrably transferable constant that scales a well - known approximate bridge function . in order to set the stage for what follows , it is useful to examine the equation used to calculate the hfe and derived , as stated above , directly from the thermodynamic formulation . the formulas and its approximation changes with different closure equations , but for the approximate kh closure one has2where kb is the boltzmann constant , t is the temperature in kelvin units , is the bulk density of solvent center ( 0.0333 for oxygen and 0.0666 for hydrogen ) , and ( ) is the deviation of the solvent particle density relative to bulk for the solvent center . the position dependent change in density can be written as o ( ) = o ( ) o where o ( ) is the calculated oxygen density distribution of water that results from the 3d - rism calculations . the function (x ) is a heaviside step function that effects the quadratic term of the density deviation when the density is less than bulk . finally , the direct correlation function for a solvent particle is noted c ( ) . the solute solvent interaction potential , used in the 3d - rism calculation , is defined as3where the i summation is over the solute centers , 0 is the vacuum permittivity , the atomic partial charges are denoted by q , and the distance between the point and the ith solute atomic center coordinate is given by ri . the standard lennard - jones potential is used where i is the energy well depth associated with the ith solute atom , is the energy well depth associated with the solvent particle , and the lennard - jones atomic radius minima are denoted by . it is useful to point out at this stage that the hfe can be formally split into its nonpolar ( np ) and electrostatic ( ele ) components4 each of these terms can be calculated using molecular simulations and the free energy perturbation ( fep ) technique using double decoupling for instance . the gnp term corresponds to the work required to create a cavity with lennard - jones attractions in water , while all the atomic charges of the solute are zero . the gele term corresponds to the work needed to restore the solute atomic partial charges in solution once the cavity is created . in other words , it is the cost ( or gain ) of the reorganization of the solvent when the solute lennard - jones volume recovers its full polarity . this separation of the total free energy commands a stepwise calculation with a specific order of decoupling in simulation where the atomic partial charges need to be zeroed before decoupling the lennard - jones parameters . these free energy quantities can also be computed individually with 3d - rism using eq 2 in two steps : gnp is calculated using eq 2 from a 3d - rism / kh run with the solute atomic partial charges set to zero , and then gele is computed using eqs 2 and 4 . we can rewrite eq 2 with the definition of a free energy density function integrand for each solvent center , noted g ( ) , as follows5which simplifies the notation when the volume of integration v is partitioned by a surface within which the solvent is excluded vin and the part of space outside the exclusion domain vext6then , for the integral over vin , gkh is split into an electrostatic and a nonpolar component . we wish to correct the behavior of the 3d - rism / kh hfes for the problematic behavior of the nonpolar component only . an effective bridge function correction to the hfe may be introduced by scaling the dcfs ( c functions ) in eq 5 by a constant yet to be determined . this leads directly to7from which one recovers eq 2 when the full dcfs are used ( = 1 ) . it is useful to note that the most positive contribution to gkh comes from conp ( ) inside the molecular core exclusion domain . also , the theoretical separation of the solvent water molecule densities into oxygen and hydrogen in the exclusion domain should be dominated by the repulsive core behavior ( exclusion ) of single water molecules . this led us to consider the oxygen correction only given that the packing is mainly represented by the oxygen l - j in most water models8 this leaves us with an equation with one adjustable parameter . we can optimize the free energy functional by adjusting the parameter following a strategy employed by others with bridge functions . we note that the bridge function correction proposed here is not computed self - consistently in the integral equation but only for the free energy form itself . the 504 molecules used in this work come from a compilation done by rizzo et al . and then enriched by mobley et al . with extensive free energy perturbation results . the atomic partial charges , computed using am1-bcc , and starting geometry come from the supporting information of mobley et al . the small molecule lennard - jones parameters were set using the moe assignment rules based on the opls parameters and functions . the solute geometries were optimized using moe and the mmff94 force field with the moe 2011.10 rfield implicit solvation model with a dielectric of 80 to account for the formation of intramolecular polar interactions . we used the c - tip3p water model that was shown to reproduce the pair correlation function of pure water in simulation . it should be noted that the results for all rism like integral equations are approximate . e , the o = 0.156 kcal / mol , o = 1.76827 , h = 0.152 kcal / mol , and h = 0.6938 . the oh distance is set to 0.9572 and the hoh angle to 104.52. the main difference with the standard tip3p water model is the addition of a hydrogen repulsive l - j term set to match the water dimer potential of the tip3p water model and the water water pair correlation functions , which eliminates the need for other approximations typically needed . the calculations were conducted using ambertools , version 1.4 . the kovalenko hirata ( kh ) closure the susceptibility response function of the pure liquid was computed using the ambertools rism1d program based on the drism method . the calculation was stopped when the direct correlation function residuals reached 10 , the temperature was set to 298 k , and the water dielectric constant was set to 78.497 . the produced correlation functions were used as the input to the 3d - rism program for which the grid spacing was set to 0.4 ; the minimum distance between any atom of the solute and the boundary of the grid box was set to 10 . the calculation was converged to a direct correlation function residual of 10 per point . in all cases , the hfes produced with these parameters had numerical errors around 0.1 kcal / mol . our choice of calculation parameters allowed computation of hfes within seconds instead of many tens of minutes as previously reported . this was achieved by reducing the grid density , by reducing the grid volume , and by lowering the convergence thresholds . the improved timings come from the combined effects of a careful scan of the parameters and the efficient mdiis algorithm . the presented approach is orders of magnitude faster than explicit solvent simulation that typically requires thousands of cpu hours . the vin region or interior volume was determined using the 3d - rism calculation grid . at each grid point , the lennard - jones interaction potential between the water oxygen and the solute was calculated . any point with a potential larger than 10 kcal / mol was considered as part of vin and included as part of the integration domain of eq 8 . beyond 5 kcal / mol , the boundary threshold does not need to be fitted . what matters is that the water density must be very low . a threshold based on the lennard - jones potential as opposed to the water oxygen density has the advantage of decoupling the choice of the boundary from the 3d - rism solution . the reported standard errors are estimated for the fitted parameters and the predicted quantities using 2000 samples in a bootstrap analysis . as mentioned in the introduction , the calculated 3d - rism / kh hfes do not correlate well with either experimental or fep values , as clearly shown in figures 1 and 2a . even more troubling , the dynamic range of the values calculated with 3d - rism is not reasonable . however , this explanation does not lead to a straightforward correction since the calculation of the entropy is not explicit in the formula . when the fep - calculated hfe is partitioned into the nonpolar and electrostatic components , the 3d - rism evaluation of the electrostatic components of the hfe correlates remarkably well , as shown in figure 2c , given that no parameter is fitted . the slope is close to one and the y - axis intercept close to zero . plots of g fep vs g 3d - rism , gnp fep vs gnp 3d - rism , and gele fep vs gele 3d - rism . the total 3d - rism / kh hydration free energy ( hfe ) does not correlate with the simulation based free energy perturbation hfe ( a ) . the nonpolar component of the hfe explains most of the errors ( b ) because the electrostatic component ( c ) of the hfe obtained with 3d - rism correlates well with the fep results . the nonpolar contribution explains most of the variance observed in the residuals of the calculated hfes as shown in figure 2b . therefore , gnp needs to be corrected . here , we use eq 8 . it seems natural to simplify the problem to the calculation of the hfe of monatomic spherical lennard - jones ( lj ) solutes , which by definition do not have atomic partial charge and bear the simplest molecular shape one can imagine . for this purpose the optimization of the parameter in eq 8 to reproduce the calculated hfes of the lj monatomic solutes shows considerable improvement as demonstrated in figure 3a and b ( see also figure 4 ) . the dcf scaling factor obtained was = 0.380 . to verify that it is not only valid for the solute of spherical shapes , table 2 ) , the two values are statistically indistinguishable , which suggests that the optimal value of is solute - shape - invariant , at least for molecules of the size and shape of our data set . once again , the correction applied on the dcf in the solvent - excluded volume markedly improves the correspondence between the 3d - rism and the calculated fep gnp as shown by the correlation graph illustrated in figure 3a . most notably , the dynamical ranges of both fep and corrected 3d - rism match ; the correlation slope is close to one and the intercept close to zero . using our corrected nonpolar 3d - rism component and the electrostatic term leads to a corrected 3d - rism hfe in good agreement with fep . indeed , with a single constant fitted to the lj solute hfe , one can not statistically distinguish fep from 3d - rism hfes as shown in figure 3b . plots of g fep vs g 3d - rism and vs g 3d - rism corrected . the optimization of the parameter in eq 8 improves the hydration free energy ( hfe ) correlation for a series of lennard - jones monatomic solutes as shown above , where ( a ) the initial 3d - rism formula ( c.f . eq 2 ) is applied and where ( b ) the correction is applied ( c.f . plots of gnp fep vs gnp 3d - rism corrected and g fep vs g 3d - rism corrected . the corrected 3d - rism nonpolar hydration free ( hfe ) energy ( c.f . eq 8) , using = 0.38 obtained by fitting monatomic lennard - jones solute hfes , shows a significant improvement for both the nonpolar component and the total hfe over the original formulation . the repulsive bridge correction ( rbc ) from kovalenko and hirata was shown to improve the hfe of inert gas atoms , and we computed the hfe using their thermodynamic perturbation theory ( tpt ) approximation . although we observed that the comparison with experimental results changed the r of the rbc - tpt model from 0.2 to 0.6 , the gnpr remains poor at a value of 0.16 . the refit was necessary because we used a more extensive data set and a different water model . it is interesting to observe that the fit on the lj solute data set leads to statistically similar constants to those fit to the mobley hfes . furthermore , the addition of a constant ( b in table 2 ) did not significantly improve the model . the fact that the corrections presented in table 2 and using either the water - excluded domain or the more empirical approach based on the partial molar volume have similar accuracy is not too surprising since the partial molar volume is related to the solvent - excluded volume given that the former represents the volume of one solute in pure water . in this study , we showed that the 3d - rism hydration free energy ( hfe ) with the kh closure formula is not accurate when used on a large data set of 504 small molecules for which experimental and double decoupling free energy perturbation ( fep ) values are available from the literature . the decomposition of the hfe into its electrostatic and nonpolar components lead us to conclude that the electrostatic component of the hfe is reasonably well reproduced without any empirical fit , i.e. , using standard molecular mechanics parameters for the lennard - jones potential and the am1-bcc partial charge model . however , the nonpolar component of the hfe was the clear source of error . we have found that scaling the nonpolar oxygen - solute ng - style direct correlation bridge function contribution to the hfe only over the solvent - excluded region using a single scaling constant brings the theoretically correct 3d - rism formula to much better agreement with the corresponding simulated fep and experimental values . it was also surprising that this single parameter is the same for lennard - jones monatomic solutes and for a diverse set of 504 small molecules . this -corrected hfe functional can simultaneously reproduce the high level fep nonpolar hfes , the fep electrostatic hfes , the fep total hfes , and the experimental hfes of the set of 504 small molecules used in this study . it is important to realize that the optimal was not fit to experimental quantities but solely to computed quantities . moreover the transferability of the parameter from a lennard - jones solute to small molecules suggests that it is a shape - independent property . ultimately , it was derived from atomistic fundamental properties of a lennard - jones force field at a relevant thermodynamic state . the ability of the -corrected 3d - rism nonpolar free energy term to reproduce the fep - calculated corresponding values is remarkable , and here we compare it to recent attempts using different methods . first , it was shown that surface area or volume based approximations do not correlate with the fep nonpolar components . previous attempts using the full dispersion solvent solute terms were shown to be challenging and demonstrated poor transferability . wagoner and baker had optimized an approach based on a combination of the weeks anderson chandler decomposition and scaled particle theory . in their work , they focus on the hfe of 11 alkanes and were able to match the fep computed hfe with an r of 0.71 and an r of 0.38 against experimental results . the -corrected 3d - rism values on this subset lead to an r of 0.72 versus fep and 0.88 versus experimental results . more recently , chen et al . have developed a method based on a differential geometry approach with three free parameters that predicted the experimental hfe of 11 alkane molecules with a root - mean - square error ( rmse ) of 0.12 kcal / mol . the corresponding -corrected 3d - rism rmse is 0.44 kcal / mol . in their article , they show that the rmse on the prediction of the experimental hfe on 19 alkanes is 0.33 kcal / mol . the results of chen et al.s method s ability is most likely due to their focus on alkanes , whereas we tried to cover a more broad range of chemical species . finally , guo et al . used a level - set based approach , called vism - cfa , to compute the nonpolar component of the hfe . using the mobley data set , they report an r of 0.76 versus experimental results and of 0.84 versus fep . our corresponding results reported in table 1 are 0.88 and 0.96 , respectively . however , the vism - cfa r for the nonpolar hfe component is only 0.42 . although encouraging , all these methods are either less accurate or more limited than the model developed here . furthermore , the herein presented semiempirical method has strong theoretical underpinnings that only rely on theory and computed quantities . finally , the correction we found for the 3d - rism formulation of hydration free energy shows that it accounts accurately for most of the physical phenomena involved in the hydration free energy of small molecules . the major asset in 3d - rism is the computational expediency at which converged hydration functions can be calculated within a few seconds for small molecules . it is reasonable to think that this accuracy can translate into more complex environments such as protein binding sites . a proper demonstration based on experimental evaluations remains challenging due to the lack of high quality data available .

we show that an ng bridge function modified version of the three - dimensional reference interaction site model ( 3d - rism - ngb ) solvation free energy method can accurately predict the hydration free energy ( hfe ) of a set of 504 organic molecules . to achieve this , a single unique constant parameter was adjusted to the computed hfe of single atom lennard - jones solutes . it is shown that 3d - rism is relatively accurate at predicting the electrostatic component of the hfe without correction but requires a modification of the nonpolar contribution that originates in the formation of the cavity created by the solute in water . we use a free energy functional with the ng scaling of the direct correlation function [ ng , k. c. j. chem . phys.1974 , 61 , 2680 ] . this produces a rapid , reliable small molecule hfe calculation for applications in drug design .

Introduction Theory Calculation Details Results Discussion

hydration mediates many processes in nature , among which are protein folding , phase partitioning of chemicals , and protein there is increasing interest in taking advantage of the water thermodynamics and structure in drug design . an intermediate approach using liquid state integral equations , called the three - dimensional reference interaction site model ( 3d - rism ) , is very attractive for applications where the high dielectric polarization , the detailed interactions with a solute , and the multibody correlations of the solvent structure matters . the 3d - rism method produces an approximate average solvent distribution around a rigid solute . the 3d - rism approach becomes an interesting alternative in many applications to the more traditional methods . however , a major weakness of the 3d - rism is its poor ability to compute , with any reasonable accuracy , the hydration free energy ( hfe ) of organic solutes . this defect is related to the problem of all extended rism theories in dealing with the thermodynamics of hydrophobicity . figure 1 shows the inadequacy of the 3d - rism with the kh closure hfes for a set of 504 small organic molecules when compared to experimental results ( c.f . comparison of the 3d - rism / kh prediction of the hydration free energy with experimental results for a set of 504 small organic molecules . it is important to be able to calculate with sufficient accuracy the hfe of small solutes given that the equilibrium for ligand binding depends on both the direct interaction of the ligand as well as the desolvation penalty . explicit solvent approaches such as watermap and gist are scoring functions not designed to compute hfes since these methods do not account , for example , for the cavity free energy . the independent particle methods and continuum approaches are also missing most of the cavity free energy and usually rely on surface area ( sa ) scaling or other approximate types of methods . it is therefore of great interest to find a theoretical formalism to compute the full hfe including the cavity free energy component . in this article , we test 3d - rism with the kh closure and the ng modified free energy functional against converged explicit solvent simulations . we show that only a single component of the energy functional is responsible for most of the error correction . in the next sections , we first briefly review important aspects of the 3d - rism / kh ; we then show results and present our calculations . the fundamental equations that lead to the practical framework we use can be derived using the variational principle in the density functional theory of liquids . the 3d - rism method makes possible applications such as computing the hfe of more elaborate solutes . the idea is to fix a single solute ( implying infinite dilution of the solute ) and compute the solvent perturbation using any of a number of closures . the 3d - rism equations with a suitable closure only need the solute and solvent potential parameters as input , typically from molecular two - body additive force fields ( e.g. once solved , the 3d - rism equations yield approximate density distribution functions , direct correlation functions ( dcf ) for each h and for o in the case of pure water . the distribution function is the three - dimensional analog to the radial distribution function ( also called pair distribution function ) . it gives the spatial density of the solvent atoms in the presence of a solute . the auxiliary dcfs are essential to relating the solvent distribution functions to the solvent free energy . it is very convenient that , using thermodynamic integration , an exact equation can be derived for the hfe . in an attempt to improve the 3d - rism hfes , the theoretical corrections had success on a limited number of molecules and focus on the improvement of the closure equation with additional so - called bridge terms . universal correction ( uc ) shows that , with a two parameter fit to the partial molar volume , the 3d - rism hfe can be reasonably well corrected . the original uc is given by1where guc is the corrected hydration free energy in kcal / mol , gkh is the 3d - rism hydration free energy using the kovalenko hirata ( kh ) closure in kcal / mol , is the number density of bulk water ( 0.0333 molecule / ) , and vm is the partial molar volume in units of / molecule . also , our proposed method stems from correcting the analytical free energy functional found in 3d - rism / kh and provides a better understanding of the nature of the hfe errors . to accomplish this , we fit with a single parameter based on a computed lennard - jones sphere hfe to obtain a demonstrably transferable constant that scales a well - known approximate bridge function . in order to set the stage for what follows , it is useful to examine the equation used to calculate the hfe and derived , as stated above , directly from the thermodynamic formulation . the position dependent change in density can be written as o ( ) = o ( ) o where o ( ) is the calculated oxygen density distribution of water that results from the 3d - rism calculations . finally , the direct correlation function for a solvent particle is noted c ( ) . the solute solvent interaction potential , used in the 3d - rism calculation , is defined as3where the i summation is over the solute centers , 0 is the vacuum permittivity , the atomic partial charges are denoted by q , and the distance between the point and the ith solute atomic center coordinate is given by ri . the standard lennard - jones potential is used where i is the energy well depth associated with the ith solute atom , is the energy well depth associated with the solvent particle , and the lennard - jones atomic radius minima are denoted by . it is useful to point out at this stage that the hfe can be formally split into its nonpolar ( np ) and electrostatic ( ele ) components4 each of these terms can be calculated using molecular simulations and the free energy perturbation ( fep ) technique using double decoupling for instance . the gnp term corresponds to the work required to create a cavity with lennard - jones attractions in water , while all the atomic charges of the solute are zero . the gele term corresponds to the work needed to restore the solute atomic partial charges in solution once the cavity is created . in other words , it is the cost ( or gain ) of the reorganization of the solvent when the solute lennard - jones volume recovers its full polarity . this separation of the total free energy commands a stepwise calculation with a specific order of decoupling in simulation where the atomic partial charges need to be zeroed before decoupling the lennard - jones parameters . these free energy quantities can also be computed individually with 3d - rism using eq 2 in two steps : gnp is calculated using eq 2 from a 3d - rism / kh run with the solute atomic partial charges set to zero , and then gele is computed using eqs 2 and 4 . we can rewrite eq 2 with the definition of a free energy density function integrand for each solvent center , noted g ( ) , as follows5which simplifies the notation when the volume of integration v is partitioned by a surface within which the solvent is excluded vin and the part of space outside the exclusion domain vext6then , for the integral over vin , gkh is split into an electrostatic and a nonpolar component . we wish to correct the behavior of the 3d - rism / kh hfes for the problematic behavior of the nonpolar component only . an effective bridge function correction to the hfe may be introduced by scaling the dcfs ( c functions ) in eq 5 by a constant yet to be determined . also , the theoretical separation of the solvent water molecule densities into oxygen and hydrogen in the exclusion domain should be dominated by the repulsive core behavior ( exclusion ) of single water molecules . we can optimize the free energy functional by adjusting the parameter following a strategy employed by others with bridge functions . we note that the bridge function correction proposed here is not computed self - consistently in the integral equation but only for the free energy form itself . the small molecule lennard - jones parameters were set using the moe assignment rules based on the opls parameters and functions . the solute geometries were optimized using moe and the mmff94 force field with the moe 2011.10 rfield implicit solvation model with a dielectric of 80 to account for the formation of intramolecular polar interactions . the oh distance is set to 0.9572 and the hoh angle to 104.52. the main difference with the standard tip3p water model is the addition of a hydrogen repulsive l - j term set to match the water dimer potential of the tip3p water model and the water water pair correlation functions , which eliminates the need for other approximations typically needed . the calculation was stopped when the direct correlation function residuals reached 10 , the temperature was set to 298 k , and the water dielectric constant was set to 78.497 . the produced correlation functions were used as the input to the 3d - rism program for which the grid spacing was set to 0.4 ; the minimum distance between any atom of the solute and the boundary of the grid box was set to 10 . the calculation was converged to a direct correlation function residual of 10 per point . the improved timings come from the combined effects of a careful scan of the parameters and the efficient mdiis algorithm . the vin region or interior volume was determined using the 3d - rism calculation grid . at each grid point , the lennard - jones interaction potential between the water oxygen and the solute was calculated . a threshold based on the lennard - jones potential as opposed to the water oxygen density has the advantage of decoupling the choice of the boundary from the 3d - rism solution . as mentioned in the introduction , the calculated 3d - rism / kh hfes do not correlate well with either experimental or fep values , as clearly shown in figures 1 and 2a . even more troubling , the dynamic range of the values calculated with 3d - rism is not reasonable . however , this explanation does not lead to a straightforward correction since the calculation of the entropy is not explicit in the formula . when the fep - calculated hfe is partitioned into the nonpolar and electrostatic components , the 3d - rism evaluation of the electrostatic components of the hfe correlates remarkably well , as shown in figure 2c , given that no parameter is fitted . plots of g fep vs g 3d - rism , gnp fep vs gnp 3d - rism , and gele fep vs gele 3d - rism . the total 3d - rism / kh hydration free energy ( hfe ) does not correlate with the simulation based free energy perturbation hfe ( a ) . the nonpolar component of the hfe explains most of the errors ( b ) because the electrostatic component ( c ) of the hfe obtained with 3d - rism correlates well with the fep results . the nonpolar contribution explains most of the variance observed in the residuals of the calculated hfes as shown in figure 2b . it seems natural to simplify the problem to the calculation of the hfe of monatomic spherical lennard - jones ( lj ) solutes , which by definition do not have atomic partial charge and bear the simplest molecular shape one can imagine . to verify that it is not only valid for the solute of spherical shapes , table 2 ) , the two values are statistically indistinguishable , which suggests that the optimal value of is solute - shape - invariant , at least for molecules of the size and shape of our data set . once again , the correction applied on the dcf in the solvent - excluded volume markedly improves the correspondence between the 3d - rism and the calculated fep gnp as shown by the correlation graph illustrated in figure 3a . most notably , the dynamical ranges of both fep and corrected 3d - rism match ; the correlation slope is close to one and the intercept close to zero . using our corrected nonpolar 3d - rism component and the electrostatic term leads to a corrected 3d - rism hfe in good agreement with fep . indeed , with a single constant fitted to the lj solute hfe , one can not statistically distinguish fep from 3d - rism hfes as shown in figure 3b . plots of g fep vs g 3d - rism and vs g 3d - rism corrected . the optimization of the parameter in eq 8 improves the hydration free energy ( hfe ) correlation for a series of lennard - jones monatomic solutes as shown above , where ( a ) the initial 3d - rism formula ( c.f . plots of gnp fep vs gnp 3d - rism corrected and g fep vs g 3d - rism corrected . the corrected 3d - rism nonpolar hydration free ( hfe ) energy ( c.f . eq 8) , using = 0.38 obtained by fitting monatomic lennard - jones solute hfes , shows a significant improvement for both the nonpolar component and the total hfe over the original formulation . the repulsive bridge correction ( rbc ) from kovalenko and hirata was shown to improve the hfe of inert gas atoms , and we computed the hfe using their thermodynamic perturbation theory ( tpt ) approximation . it is interesting to observe that the fit on the lj solute data set leads to statistically similar constants to those fit to the mobley hfes . the fact that the corrections presented in table 2 and using either the water - excluded domain or the more empirical approach based on the partial molar volume have similar accuracy is not too surprising since the partial molar volume is related to the solvent - excluded volume given that the former represents the volume of one solute in pure water . in this study , we showed that the 3d - rism hydration free energy ( hfe ) with the kh closure formula is not accurate when used on a large data set of 504 small molecules for which experimental and double decoupling free energy perturbation ( fep ) values are available from the literature . the decomposition of the hfe into its electrostatic and nonpolar components lead us to conclude that the electrostatic component of the hfe is reasonably well reproduced without any empirical fit , i.e. however , the nonpolar component of the hfe was the clear source of error . we have found that scaling the nonpolar oxygen - solute ng - style direct correlation bridge function contribution to the hfe only over the solvent - excluded region using a single scaling constant brings the theoretically correct 3d - rism formula to much better agreement with the corresponding simulated fep and experimental values . it was also surprising that this single parameter is the same for lennard - jones monatomic solutes and for a diverse set of 504 small molecules . this -corrected hfe functional can simultaneously reproduce the high level fep nonpolar hfes , the fep electrostatic hfes , the fep total hfes , and the experimental hfes of the set of 504 small molecules used in this study . moreover the transferability of the parameter from a lennard - jones solute to small molecules suggests that it is a shape - independent property . ultimately , it was derived from atomistic fundamental properties of a lennard - jones force field at a relevant thermodynamic state . the ability of the -corrected 3d - rism nonpolar free energy term to reproduce the fep - calculated corresponding values is remarkable , and here we compare it to recent attempts using different methods . first , it was shown that surface area or volume based approximations do not correlate with the fep nonpolar components . in their work , they focus on the hfe of 11 alkanes and were able to match the fep computed hfe with an r of 0.71 and an r of 0.38 against experimental results . the -corrected 3d - rism values on this subset lead to an r of 0.72 versus fep and 0.88 versus experimental results . have developed a method based on a differential geometry approach with three free parameters that predicted the experimental hfe of 11 alkane molecules with a root - mean - square error ( rmse ) of 0.12 kcal / mol . the corresponding -corrected 3d - rism rmse is 0.44 kcal / mol . in their article , they show that the rmse on the prediction of the experimental hfe on 19 alkanes is 0.33 kcal / mol . used a level - set based approach , called vism - cfa , to compute the nonpolar component of the hfe . finally , the correction we found for the 3d - rism formulation of hydration free energy shows that it accounts accurately for most of the physical phenomena involved in the hydration free energy of small molecules . the major asset in 3d - rism is the computational expediency at which converged hydration functions can be calculated within a few seconds for small molecules .

hydration mediates many processes in nature , among which are protein folding , phase partitioning of chemicals , and protein there is increasing interest in taking advantage of the water thermodynamics and structure in drug design . indeed , methods such as watermap and gist use explicit solvent simulations to score the stability of specific water sites in an enzymatic active site by the means of explicit solvent simulations . these methods have the disadvantage of being relatively slow , which opens the way for more approximate methods including grid or szmap that treat water as a fluid lacking correlations interacting with a solute via an effective solvent potential as in the case of szmap . more approximate methods based on continuum solvent models can also capture certain aspects of hydration such as the high dielectric polarization of water . they have the advantage of being orders of magnitude faster than molecular simulations , but lack the packing and h - bonding structure of water , which greatly limits the behavior of the model where the size and anisotropy of water molecules matter . an intermediate approach using liquid state integral equations , called the three - dimensional reference interaction site model ( 3d - rism ) , is very attractive for applications where the high dielectric polarization , the detailed interactions with a solute , and the multibody correlations of the solvent structure matters . it is orders of magnitude faster than molecular simulations and does not share the sequestered - water sampling problem that molecular simulations have . compared to the independent particle approximation or continuum models , it has the advantage of treating water in a liquid state , incorporating the molecular correlations in an effective way . the 3d - rism approach becomes an interesting alternative in many applications to the more traditional methods . however , a major weakness of the 3d - rism is its poor ability to compute , with any reasonable accuracy , the hydration free energy ( hfe ) of organic solutes . this defect is related to the problem of all extended rism theories in dealing with the thermodynamics of hydrophobicity . figure 1 shows the inadequacy of the 3d - rism with the kh closure hfes for a set of 504 small organic molecules when compared to experimental results ( c.f . comparison of the 3d - rism / kh prediction of the hydration free energy with experimental results for a set of 504 small organic molecules . it is important to be able to calculate with sufficient accuracy the hfe of small solutes given that the equilibrium for ligand binding depends on both the direct interaction of the ligand as well as the desolvation penalty . explicit solvent approaches such as watermap and gist are scoring functions not designed to compute hfes since these methods do not account , for example , for the cavity free energy . the independent particle methods and continuum approaches are also missing most of the cavity free energy and usually rely on surface area ( sa ) scaling or other approximate types of methods . in this article , we test 3d - rism with the kh closure and the ng modified free energy functional against converged explicit solvent simulations . we show that only a single component of the energy functional is responsible for most of the error correction . in the next sections , we first briefly review important aspects of the 3d - rism / kh ; we then show results and present our calculations . the fundamental equations that lead to the practical framework we use can be derived using the variational principle in the density functional theory of liquids . , the interaction potential coming from a solute ) , the true physical solvent distribution density minimizes the grand canonical potential functional . the ornstein zernike type of integral equation such as the rism approximation for molecular solvents , may be used to compute solvent density and thermodynamic functions that can be solved on a spatial grid . the 3d - rism method makes possible applications such as computing the hfe of more elaborate solutes . the idea is to fix a single solute ( implying infinite dilution of the solute ) and compute the solvent perturbation using any of a number of closures . the 3d - rism equations with a suitable closure only need the solute and solvent potential parameters as input , typically from molecular two - body additive force fields ( e.g. once solved , the 3d - rism equations yield approximate density distribution functions , direct correlation functions ( dcf ) for each h and for o in the case of pure water . the distribution function is the three - dimensional analog to the radial distribution function ( also called pair distribution function ) . it gives the spatial density of the solvent atoms in the presence of a solute . the auxiliary dcfs are essential to relating the solvent distribution functions to the solvent free energy . however , as previously shown , the approximate 3d - rism hfes lack sufficient accuracy when compared to either experiments or more rigorous simulation methods . in an attempt to improve the 3d - rism hfes , the theoretical corrections had success on a limited number of molecules and focus on the improvement of the closure equation with additional so - called bridge terms . universal correction ( uc ) shows that , with a two parameter fit to the partial molar volume , the 3d - rism hfe can be reasonably well corrected . the original uc is given by1where guc is the corrected hydration free energy in kcal / mol , gkh is the 3d - rism hydration free energy using the kovalenko hirata ( kh ) closure in kcal / mol , is the number density of bulk water ( 0.0333 molecule / ) , and vm is the partial molar volume in units of / molecule . we find a correction optimized on computed quantities that ultimately only depends on fundamental atomic quantities derived from simulation and the thermodynamic state of the system . also , our proposed method stems from correcting the analytical free energy functional found in 3d - rism / kh and provides a better understanding of the nature of the hfe errors . to accomplish this , we fit with a single parameter based on a computed lennard - jones sphere hfe to obtain a demonstrably transferable constant that scales a well - known approximate bridge function . in order to set the stage for what follows , it is useful to examine the equation used to calculate the hfe and derived , as stated above , directly from the thermodynamic formulation . the formulas and its approximation changes with different closure equations , but for the approximate kh closure one has2where kb is the boltzmann constant , t is the temperature in kelvin units , is the bulk density of solvent center ( 0.0333 for oxygen and 0.0666 for hydrogen ) , and ( ) is the deviation of the solvent particle density relative to bulk for the solvent center . the position dependent change in density can be written as o ( ) = o ( ) o where o ( ) is the calculated oxygen density distribution of water that results from the 3d - rism calculations . the function (x ) is a heaviside step function that effects the quadratic term of the density deviation when the density is less than bulk . the solute solvent interaction potential , used in the 3d - rism calculation , is defined as3where the i summation is over the solute centers , 0 is the vacuum permittivity , the atomic partial charges are denoted by q , and the distance between the point and the ith solute atomic center coordinate is given by ri . the standard lennard - jones potential is used where i is the energy well depth associated with the ith solute atom , is the energy well depth associated with the solvent particle , and the lennard - jones atomic radius minima are denoted by . it is useful to point out at this stage that the hfe can be formally split into its nonpolar ( np ) and electrostatic ( ele ) components4 each of these terms can be calculated using molecular simulations and the free energy perturbation ( fep ) technique using double decoupling for instance . the gnp term corresponds to the work required to create a cavity with lennard - jones attractions in water , while all the atomic charges of the solute are zero . the gele term corresponds to the work needed to restore the solute atomic partial charges in solution once the cavity is created . in other words , it is the cost ( or gain ) of the reorganization of the solvent when the solute lennard - jones volume recovers its full polarity . this separation of the total free energy commands a stepwise calculation with a specific order of decoupling in simulation where the atomic partial charges need to be zeroed before decoupling the lennard - jones parameters . these free energy quantities can also be computed individually with 3d - rism using eq 2 in two steps : gnp is calculated using eq 2 from a 3d - rism / kh run with the solute atomic partial charges set to zero , and then gele is computed using eqs 2 and 4 . we can rewrite eq 2 with the definition of a free energy density function integrand for each solvent center , noted g ( ) , as follows5which simplifies the notation when the volume of integration v is partitioned by a surface within which the solvent is excluded vin and the part of space outside the exclusion domain vext6then , for the integral over vin , gkh is split into an electrostatic and a nonpolar component . we wish to correct the behavior of the 3d - rism / kh hfes for the problematic behavior of the nonpolar component only . an effective bridge function correction to the hfe may be introduced by scaling the dcfs ( c functions ) in eq 5 by a constant yet to be determined . also , the theoretical separation of the solvent water molecule densities into oxygen and hydrogen in the exclusion domain should be dominated by the repulsive core behavior ( exclusion ) of single water molecules . we can optimize the free energy functional by adjusting the parameter following a strategy employed by others with bridge functions . we note that the bridge function correction proposed here is not computed self - consistently in the integral equation but only for the free energy form itself . the solute geometries were optimized using moe and the mmff94 force field with the moe 2011.10 rfield implicit solvation model with a dielectric of 80 to account for the formation of intramolecular polar interactions . we used the c - tip3p water model that was shown to reproduce the pair correlation function of pure water in simulation . the oh distance is set to 0.9572 and the hoh angle to 104.52. the main difference with the standard tip3p water model is the addition of a hydrogen repulsive l - j term set to match the water dimer potential of the tip3p water model and the water water pair correlation functions , which eliminates the need for other approximations typically needed . the kovalenko hirata ( kh ) closure the susceptibility response function of the pure liquid was computed using the ambertools rism1d program based on the drism method . the calculation was stopped when the direct correlation function residuals reached 10 , the temperature was set to 298 k , and the water dielectric constant was set to 78.497 . the produced correlation functions were used as the input to the 3d - rism program for which the grid spacing was set to 0.4 ; the minimum distance between any atom of the solute and the boundary of the grid box was set to 10 . the improved timings come from the combined effects of a careful scan of the parameters and the efficient mdiis algorithm . a threshold based on the lennard - jones potential as opposed to the water oxygen density has the advantage of decoupling the choice of the boundary from the 3d - rism solution . as mentioned in the introduction , the calculated 3d - rism / kh hfes do not correlate well with either experimental or fep values , as clearly shown in figures 1 and 2a . even more troubling , the dynamic range of the values calculated with 3d - rism is not reasonable . when the fep - calculated hfe is partitioned into the nonpolar and electrostatic components , the 3d - rism evaluation of the electrostatic components of the hfe correlates remarkably well , as shown in figure 2c , given that no parameter is fitted . plots of g fep vs g 3d - rism , gnp fep vs gnp 3d - rism , and gele fep vs gele 3d - rism . the total 3d - rism / kh hydration free energy ( hfe ) does not correlate with the simulation based free energy perturbation hfe ( a ) . the nonpolar component of the hfe explains most of the errors ( b ) because the electrostatic component ( c ) of the hfe obtained with 3d - rism correlates well with the fep results . the nonpolar contribution explains most of the variance observed in the residuals of the calculated hfes as shown in figure 2b . it seems natural to simplify the problem to the calculation of the hfe of monatomic spherical lennard - jones ( lj ) solutes , which by definition do not have atomic partial charge and bear the simplest molecular shape one can imagine . for this purpose the optimization of the parameter in eq 8 to reproduce the calculated hfes of the lj monatomic solutes shows considerable improvement as demonstrated in figure 3a and b ( see also figure 4 ) . to verify that it is not only valid for the solute of spherical shapes , table 2 ) , the two values are statistically indistinguishable , which suggests that the optimal value of is solute - shape - invariant , at least for molecules of the size and shape of our data set . once again , the correction applied on the dcf in the solvent - excluded volume markedly improves the correspondence between the 3d - rism and the calculated fep gnp as shown by the correlation graph illustrated in figure 3a . most notably , the dynamical ranges of both fep and corrected 3d - rism match ; the correlation slope is close to one and the intercept close to zero . using our corrected nonpolar 3d - rism component and the electrostatic term leads to a corrected 3d - rism hfe in good agreement with fep . the optimization of the parameter in eq 8 improves the hydration free energy ( hfe ) correlation for a series of lennard - jones monatomic solutes as shown above , where ( a ) the initial 3d - rism formula ( c.f . the repulsive bridge correction ( rbc ) from kovalenko and hirata was shown to improve the hfe of inert gas atoms , and we computed the hfe using their thermodynamic perturbation theory ( tpt ) approximation . although we observed that the comparison with experimental results changed the r of the rbc - tpt model from 0.2 to 0.6 , the gnpr remains poor at a value of 0.16 . the fact that the corrections presented in table 2 and using either the water - excluded domain or the more empirical approach based on the partial molar volume have similar accuracy is not too surprising since the partial molar volume is related to the solvent - excluded volume given that the former represents the volume of one solute in pure water . in this study , we showed that the 3d - rism hydration free energy ( hfe ) with the kh closure formula is not accurate when used on a large data set of 504 small molecules for which experimental and double decoupling free energy perturbation ( fep ) values are available from the literature . the decomposition of the hfe into its electrostatic and nonpolar components lead us to conclude that the electrostatic component of the hfe is reasonably well reproduced without any empirical fit , i.e. we have found that scaling the nonpolar oxygen - solute ng - style direct correlation bridge function contribution to the hfe only over the solvent - excluded region using a single scaling constant brings the theoretically correct 3d - rism formula to much better agreement with the corresponding simulated fep and experimental values . this -corrected hfe functional can simultaneously reproduce the high level fep nonpolar hfes , the fep electrostatic hfes , the fep total hfes , and the experimental hfes of the set of 504 small molecules used in this study . the ability of the -corrected 3d - rism nonpolar free energy term to reproduce the fep - calculated corresponding values is remarkable , and here we compare it to recent attempts using different methods . in their work , they focus on the hfe of 11 alkanes and were able to match the fep computed hfe with an r of 0.71 and an r of 0.38 against experimental results . have developed a method based on a differential geometry approach with three free parameters that predicted the experimental hfe of 11 alkane molecules with a root - mean - square error ( rmse ) of 0.12 kcal / mol . in their article , they show that the rmse on the prediction of the experimental hfe on 19 alkanes is 0.33 kcal / mol . finally , the correction we found for the 3d - rism formulation of hydration free energy shows that it accounts accurately for most of the physical phenomena involved in the hydration free energy of small molecules . the major asset in 3d - rism is the computational expediency at which converged hydration functions can be calculated within a few seconds for small molecules .

puerto rico receives the impact of the global phenomenon known as african dust events ( ade)or storms [ 13 ] . this atmospheric formation is one of the largest in the world capable of transporting billion of tons of mineral dust from the northwest african region of sahara - sahel to the caribbean and the americas . it also contributes to dust transport into the south region of europe and to the east coast of asia . dust particles comprise a wide range of components derived from biogenic materials such as pollen , microorganisms , insects ( e.g. , locusts ) ; fossil fuel , biomass burning and mostly eroded mineral soils [ 1 , 4 , 5 ] . pm10 particle pollution is mainly derived from anthropogenic and natural sources such as ade influenced by input of warm air masses ; and traffic and industry emissions from urban environments and secondary atmospheric processes [ 68 ] . it has been reported that approximately half of the bacterial population isolated from ade particles are gram - negative containing enx , also known as lipopolysaccharide ( lps ) [ 1 , 9 ] . an estimate of ~10 bacteria is found per gram of top soil ; thus ade can mobilize high microbial masses downwind ; and given that dust episodes are common , high incidences of asthma related to lps may be encountered [ 1 , 9 ] . enx levels present in pm10 ( whole extracts ) collected throughout the atlantic ocean were reported as high as 6.41 eu / m during an ade compared to 0.50 eu / m and 0.80 eu / m before and after the respective ade . ade pm10 pollution and the high enx concentration found in particles suggests that it could very well be an important pm biological component influencing respiratory and cardiovascular diseases [ 3 , 5 , 10 , 11 ] . in fact , the united states environmental protection agency ( 2009 ) stated that there was suggestive evidence about a causal relationship between short - term coarse particle exposure and cardiovascular / respiratory / mortality effects . moreover , pm10 concentration can reach high levels ( up to 263 g / m ) during dust storm days and be associated to increased mortality compared to clear days ( up to 42 g / m ) , increased respiratory hospital admissions , and outpatient visits for bronchitis , asthma , and upper respiratory tract infections . exposures to enx along with other environmental agents such as indoor allergens , viruses , and air pollutants ( e.g. , particulate matter or pm ) have been linked to worsened lung function , atopy , enhancement of asthma pathogenesis , and/or already established inflammation [ 14 , 15 ] . these molecules have also been associated with the biological action ( e.g. , inflammatory effects ) of ambient particles in in vitro and in vivo models [ 3 , 1618 ] . the bronchial epithelium represents the first line of pulmonary defense , functioning as a physicochemical barrier to the external environment and initiating the required signals for systemic response [ 19 , 20 ] . it is capable of recognizing microorganisms and their products , secreting antimicrobial factors and other molecules involved in inflammatory responses [ 19 , 20 ] . enx can stimulate the innate immune system , causing the production of inflammatory cytokines , maturation of antigen - presenting cells , and upregulation of receptors , and even can cause influx of macrophage like cells to the alveolar space , which can enhance inflammation in the asthmatic lung [ 2123 ] . the discovery of pattern recognition receptors ( capable of sensing pathogen associated molecular patterns or pamps ) , toll - like receptors ( tlrs ) functional expression in respiratory airway epithelial cells , which are involved in allergic sensitization , underlines their importance in inflammation and the pathogenesis of respiratory disorders such as asthma and allergy [ 2428 ] . these receptors have the capacity of signaling to secrete diffusible chemotactic molecules and cytokines , upregulate surface adhesion molecules , and enhance expression of antimicrobial peptides . expression of tlr110 has been determined in primary as well as human bronchial epithelial cells , beas-2b [ 24 , 30 , 32 ] , where tlr7 , tlr8 , and tlr10 are the least expressed . tlr2 can recognize some atypical lps as long as peptidoglycans or pgn and lipopeptides derived from gram - positive bacteria and fungal molecules . tlr4 responds to lps via interaction with accessory proteins such as cd14 , a glycophosphatidyl inositol - anchored coreceptor , which can exist in soluble and membrane bound forms [ 29 , 35 ] . two signaling pathways have been described for tlr4 : the myeloid differentiation factor 88 , myd88-dependent ( also used by tlr2 heterodimerized with tlr1 and tlr6 ) , and myd88-independent . both pathways lead to the degradation of inhibitor of nf-b ( ib ) and transcription factor , nf-b translocation to the nucleus in order to induce the expression of proinflammatory and host defense genes [ 20 , 30 , 36 ] . some of the proinflammatory mediators induced by the myd88-dependent pathway are cytokines and chemokines ( e.g. , il-6 and il-8 ) [ 20 , 24 , 30 , 37 ] . il-6 can stimulate b cells and promote t cell activation , growth and differentiation and il-8 is a chemokine responsible for neutrophil chemoattraction and activation [ 29 , 3840 ] . ultimately , neutrophils induce goblet cells growth , mucus production , and alveolar wall damage . our laboratory has studied the toxic and proinflammatory effects of rural ade aqueous and organic pm10 and pm2.5 pr extracts in vitro using human bronchial epithelial cells , beas-2b [ 2 , 3 , 4143 ] . in addition , chemical characterization ( trace elements quantification ) of african dust storms ( pm2.5 ) collected in the aerosol and oceanographic science expedition ( aerose ) during march 2004 was also evaluated . other investigators have also studied the contribution of airborne particulate matter to proinflammatory outcomes in that type of cells [ 4548 ] . this study aims to study the toxicological and proinflammatory contribution of biological constituents ( enx ) present in puerto rican ade pm10 collected at a rural and urban environment in vitro . the beas-2b cell line was employed for that purpose since it is suitable and one of the models most affected by the interaction , deposition , and toxic effects of air pollution particles has an important role in surveillance being easily activated by superficial exposure to microbial factors and is the most promising model to assess respiratory sensitization of chemical compounds [ 49 , 50 ] . therefore , this work aims to characterize the pm10 carried by ade to puerto rico during 2004 . then , particular emphasis is given to the study of the in vitro proinflammatory potential of natural enx carried to the island . pm10 from a rural site is employed and the input of anthropogenic enx added from local urban sources is specially considered throughout the analysis . ade and non - ade samples ( pm10 quartz filters ) were determined based on parameters and selection criteria previously reported [ 2 , 3 ] . the pm filters were obtained from stations located at the municipalities of fajardo / rural ( reference ) and guaynabo / urban site ( figure 1 ) . briefly , pm10 data reported by the puerto rico environmental quality board in conjunction with the aerosol index satellite image records ( total ozone mapping spectrometer , toms ) for each day was used to identify ade and non - ade days at a considerable confidence level . all the dust events were classified and the mean duration through the year period of 20002012 was determined . an ade day was defined as a day characterized by a pm10 concentration above the background level of 18.3 g / m with a concurrent presence of aerosol clouds over pr ( as showed by toms ) . a day before and one to two days after the event were also considered as ade . ade input was analyzed to assess the frequency event arrival pattern to pr . in an effort to study a possible correlation between ade and respiratory health outcomes in pr , pediatric hospitalization cases due to respiratory conditions ( by means of total services offered to asthma cases ) were retrieved from the health insurance administration ( ases ) of pr and evaluated . data was meticulously revised and individuals affected by asthma were considered for one service per day , allowing for an appropriate distribution of total and age - categorized cases for statistical analyses ( see section 2.8 ahead ) . organic extracts from pm10 quartz filters classified as ade and non - ade from march 2004 were soxhlet extracted following the procedure described in rodrguez - cotto et al . . briefly , filters were submitted to a 48 hr extraction period in a 1 : 1 hexane to acetone solution ( fisher , waltham , ma ) ; the resulting extracts were dried under a gentle nitrogen stream and dissolved in dimethylsulfoxide ( dmso , sigma , st . two organic composite samples at a final concentration of 100 mg / ml were prepared as stocks from each site corresponding to the same month . a 1/1000 dilution of the organic extracts was used to determine endotoxin concentrations employing the kinetic limulus amebocyte lysate assay ( kinetic - qcl , lonza , walkersville , md ) as recommended by the manufacturer . the assay is based on the activation of a proteolytic cascade by enx in the serum of the horseshoe crab limulus polyphemus . a standard curve ( 500.005 eu / ml ) was prepared from escherichia coli o55:b5 endotoxin . absorbance readings ( 405 nm ) at 37c were recorded using an elx 808 microplate reader ( biotek instruments , winooski , vt ) and data analyzed using the winkqcl software ( lonza , walkersville , md ) . human bronchial epithelial cells ( beas-2b , atcc # crl-9609 ) were cultured and maintained with keratinocyte growth medium ( kgm-2 , walkersville , md ) at 37c , 5% co2 . cells from passages 4469 were grown in 96-well plates and exposed for 24 hr to different ade / non - ade pm10 organic ( 510 g / ml range ) extract concentrations in three independent experiments . negative controls were verified in three different ways : unexposed ( media alone ) , dilution ( vehicle : dmso used in the organic extracts ) , and process ( organic extract derived from a blank filter ) . endotoxin inhibition experiments were conducted by preincubating the extracts to 10 g / ml polymyxin b sulfate ( pmb , sigma , st . louis , mo ) in a sonic water bath for 30 min prior to cell treatment in order to prevent any aggregates and allow time for inhibition . other negative and positive controls were exposed to media preincubated with pmb , lps ( 0111:b4 , sigma , st . cell supernatants were collected after each exposure and stored at 80c until further cytokine analyses . cytotoxic effects of pm10 on exposed cells were assessed using the neutral red bioassay ( sigma , st . after exposures , media were removed and neutral red dye was added at 100 g / ml for 3 hr . following this incubation , cells were fixed with 1% calcium chloride ( sigma , st . louis , mo ) and 0.5% formaldehyde ( invitrogen , life technologies , grand island , ny ) , rinsed with phosphate buffered saline , and solubilized with 1% acetic acid ( fisher , waltham , ma ) and 50% ethanol . cell viability was determined by absorbance at 540 nm in an ultramark microplate reader ( bio - rad , hercules , ca ) . triton x-100 ( fisher , walthman , ma ) at 25 g / ml was used as a positive control for cytotoxicity . the levels of proinflammatory cytokines ( il-6 and il-8 ) in cell supernatants of three independent experiments were simultaneously determined in cell supernatants using a multiplex bead assay ( millipore ) in the dual laser flow analyzer with a luminex 200 software ( luminex corp . , basically , multiplex bead assays are solid phase immunoassays that allow simultaneous quantitative detection theoretically of up to 100 analytes in a single microtiter by flow cytometry . the median fluorescence intensities of fluorochrome - conjugated antibodies bound to individual microspheres are derived from the flow analysis of 100 microspheres per well ( in duplicate ) . cytokine standard curves were constructed using a 5-parameter logistic fit [ 2 , 3 ] . beas-2b cells were seeded in 96-well plates and exposed to ade / non - ade pm10 extracts ( fajardo and guaynabo ) with and without the enx inhibitor ( pmb ) . the mrna expression of tlrs and coreceptor cd14 were determined using quantitative real - time pcr at exposure times of 12 hr ( tlr2 ) and 7 hr ( tlr4 ) , specific time points selected based on previous time course data which revealed peak expression of the receptors . total rna was isolated using trizol reagent ( invitrogen , life technologies , grand island , ny ) and its integrity and purity were verified by gel electrophoresis . the cdna was synthesized using high capacity cdna reverse transcription kit ( applied biosystems , foster city , ca ) and quantitation of 100 g / ml samples performed using a stepone pcr instrument ( applied biosystems , foster city , ca ) based on commercial taqman assays and gene expression master mix ( applied biosystems , foster city , ca ) . the pcr program used consisted of 2 min at 50c , 10 min at 95c followed by 45 cycles of 15 s at 95c , and 1 min at 60c . ct data was analyzed employing the stepone software ( applied biosystems , foster city , ca ) and normalized to the glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) housekeeping gene . beas-2b cells were seeded in 6-well plates and exposed to ade / non - ade pm10 organic extracts ( fajardo and guaynabo ) with and without pmb ( enx inhibitor ) and incubated for 4 hr . nuclear extracts were prepared using the nuclear extract kit ( active motif , carlsbad , ca ) following the manufacturer 's guidelines . protein concentrations were determined using the bradford - based assay ( protein assay , bio - rad , hercules , ca ) by measuring absorbance at 595 nm in an ultramark microplate reader ( bio - rad , hercules , ca ) . nuclear extracts ( 5 g protein ) were evaluated in three to four independent experiments using the transam nf-b enzyme - linked immunosorbent assay ( elisa ) based kit ( active motif , carlsbad , ca ) for nf-b activation . the kit functions through the use of oligonucleotides containing the nf-b consensus binding site which are immobilized in a 96-well plate and the range of detection is 0.210 g of nuclear extract . absorbance was measured on a spectrophotometer ( bio - rad , hercules , ca ) at 450 nm . statistical significance was set at p 0.05 and results were graphed using the graphpad instat 3 software . a possible association between the pediatric asthma cases ( total and subdivided in age groups ) and three variables was tested through examining proportion differences and population homogeneity followed by a correlation analysis . the variables were ( 1 ) pm10 concentration ; ( 2 ) pm10 increment ( calculated from subtraction : average high concentration values average low concentration values ) ; and ( 3 ) ade days reported for the rural ( fajardo ) and urban ( san juan ) site during 2004 and 2005 for comparison . the analysis of pm10 concentration data for a period of 12 years in conjunction with satellite imaging indicated that the mean ade duration increased from 2001 until a peak in 2003 and 2004 , decreasing to its background levels in 2006 followed by fairly constant mean values until 2012 ( figure 2(a ) ) . when the average storm durations of all these years are analyzed in a per month basis a remarkable finding reveals that march is characterized by the longest ade ( 11.8 days ) , followed by may ( 11.4 days ) , june ( 9.3 days ) , and september ( 5 days ) . clearly , ade duration varies throughout the year with a peak in march and decreasing after june ( figure 2(b ) ) . the mean pm10 concentration for the urban site during 2004 also showed increase during the months of march and june ( figure 2(c ) ) . the asthma cases maintain a similar pattern ( urban and rural ) throughout the island for this year with the highest incidence occurring during the months of march 2004 ( figure 3(a ) ) . a reevaluation of pediatric claims for 2004 shows that march has the highest number of asthma cases with the most significant affected category being children between 0 and 5 years of age , followed by those of 612 and 1318 ( figure 3(b ) ) . the number of 05 years cases for san juan was significantly higher than those for fajardo ; however that was not observed for the 612 and 1318 age ranges ( z = 6.09 ; p value = 0.00 for 05 years cases / z = 4.83 ; p value = 1.00 for 612 years cases / z = 2.56 ; p value = 0.995 ) . a statistical analysis of the annual distribution of asthma cases during 2004 at the rural and urban sites revealed that case frequency distribution was different in all the 2004 months ( = 82.84 and 56.75 ; p value = 0.00 ) . a correlation analysis revealed a relation between total asthma cases and both pm10 concentration ( strong association with a correlation coefficient , r = 0.715 and p value = 0.009 ) and increment in the rural site , fajardo , pr ( table 1 ) . the pm10 concentration was related to asthma cases in the rural site distributed in all the 3 categories , the 05 years being the strongest one , but pm10 increment was only significantly correlated to that same category . asthma cases during ade days were also associated to the rural site ( r = 0.548 ; p value = 0.042 ) and somewhat marginally to the urban site , san juan , pr ( r = 0.470 ; p value = 0.089 ) . adjustments made by location and year of event ( 2004 versus 2005 ) did not exhibit any significant correlations ( data not shown ) . urban and rural organic extracts were nontoxic at 5 g / ml and toxic at 10 g / ml ( figure 4 ) . the positive control , lipopolysaccharide ( lps ) , was tested at 10 g / ml and was also nontoxic ( data not shown ) . a concentration of 168 eu / mg was measured in the pm10 ade sample from the urban compared to 116 in the non - ade counterpart ( table 2 ) . endotoxin concentration in the non - ade extract was comparable between the rural and urban sites but higher in the ade extract from the urban site . pmb pretreatment to the extract reduced significantly ( ~4550% ) the inherent toxicity of the urban site both derived from ade and non - ade material ( figure 5 ) . endotoxins were present in a considerable level and responsible for most of the toxicity at the urban site . urban ade extract is a strong inducer of il-6 secretion in beas-2b lung epithelial cells resulting in 200-fold higher than the non - ade extract ( figure 6(a ) ) while il-8 was around 30-fold ( figure 6(b ) ) . enx inhibition was able to significantly decrease both interleukin levels induced by the urban ade organic extract . although il-6 and il-8 were also found to increase in the non - ade extract at the urban site , the effect was significantly lower than that obtained from extracts during ade ( figure 6(b ) ) . pm10 organic extracts were capable of inducing the mrna levels of tlr2 and tlr4 at both the urban and rural sites . all ade extracts induced tlr expression with the exception of tlr4 by the rural extract ( figure 7 ) . however , the ade urban extract had a significant influence on the expression of both tlr2 and tlr4 ( figures 7(a ) and 7(b ) ) . neither of the non - ade extracts was able to induce any tlr expression ( figure 7(a ) ) . the use of pmb was not very effective in reducing tlr expression in non - ade samples at either site . conversely , the use of enx inhibitor was effective in reducing only the tlr4 expression induced by the ade extract from the urban site ( figure 7(b ) ) . the enx found in the urban ade extract have the greatest effects on tlr2 ( although not significant ) and on tlr4 ( 3-fold ) mrna inductions . cd14 mrna expression was not detected in beas-2b even in the positive control ( lps ) treated samples . cells exposed to ade / non - ade organic extracts from both urban and rural sites showed nuclear factor kappa b ( nf-b ) activation at 4 hr ( figure 8) only for the ade urban extract . the use of the endotoxin inhibitor , pmb , significantly reduced that activation to basal levels . high african dust ( based on ground pm10 concentration and aerosol images ) was reported for 2004 and specifically for the month of march and june [ 2 , 3 , 41 ] . the analysis of mean ade duration per year and monthly mean from 20002012 , compared to pm10 concentration for the urban site , undoubtedly supports the use of pm10 derived from march 2004 . this particular year was one of the few with the most extended ade and march was characterized by elevated mean ade duration during the entire period added to an increase in pm10 concentration . this is the first time that an analysis for ade duration in puerto rico has been reported . furthermore , analysis of asthmatic data also showed an increase in pediatric cases during march , but this was not the case for june , even though it was a month with high pm10 concentration . this is consistent with studies from montealegre et al . in pr showing a lower occurrence of asthma attacks during june . the difference observed between march and june could be explained by various factors such as the difference between ade occurrence , stability and composition , the length of cloud exposure days , and individual protection during a known dust season . in addition , it was found that asthma claims correlate to average pm10 concentration ( in all three age categories ) , pm10 increment , and ade days in 2004 in the rural site . the urban site extract showed a marginally significant correlation between asthma cases and ade days . these associations are in agreement with what was reported for the caribbean island of trinidad back in 2001 - 2002 . the pediatric asthma cases reported at the urban site were significantly higher compared to the rural site , suggesting that urban activities are contributing to the exacerbation of this illness . local air pollution at the urban site sets the conditions that are accentuated during the advent of african dust storms . evidence of this is stated in a case - control study conducted by loyo - berros et al . which discovered that the risk of asthma attacks in children from catao ( close to san juan ) , pr , was associated to residing in the proximity of anthropogenic air pollution sources . others have suggested the influence of high traffic and industries in risk to pediatric asthma . the high number of cases reported during the fall and winter months , characterized by reduction in ade and lower pm10 mass concentration [ 2 , 3 ] , could be attributed to other biological or chemical components such as pollen , dust mites , and fungal spores , which increase with humidity changes . endotoxin concentrations have been reported during the warmer spring and summer seasons , where bacteria and plants develop more [ 1 , 57 , 58 ] . the biological constituents of endotoxins are more prevalent and exhibit greater biological responses in the coarse pm than fine and ultrafine . the use of the enx inhibitor ( pmb ) demonstrated the importance of endotoxins on lung cell toxicity to urban pm10 organic extracts . the effect was not observed in the rural extracts , indicating that enx derived from urban local emissions are potent mediators of toxicity . moreover , endotoxins found in both extracts ( ade / non - ade ) were similarly capable of causing cytotoxicity to the level of reaching basal cell viability , indicating that the potency of enx is dependent on type rather than quantity itself , which also explains the difference between rural and organic fractions . investigators studying the effect of urban air pm have found cytotoxic effects caused by enx ( in pm10 - 2.5 fraction ) and have suggested that the inhibitory action of pmb may be slightly nonspecific but nonetheless measure of the microbial contribution to cellular responses [ 59 , 60 ] . the authors found an inhibition of cytotoxicity upon use of pmb in gram - negative but not in gram - positive bacteria ; however in the case of inflammatory responses ( which are discussed later ) a higher reduction was observed with gram - negative bacteria . endotoxins appear to affect cellular responses irrespective of the content , which could explain the similar effect obtained between ade / non - ade extracts . these molecules are one of the most important and studied pm constituents to which many respiratory health problems ( respiratory symptoms , atopic reactions , airway and systemic inflammation , and pulmonary function decline ) have been attributed [ 14 , 61 ] . asthma is thought to worsen to increased enx exposure , particularly in individuals sensitized against allergens . also , these toxins can intensify existing sensitization , eosinophilic influx into lung tissue and mucus production , among others . previous reports have acknowledged the presence of gram - negative bacteria in african dust storms , supporting the presence of enx in transatlantic material [ 1 , 9 ] . endotoxins in ade urban organic extracts were higher than in rural extracts ( 168 vs 114 eu / mg ) reported by rodrguez - cotto et al . some state that human activity increases microbial matter ( increasing aero - dispersed enx ) suspended in the atmosphere [ 6163 ] . others argue that enx levels tend to be lower in urban settings [ 14 , 64 ] . these reports suggest that enx in air will depend on the characteristics of the environment . in the present work , the toxins appear higher in the puerto rican urban environment , which supports the hypothesis that human activity modifies the environment enhancing enx enrichment . this local urban load is augmented by incoming contributions from african dust , which are prolonged during march , april , and may months . pm10 have been reported to induce the release of cytokines in beas-2b and alveolar macrophages [ 46 , 6567 ] . our study reports that ade pm10 organic extracts recovered in pr also induce the release of il-6 and il-8 , illustrating their inflammatory strength on human bronchial epithelial cells . the release of il-6 and il-8 was partial but predominantly reduced upon pmb pretreatment demonstrating the importance of enx involvement in this response . this effect has also been described after pmb pretreatment in pm10 using human alveolar and mouse macrophages [ 65 , 68 , 69 ] . in the case of human alveolar macrophages a dramatic il-6 reduction of 59% was found upon air pollution ( insoluble pm10 characterized by 4 eu / ml ) treatment with pmb ; and on the other side , mouse macrophages exposed to urban particles exhibited il-6 reduction after pmb treatment . the proinflammatory effect of endotoxins in pm10 was also previously described in alveolar epithelial cells ( a549 ) exposed to urban versus rural pm10 , where even though rural particles were more il-6 and il-8 inducers , an association was observed with higher endotoxin content in the particles . other studies have indicated that endotoxin content is not directly correlated with inflammatory response [ 59 , 60 ] , giving importance to enx types and other microbial and organic components in the extracts . the use of pmb increased the secretion of il-6 and il-8 for the non - ade urban extract instead of reducing it . this could be due to differences in enx types extracted and present during different times and locations . for example , it has been shown that lung cell responses vary depending on the e. coli serotype or bacteria species present . it is also possible that other biological agents such as peptidoglycans and fungal spores , not inactivated by pmb , may contribute to il release [ 18 , 72 ] . the enx may interact with some of those other agents , possibly preventing them to effectively induce the release of proinflammatory cytokines . upon pmb pretreatment differences in the contribution of enx to the proinflammatory response could also be explained by distinct components adsorbed to pm and the particle sources . interactions between pm constituents and enx have been proposed between , for example , enx / metals and enx / inactive fungi ( e.g. , -glucans ) [ 15 , 65 , 73 ] . enx are recognized by tlrs ( mostly tlr4 and partially tlr2 ) on the bronchial epithelial cell surface , activating intracellular signal transduction that will ultimately lead to the induction of proinflammatory genes such as il-6 and il-8 via the transcription factor , nf-b nuclear translocation . both tlrs were found to be induced by organic extracts , but the tlr4 exhibited the stronger effect in beas-2b after exposure to the urban ade extract . pretreatment with pmb was able to reduce tlr4 expression , indicating the importance of enx in positively regulating its own receptor expression . biological constituents in pm have been recognized to be important agents in tlr4/2 signaling since heat inactivation of microbial materials is able to diminish tlr expression [ 72 , 74 ] . the tlr2 was induced by both rural and urban ade extracts showing cell responsiveness to other molecular patterns ( e.g. , other bacterial products : peptidoglycans and lipopeptides ) recognized by tlr2 possibly present in ade , despite the fact that tlr2 expression is lower in this cell line . moreover , this increase in expression was not due to atypical enx as proven by the lack of response due to enx inhibition ( pmb ) . pm10 extracts are largely tlr4-dependent and activate alveolar macrophages to produce cytokines while pm2.5 tends to be tlr2-dependent and activates bronchial epithelial cells for the same outcome . reinforcing these distinct findings , expression of both tlr2 and tlr4 mrnas has been reported by others in beas-2b [ 25 , 76 ] and tlr2 expression has been found to be ligand - dependent . overall , we found that pm10 extracts induce tlr2 expression in rural and urban environments while tlr4 expression is only induced by urban dust . expression of these tlrs in bronchial epithelial cells supports the triggering of innate immunity in the respiratory epithelium . contradictory results have been shown by different studies in terms of its mrna expression ( low levels or none ) in beas-2b [ 77 , 78 ] . a cd14-independent mechanism of cell activation has been suggested as possible in bronchial epithelial cells [ 26 , 35 ] . these differences could be explained by distinct basal activation or the differentiation state of the epithelial cells employed . in addition , soluble forms of cd14 from serum plasma can mediate lps - induced cell activation in epithelial cells under low cd14 membrane expression . nf-b transcription factor is a key player for cytokine induction ; it was stimulated after 4 hrs by ade organic extracts only from the urban site . more importantly it was shown that this activation was directly related to enx since pmb was able to significantly reduce nf-b activation . the il-8 promoter contains the nf-b response element indicating that at least part of il-8 transcriptional activity is dependent on nf-b as well as il-6 . diesel exhaust particles have also been reported to activate this factor in beas-2b , which demonstrates that human activity can modify the environment activating immunological responses in humans . it appears that pm10 is able to promote inflammatory responses through nf-b . on the contrary , a previous study using pm2.5 organic extracts from the same ade source did not find any nf-b activation in beas-2b . instead this work establishes the importance of differences in bronchial epithelial responses by extracts derived from pm10 ade collected from distinct puerto rican areas ( urban / guaynabo versus rural / fajardo ) . particles from different environments differ in their potential to induce inflammatory biomarkers due to different chemical ( e.g. , organic , inorganic compounds ) , biological ( e.g. , enx ) , and physical agents ( e.g. , particle size , seasons , and emissions ) [ 66 , 82 ] . african dust storms can take 3 to 5 days before it reaches the caribbean . as the dust is transported and settles it becomes mixed with adjacent air masses that contain particular aerosols including biomass - burning products and anthropogenic compounds from various sources . local urban pm10 contains a mixture of microbes and organic and inorganic constituents , which are unique to the site and season . it was noted that african dust reaching pr increases the concentration load of enx and inorganic and other organic constituents that uniquely interact in urban environments stimulating the secretion of proinflammatory cytokines in targeted cells . by studying african dust events using satellite imaging and onsite specific pm10 concentrations it was possible to learn more about the duration and occurrence of this global storm phenomenon in puerto rico . furthermore , ade material at an urban and rural site was successfully obtained to conduct specific in vitro testing . the enx were identified as one of the unique components being incorporated in the local puerto rican environment which is biologically active through specific exposure pathways . the enx interactive complex formed in the environment associates with pm10 and strongly activates and induces tlr4 expression while weakly affecting tlr2 . this tlr interaction triggers the signal transduction pathway of nf-b leading to the release of proinflammatory il-6 and il-8 . for the first time epidemiological asthma data is directly linked to african dust storms events ( march 2004 ) in puerto rico . using data from a 12-year period , in addition , a direct correlation between children of 05 years suffering from asthma and ade in the month of march 2004 was reported . moreover , asthma cases during this period were highly prevalent and amplified at the urban site . the in vitro toxicological testing of ade material from that same month supports these findings . using the correct treatments to avoid respiratory and cardiovascular exacerbations during a pollution episode ( e.g. , ade ) the present study contributes in evaluating the effect of enx derived from african dust reaching puerto rico . it points out that adverse health outcomes depend not only on pm mass concentration but also on biological constituents as part of a complex mixture which is modulated by urban emissions . as a matter of public health , these findings greatly recommend preventive measures for the puerto rican population since asthma in puerto rico is highly prevalent and patients constantly complain of having exacerbations during ade . more importantly , this research highlights the tlrs as potential targets to consider in the future road of asthma drug therapy and even personalized medicine .

african dust events ( ade ) are a seasonal phenomenon that has been suggested to exacerbate respiratory and proinflammatory diseases in puerto rico ( pr ) . increases in pm10 concentration and the effects of biological endotoxins ( enx ) are critical factors to consider during these storms . enx promote proinflammatory responses in lungs of susceptible individuals through activation of the toll - like receptors ( tlr2/4 ) signaling pathways . the objective of the study was to evaluate the toxicological and proinflammatory responses stimulated by ade pm10 enx reaching pr using human bronchial epithelial cells . pm10 organic extracts from a rural and urban site in pr ( march 2004 ) were obtained from ade and non - ade and compared . a retrospective data analysis ( pm10 concentration , aerosol images , and pediatric asthma claims ) was performed from 2000 to 2012 with particular emphasis in 2004 to classify pm samples . urban extracts were highly toxic , proinflammatory ( il-6/il-8 secretion ) , and induced higher tlr4 expression and nf-b activation compared to rural extracts . enx were found to contribute to cytotoxicity and inflammatory responses provoked by urban ade pm10 exposure suggesting a synergistic potency of local and natural enx incoming from ade . the contribution of ade pm10 enx is valuable in order to understand interactions and action mechanisms of airborne pollutants as asthma triggers in pr .

1. Introduction 2. Methodology 3. Results 4. Discussion 5. Conclusions

puerto rico receives the impact of the global phenomenon known as african dust events ( ade)or storms [ 13 ] . this atmospheric formation is one of the largest in the world capable of transporting billion of tons of mineral dust from the northwest african region of sahara - sahel to the caribbean and the americas . pm10 particle pollution is mainly derived from anthropogenic and natural sources such as ade influenced by input of warm air masses ; and traffic and industry emissions from urban environments and secondary atmospheric processes [ 68 ] . it has been reported that approximately half of the bacterial population isolated from ade particles are gram - negative containing enx , also known as lipopolysaccharide ( lps ) [ 1 , 9 ] . enx levels present in pm10 ( whole extracts ) collected throughout the atlantic ocean were reported as high as 6.41 eu / m during an ade compared to 0.50 eu / m and 0.80 eu / m before and after the respective ade . ade pm10 pollution and the high enx concentration found in particles suggests that it could very well be an important pm biological component influencing respiratory and cardiovascular diseases [ 3 , 5 , 10 , 11 ] . moreover , pm10 concentration can reach high levels ( up to 263 g / m ) during dust storm days and be associated to increased mortality compared to clear days ( up to 42 g / m ) , increased respiratory hospital admissions , and outpatient visits for bronchitis , asthma , and upper respiratory tract infections . the discovery of pattern recognition receptors ( capable of sensing pathogen associated molecular patterns or pamps ) , toll - like receptors ( tlrs ) functional expression in respiratory airway epithelial cells , which are involved in allergic sensitization , underlines their importance in inflammation and the pathogenesis of respiratory disorders such as asthma and allergy [ 2428 ] . expression of tlr110 has been determined in primary as well as human bronchial epithelial cells , beas-2b [ 24 , 30 , 32 ] , where tlr7 , tlr8 , and tlr10 are the least expressed . two signaling pathways have been described for tlr4 : the myeloid differentiation factor 88 , myd88-dependent ( also used by tlr2 heterodimerized with tlr1 and tlr6 ) , and myd88-independent . both pathways lead to the degradation of inhibitor of nf-b ( ib ) and transcription factor , nf-b translocation to the nucleus in order to induce the expression of proinflammatory and host defense genes [ 20 , 30 , 36 ] . our laboratory has studied the toxic and proinflammatory effects of rural ade aqueous and organic pm10 and pm2.5 pr extracts in vitro using human bronchial epithelial cells , beas-2b [ 2 , 3 , 4143 ] . in addition , chemical characterization ( trace elements quantification ) of african dust storms ( pm2.5 ) collected in the aerosol and oceanographic science expedition ( aerose ) during march 2004 was also evaluated . other investigators have also studied the contribution of airborne particulate matter to proinflammatory outcomes in that type of cells [ 4548 ] . this study aims to study the toxicological and proinflammatory contribution of biological constituents ( enx ) present in puerto rican ade pm10 collected at a rural and urban environment in vitro . the beas-2b cell line was employed for that purpose since it is suitable and one of the models most affected by the interaction , deposition , and toxic effects of air pollution particles has an important role in surveillance being easily activated by superficial exposure to microbial factors and is the most promising model to assess respiratory sensitization of chemical compounds [ 49 , 50 ] . therefore , this work aims to characterize the pm10 carried by ade to puerto rico during 2004 . then , particular emphasis is given to the study of the in vitro proinflammatory potential of natural enx carried to the island . pm10 from a rural site is employed and the input of anthropogenic enx added from local urban sources is specially considered throughout the analysis . ade and non - ade samples ( pm10 quartz filters ) were determined based on parameters and selection criteria previously reported [ 2 , 3 ] . the pm filters were obtained from stations located at the municipalities of fajardo / rural ( reference ) and guaynabo / urban site ( figure 1 ) . briefly , pm10 data reported by the puerto rico environmental quality board in conjunction with the aerosol index satellite image records ( total ozone mapping spectrometer , toms ) for each day was used to identify ade and non - ade days at a considerable confidence level . all the dust events were classified and the mean duration through the year period of 20002012 was determined . an ade day was defined as a day characterized by a pm10 concentration above the background level of 18.3 g / m with a concurrent presence of aerosol clouds over pr ( as showed by toms ) . in an effort to study a possible correlation between ade and respiratory health outcomes in pr , pediatric hospitalization cases due to respiratory conditions ( by means of total services offered to asthma cases ) were retrieved from the health insurance administration ( ases ) of pr and evaluated . organic extracts from pm10 quartz filters classified as ade and non - ade from march 2004 were soxhlet extracted following the procedure described in rodrguez - cotto et al . a 1/1000 dilution of the organic extracts was used to determine endotoxin concentrations employing the kinetic limulus amebocyte lysate assay ( kinetic - qcl , lonza , walkersville , md ) as recommended by the manufacturer . the assay is based on the activation of a proteolytic cascade by enx in the serum of the horseshoe crab limulus polyphemus . human bronchial epithelial cells ( beas-2b , atcc # crl-9609 ) were cultured and maintained with keratinocyte growth medium ( kgm-2 , walkersville , md ) at 37c , 5% co2 . cells from passages 4469 were grown in 96-well plates and exposed for 24 hr to different ade / non - ade pm10 organic ( 510 g / ml range ) extract concentrations in three independent experiments . negative controls were verified in three different ways : unexposed ( media alone ) , dilution ( vehicle : dmso used in the organic extracts ) , and process ( organic extract derived from a blank filter ) . louis , mo ) in a sonic water bath for 30 min prior to cell treatment in order to prevent any aggregates and allow time for inhibition . cytotoxic effects of pm10 on exposed cells were assessed using the neutral red bioassay ( sigma , st . louis , mo ) and 0.5% formaldehyde ( invitrogen , life technologies , grand island , ny ) , rinsed with phosphate buffered saline , and solubilized with 1% acetic acid ( fisher , waltham , ma ) and 50% ethanol . beas-2b cells were seeded in 96-well plates and exposed to ade / non - ade pm10 extracts ( fajardo and guaynabo ) with and without the enx inhibitor ( pmb ) . the mrna expression of tlrs and coreceptor cd14 were determined using quantitative real - time pcr at exposure times of 12 hr ( tlr2 ) and 7 hr ( tlr4 ) , specific time points selected based on previous time course data which revealed peak expression of the receptors . beas-2b cells were seeded in 6-well plates and exposed to ade / non - ade pm10 organic extracts ( fajardo and guaynabo ) with and without pmb ( enx inhibitor ) and incubated for 4 hr . nuclear extracts ( 5 g protein ) were evaluated in three to four independent experiments using the transam nf-b enzyme - linked immunosorbent assay ( elisa ) based kit ( active motif , carlsbad , ca ) for nf-b activation . the variables were ( 1 ) pm10 concentration ; ( 2 ) pm10 increment ( calculated from subtraction : average high concentration values average low concentration values ) ; and ( 3 ) ade days reported for the rural ( fajardo ) and urban ( san juan ) site during 2004 and 2005 for comparison . the analysis of pm10 concentration data for a period of 12 years in conjunction with satellite imaging indicated that the mean ade duration increased from 2001 until a peak in 2003 and 2004 , decreasing to its background levels in 2006 followed by fairly constant mean values until 2012 ( figure 2(a ) ) . when the average storm durations of all these years are analyzed in a per month basis a remarkable finding reveals that march is characterized by the longest ade ( 11.8 days ) , followed by may ( 11.4 days ) , june ( 9.3 days ) , and september ( 5 days ) . the mean pm10 concentration for the urban site during 2004 also showed increase during the months of march and june ( figure 2(c ) ) . a statistical analysis of the annual distribution of asthma cases during 2004 at the rural and urban sites revealed that case frequency distribution was different in all the 2004 months ( = 82.84 and 56.75 ; p value = 0.00 ) . a correlation analysis revealed a relation between total asthma cases and both pm10 concentration ( strong association with a correlation coefficient , r = 0.715 and p value = 0.009 ) and increment in the rural site , fajardo , pr ( table 1 ) . the pm10 concentration was related to asthma cases in the rural site distributed in all the 3 categories , the 05 years being the strongest one , but pm10 increment was only significantly correlated to that same category . asthma cases during ade days were also associated to the rural site ( r = 0.548 ; p value = 0.042 ) and somewhat marginally to the urban site , san juan , pr ( r = 0.470 ; p value = 0.089 ) . urban and rural organic extracts were nontoxic at 5 g / ml and toxic at 10 g / ml ( figure 4 ) . a concentration of 168 eu / mg was measured in the pm10 ade sample from the urban compared to 116 in the non - ade counterpart ( table 2 ) . endotoxin concentration in the non - ade extract was comparable between the rural and urban sites but higher in the ade extract from the urban site . pmb pretreatment to the extract reduced significantly ( ~4550% ) the inherent toxicity of the urban site both derived from ade and non - ade material ( figure 5 ) . endotoxins were present in a considerable level and responsible for most of the toxicity at the urban site . urban ade extract is a strong inducer of il-6 secretion in beas-2b lung epithelial cells resulting in 200-fold higher than the non - ade extract ( figure 6(a ) ) while il-8 was around 30-fold ( figure 6(b ) ) . although il-6 and il-8 were also found to increase in the non - ade extract at the urban site , the effect was significantly lower than that obtained from extracts during ade ( figure 6(b ) ) . pm10 organic extracts were capable of inducing the mrna levels of tlr2 and tlr4 at both the urban and rural sites . neither of the non - ade extracts was able to induce any tlr expression ( figure 7(a ) ) . the use of pmb was not very effective in reducing tlr expression in non - ade samples at either site . conversely , the use of enx inhibitor was effective in reducing only the tlr4 expression induced by the ade extract from the urban site ( figure 7(b ) ) . the enx found in the urban ade extract have the greatest effects on tlr2 ( although not significant ) and on tlr4 ( 3-fold ) mrna inductions . cells exposed to ade / non - ade organic extracts from both urban and rural sites showed nuclear factor kappa b ( nf-b ) activation at 4 hr ( figure 8) only for the ade urban extract . high african dust ( based on ground pm10 concentration and aerosol images ) was reported for 2004 and specifically for the month of march and june [ 2 , 3 , 41 ] . the analysis of mean ade duration per year and monthly mean from 20002012 , compared to pm10 concentration for the urban site , undoubtedly supports the use of pm10 derived from march 2004 . this particular year was one of the few with the most extended ade and march was characterized by elevated mean ade duration during the entire period added to an increase in pm10 concentration . this is the first time that an analysis for ade duration in puerto rico has been reported . in pr showing a lower occurrence of asthma attacks during june . in addition , it was found that asthma claims correlate to average pm10 concentration ( in all three age categories ) , pm10 increment , and ade days in 2004 in the rural site . the urban site extract showed a marginally significant correlation between asthma cases and ade days . the pediatric asthma cases reported at the urban site were significantly higher compared to the rural site , suggesting that urban activities are contributing to the exacerbation of this illness . local air pollution at the urban site sets the conditions that are accentuated during the advent of african dust storms . the high number of cases reported during the fall and winter months , characterized by reduction in ade and lower pm10 mass concentration [ 2 , 3 ] , could be attributed to other biological or chemical components such as pollen , dust mites , and fungal spores , which increase with humidity changes . the use of the enx inhibitor ( pmb ) demonstrated the importance of endotoxins on lung cell toxicity to urban pm10 organic extracts . moreover , endotoxins found in both extracts ( ade / non - ade ) were similarly capable of causing cytotoxicity to the level of reaching basal cell viability , indicating that the potency of enx is dependent on type rather than quantity itself , which also explains the difference between rural and organic fractions . investigators studying the effect of urban air pm have found cytotoxic effects caused by enx ( in pm10 - 2.5 fraction ) and have suggested that the inhibitory action of pmb may be slightly nonspecific but nonetheless measure of the microbial contribution to cellular responses [ 59 , 60 ] . endotoxins appear to affect cellular responses irrespective of the content , which could explain the similar effect obtained between ade / non - ade extracts . these molecules are one of the most important and studied pm constituents to which many respiratory health problems ( respiratory symptoms , atopic reactions , airway and systemic inflammation , and pulmonary function decline ) have been attributed [ 14 , 61 ] . endotoxins in ade urban organic extracts were higher than in rural extracts ( 168 vs 114 eu / mg ) reported by rodrguez - cotto et al . this local urban load is augmented by incoming contributions from african dust , which are prolonged during march , april , and may months . our study reports that ade pm10 organic extracts recovered in pr also induce the release of il-6 and il-8 , illustrating their inflammatory strength on human bronchial epithelial cells . this effect has also been described after pmb pretreatment in pm10 using human alveolar and mouse macrophages [ 65 , 68 , 69 ] . the proinflammatory effect of endotoxins in pm10 was also previously described in alveolar epithelial cells ( a549 ) exposed to urban versus rural pm10 , where even though rural particles were more il-6 and il-8 inducers , an association was observed with higher endotoxin content in the particles . the use of pmb increased the secretion of il-6 and il-8 for the non - ade urban extract instead of reducing it . upon pmb pretreatment differences in the contribution of enx to the proinflammatory response could also be explained by distinct components adsorbed to pm and the particle sources . both tlrs were found to be induced by organic extracts , but the tlr4 exhibited the stronger effect in beas-2b after exposure to the urban ade extract . the tlr2 was induced by both rural and urban ade extracts showing cell responsiveness to other molecular patterns ( e.g. pm10 extracts are largely tlr4-dependent and activate alveolar macrophages to produce cytokines while pm2.5 tends to be tlr2-dependent and activates bronchial epithelial cells for the same outcome . reinforcing these distinct findings , expression of both tlr2 and tlr4 mrnas has been reported by others in beas-2b [ 25 , 76 ] and tlr2 expression has been found to be ligand - dependent . overall , we found that pm10 extracts induce tlr2 expression in rural and urban environments while tlr4 expression is only induced by urban dust . expression of these tlrs in bronchial epithelial cells supports the triggering of innate immunity in the respiratory epithelium . a cd14-independent mechanism of cell activation has been suggested as possible in bronchial epithelial cells [ 26 , 35 ] . these differences could be explained by distinct basal activation or the differentiation state of the epithelial cells employed . in addition , soluble forms of cd14 from serum plasma can mediate lps - induced cell activation in epithelial cells under low cd14 membrane expression . nf-b transcription factor is a key player for cytokine induction ; it was stimulated after 4 hrs by ade organic extracts only from the urban site . diesel exhaust particles have also been reported to activate this factor in beas-2b , which demonstrates that human activity can modify the environment activating immunological responses in humans . on the contrary , a previous study using pm2.5 organic extracts from the same ade source did not find any nf-b activation in beas-2b . , enx ) , and physical agents ( e.g. it was noted that african dust reaching pr increases the concentration load of enx and inorganic and other organic constituents that uniquely interact in urban environments stimulating the secretion of proinflammatory cytokines in targeted cells . by studying african dust events using satellite imaging and onsite specific pm10 concentrations it was possible to learn more about the duration and occurrence of this global storm phenomenon in puerto rico . the enx were identified as one of the unique components being incorporated in the local puerto rican environment which is biologically active through specific exposure pathways . for the first time epidemiological asthma data is directly linked to african dust storms events ( march 2004 ) in puerto rico . using data from a 12-year period , in addition , a direct correlation between children of 05 years suffering from asthma and ade in the month of march 2004 was reported . moreover , asthma cases during this period were highly prevalent and amplified at the urban site . , ade ) the present study contributes in evaluating the effect of enx derived from african dust reaching puerto rico . it points out that adverse health outcomes depend not only on pm mass concentration but also on biological constituents as part of a complex mixture which is modulated by urban emissions . as a matter of public health , these findings greatly recommend preventive measures for the puerto rican population since asthma in puerto rico is highly prevalent and patients constantly complain of having exacerbations during ade .

puerto rico receives the impact of the global phenomenon known as african dust events ( ade)or storms [ 13 ] . this atmospheric formation is one of the largest in the world capable of transporting billion of tons of mineral dust from the northwest african region of sahara - sahel to the caribbean and the americas . pm10 particle pollution is mainly derived from anthropogenic and natural sources such as ade influenced by input of warm air masses ; and traffic and industry emissions from urban environments and secondary atmospheric processes [ 68 ] . an estimate of ~10 bacteria is found per gram of top soil ; thus ade can mobilize high microbial masses downwind ; and given that dust episodes are common , high incidences of asthma related to lps may be encountered [ 1 , 9 ] . enx levels present in pm10 ( whole extracts ) collected throughout the atlantic ocean were reported as high as 6.41 eu / m during an ade compared to 0.50 eu / m and 0.80 eu / m before and after the respective ade . ade pm10 pollution and the high enx concentration found in particles suggests that it could very well be an important pm biological component influencing respiratory and cardiovascular diseases [ 3 , 5 , 10 , 11 ] . in fact , the united states environmental protection agency ( 2009 ) stated that there was suggestive evidence about a causal relationship between short - term coarse particle exposure and cardiovascular / respiratory / mortality effects . moreover , pm10 concentration can reach high levels ( up to 263 g / m ) during dust storm days and be associated to increased mortality compared to clear days ( up to 42 g / m ) , increased respiratory hospital admissions , and outpatient visits for bronchitis , asthma , and upper respiratory tract infections . , particulate matter or pm ) have been linked to worsened lung function , atopy , enhancement of asthma pathogenesis , and/or already established inflammation [ 14 , 15 ] . , inflammatory effects ) of ambient particles in in vitro and in vivo models [ 3 , 1618 ] . the bronchial epithelium represents the first line of pulmonary defense , functioning as a physicochemical barrier to the external environment and initiating the required signals for systemic response [ 19 , 20 ] . enx can stimulate the innate immune system , causing the production of inflammatory cytokines , maturation of antigen - presenting cells , and upregulation of receptors , and even can cause influx of macrophage like cells to the alveolar space , which can enhance inflammation in the asthmatic lung [ 2123 ] . the discovery of pattern recognition receptors ( capable of sensing pathogen associated molecular patterns or pamps ) , toll - like receptors ( tlrs ) functional expression in respiratory airway epithelial cells , which are involved in allergic sensitization , underlines their importance in inflammation and the pathogenesis of respiratory disorders such as asthma and allergy [ 2428 ] . these receptors have the capacity of signaling to secrete diffusible chemotactic molecules and cytokines , upregulate surface adhesion molecules , and enhance expression of antimicrobial peptides . expression of tlr110 has been determined in primary as well as human bronchial epithelial cells , beas-2b [ 24 , 30 , 32 ] , where tlr7 , tlr8 , and tlr10 are the least expressed . two signaling pathways have been described for tlr4 : the myeloid differentiation factor 88 , myd88-dependent ( also used by tlr2 heterodimerized with tlr1 and tlr6 ) , and myd88-independent . both pathways lead to the degradation of inhibitor of nf-b ( ib ) and transcription factor , nf-b translocation to the nucleus in order to induce the expression of proinflammatory and host defense genes [ 20 , 30 , 36 ] . our laboratory has studied the toxic and proinflammatory effects of rural ade aqueous and organic pm10 and pm2.5 pr extracts in vitro using human bronchial epithelial cells , beas-2b [ 2 , 3 , 4143 ] . in addition , chemical characterization ( trace elements quantification ) of african dust storms ( pm2.5 ) collected in the aerosol and oceanographic science expedition ( aerose ) during march 2004 was also evaluated . other investigators have also studied the contribution of airborne particulate matter to proinflammatory outcomes in that type of cells [ 4548 ] . this study aims to study the toxicological and proinflammatory contribution of biological constituents ( enx ) present in puerto rican ade pm10 collected at a rural and urban environment in vitro . the beas-2b cell line was employed for that purpose since it is suitable and one of the models most affected by the interaction , deposition , and toxic effects of air pollution particles has an important role in surveillance being easily activated by superficial exposure to microbial factors and is the most promising model to assess respiratory sensitization of chemical compounds [ 49 , 50 ] . therefore , this work aims to characterize the pm10 carried by ade to puerto rico during 2004 . then , particular emphasis is given to the study of the in vitro proinflammatory potential of natural enx carried to the island . briefly , pm10 data reported by the puerto rico environmental quality board in conjunction with the aerosol index satellite image records ( total ozone mapping spectrometer , toms ) for each day was used to identify ade and non - ade days at a considerable confidence level . an ade day was defined as a day characterized by a pm10 concentration above the background level of 18.3 g / m with a concurrent presence of aerosol clouds over pr ( as showed by toms ) . in an effort to study a possible correlation between ade and respiratory health outcomes in pr , pediatric hospitalization cases due to respiratory conditions ( by means of total services offered to asthma cases ) were retrieved from the health insurance administration ( ases ) of pr and evaluated . a 1/1000 dilution of the organic extracts was used to determine endotoxin concentrations employing the kinetic limulus amebocyte lysate assay ( kinetic - qcl , lonza , walkersville , md ) as recommended by the manufacturer . the assay is based on the activation of a proteolytic cascade by enx in the serum of the horseshoe crab limulus polyphemus . cells from passages 4469 were grown in 96-well plates and exposed for 24 hr to different ade / non - ade pm10 organic ( 510 g / ml range ) extract concentrations in three independent experiments . negative controls were verified in three different ways : unexposed ( media alone ) , dilution ( vehicle : dmso used in the organic extracts ) , and process ( organic extract derived from a blank filter ) . the levels of proinflammatory cytokines ( il-6 and il-8 ) in cell supernatants of three independent experiments were simultaneously determined in cell supernatants using a multiplex bead assay ( millipore ) in the dual laser flow analyzer with a luminex 200 software ( luminex corp . beas-2b cells were seeded in 96-well plates and exposed to ade / non - ade pm10 extracts ( fajardo and guaynabo ) with and without the enx inhibitor ( pmb ) . the mrna expression of tlrs and coreceptor cd14 were determined using quantitative real - time pcr at exposure times of 12 hr ( tlr2 ) and 7 hr ( tlr4 ) , specific time points selected based on previous time course data which revealed peak expression of the receptors . beas-2b cells were seeded in 6-well plates and exposed to ade / non - ade pm10 organic extracts ( fajardo and guaynabo ) with and without pmb ( enx inhibitor ) and incubated for 4 hr . nuclear extracts ( 5 g protein ) were evaluated in three to four independent experiments using the transam nf-b enzyme - linked immunosorbent assay ( elisa ) based kit ( active motif , carlsbad , ca ) for nf-b activation . the kit functions through the use of oligonucleotides containing the nf-b consensus binding site which are immobilized in a 96-well plate and the range of detection is 0.210 g of nuclear extract . a possible association between the pediatric asthma cases ( total and subdivided in age groups ) and three variables was tested through examining proportion differences and population homogeneity followed by a correlation analysis . the analysis of pm10 concentration data for a period of 12 years in conjunction with satellite imaging indicated that the mean ade duration increased from 2001 until a peak in 2003 and 2004 , decreasing to its background levels in 2006 followed by fairly constant mean values until 2012 ( figure 2(a ) ) . when the average storm durations of all these years are analyzed in a per month basis a remarkable finding reveals that march is characterized by the longest ade ( 11.8 days ) , followed by may ( 11.4 days ) , june ( 9.3 days ) , and september ( 5 days ) . clearly , ade duration varies throughout the year with a peak in march and decreasing after june ( figure 2(b ) ) . the mean pm10 concentration for the urban site during 2004 also showed increase during the months of march and june ( figure 2(c ) ) . the asthma cases maintain a similar pattern ( urban and rural ) throughout the island for this year with the highest incidence occurring during the months of march 2004 ( figure 3(a ) ) . a reevaluation of pediatric claims for 2004 shows that march has the highest number of asthma cases with the most significant affected category being children between 0 and 5 years of age , followed by those of 612 and 1318 ( figure 3(b ) ) . the number of 05 years cases for san juan was significantly higher than those for fajardo ; however that was not observed for the 612 and 1318 age ranges ( z = 6.09 ; p value = 0.00 for 05 years cases / z = 4.83 ; p value = 1.00 for 612 years cases / z = 2.56 ; p value = 0.995 ) . a statistical analysis of the annual distribution of asthma cases during 2004 at the rural and urban sites revealed that case frequency distribution was different in all the 2004 months ( = 82.84 and 56.75 ; p value = 0.00 ) . a correlation analysis revealed a relation between total asthma cases and both pm10 concentration ( strong association with a correlation coefficient , r = 0.715 and p value = 0.009 ) and increment in the rural site , fajardo , pr ( table 1 ) . the pm10 concentration was related to asthma cases in the rural site distributed in all the 3 categories , the 05 years being the strongest one , but pm10 increment was only significantly correlated to that same category . asthma cases during ade days were also associated to the rural site ( r = 0.548 ; p value = 0.042 ) and somewhat marginally to the urban site , san juan , pr ( r = 0.470 ; p value = 0.089 ) . a concentration of 168 eu / mg was measured in the pm10 ade sample from the urban compared to 116 in the non - ade counterpart ( table 2 ) . endotoxin concentration in the non - ade extract was comparable between the rural and urban sites but higher in the ade extract from the urban site . pmb pretreatment to the extract reduced significantly ( ~4550% ) the inherent toxicity of the urban site both derived from ade and non - ade material ( figure 5 ) . urban ade extract is a strong inducer of il-6 secretion in beas-2b lung epithelial cells resulting in 200-fold higher than the non - ade extract ( figure 6(a ) ) while il-8 was around 30-fold ( figure 6(b ) ) . although il-6 and il-8 were also found to increase in the non - ade extract at the urban site , the effect was significantly lower than that obtained from extracts during ade ( figure 6(b ) ) . pm10 organic extracts were capable of inducing the mrna levels of tlr2 and tlr4 at both the urban and rural sites . however , the ade urban extract had a significant influence on the expression of both tlr2 and tlr4 ( figures 7(a ) and 7(b ) ) . conversely , the use of enx inhibitor was effective in reducing only the tlr4 expression induced by the ade extract from the urban site ( figure 7(b ) ) . the enx found in the urban ade extract have the greatest effects on tlr2 ( although not significant ) and on tlr4 ( 3-fold ) mrna inductions . cells exposed to ade / non - ade organic extracts from both urban and rural sites showed nuclear factor kappa b ( nf-b ) activation at 4 hr ( figure 8) only for the ade urban extract . high african dust ( based on ground pm10 concentration and aerosol images ) was reported for 2004 and specifically for the month of march and june [ 2 , 3 , 41 ] . the analysis of mean ade duration per year and monthly mean from 20002012 , compared to pm10 concentration for the urban site , undoubtedly supports the use of pm10 derived from march 2004 . this particular year was one of the few with the most extended ade and march was characterized by elevated mean ade duration during the entire period added to an increase in pm10 concentration . the difference observed between march and june could be explained by various factors such as the difference between ade occurrence , stability and composition , the length of cloud exposure days , and individual protection during a known dust season . in addition , it was found that asthma claims correlate to average pm10 concentration ( in all three age categories ) , pm10 increment , and ade days in 2004 in the rural site . the pediatric asthma cases reported at the urban site were significantly higher compared to the rural site , suggesting that urban activities are contributing to the exacerbation of this illness . local air pollution at the urban site sets the conditions that are accentuated during the advent of african dust storms . which discovered that the risk of asthma attacks in children from catao ( close to san juan ) , pr , was associated to residing in the proximity of anthropogenic air pollution sources . the high number of cases reported during the fall and winter months , characterized by reduction in ade and lower pm10 mass concentration [ 2 , 3 ] , could be attributed to other biological or chemical components such as pollen , dust mites , and fungal spores , which increase with humidity changes . the use of the enx inhibitor ( pmb ) demonstrated the importance of endotoxins on lung cell toxicity to urban pm10 organic extracts . moreover , endotoxins found in both extracts ( ade / non - ade ) were similarly capable of causing cytotoxicity to the level of reaching basal cell viability , indicating that the potency of enx is dependent on type rather than quantity itself , which also explains the difference between rural and organic fractions . investigators studying the effect of urban air pm have found cytotoxic effects caused by enx ( in pm10 - 2.5 fraction ) and have suggested that the inhibitory action of pmb may be slightly nonspecific but nonetheless measure of the microbial contribution to cellular responses [ 59 , 60 ] . the authors found an inhibition of cytotoxicity upon use of pmb in gram - negative but not in gram - positive bacteria ; however in the case of inflammatory responses ( which are discussed later ) a higher reduction was observed with gram - negative bacteria . endotoxins appear to affect cellular responses irrespective of the content , which could explain the similar effect obtained between ade / non - ade extracts . these molecules are one of the most important and studied pm constituents to which many respiratory health problems ( respiratory symptoms , atopic reactions , airway and systemic inflammation , and pulmonary function decline ) have been attributed [ 14 , 61 ] . previous reports have acknowledged the presence of gram - negative bacteria in african dust storms , supporting the presence of enx in transatlantic material [ 1 , 9 ] . endotoxins in ade urban organic extracts were higher than in rural extracts ( 168 vs 114 eu / mg ) reported by rodrguez - cotto et al . in the present work , the toxins appear higher in the puerto rican urban environment , which supports the hypothesis that human activity modifies the environment enhancing enx enrichment . our study reports that ade pm10 organic extracts recovered in pr also induce the release of il-6 and il-8 , illustrating their inflammatory strength on human bronchial epithelial cells . in the case of human alveolar macrophages a dramatic il-6 reduction of 59% was found upon air pollution ( insoluble pm10 characterized by 4 eu / ml ) treatment with pmb ; and on the other side , mouse macrophages exposed to urban particles exhibited il-6 reduction after pmb treatment . the proinflammatory effect of endotoxins in pm10 was also previously described in alveolar epithelial cells ( a549 ) exposed to urban versus rural pm10 , where even though rural particles were more il-6 and il-8 inducers , an association was observed with higher endotoxin content in the particles . the use of pmb increased the secretion of il-6 and il-8 for the non - ade urban extract instead of reducing it . upon pmb pretreatment differences in the contribution of enx to the proinflammatory response could also be explained by distinct components adsorbed to pm and the particle sources . enx are recognized by tlrs ( mostly tlr4 and partially tlr2 ) on the bronchial epithelial cell surface , activating intracellular signal transduction that will ultimately lead to the induction of proinflammatory genes such as il-6 and il-8 via the transcription factor , nf-b nuclear translocation . both tlrs were found to be induced by organic extracts , but the tlr4 exhibited the stronger effect in beas-2b after exposure to the urban ade extract . biological constituents in pm have been recognized to be important agents in tlr4/2 signaling since heat inactivation of microbial materials is able to diminish tlr expression [ 72 , 74 ] . reinforcing these distinct findings , expression of both tlr2 and tlr4 mrnas has been reported by others in beas-2b [ 25 , 76 ] and tlr2 expression has been found to be ligand - dependent . expression of these tlrs in bronchial epithelial cells supports the triggering of innate immunity in the respiratory epithelium . instead this work establishes the importance of differences in bronchial epithelial responses by extracts derived from pm10 ade collected from distinct puerto rican areas ( urban / guaynabo versus rural / fajardo ) . it was noted that african dust reaching pr increases the concentration load of enx and inorganic and other organic constituents that uniquely interact in urban environments stimulating the secretion of proinflammatory cytokines in targeted cells . by studying african dust events using satellite imaging and onsite specific pm10 concentrations it was possible to learn more about the duration and occurrence of this global storm phenomenon in puerto rico . , ade ) the present study contributes in evaluating the effect of enx derived from african dust reaching puerto rico . as a matter of public health , these findings greatly recommend preventive measures for the puerto rican population since asthma in puerto rico is highly prevalent and patients constantly complain of having exacerbations during ade .

the international continence society defines overactive bladder ( oab ) as : urgency , with or without urge incontinence , usually with frequency and nocturia in the absence of infection or any other proven causative pathology.1 thus the oab population includes both patients with urge incontinence ( oab wet ) as well as without urge incontinence ( oab dry ) . indeed , the incidence of oab dry has been reported at 66% of oab patients while 33% have oab wet and suffer from leakage.2 with prevalence rates similar to those of asthma and chronic bronchitis , the burden of this disease is enormous and presents great cost to society.3 the national overactive bladder evaluation ( noble ) program was a large scale , well designed epidemiological study which reported the prevalence of oab in the us at approximately 16% with the distribution between men and women being roughly equal and with the prevalence mounting with increasing age.3 similar results were seen in epidemiological studies in europe with prevalence rates of oab in the 12% to 17% range.4 the pathophysiology of oab is not completely understood and likely there are multiple pathways leading to the end disease state . normal bladder storage and micturition function requires precise neurologic and muscular coordination ; possible alterations at the myogenic , mucosal and neurogenic levels may contribute to the condition . while causative myogenic and mucosal alterations would take place at the end organ ( bladder and urethra ) , adverse changes in bladder innervations in such neurogenic theories , alterations in the neurologic pathways that normally provide tonic inhibition of detrusor contractions may account for the disease state.5 alternatively , sensory nerve endings in the bladder may become hypersensitive , resulting in detrusor overactivity.5 indeed , research studying the role of the urothelium in normal and abnormal micturitional states has advanced dramatically and shaped the field of oab . it has long been understood that the autonomic nervous system plays a critical role in micturition . the sacral parasympathetic outflow provides excitatory input to the bladder via the pelvic nerve , which results in detrusor contraction . bladder ganglia cells excite detrusor smooth muscle with release of both cholinergic neurotransmitter in the form of acetylcholine and also non - cholinergic , non - adrenergic neurotransmitters.5 acetylcholine activates muscarinic receptors on the detrusor smooth muscle and is considered a key neurotransmitter for detrusor function . there are 5 distinct subtypes of the muscarinic receptor throughout the body ( m1m5 ) , the m2 and m3 sub - types predominating in the bladder . although the density of m2 receptor is higher than the m3 receptor in the human bladder , m3 is thought to be more important in the manifestation of detrusor contractions.6 given this basic understanding of neuroanatomy , it is not surprising that muscarinic cholinergic receptors became early therapeutic targets in oab treatment . oxybutynin immediate release ( ir ) represents the first such medication approved by the fda specifically for the treatment of oab in 1975.7 it was originally investigated in the 1960s for the potential treatment of gastrointestinal hypermotility . researchers incidentally discovered its usefulness as an agent for oab and it has been since widely used for decades . in addition to its anticholinergics effects oxybutynin also shows some in vitro activity as a direct smooth muscle relaxant8 and early in vitro studies on rabbit detrusor also noted moderate local anesthetic action.9 clinical efficacy for treatment of oab symptoms was well documented in the 1990s.10 despite proven efficacy , oxybutynin ir shows some major shortcomings as a therapeutic drug , namely high side effects , some so bothersome that some patients prefer to discontinue treatment and endure the disease itself rather than deal with the side effects . lawrence et al showed a 6-month treatment adherence rate of only 32% in a retrospective analysis 515 patients prescribed oxybutynin ir in a pharmacy claims database.11 these pitfalls stem from fact that oxybutynin has a relatively nonspecific affinity for all of the various muscarinic receptors which are widespread throughout the human body . in addition to muscarinic receptors in the bladder , oxybutynin has particular affinity for muscarinic receptors in the central nervous system ( cns ) , gastrointestinal ( gi ) tract , parotid gland and eye which may account for troublesome cognitive impairment , constipation , dry mouth and difficulty with papillary accommodation . of these , dry mouth appears to be most prominent , occurring in up to 80% of patients taking oxybutynin.12 beyond bother , decreased salivation may predispose to oral infections and dental caries as well as inhibit proper mastication and swallowing , particularly in the elderly.13,14 given the limitations of oxybutynin ir , it comes as no surprise that alternative agents would be explored in the hopes that tolerability could be improved while maintaining efficacy in the treatment of oab . other antimuscarinics such as propantheline , methantheline , emepronium , dicyclomine , and terodiline were once employed as alternative oab treatment options as they were perceived to be more tolerable , however they were eventually abandoned due to either lack of efficacy and/or poor tolerability.15,16 currently , there are five different antimuscarinic drug formulations on the market for treatment of oab in addition to oxybutynin . tolterodine , propiverine , trospium , darifenacin and solifenacin all possess differing pharmacokinetic properties relating to organ and receptor subtype selectivities as well as structural differences . in addition , a separate strategy for changing the side effect profile of oxybutynin was employed by using an extended release ( er ) formulation . in addition , a patch formulation of oxybutynin was developed in hopes that tolerability could be improved through of use of a different delivery system which altered drug metabolism and lengthened the drug dosing interval . an oxybutynin gel formulation was recently introduced into the us market with similar aims . in this article we focus our attention on oxybutynin er formulation likewise meant to simplify drug dosing and improve on the side effect profile of the older oxybutynin ir . oxybutynin er came to market in the united states in 199817 with intent to improve upon tolerability , convenience and compliance while maintaining efficacy by offering once daily dosing . oros oxybutynin chloride ( oxybutynin er , ditropan xl ) is an extended - release oral tablet formulation ( oros ) which utilizes osmotic pressure to deliver the drug at a controlled rate over 24 hours . the tablet itself consists of a semi - permeable membrane covering a drug layer which overlies an osmotically active push layer . in the gi tract , the absorption of water by this osmotically active layer results in the expulsion of the drug ( which has likewise absorbed water forming a suspension ) out a small opening in the membrane . pharmacokinetic studies show that after the first dose of oxybutynin er , plasma drug concentration rises for 6 hours and then maintains a constant concentration for up to 24 hours with steady state plasma concentrations achieved by day three of treatment.18 normally , when absorbed in the upper gi tract , oxybutynin ir undergoes first pass metabolism by the cytochrome p450 system into its active metabolite n - desethyloxybutynin ( ndo ) . this may be particularly important as ndo has been proposed to be responsible for more dry mouth than oxybutynin itself.19 however , because the oros system delays the release of oxybutynin in the er formulation , it is thought that the parent drug is not biologically available until the tablet reaches the large intestine . by this point , the drug has bypassed the upper gi s first pass metabolism system and is left to be absorbed in the colon where conversion to ndo is less likely and absorption is mainly of the parent drug itself . biologically inert components of the tablet including the tablet itself are ultimately eliminated in the feces . the drug did show at least 40% greater decrease in urinary urge incontinence versus placebo in a 6-week trial though the results are recorded in abstract form only.20 in an early industry sponsored study anderson et al for the ditropan study group enrolled 105 patients with urge incontinence into a randomized , double blind study of dose titration to compare the efficacy and safety of controlled and immediate release oxybutynin . they concluded that both groups shared a similar rate of reductions in urge incontinence and total incontinence episodes while the oxybutynin er group reported significantly less dry mouth at 68% compared to 87% for any degree of complaint.21 another study published in the year 2000 sought to compare the efficacy and safety of oxybutynin ir as well as to determine rates of dry mouth . the authors enrolled known anticholinergic responders with seven or more urge incontinence episodes per week into a dose escalation trial of the ir and er drug forms starting at 5 mg per day and increasing to 20 mg per day as limited by intolerable side effects . they likewise found comparable rates of reduction in incontinence episodes with similar rates of dose - dependent dry mouth , but moderate to severe dry mouth was significantly less prominent in the group receiving oxybutynin er . stratified by dosage , the greatest differences were at the 10 mg per day ( 8.5% for the er group vs 25.6% for the ir group ) and 15 mg per day doses ( 19.4% for the er group vs 38.9% for the ir group ) groups.22 nevertheless , the early data on direct comparisons between oxybutynin ir and er remain limited by trial design shortcomings including studying enriched populations such as known responders and reporting results on a completer basis . some are reported only in abstract form at meetings and many are industry sponsored , thus limiting the ability to make evidence - ased conclusions of the two drug formulations . in 2005 individualized doses of oxybutynin er for urinary incontinence.23 his group combined data from three flexible - dosing studies of patients with urge urinary incontinence or mixed incontinence ; two of the data sets used were from trials completed in the late 1990s on er versus ir oxybutynin while the third was from a study published in 1999 by the ditropan xl study group on evaluation of a new once - daily formulation of oxybutynin for the treatment of urge urinary incontinence.19,20,24 a total of 420 patients combined were assigned to treatment with oxybutynin er . in the combined dataset , 420 patients were assigned to treatment with oxybutynin er . treatment began with 5 mg daily of oxybutynin er and was increased in 5-mg increments at intervals of approximately 1 week until the patient either achieved complete continence or experienced intolerable side effects . the patients remained on maxiumum dose for 2 to 12 weeks according to which study they were participating in . of these patients , some 14% had withdrawn prior to completion of their respective trial ( some 6.7% due to adverse events ) , leaving 368 on drug at the end of the study periods . from the pooled data , 40% of these patients achieved total dryness with 80% sustaining 70% or better improvement in incontinence at their respective doses . dry mouth was the most prevalent adverse event , but some of the studied patients experienced only mild or no dry mouth at all . the authors made note of the fact that 7% of the patients selected the dose of 30 mg per day with only slightly higher rates of dry mouth and similar rates of treatment discontinuation due to adverse events as subjects at lower daily doses . the authors freely acknowledge the limitations of post - hoc analysis of pooled data from studies that employed varying methodologies . in addition they recognize that the three studies each contained some of the previously discussed study design flaws common to early studies of oxybutynin er versus ir , further weakening conclusions drawn in this paper.21 the next round of clinical trials involving oxybutynin mainly focused on comparison studies to other oab medications . two large , multi - center , prospective , randomized clinical trials comparing oxybutynin er and tolterodine tartrate in oab patients stand out for their sound methodology and power relative to all other clinical trials employing oxybutynin er : the overactive bladder : judging effective control and treatment ( object ) study25 and the overactive bladder : performance of extended release agents ( opera ) trial.26 the object study was conducted between march and october 2000 at 37 us study sites with the objective to compare the efficacy and tolerability of fixed dose extended - release oxybutynin and tolterodine tartrate at 12 weeks . the study enrolled and randomized 315 women and 63 men with overactive bladder who received either oxybutynin er 10 mg daily or tolterodine tartrate 2 mg q 12 hours in a double - blind , double - dummy fashion . trial completion was accomplished by 276 women and 56 men while a total 46 discontinued early , 29 because of adverse events ( 14 in the oxybutynin er and 15 in the tolterodine group ) . efficacy was assessed using self ( diary ) reported incontinence episodes as a primary outcome measure . the diaries documented number of voids as well as number and nature of incontinence episodes . study findings included similar tolerability and discontinuation rates as well as similar adverse event rates for both drugs . in the object study , oxybutynin er proved significantly more effective than tolterodine in reducing the mean number of weekly urge incontinence episodes at end of study as well as total incontinence episodes and total urinary frequency . urge incontinence episodes improved from 25.6 14.7 to 6.1 9.7 for oxybutynin er and 24.1 14.5 to 7.8 11.1 for tolterodine . total number of incontinence episodes per week decreased from 28.6 17.9 to 7.1 12 for oxybutynin er and from 27 17 to 9.3 13.4 for tolterodine . micturitions per week decreased from 91.8 22.6 to 67.1 22.1 for oxybutynin er and from 91.6 20.2 to 71.5 20.5 for tolterodine . both drugs significantly improved all studied outcome parameters . the authors made note of the limited applicability of 12 week results with regards to a chronic condition such as oab . they also noted that the study population was selected by specialists rather than primary care physicians who treat most oab and that study population was not racially diverse , though this is a common limitation in most oab studies . it should be noted that object compared once daily oxybutynin er to twice daily tolterodine tartrate , though this formulation of tolterodine has essentially been replaced in most cases by the once daily formulation of tolterodine which is expected to improve patient compliance . regardless , object did compare oxybutynin er with the most widely prescribed non - generic anticholinergic medication at that time in a blinded head to head randomized clinical trial in the us.23 it must be noted that the object study was funded by alza corporation in mountain view , california , a bio - pharmaceutical company with specialization in drug delivery systems and developer of the oros drug delivery system . the authors include advisors , investigators and speakers for alza corporation as well as one employee stockholder of alza corporation . the second large multicenter trial , opera , was likewise funded by the alza corporation as well as ortho - mcneil pharmaceutical in raritan , nj , makers of oxybutynin er . opera authors likewise disclosed relationships including : medical consultant , investigator , advisor as well as an employee and stockholder of ortho - mcneil and an employee and stockholder of azla . the opera trial sought to compare the efficacy and tolerability of extended - release formulations of both oxybutynin chloride and tolterodine tartrate in women with overactive bladder . the trial ran from november 2000 to october 2001 and pitted oxybutynin er head to head with the most prescribed once daily anticholinergic in a prospective , randomized , double - blind study conducted at 71 us centers . us women aged 18 years or older who documented 21 to 60 episodes of urinary urge incontinence per week and an average of 10 or more voids per 24 hours were recruited . a total of 790 women were randomized to receive either 10 mg of oxybutynin er or 4 mg of tolterodine er per day . randomized groups were similar in both demographics and severity of oab while prior treatment for oab was not an exclusion criteria . patients kept 24-hour voiding diaries at baseline and during weeks 2 , 4 , 8 and 12 of treatment . outcome measures were reduction from baseline in the number of urinary urge incontinent episodes / week , weekly total incontinence episodes and weekly micturition frequency numbers . a similar number of patients completed the study in both groups ( 87% for the oxybutynin er group versus 89% for the tolterodine er group . ) likewise , rates of discontinuation due to adverse events were essentially equal with 20 oxybutynin patients and 19 tolterodine er patients . there was no statistical difference in the number of weekly urge incontinent episodes at the end of study for the competing groups . after 12 weeks of treatment , the oxybutynin er group saw a reduction from a mean of 37.1 to 10.8 urge incontinent episodes per week while the tolterodine er group dropped from 36.7 to 11.2 . the oxybutynin er group performed slightly for reducing total mean number of weekly incontinent episodes and weekly micturition frequency . for total incontinent episodes per week , the oxybutynin group went from 43.4 to 12.3 and the tolterodine group from 42.4 to 13.8 . total decrease in mean number of voids per week was 28.4 for the oxybutynin group versus 25.2 for the tolterodine group ; respective means were 66.4 and 71.7 at the end of 12 weeks treatment . slightly more patients in the oxybutynin er group reported no incontinence episodes in their final diary versus the tolterodine er group at 23% versus 16.8% completely dry . dry mouth was more common in the oxybutynin er group at 29.7% versus 22.3% in the tolterodine group ; while statistically significant , the majority of the dry mouth effects were categorized as mild ( 93% of oxybutynin er participants and 95% of tolterodine er participants.24 overall , the opera study shows oxybutynin er to have modestly greater efficacy than er tolterodine at its most commonly prescribed dose . the authors point out that the flexible dosing allowed for oxybutynin provides a wider range of fda approved dosages allowing individuals to balance their respective efficacy and tolerability without turning to off label dosages as would be required with tolterodine er of 4 mg daily . one obvious limitation of the opera study is its study population of women only . while women do constitute the majority of patients presenting with complaints of overactive bladder , the condition is certainly not limited to women . some epidemiological studies would argue that the prevalence in men is nearly equal.3 in 2006 , anderson et al published a post hoc analysis of data from the opera trial specifically comparing the efficacy and tolerability of oxybutynin er and tolterodine er in women with or without prior anticholinergic treatment for oab . they found that oxybutynin er was significantly more effective in reducing micturition frequency by week 12 among the population of patients with prior anti - cholinergic treatment experience . likewise in this particular subgroup , the participants taking oxybutynin er reported a statistically significant greater chance of being incontinence free and they had a greater reduction in number of urge incontinence episodes . in contrast , the anticholinergic - naive group showed statistically equal efficacy for all study parameters with the exception of average number of micturitions per week at study completion ; this parameter favored the participants taking oxybutynin er . dry mouth was more common in the oxybutynin er study participants , but only in the group with history of prior anticholinergic treatment.27 several clinical trials address the use of oxybutynin er in special populations , including the elderly , children , and neurogenic bladder . interestingly , while the concept that oab may contribute to male lower urinary tract symptoms has become more widely accepted28 we were unable to identify any studies in the literature focusing on oxybutynin er in the male subpopulation . oxybutynin er is considered : pregnancy catetory b , reproduction studies in the mouse , rat , hamster and rabbit showing no definite evidence of impaired fertility or harm to animal fetus . according to the manufacturer s package insert : the safety of ditropan xl administration to women who are or may become pregnant has not been established . therefore ditropan xl should not be given to pregnant women unless , in the judgement of the physician , the probable clinical benefits outweigh the possible hazards . it is unlikely that studies of oxybutynin in pregnant women will ever be undertaken . the elderly represent a special population in oab treatment particularly related to risk of negative effects on the cns such as cognitive impairment and memory problems . they tend to be at risk for poly - pharmacy , often taking a variety of medicines besides anticholinergics which may also have adverse effects on the cns . as the number of medications use increases , so does the risk to the older patient including problems with compliance as well as potential drug - drug interactions which can cause adverse events or alter drug efficacy.29 older patients are at risk for falls and sleep disturbances , and they often already suffer from dry mouth , dry eye , and vision problems as well as gi motility problems including constipation.30 kay et al studied the differential effects oxybutynin er versus the drug darifenacin on memory in older patients . in a multicenter , double blind study , 150 healthy individuals over the age of 60 with oab were randomized to receive oxybutynin er , darifenacin or placebo . initial dose for oxybutynin er was 10 mg daily , and increases in 5-mg increments was permitted up to 20 mg by the end of this 3-week study . initial dose for darifenacin was 7.5 mg each day for 2 weeks , with permitted increase to 15 mg each day for the final week . primary outcome was the effect of each antimuscarinic versus placebo at 3 weeks on recent memory as measured by the delayed recall name - face association test . secondary end points included delayed recall on the first - last name association test and the misplaced objects test . results of this study showed that oxybutynin er caused significant memory deterioration , while darifenacin did not cause memory changes significantly different from placebo . while oxybutynin er may be fda approved to 30 mg daily dose , the most commonly prescribed dosage is 10 mg daily which is the same dose selected for object and opera trials . despite this potential dosage mismatch , interval evaluation of study subjects does show a drop in measured cognitive function in the oxybutynin er group after one week even at 10 mg daily . despite the short duration of the study , it does show that older patients with pharmacological steady state plasma levels of oxybutynin er may have an increased risk of impaired cognitive function relative to those in steady state taking darifenacin.31 contrary to the relatively high dose oxybutynin er used in previous study , lackner reported a randomized , placebo - controlled trial of the cognitive effect , safety and tolerability of 5 mg daily dosage of oxybutynin er in a population of cognitively impaired nursing home residents with urge incontinence . the authors cite that oab is undertreated in this population due to the fear that the frail older patient is more susceptible to anticholinergic associated adverse reactions . they note the lack of studies to document safety and efficacy of anticholinergics in the frail elderly population despite the high prevalence of urinary urge incontinence in particular . in this trial , 50 women aged 65 and older with urge incontinence and documented cognitive impairment were randomized to oxybutynin er versus placebo and followed closely for 4 weeks . primary outcome measures included the confusion assessment method ( cam , ) an accepted , validated and widely used diagnostic algorithm to measure cognitive function . changes in cam score from baseline were the primary outcome with presence or absence of delirium as determined by the cam . other secondary endpoints were further standardized measurements of cognition . in short , the study showed that oxybutynin er 5 mg daily was well tolerated in elderly female nursing home patients with mild to severe dementia . there was no significant impairment in cognitive function in the treatment group by any measured outcome at any measured point in the study . there were no more withdrawals in the treatment versus placebo groups nor was there any difference in adverse events . rates of agitation and falls were equal with 54% of the participants in both groups experiencing at least one fall during the months preceding , including and following the trial . study limitations include the short duration , homogenous caucasian female population as well as lack of information about efficacy at this dosage.32 however , these results do contradict the guidelines of the american neurological association , the american psychiatric association and the beers criteria for potentially inappropriate medication use in older adults33 and conclusions about their use in this population remain guarded . cns side effects do represent a cause of potential severe consequences in the elderly in particular . current us prescribing information applying to all forms of oxybutynin states under precautions : a variety of cns effects have been reported including hallucinations , agitation , confusion and somnolence . patients should be monitored for signs of anticholinergic cns effects , particularly in the first few months after beginning treatment or increasing the dose . it also cautions that the drug should be used with caution in patients with pre - existing dementia treated with cholin - esterase inhibitors due to risk of aggravation of symptoms and that the drug should be used with caution in the frail elderly . children represent another highly unique special population to consider when considering drug therapy . drug treatment of neurologically intact children with oab type symptoms generally focuses on incontinence , which can , in older children have marked effects on the child s social and psychological development and well being in addition to causing great stress and anxiety in parents . beyond behavioral therapy and biofeedback , clinicians , parents and patients often wish to turn to drug therapy to treat oab . while studies on oab drugs in adults are limited and conflicting , only one non - randomized clinical study was identified investigating therapeutic efficacy and tolerability of oxybutynin er in children . the authors identified 86 girls and 46 boys between the ages of 5 and 18 years of age who were referred to a pediatric urology clinic with diurnal urinary incontinence and oab symptoms including urinary frequency , urgency , posturing associated with urge and/or spontaneous urinary incontinence . these children were arbitrarily assigned to receive tolterodine ir , tolterodine er or oxybutynin er based primarily on the formulary restrictions of their health care plans . medications were started at the lowest dose available in non - liquid form ( tolterodine ir and er at 2 mg , oxybutynin er 2 mg ) and titrated according to response and tolerability . after thorough baseline evaluations , study nurses assessed efficacy by voiding diary and side effects on the basis of direct questioning to parents and patients . validated instruments were not used and it appears that assessments did not occur at set , prescribed points in time . in 2003 the authors reported their retrospective analysis citing that all medications appeared well tolerated and that the er formulations of both oxybutynin and tolterodine were significantly more effective than ir tolterodine . oxybutynin er was again significantly more effective than either formulation of tolterodine for control of daytime urinary incontinence and micturition frequency.34 while this non - randomized , non - blinded retrospective study using non - validated instruments contains serious flaws , it nevertheless represents the very small amount of literature devoted to use of oxybutynin er in children and suggests that the use of this type of medication is safe and effective in children and that oxybutynin er is at least as effective as either formulation of tolterodine . one other study focused on the use of oxybutynin er in children with neurogenic bladder ( ngb ) . this is a distinct and important subset of patients who frequently require aggressive management of bladder issues to maximize social functionality as well as to protect renal function . while published studies as early as 1977 address the use of oxybutynin ir in this population , very little is available regarding oxybutynin er in children with ngb.35 interestingly , no studies were identified which reported specifically on use of oxybutynin er in adults with neurogenic bladder . franco et al reported on use of three different formulations of oxybutynin in children aged 615 years of age who performed clean intermittent catheterization ( cic ) and who had documented neurogenic detrusor overactivity at 24 different centers in the us and europe . sixty one patients received oxybutynin er while 28 received oxybutynin ir and 30 were treated with oxybutynin syrup . improvement of urodynamic parameters was seen in all treatment arms as well as increased continence . likewise all three drug formulations were well tolerated and no patient discontinued treatment due to adverse events related to oxybutynin . in contrast to other studies , constipation represented the most frequently reported adverse event in this population while dry mouth is not even listed as occurring at 5%.36 despite flaws , this report does provide some evidence for tolerability of oxybutynin er in this small but important patient population . first , one must understand the staggering financial cost of oab in modern society ; estimates of dollars spent annually in the us alone run to us$12 billion.37 while non - pharmacological treatment methods may be employed , anticholinergic medications represent the mainstay of treatment for oab and drug costs contribute considerably to the overall burden of cost related to oab . however , some studies do indicate that drug therapy for oab may be associated with decreased utilization of other healthcare resources resulting in cost savings elsewhere;38 naturally , the cost of therapeutic medication for oab may offset these net savings . considerable literature in the managed care and pharmacoeconomics literature is devoted to these issues including evaluations of oxybutynin er as a therapeutic choice for oab . the economic modeling is quite complex and unique in each case due to the regional or geographic variation with myriads of variables affecting cost of treatment and cost of disease state . one european study sought to study the cost - effectiveness of long - term pharmacological management of urge urinary incontinence using ir oxybutynin and tolterodine as well as er formulations of the two drugs in the united kingdom . the study included estimated costs of incontinence products such as pads which are often paid out of pocket in the us and often overlooked . they concluded that tolterodine ir was less cost effective than the other three drug choices.39 in contrast , two large us retrospective studies designed to determine financial cost and benefit of treatment with er formulations of oxybutynin versus tolterodine estimated healthcare costs based on reimbursement for services ( hence only covered benefits ) by using enormous patient databases related to us health insurers.40,41 both studies found cost savings in total reimbursement costs over one year for patients treated with tolterodine er versus oxybutynin er . antimuscarinic agents remain a mainstay of therapy for the treatment of oab . while this medication class has proven efficacious for this condition , decreased tolerability due to antimuscarinic side effects oxybutynin has proven to be effective for the treatment of oab however antimuscarinic side effects such as dry mouth and constipation often limit patients compliance with it . the use of the oros delayed release delivery system is an excellent example of how altering the delivery of the active agent can improve oxybutynin s tolerability while still maintaining its effectiveness . this transformation has allowed oxybutynin to remain a formidable treatment option for oab despite the introduction of newer and more tissue - specific antimuscarinic agents . oxybutynin er has clearly demonstrated its applicability in the treatment of oab and voiding dysfunction in the adult population . oxybutynin ir has been widely used in the pediatric population for neurogenic bladder and voiding dysfunction though there are limited studies looking at the oxybutynin er formulation in children . the incidence of oab and urinary incontinence increases with age yet the use of oxybutynin in this population is cautioned due to concerns that older patients may have problems with increased side effects especially for cns , memory and cognition .

overactive bladder ( oab ) is a common condition which negatively impacts the quality of life of afflicted patients . this can result in alterations in social interactions at home , in the workplace and in the community , often leading to depression and poor self esteem as well as loss of productivity . traditional mainstays of treatment include both behavioral therapy and pharmacotherapy . oxybutynin immediate release ( ir ) represents the first such medication approved by the fda specifically for treatment of oab in 1975 . nevertheless , bothersome side effects in addition to thrice daily dosing often led to treatment cessation which raised the question that patients may actually prefer to live with their oab symptoms rather than incur side effects or complex dosing schemes . pharmacological advances ultimately led to development of a long - acting formulation of oxybutynin in the form of oxybutynin extended release ( er ) with the hope that this drug would maintain efficacy while decreasing bothersome side effects and improve compliance with the convenience of once daily dosing regimen . this paper will review the major clinical studies involving oxybutynin er as well as its role in different patient populations and potential concerns with its use .

Introduction History of oxybutynin ER Early studies: extended release vs placebo and immediate release vs extended release Oxybutynin ER vs other OAB medications Special populations and other trials Conclusions

the international continence society defines overactive bladder ( oab ) as : urgency , with or without urge incontinence , usually with frequency and nocturia in the absence of infection or any other proven causative pathology.1 thus the oab population includes both patients with urge incontinence ( oab wet ) as well as without urge incontinence ( oab dry ) . indeed , the incidence of oab dry has been reported at 66% of oab patients while 33% have oab wet and suffer from leakage.2 with prevalence rates similar to those of asthma and chronic bronchitis , the burden of this disease is enormous and presents great cost to society.3 the national overactive bladder evaluation ( noble ) program was a large scale , well designed epidemiological study which reported the prevalence of oab in the us at approximately 16% with the distribution between men and women being roughly equal and with the prevalence mounting with increasing age.3 similar results were seen in epidemiological studies in europe with prevalence rates of oab in the 12% to 17% range.4 the pathophysiology of oab is not completely understood and likely there are multiple pathways leading to the end disease state . while causative myogenic and mucosal alterations would take place at the end organ ( bladder and urethra ) , adverse changes in bladder innervations in such neurogenic theories , alterations in the neurologic pathways that normally provide tonic inhibition of detrusor contractions may account for the disease state.5 alternatively , sensory nerve endings in the bladder may become hypersensitive , resulting in detrusor overactivity.5 indeed , research studying the role of the urothelium in normal and abnormal micturitional states has advanced dramatically and shaped the field of oab . bladder ganglia cells excite detrusor smooth muscle with release of both cholinergic neurotransmitter in the form of acetylcholine and also non - cholinergic , non - adrenergic neurotransmitters.5 acetylcholine activates muscarinic receptors on the detrusor smooth muscle and is considered a key neurotransmitter for detrusor function . oxybutynin immediate release ( ir ) represents the first such medication approved by the fda specifically for the treatment of oab in 1975.7 it was originally investigated in the 1960s for the potential treatment of gastrointestinal hypermotility . in addition to its anticholinergics effects oxybutynin also shows some in vitro activity as a direct smooth muscle relaxant8 and early in vitro studies on rabbit detrusor also noted moderate local anesthetic action.9 clinical efficacy for treatment of oab symptoms was well documented in the 1990s.10 despite proven efficacy , oxybutynin ir shows some major shortcomings as a therapeutic drug , namely high side effects , some so bothersome that some patients prefer to discontinue treatment and endure the disease itself rather than deal with the side effects . in addition to muscarinic receptors in the bladder , oxybutynin has particular affinity for muscarinic receptors in the central nervous system ( cns ) , gastrointestinal ( gi ) tract , parotid gland and eye which may account for troublesome cognitive impairment , constipation , dry mouth and difficulty with papillary accommodation . of these , dry mouth appears to be most prominent , occurring in up to 80% of patients taking oxybutynin.12 beyond bother , decreased salivation may predispose to oral infections and dental caries as well as inhibit proper mastication and swallowing , particularly in the elderly.13,14 given the limitations of oxybutynin ir , it comes as no surprise that alternative agents would be explored in the hopes that tolerability could be improved while maintaining efficacy in the treatment of oab . other antimuscarinics such as propantheline , methantheline , emepronium , dicyclomine , and terodiline were once employed as alternative oab treatment options as they were perceived to be more tolerable , however they were eventually abandoned due to either lack of efficacy and/or poor tolerability.15,16 currently , there are five different antimuscarinic drug formulations on the market for treatment of oab in addition to oxybutynin . tolterodine , propiverine , trospium , darifenacin and solifenacin all possess differing pharmacokinetic properties relating to organ and receptor subtype selectivities as well as structural differences . in addition , a separate strategy for changing the side effect profile of oxybutynin was employed by using an extended release ( er ) formulation . in addition , a patch formulation of oxybutynin was developed in hopes that tolerability could be improved through of use of a different delivery system which altered drug metabolism and lengthened the drug dosing interval . in this article we focus our attention on oxybutynin er formulation likewise meant to simplify drug dosing and improve on the side effect profile of the older oxybutynin ir . oxybutynin er came to market in the united states in 199817 with intent to improve upon tolerability , convenience and compliance while maintaining efficacy by offering once daily dosing . oros oxybutynin chloride ( oxybutynin er , ditropan xl ) is an extended - release oral tablet formulation ( oros ) which utilizes osmotic pressure to deliver the drug at a controlled rate over 24 hours . pharmacokinetic studies show that after the first dose of oxybutynin er , plasma drug concentration rises for 6 hours and then maintains a constant concentration for up to 24 hours with steady state plasma concentrations achieved by day three of treatment.18 normally , when absorbed in the upper gi tract , oxybutynin ir undergoes first pass metabolism by the cytochrome p450 system into its active metabolite n - desethyloxybutynin ( ndo ) . this may be particularly important as ndo has been proposed to be responsible for more dry mouth than oxybutynin itself.19 however , because the oros system delays the release of oxybutynin in the er formulation , it is thought that the parent drug is not biologically available until the tablet reaches the large intestine . they concluded that both groups shared a similar rate of reductions in urge incontinence and total incontinence episodes while the oxybutynin er group reported significantly less dry mouth at 68% compared to 87% for any degree of complaint.21 another study published in the year 2000 sought to compare the efficacy and safety of oxybutynin ir as well as to determine rates of dry mouth . they likewise found comparable rates of reduction in incontinence episodes with similar rates of dose - dependent dry mouth , but moderate to severe dry mouth was significantly less prominent in the group receiving oxybutynin er . in 2005 individualized doses of oxybutynin er for urinary incontinence.23 his group combined data from three flexible - dosing studies of patients with urge urinary incontinence or mixed incontinence ; two of the data sets used were from trials completed in the late 1990s on er versus ir oxybutynin while the third was from a study published in 1999 by the ditropan xl study group on evaluation of a new once - daily formulation of oxybutynin for the treatment of urge urinary incontinence.19,20,24 a total of 420 patients combined were assigned to treatment with oxybutynin er . in the combined dataset , 420 patients were assigned to treatment with oxybutynin er . treatment began with 5 mg daily of oxybutynin er and was increased in 5-mg increments at intervals of approximately 1 week until the patient either achieved complete continence or experienced intolerable side effects . in addition they recognize that the three studies each contained some of the previously discussed study design flaws common to early studies of oxybutynin er versus ir , further weakening conclusions drawn in this paper.21 the next round of clinical trials involving oxybutynin mainly focused on comparison studies to other oab medications . two large , multi - center , prospective , randomized clinical trials comparing oxybutynin er and tolterodine tartrate in oab patients stand out for their sound methodology and power relative to all other clinical trials employing oxybutynin er : the overactive bladder : judging effective control and treatment ( object ) study25 and the overactive bladder : performance of extended release agents ( opera ) trial.26 the object study was conducted between march and october 2000 at 37 us study sites with the objective to compare the efficacy and tolerability of fixed dose extended - release oxybutynin and tolterodine tartrate at 12 weeks . trial completion was accomplished by 276 women and 56 men while a total 46 discontinued early , 29 because of adverse events ( 14 in the oxybutynin er and 15 in the tolterodine group ) . the diaries documented number of voids as well as number and nature of incontinence episodes . study findings included similar tolerability and discontinuation rates as well as similar adverse event rates for both drugs . in the object study , oxybutynin er proved significantly more effective than tolterodine in reducing the mean number of weekly urge incontinence episodes at end of study as well as total incontinence episodes and total urinary frequency . they also noted that the study population was selected by specialists rather than primary care physicians who treat most oab and that study population was not racially diverse , though this is a common limitation in most oab studies . it should be noted that object compared once daily oxybutynin er to twice daily tolterodine tartrate , though this formulation of tolterodine has essentially been replaced in most cases by the once daily formulation of tolterodine which is expected to improve patient compliance . regardless , object did compare oxybutynin er with the most widely prescribed non - generic anticholinergic medication at that time in a blinded head to head randomized clinical trial in the us.23 it must be noted that the object study was funded by alza corporation in mountain view , california , a bio - pharmaceutical company with specialization in drug delivery systems and developer of the oros drug delivery system . the second large multicenter trial , opera , was likewise funded by the alza corporation as well as ortho - mcneil pharmaceutical in raritan , nj , makers of oxybutynin er . opera authors likewise disclosed relationships including : medical consultant , investigator , advisor as well as an employee and stockholder of ortho - mcneil and an employee and stockholder of azla . the trial ran from november 2000 to october 2001 and pitted oxybutynin er head to head with the most prescribed once daily anticholinergic in a prospective , randomized , double - blind study conducted at 71 us centers . a total of 790 women were randomized to receive either 10 mg of oxybutynin er or 4 mg of tolterodine er per day . after 12 weeks of treatment , the oxybutynin er group saw a reduction from a mean of 37.1 to 10.8 urge incontinent episodes per week while the tolterodine er group dropped from 36.7 to 11.2 . slightly more patients in the oxybutynin er group reported no incontinence episodes in their final diary versus the tolterodine er group at 23% versus 16.8% completely dry . dry mouth was more common in the oxybutynin er group at 29.7% versus 22.3% in the tolterodine group ; while statistically significant , the majority of the dry mouth effects were categorized as mild ( 93% of oxybutynin er participants and 95% of tolterodine er participants.24 overall , the opera study shows oxybutynin er to have modestly greater efficacy than er tolterodine at its most commonly prescribed dose . some epidemiological studies would argue that the prevalence in men is nearly equal.3 in 2006 , anderson et al published a post hoc analysis of data from the opera trial specifically comparing the efficacy and tolerability of oxybutynin er and tolterodine er in women with or without prior anticholinergic treatment for oab . in contrast , the anticholinergic - naive group showed statistically equal efficacy for all study parameters with the exception of average number of micturitions per week at study completion ; this parameter favored the participants taking oxybutynin er . dry mouth was more common in the oxybutynin er study participants , but only in the group with history of prior anticholinergic treatment.27 several clinical trials address the use of oxybutynin er in special populations , including the elderly , children , and neurogenic bladder . interestingly , while the concept that oab may contribute to male lower urinary tract symptoms has become more widely accepted28 we were unable to identify any studies in the literature focusing on oxybutynin er in the male subpopulation . oxybutynin er is considered : pregnancy catetory b , reproduction studies in the mouse , rat , hamster and rabbit showing no definite evidence of impaired fertility or harm to animal fetus . therefore ditropan xl should not be given to pregnant women unless , in the judgement of the physician , the probable clinical benefits outweigh the possible hazards . it is unlikely that studies of oxybutynin in pregnant women will ever be undertaken . as the number of medications use increases , so does the risk to the older patient including problems with compliance as well as potential drug - drug interactions which can cause adverse events or alter drug efficacy.29 older patients are at risk for falls and sleep disturbances , and they often already suffer from dry mouth , dry eye , and vision problems as well as gi motility problems including constipation.30 kay et al studied the differential effects oxybutynin er versus the drug darifenacin on memory in older patients . initial dose for oxybutynin er was 10 mg daily , and increases in 5-mg increments was permitted up to 20 mg by the end of this 3-week study . despite this potential dosage mismatch , interval evaluation of study subjects does show a drop in measured cognitive function in the oxybutynin er group after one week even at 10 mg daily . despite the short duration of the study , it does show that older patients with pharmacological steady state plasma levels of oxybutynin er may have an increased risk of impaired cognitive function relative to those in steady state taking darifenacin.31 contrary to the relatively high dose oxybutynin er used in previous study , lackner reported a randomized , placebo - controlled trial of the cognitive effect , safety and tolerability of 5 mg daily dosage of oxybutynin er in a population of cognitively impaired nursing home residents with urge incontinence . study limitations include the short duration , homogenous caucasian female population as well as lack of information about efficacy at this dosage.32 however , these results do contradict the guidelines of the american neurological association , the american psychiatric association and the beers criteria for potentially inappropriate medication use in older adults33 and conclusions about their use in this population remain guarded . cns side effects do represent a cause of potential severe consequences in the elderly in particular . patients should be monitored for signs of anticholinergic cns effects , particularly in the first few months after beginning treatment or increasing the dose . drug treatment of neurologically intact children with oab type symptoms generally focuses on incontinence , which can , in older children have marked effects on the child s social and psychological development and well being in addition to causing great stress and anxiety in parents . beyond behavioral therapy and biofeedback , clinicians , parents and patients often wish to turn to drug therapy to treat oab . while studies on oab drugs in adults are limited and conflicting , only one non - randomized clinical study was identified investigating therapeutic efficacy and tolerability of oxybutynin er in children . oxybutynin er was again significantly more effective than either formulation of tolterodine for control of daytime urinary incontinence and micturition frequency.34 while this non - randomized , non - blinded retrospective study using non - validated instruments contains serious flaws , it nevertheless represents the very small amount of literature devoted to use of oxybutynin er in children and suggests that the use of this type of medication is safe and effective in children and that oxybutynin er is at least as effective as either formulation of tolterodine . one other study focused on the use of oxybutynin er in children with neurogenic bladder ( ngb ) . this is a distinct and important subset of patients who frequently require aggressive management of bladder issues to maximize social functionality as well as to protect renal function . while published studies as early as 1977 address the use of oxybutynin ir in this population , very little is available regarding oxybutynin er in children with ngb.35 interestingly , no studies were identified which reported specifically on use of oxybutynin er in adults with neurogenic bladder . franco et al reported on use of three different formulations of oxybutynin in children aged 615 years of age who performed clean intermittent catheterization ( cic ) and who had documented neurogenic detrusor overactivity at 24 different centers in the us and europe . improvement of urodynamic parameters was seen in all treatment arms as well as increased continence . in contrast to other studies , constipation represented the most frequently reported adverse event in this population while dry mouth is not even listed as occurring at 5%.36 despite flaws , this report does provide some evidence for tolerability of oxybutynin er in this small but important patient population . first , one must understand the staggering financial cost of oab in modern society ; estimates of dollars spent annually in the us alone run to us$12 billion.37 while non - pharmacological treatment methods may be employed , anticholinergic medications represent the mainstay of treatment for oab and drug costs contribute considerably to the overall burden of cost related to oab . considerable literature in the managed care and pharmacoeconomics literature is devoted to these issues including evaluations of oxybutynin er as a therapeutic choice for oab . one european study sought to study the cost - effectiveness of long - term pharmacological management of urge urinary incontinence using ir oxybutynin and tolterodine as well as er formulations of the two drugs in the united kingdom . they concluded that tolterodine ir was less cost effective than the other three drug choices.39 in contrast , two large us retrospective studies designed to determine financial cost and benefit of treatment with er formulations of oxybutynin versus tolterodine estimated healthcare costs based on reimbursement for services ( hence only covered benefits ) by using enormous patient databases related to us health insurers.40,41 both studies found cost savings in total reimbursement costs over one year for patients treated with tolterodine er versus oxybutynin er . antimuscarinic agents remain a mainstay of therapy for the treatment of oab . while this medication class has proven efficacious for this condition , decreased tolerability due to antimuscarinic side effects oxybutynin has proven to be effective for the treatment of oab however antimuscarinic side effects such as dry mouth and constipation often limit patients compliance with it . oxybutynin er has clearly demonstrated its applicability in the treatment of oab and voiding dysfunction in the adult population . oxybutynin ir has been widely used in the pediatric population for neurogenic bladder and voiding dysfunction though there are limited studies looking at the oxybutynin er formulation in children . the incidence of oab and urinary incontinence increases with age yet the use of oxybutynin in this population is cautioned due to concerns that older patients may have problems with increased side effects especially for cns , memory and cognition .

the international continence society defines overactive bladder ( oab ) as : urgency , with or without urge incontinence , usually with frequency and nocturia in the absence of infection or any other proven causative pathology.1 thus the oab population includes both patients with urge incontinence ( oab wet ) as well as without urge incontinence ( oab dry ) . indeed , the incidence of oab dry has been reported at 66% of oab patients while 33% have oab wet and suffer from leakage.2 with prevalence rates similar to those of asthma and chronic bronchitis , the burden of this disease is enormous and presents great cost to society.3 the national overactive bladder evaluation ( noble ) program was a large scale , well designed epidemiological study which reported the prevalence of oab in the us at approximately 16% with the distribution between men and women being roughly equal and with the prevalence mounting with increasing age.3 similar results were seen in epidemiological studies in europe with prevalence rates of oab in the 12% to 17% range.4 the pathophysiology of oab is not completely understood and likely there are multiple pathways leading to the end disease state . while causative myogenic and mucosal alterations would take place at the end organ ( bladder and urethra ) , adverse changes in bladder innervations in such neurogenic theories , alterations in the neurologic pathways that normally provide tonic inhibition of detrusor contractions may account for the disease state.5 alternatively , sensory nerve endings in the bladder may become hypersensitive , resulting in detrusor overactivity.5 indeed , research studying the role of the urothelium in normal and abnormal micturitional states has advanced dramatically and shaped the field of oab . bladder ganglia cells excite detrusor smooth muscle with release of both cholinergic neurotransmitter in the form of acetylcholine and also non - cholinergic , non - adrenergic neurotransmitters.5 acetylcholine activates muscarinic receptors on the detrusor smooth muscle and is considered a key neurotransmitter for detrusor function . there are 5 distinct subtypes of the muscarinic receptor throughout the body ( m1m5 ) , the m2 and m3 sub - types predominating in the bladder . although the density of m2 receptor is higher than the m3 receptor in the human bladder , m3 is thought to be more important in the manifestation of detrusor contractions.6 given this basic understanding of neuroanatomy , it is not surprising that muscarinic cholinergic receptors became early therapeutic targets in oab treatment . oxybutynin immediate release ( ir ) represents the first such medication approved by the fda specifically for the treatment of oab in 1975.7 it was originally investigated in the 1960s for the potential treatment of gastrointestinal hypermotility . in addition to its anticholinergics effects oxybutynin also shows some in vitro activity as a direct smooth muscle relaxant8 and early in vitro studies on rabbit detrusor also noted moderate local anesthetic action.9 clinical efficacy for treatment of oab symptoms was well documented in the 1990s.10 despite proven efficacy , oxybutynin ir shows some major shortcomings as a therapeutic drug , namely high side effects , some so bothersome that some patients prefer to discontinue treatment and endure the disease itself rather than deal with the side effects . lawrence et al showed a 6-month treatment adherence rate of only 32% in a retrospective analysis 515 patients prescribed oxybutynin ir in a pharmacy claims database.11 these pitfalls stem from fact that oxybutynin has a relatively nonspecific affinity for all of the various muscarinic receptors which are widespread throughout the human body . in addition to muscarinic receptors in the bladder , oxybutynin has particular affinity for muscarinic receptors in the central nervous system ( cns ) , gastrointestinal ( gi ) tract , parotid gland and eye which may account for troublesome cognitive impairment , constipation , dry mouth and difficulty with papillary accommodation . of these , dry mouth appears to be most prominent , occurring in up to 80% of patients taking oxybutynin.12 beyond bother , decreased salivation may predispose to oral infections and dental caries as well as inhibit proper mastication and swallowing , particularly in the elderly.13,14 given the limitations of oxybutynin ir , it comes as no surprise that alternative agents would be explored in the hopes that tolerability could be improved while maintaining efficacy in the treatment of oab . other antimuscarinics such as propantheline , methantheline , emepronium , dicyclomine , and terodiline were once employed as alternative oab treatment options as they were perceived to be more tolerable , however they were eventually abandoned due to either lack of efficacy and/or poor tolerability.15,16 currently , there are five different antimuscarinic drug formulations on the market for treatment of oab in addition to oxybutynin . in addition , a patch formulation of oxybutynin was developed in hopes that tolerability could be improved through of use of a different delivery system which altered drug metabolism and lengthened the drug dosing interval . in this article we focus our attention on oxybutynin er formulation likewise meant to simplify drug dosing and improve on the side effect profile of the older oxybutynin ir . in the gi tract , the absorption of water by this osmotically active layer results in the expulsion of the drug ( which has likewise absorbed water forming a suspension ) out a small opening in the membrane . pharmacokinetic studies show that after the first dose of oxybutynin er , plasma drug concentration rises for 6 hours and then maintains a constant concentration for up to 24 hours with steady state plasma concentrations achieved by day three of treatment.18 normally , when absorbed in the upper gi tract , oxybutynin ir undergoes first pass metabolism by the cytochrome p450 system into its active metabolite n - desethyloxybutynin ( ndo ) . this may be particularly important as ndo has been proposed to be responsible for more dry mouth than oxybutynin itself.19 however , because the oros system delays the release of oxybutynin in the er formulation , it is thought that the parent drug is not biologically available until the tablet reaches the large intestine . by this point , the drug has bypassed the upper gi s first pass metabolism system and is left to be absorbed in the colon where conversion to ndo is less likely and absorption is mainly of the parent drug itself . the drug did show at least 40% greater decrease in urinary urge incontinence versus placebo in a 6-week trial though the results are recorded in abstract form only.20 in an early industry sponsored study anderson et al for the ditropan study group enrolled 105 patients with urge incontinence into a randomized , double blind study of dose titration to compare the efficacy and safety of controlled and immediate release oxybutynin . they concluded that both groups shared a similar rate of reductions in urge incontinence and total incontinence episodes while the oxybutynin er group reported significantly less dry mouth at 68% compared to 87% for any degree of complaint.21 another study published in the year 2000 sought to compare the efficacy and safety of oxybutynin ir as well as to determine rates of dry mouth . they likewise found comparable rates of reduction in incontinence episodes with similar rates of dose - dependent dry mouth , but moderate to severe dry mouth was significantly less prominent in the group receiving oxybutynin er . stratified by dosage , the greatest differences were at the 10 mg per day ( 8.5% for the er group vs 25.6% for the ir group ) and 15 mg per day doses ( 19.4% for the er group vs 38.9% for the ir group ) groups.22 nevertheless , the early data on direct comparisons between oxybutynin ir and er remain limited by trial design shortcomings including studying enriched populations such as known responders and reporting results on a completer basis . in 2005 individualized doses of oxybutynin er for urinary incontinence.23 his group combined data from three flexible - dosing studies of patients with urge urinary incontinence or mixed incontinence ; two of the data sets used were from trials completed in the late 1990s on er versus ir oxybutynin while the third was from a study published in 1999 by the ditropan xl study group on evaluation of a new once - daily formulation of oxybutynin for the treatment of urge urinary incontinence.19,20,24 a total of 420 patients combined were assigned to treatment with oxybutynin er . the authors made note of the fact that 7% of the patients selected the dose of 30 mg per day with only slightly higher rates of dry mouth and similar rates of treatment discontinuation due to adverse events as subjects at lower daily doses . in addition they recognize that the three studies each contained some of the previously discussed study design flaws common to early studies of oxybutynin er versus ir , further weakening conclusions drawn in this paper.21 the next round of clinical trials involving oxybutynin mainly focused on comparison studies to other oab medications . two large , multi - center , prospective , randomized clinical trials comparing oxybutynin er and tolterodine tartrate in oab patients stand out for their sound methodology and power relative to all other clinical trials employing oxybutynin er : the overactive bladder : judging effective control and treatment ( object ) study25 and the overactive bladder : performance of extended release agents ( opera ) trial.26 the object study was conducted between march and october 2000 at 37 us study sites with the objective to compare the efficacy and tolerability of fixed dose extended - release oxybutynin and tolterodine tartrate at 12 weeks . the study enrolled and randomized 315 women and 63 men with overactive bladder who received either oxybutynin er 10 mg daily or tolterodine tartrate 2 mg q 12 hours in a double - blind , double - dummy fashion . in the object study , oxybutynin er proved significantly more effective than tolterodine in reducing the mean number of weekly urge incontinence episodes at end of study as well as total incontinence episodes and total urinary frequency . regardless , object did compare oxybutynin er with the most widely prescribed non - generic anticholinergic medication at that time in a blinded head to head randomized clinical trial in the us.23 it must be noted that the object study was funded by alza corporation in mountain view , california , a bio - pharmaceutical company with specialization in drug delivery systems and developer of the oros drug delivery system . the opera trial sought to compare the efficacy and tolerability of extended - release formulations of both oxybutynin chloride and tolterodine tartrate in women with overactive bladder . a similar number of patients completed the study in both groups ( 87% for the oxybutynin er group versus 89% for the tolterodine er group . ) after 12 weeks of treatment , the oxybutynin er group saw a reduction from a mean of 37.1 to 10.8 urge incontinent episodes per week while the tolterodine er group dropped from 36.7 to 11.2 . total decrease in mean number of voids per week was 28.4 for the oxybutynin group versus 25.2 for the tolterodine group ; respective means were 66.4 and 71.7 at the end of 12 weeks treatment . slightly more patients in the oxybutynin er group reported no incontinence episodes in their final diary versus the tolterodine er group at 23% versus 16.8% completely dry . dry mouth was more common in the oxybutynin er group at 29.7% versus 22.3% in the tolterodine group ; while statistically significant , the majority of the dry mouth effects were categorized as mild ( 93% of oxybutynin er participants and 95% of tolterodine er participants.24 overall , the opera study shows oxybutynin er to have modestly greater efficacy than er tolterodine at its most commonly prescribed dose . the authors point out that the flexible dosing allowed for oxybutynin provides a wider range of fda approved dosages allowing individuals to balance their respective efficacy and tolerability without turning to off label dosages as would be required with tolterodine er of 4 mg daily . some epidemiological studies would argue that the prevalence in men is nearly equal.3 in 2006 , anderson et al published a post hoc analysis of data from the opera trial specifically comparing the efficacy and tolerability of oxybutynin er and tolterodine er in women with or without prior anticholinergic treatment for oab . they found that oxybutynin er was significantly more effective in reducing micturition frequency by week 12 among the population of patients with prior anti - cholinergic treatment experience . likewise in this particular subgroup , the participants taking oxybutynin er reported a statistically significant greater chance of being incontinence free and they had a greater reduction in number of urge incontinence episodes . in contrast , the anticholinergic - naive group showed statistically equal efficacy for all study parameters with the exception of average number of micturitions per week at study completion ; this parameter favored the participants taking oxybutynin er . dry mouth was more common in the oxybutynin er study participants , but only in the group with history of prior anticholinergic treatment.27 several clinical trials address the use of oxybutynin er in special populations , including the elderly , children , and neurogenic bladder . as the number of medications use increases , so does the risk to the older patient including problems with compliance as well as potential drug - drug interactions which can cause adverse events or alter drug efficacy.29 older patients are at risk for falls and sleep disturbances , and they often already suffer from dry mouth , dry eye , and vision problems as well as gi motility problems including constipation.30 kay et al studied the differential effects oxybutynin er versus the drug darifenacin on memory in older patients . despite this potential dosage mismatch , interval evaluation of study subjects does show a drop in measured cognitive function in the oxybutynin er group after one week even at 10 mg daily . despite the short duration of the study , it does show that older patients with pharmacological steady state plasma levels of oxybutynin er may have an increased risk of impaired cognitive function relative to those in steady state taking darifenacin.31 contrary to the relatively high dose oxybutynin er used in previous study , lackner reported a randomized , placebo - controlled trial of the cognitive effect , safety and tolerability of 5 mg daily dosage of oxybutynin er in a population of cognitively impaired nursing home residents with urge incontinence . study limitations include the short duration , homogenous caucasian female population as well as lack of information about efficacy at this dosage.32 however , these results do contradict the guidelines of the american neurological association , the american psychiatric association and the beers criteria for potentially inappropriate medication use in older adults33 and conclusions about their use in this population remain guarded . it also cautions that the drug should be used with caution in patients with pre - existing dementia treated with cholin - esterase inhibitors due to risk of aggravation of symptoms and that the drug should be used with caution in the frail elderly . drug treatment of neurologically intact children with oab type symptoms generally focuses on incontinence , which can , in older children have marked effects on the child s social and psychological development and well being in addition to causing great stress and anxiety in parents . while studies on oab drugs in adults are limited and conflicting , only one non - randomized clinical study was identified investigating therapeutic efficacy and tolerability of oxybutynin er in children . the authors identified 86 girls and 46 boys between the ages of 5 and 18 years of age who were referred to a pediatric urology clinic with diurnal urinary incontinence and oab symptoms including urinary frequency , urgency , posturing associated with urge and/or spontaneous urinary incontinence . medications were started at the lowest dose available in non - liquid form ( tolterodine ir and er at 2 mg , oxybutynin er 2 mg ) and titrated according to response and tolerability . oxybutynin er was again significantly more effective than either formulation of tolterodine for control of daytime urinary incontinence and micturition frequency.34 while this non - randomized , non - blinded retrospective study using non - validated instruments contains serious flaws , it nevertheless represents the very small amount of literature devoted to use of oxybutynin er in children and suggests that the use of this type of medication is safe and effective in children and that oxybutynin er is at least as effective as either formulation of tolterodine . while published studies as early as 1977 address the use of oxybutynin ir in this population , very little is available regarding oxybutynin er in children with ngb.35 interestingly , no studies were identified which reported specifically on use of oxybutynin er in adults with neurogenic bladder . franco et al reported on use of three different formulations of oxybutynin in children aged 615 years of age who performed clean intermittent catheterization ( cic ) and who had documented neurogenic detrusor overactivity at 24 different centers in the us and europe . first , one must understand the staggering financial cost of oab in modern society ; estimates of dollars spent annually in the us alone run to us$12 billion.37 while non - pharmacological treatment methods may be employed , anticholinergic medications represent the mainstay of treatment for oab and drug costs contribute considerably to the overall burden of cost related to oab . however , some studies do indicate that drug therapy for oab may be associated with decreased utilization of other healthcare resources resulting in cost savings elsewhere;38 naturally , the cost of therapeutic medication for oab may offset these net savings . one european study sought to study the cost - effectiveness of long - term pharmacological management of urge urinary incontinence using ir oxybutynin and tolterodine as well as er formulations of the two drugs in the united kingdom . they concluded that tolterodine ir was less cost effective than the other three drug choices.39 in contrast , two large us retrospective studies designed to determine financial cost and benefit of treatment with er formulations of oxybutynin versus tolterodine estimated healthcare costs based on reimbursement for services ( hence only covered benefits ) by using enormous patient databases related to us health insurers.40,41 both studies found cost savings in total reimbursement costs over one year for patients treated with tolterodine er versus oxybutynin er . while this medication class has proven efficacious for this condition , decreased tolerability due to antimuscarinic side effects oxybutynin has proven to be effective for the treatment of oab however antimuscarinic side effects such as dry mouth and constipation often limit patients compliance with it . the use of the oros delayed release delivery system is an excellent example of how altering the delivery of the active agent can improve oxybutynin s tolerability while still maintaining its effectiveness . the incidence of oab and urinary incontinence increases with age yet the use of oxybutynin in this population is cautioned due to concerns that older patients may have problems with increased side effects especially for cns , memory and cognition .

celiac disease is a prevalent autoimmune disorder , with documented presence in populations of north and south america , europe , north africa , south and west asia , and australia . the symptoms of the disease are triggered in genetically susceptible individuals by ingestion of wheat and related cereal proteins of rye and barley . the ensuing innate and adaptive immune responses to the ingested proteins are responsible for inflammation , villous atrophy , and crypt hyperplasia in the small intestine , as well as the production of autoantibodies against the transglutaminase 2 ( tg2 ) enzyme . in addition to intestinal symptoms , celiac disease may involve extra - intestinal complications . dermatitis herpetiformis is the skin manifestation of celiac disease , affecting about 1020% of celiac disease patients and is characterized by papulovesicular lesions and presence of granular deposits of iga in the dermal papillae . elimination of the offending cereals from diet is currently the only effective mode of treatment for celiac disease . willem karel dicke was the first to recognize the importance of the removal of wheat and related cereals from diets of celiac disease patients in 1950 . shortly after that , the alcohol - soluble subfraction containing the gliadin proteins was determined to contain the main toxic factor in the offending grains . gliadin proteins were found to trigger b and t cell - mediated immune responses , which were thought to play a significant role in the inflammatory cascade in celiac disease . antibodies against gliadin proteins were demonstrated to be closely associated with celiac disease and were widely utilized as serologic markers of the condition prior to the discovery of anti - tg2 autoantibodies . antibodies against specific deamidated sequences of gliadin were eventually found to be more specifically associated with celiac disease than those against unmodified native gliadin . in addition to the gliadins , glutenin proteins of wheat , initially thought to be harmless , were later reported to trigger antibody and t cell immune responses in celiac disease patients . together , gliadins and glutenins comprise approximately 70 different proteins , collectively referred to as gluten . they are the major storage proteins of wheat and related cereals , representing about 75% of the total protein content of wheat grain . the nongluten proteins comprise the remainder of the wheat proteome and include several minor storage proteins , -amylase / protease inhibitors , and a variety of other enzymes . these proteins are generally much more soluble in water or aqueous salt solutions than gluten proteins and have been historically referred to as albumins / globulins . several nongluten proteins , including -amylase / protease inhibitor , thiol reductase , serine protease inhibitor ( serpin ) , and -amylase have been identified as potent allergens in ige - mediated wheat allergy and/or baker s asthma . however , the nongluten proteins of wheat and related cereals generally have been considered to be nontoxic and to lack immunogenic potential in the context of celiac disease . a few studies on small numbers of patients have examined immune reactivity to crude albumin / globulin extracts in celiac disease , with divergent results . the earliest of these investigated antibody reactivities in 24 untreated celiac disease patients ( but no healthy controls ) by an immunodiffusion assay , reporting antibodies to gliadin in six and to a crude pbs extract of wheat flour in seven patients . a subsequently published article reported elevated igg reactivity to both gliadin and albumin / globulin protein extracts in 15 untreated celiac disease patients when compared with three normal controls , although the patients exhibited significantly lower titers of antibody to the albumin / globulin extract than to gliadin . another report on three patients with celiac disease described increased antibodies to an extract of albumins / globulins in comparison to two controls , but the utilized extract appeared to contain gluten contamination . a study of six children with celiac disease found increased antibodies to albumin and globulin fractions in comparison to six unaffected controls , although information about the relative purity of the antigenic mixtures used in the immunoassays was not provided . in contrast , the latest of these studies with 10 celiac disease patients found no significant cellular or humoral response to the total extracts of albumins or globulins in comparison to 10 unaffected controls . the authors of this study attributed the contradiction between their and others observations to the presence of extensive gluten contamination in the earlier studies . a recently published report suggested that the nongluten -amylase / protease inhibitor proteins play a role in triggering the inflammatory response associated with celiac disease as innate immune activators through the engagement of the tlr4-md2-cd14 complex , but probably without concomitant b or t cell involvement . apart from these specific studies , the nongluten proteins of wheat and related cereals have not been examined in the context of celiac disease , and their potential for immunogenicity remains unclear . in this study , we use high - resolution two - dimensional gel electrophoresis , immunoblotting , proteome mapping , and tandem mass spectrometry ( ms / ms ) to show that specific nongluten proteins of wheat trigger a robust humoral immune response in patients with celiac disease or dermatitis herpetiformis . serum samples were from 120 individuals , including 50 patients with celiac disease ( 18 male , 46 white race , mean [ sd ] age 44.6 [ 17.5 ] years ) , 20 patients with dermatitis herpetiformis ( 11 male , 20 white race , mean age 43.1 [ 20.8 ] years ) , and 50 unaffected controls ( 20 male , 46 white race , mean age 37.1 [ 11.3 ] years ) . all cases of celiac disease were biopsy - proven and diagnosed according to previously described criteria . all patients with dermatitis herpetiformis had biopsies demonstrating classic histology , as well as the characteristic immunofluorescence pattern showing clear granular iga deposits in the dermal papillae . the celiac disease and dermatitis herpetiformis patients were on a gluten - containing diet . serum samples were obtained from patients and healthy controls under institutional review board - approved protocols at columbia university and at university of utah . this study was approved by the institutional review board of columbia university medical center . hard red spring wheat triticum aestivum butte 86 flour was suspended in 1 ml of 40% ethanol and mixed for 30 min at room temperature . the supernate was removed , chilled at 4 c for 1 h , combined with 1.9 ml of 1.5 m nacl , and stored at 4 c overnight . the precipitate was removed by centrifugation , rinsed with h2o , and dissolved in 0.2 ml of 0.1 m glacial acetic acid . the solution , containing gluten proteins , was lyophilized and stored at 20 c . fifty milligrams of flour was suspended in 200 l of buffer ( 50 mm tris - hcl , 100 mm kcl , 5 mm edta , ph 7.8 ) at 4 c and incubated for 5 min with intermittent vortex mixing . samples were centrifuged at 4 c for 15 min at 14,500 g. the supernate was collected , and proteins were precipitated by the addition of 4 volumes of cold ( 20 c ) acetone . following incubation overnight at 20 c , samples were centrifuged at 14,000 g for 15 min at 4 c . the pellet was rinsed with cold acetone , air - dried , and stored at 20 c . all patients and controls were tested for the currently recommended full panel of the most sensitive and specific serologic markers of celiac disease , including iga antibody to tg2 , igg antibody to deamidated gliadin , and iga antibody to deamidated gliadin . iga antibody to recombinant human tg2 was measured by elisa , according to the manufacturer s protocol ( euroimmun ag , luebeck , germany ) . igg and iga antibody reactivities to deamidated gliadin , as represented by a previously described glutamine glutamate substituted trimer of a fusion peptide containing the sequences plqpeqpfp and peqlpqfee , were measured by separate elisas , according to the manufacturer s protocols ( euroimmun ag ) . serum igg and iga antibodies to the gluten and nongluten protein extracts were measured separately by elisa as previously described , with some modifications . prior to the elisa analyses , the protein profile of each extract was assessed by sds - page , using the xcell surelock mini - cell electrophoresis system , 412% nupage bis - tris precast gels , and 2-(n - morpholino)ethanesulfonic acid ( mes ) buffer ( life technologies , carlsbad , calif . ) . a 2 mg / ml stock solution of the gluten extract in 70% ethanol or the nongluten protein extract in pbs was prepared . wells of 96-well maxisorp round - bottom polystyrene plates ( nunc , roskilde , denmark ) were coated with 50 l / well of a 0.01 mg / ml solution of protein extract in 0.1 m carbonate buffer ( ph 9.6 ) or left uncoated to serve as controls . after incubation at 37 c for 1 h , all wells were washed and blocked by incubation with 1% bovine serum albumin ( bsa ) in pbs containing 0.05% tween-20 ( pbst ) for 1.5 h at room temperature . serum samples were diluted at 1:200 for iga and at 1:800 for igg measurement , added at 50 l / well in duplicate , and incubated for 1 h. each plate contained a positive control sample from a patient with biopsy - proven celiac disease and elevated igg and iga antibodies to each protein extract . after washing , the wells were incubated with hrp - conjugated antihuman igg ( ge healthcare , piscataway , n.j . ) or iga ( mp biomedicals , santa ana , calif . ) the plates were washed and 50 l of developing solution , containing 27 mm citric acid , 50 mm na2hpo4 , 5.5 mm o - phenylenediamine , and 0.01% h2o2 ( ph 5 ) , was added to each well . after incubating the plates at room temperature for 20 min , absorbance was measured at 450 nm . absorbance values were corrected for nonspecific binding by subtraction of the mean absorbance of the associated uncoated wells . the corrected values were first normalized according to the mean value of the positive control duplicate on each plate . the mean antibody level for the unrelated healthy control cohort was then set as 1.0 au , and all other results were normalized accordingly . the kcl - soluble proteins were separated by two - dimensional electrophoresis as previously described . mg / ml in a solution containing 9 m urea , 4% nonidet p-40 , 1% dithiothreitol , and 2% servalyt 3 - 10 iso - dalt the first dimension capillary tube gels contained 9.2 m urea , 4% ( total monomer ) acrylamide / bis , 2% nonidet p-40 , 2% servalyt 3 - 10 iso - dalt , 0.015% ammonium persulfate , and 0.125% temed . isoelectric focusing was performed using a mini protean ii tube cell ( bio - rad , hercules , calif . ) . eighteen micrograms of protein was loaded for gels that were to be coomassie - stained for visualization of the separated proteins , while 3.6 g of protein was loaded for gels that were to be used for subsequent transfer and immunoblotting . proteins were separated in the second dimension by sds polyacrylamide gel electrophoresis , using the xcell surelock mini - cell electrophoresis system , 412% nupage bis - tris precast gels , and mes buffer . protein transfer onto nitrocellulose membranes was carried out with the iblot dry blotting system ( life technologies ) . the membranes were incubated for 1 h in a blocking solution made of 5% milk and 0.5% bsa in a solution of tris - buffered saline containing 0.05% tween-20 ( tbst ) . incubation with patient and control serum specimens ( 1:2000 for iga and 1:4000 for igg determination in dilution buffer containing 10% blocking solution and 10% fetal bovine serum in tbst ) was done for 1 h. serum samples from celiac disease ( n = 14 ) and dermatitis herpetiformis ( n = 6 ) patients with elevated iga and/or igg antibody reactivity to nongluten proteins , in addition to 5 healthy controls , were included . detection of bound antibodies was by the ecl system ( millipore , billerica , mass . ) and autoradiography film ( crystalgen , commack , n.y . ) . following immunodetection , bound antibodies were removed from the nitrocellulose membranes with restore western blot stripping buffer ( thermo scientific , rockford , ill . ) , and the membrane proteins were visualized using colloidal gold stain ( bio - rad ) . each immunoblot was aligned to its corresponding colloidal gold - stained membrane using the samespots software ( version 4.5 ) ( totallab ltd . proteins in the two - dimensional electrophoresis spots that were the main targets of the antibody response were identified initially by comparison to a previously generated proteomic map of butte 86 flour . spots were excised from gels and placed in wells of a 96-well reaction plate , leaving a blank well between each sample . proteins in each sample - well were reduced , alkylated , and then digested with trypsin using a digestpro instrument ( intavis , koeln , germany ) according to the manufacturer s instructions . the resulting tryptic peptides were eluted into a collection tray that was then placed into the autosampler compartment of an easy - nlc ii ( thermo scientific , waltham , mass . ) that was interfaced by a nanoelectrospray source to an orbitrap elite mass spectrometer ( thermo scientific ) . four microliter fractions were loaded by the autosampler onto an integrafrit trap column ( 100 m 200 mm , with 5 m , 120 , reprosil - pur c18 aq packing ) ( new objective , woburn , mass . ) , washed with 20 l of solvent a to remove salts , then switched in - line with the analytical column ( 75 m 10 cm of 3 m , 120 , reprosil - pur c18 aq packing ) ( new objective ) and eluted with a gradient of 100% solvent a to 30% solvent b over 30 min into the orbitrap elite mass spectrometer . solvent a was water and solvent b was acetonitrile , both optima lc / ms grade , containing 0.1% formic acid ( thermo scientific ) . peptides were detected in the orbitrap with the ft survey scan set to scan a range from 300 to 2000 m / z at a resolution of 60,000 . the 10 most intense peaks were automatically subjected to collision - induced dissociation ( cid ) . the mass range for the cid scans was set to high and the minimal signal threshold to 10,000 . dynamic exclusion with a repeat count of 1 was enabled for a duration of 10 s. normalized collision energy was set to 35 . processing of raw files to mascot generic format ( mgf ) files was carried out using msconvert from the proteowizard open - source project [ http://proteowizard.sourceforge.net/downloads.shtml ] . a first pass search of the ms / ms spectra was conducted against the superwheat database ( version # 140828 ) . for this study , the superwheat database ( version # 100211 ) described in dupont et al . was updated with ncbi triticeae ( taxid : 147389 ) protein sequences ( download date : 08/28/2014 ) as well as with the translated sequences of 137 ests , contigs , or pcr products from butte 86 ( supplemental file 1 ) . mascot version 2.3 [ www.matrixscience.com ] and x ! tandem version 2010.12.01.1 [ http://www.thegpm.org/tandem/ ] were used to match instrument - generated spectra to fasta sequences in the superwheat database . for the first and second pass search , the parent mass tolerance was set to 15 ppm and the fragment error to 0.4 da . charge state screening allowed + 1 , + 2 , and + 3 charge states to be selected , and one missed cleavage was allowed . analysis , validation , and display of the data were carried out using scaffold version 4.3.2 [ http://www.proteomesoftware.com ] . the output results files from the two different search engines were combined in individual folders and analyzed as separate mudpit experiments . a second database of fasta files was generated from the first pass search by exporting from scaffold all proteins that contained one peptide hit and had a protein probability of 20% and peptide probability of 0% . a reverse - concatenated database was created from these sequences . the resulting subset database , containing 14,776 sequences , was used for the second pass search . criteria for protein acceptance in the second pass search was set in the scaffold validation software package to a protein probability of 99% and a requirement for four matching peptides having a parent peptide mass accuracy of 2 ppm and a calculated 95% probability . the list of proteins identified in each spot is shown in supplemental file 2 . the protein with the greatest number of exclusive unique spectra sequence coverages determined by scaffold were adjusted for the presence of the signal peptides as predicted by signalp 4.1 [ http://www.cbs.dtu.dk/services/signalp/ ] . the data files associated with this study were archived at ucsd center for computational mass spectrometry in the massive data set project . the raw mass spectrum files , the sequence database files , and the scaffold results report can be downloaded from ftp://msv000078887:a@massive.ucsd.edu/. in order to visualize scaffold report results , a free viewer can be downloaded from http://www.proteomesoftware.com/products/free-viewer . to visualize the instrument raw files , the cdnas encoding the identified serpin protein in spot 1a ( gi : 224589270 ) and the purinin protein in spot 2a ( bu_purinin#3 ) ( table 2 ) were synthesized ( after codon optimization for an e. coli expression system ) based on amino acid sequences shown in supplemental file 4 . for the purinin , a potential signal peptide cleavage site was detected at position 19 and amino acids 119 were therefore excluded . the synthesized genes were inserted between the restriction sites ndei and hindiii of the tag - free vector e3 to create the e. coli expression vectors e3 - 224589270 and e3-bu_purinin#3 . linearized vectors were transformed into e. coli , and protein expression was induced by adding 100 mm isopropyl -d-1-thiogalactopyranoside to the culture for 4 h at 37 c . cells were lysed in lysis buffer ( 50 mm tris , 1 mm pmsf , ph 8.0 ) , and inclusion bodies , containing insoluble proteins , were collected for purification . after washing ( 50 mm tris , 1% triton x-100 , 300 mm nacl , 2 mm dtt , 1 mm edta , ph 8.0 ) , pellets were dissolved in 8 m urea . the purified proteins were suspended in refolding buffer ( 50 mm tris , 10% glycerol , 150 mm nacl , ph 8.0 ) . the molecular weight and relative purity of the proteins were assessed by sds - page , following the above protocol . the identity of each protein was confirmed by mass spectrometry - assisted peptide mass mapping , as previously explained . antibody reactivity to the generated recombinant serpin and purinin proteins was assessed by immunoblotting . recombinant proteins ( 0.2 g ) were separated by sds - page and transferred onto nitrocellulose membrane . detection of bound hrp - conjugated secondary antibodies was by ecl and the fluorochem m imaging system ( protein simple , santa clara , calif . ) . differences between groups were analyzed by parametric or nonparametric one - way analysis of variance ( anova ) with posthoc testing for multiple comparisons . statistical analyses were performed with prism 6 ( graphpad , san diego , calif . ) . iga antibody to tg2 and igg / iga antibodies to deamidated gliadin , which are considered to be highly specific and sensitive for celiac disease , were measured in all patients and controls . celiac disease and dermatitis herpetiformis groups displayed significantly higher mean levels of iga anti - tg2 antibody ( figure 1a ) , iga antideamidated gliadin antibody ( figure 1b ) , and igg antideamidated gliadin antibody ( figure 1b ) than those of healthy controls ( p < 0.0001 for all comparisons ) . mean levels of antibody to ( a ) human tg2 ( iga ) and ( b ) deamidated gliadin fusion peptide ( iga and igg ) in patients with celiac disease and dermatitis herpetiformis in comparison with unaffected controls , as determined by elisa . the gel electrophoresis profiles of the gluten and nongluten protein extracts used for the elisa analyses are shown in figure 2a . levels of iga and igg class antibodies to the extracted gluten and nongluten protein fractions were measured in all patients and controls . compared with healthy controls , the two patient groups displayed significantly higher mean levels of iga ( p < 0.0001 for celiac disease ; p < 0.001 for dermatitis herpetiformis ) and igg ( p < 0.0001 for both ) antibody to gluten proteins ( figure 2b ) . in addition , compared with healthy controls , the celiac disease and dermatitis herpetiformis patient cohorts exhibited significantly increased serum iga ( p < 0.0001 for both ) and igg ( p < 0.0001 for both ) antibody reactivity to the proteins of the nongluten extract ( figure 2c ) . ( a ) gel electrophoresis profile of the butte 86 protein preparations used for the antibody assays : ( 1 ) gluten extract ( 5 g ) and ( 2 ) nongluten protein extract ( 5 g ) . molecular weight markers , shown to the left of the panel , are in kda . ( b , c ) comparison of mean levels of iga and igg antibodies to gluten ( b ) and nongluten ( c ) proteins in patients with celiac disease or dermatitis herpetiformis in comparison with unaffected controls , as determined by elisa . error bars represent the standard error of the mean . in order to identify the molecular targets of the detected increased antibody response to nongluten proteins in celiac disease and dermatitis herpetiformis patients , a randomly selected subset of antibody - positive sera was further analyzed . antibody reactivity was characterized by immunoblotting following two - dimensional separation of the nongluten proteins of the wheat cultivar butte 86 . the two - dimensional electrophoresis pattern of the nongluten proteins following coomassie staining is shown in figure 3a . the immunoblotting analyses demonstrated antibody reactivity to specific nongluten proteins of wheat in 19 of 20 analyzed patient sera . these included 10 of 11 celiac disease and 2 of 3 dermatitis herpetiformis samples tested for iga reactivity , and 9 of 9 celiac disease and 6 of 6 dermatitis herpetiformis samples tested for igg reactivity . none of the control sera exhibited antibody binding to any proteins at the dilutions and exposures tested . ( a ) pattern of proteins of the nongluten extract after separation by two - dimensional gel electrophoresis and coomassie staining . proteins in five groups , shown in boxes , were found to be the main nongluten antigen targets of the antibody response : serpins , purinins , -amylase / protease inhibitors , globulins , and farinins . ( b e ) immunoblots showing serum antibody reactivity to the two - dimensionally separated proteins in representative patients and healthy controls : ( b ) igg reactivity to serpins , purinins , -amylase / protease inhibitors , globulins , and farinins in a patient with celiac disease ; ( c ) igg reactivity to serpins , purinins , and -amylase / protease inhibitors in a patient with celiac disease ; ( d ) igg reactivity to serpins and globulins in a patient with dermatitis herpetiformis ; ( e ) iga reactivity to serpins and farinins in a patient with dermatitis herpetiformis ; ( f ) igg and ( g ) iga reactivity in healthy controls , indicating lack of binding to nongluten proteins . molecular weight markers ( in kda ) proteins within five regions of the two - dimensional gels reacted with patient sera ( figure 3 ) . comparison of the positions of the reactive proteins with a proteomic map of butte 86 flour suggested that the reactive proteins belonged to the following distinct protein groups : serpins , purinins , -amylase / protease inhibitors , globulins , and farinins ( figure 3 and table 1 ) . identities of proteins in individual spots that reacted with sera were verified by ms / ms using an orbitrap elite mass spectrometer and subsequent database search . peptide data for the predominant protein in each spot are displayed in supplemental file 3 , along with the sequence coverage of each protein . supplemental file 4 contains the amino acid sequences of the identified target proteins from table 2 . because of the sensitivity of the instrument , the analysis indicated that most spots contained multiple proteins ( supplemental file 2 ) . however , the majority of the spectra obtained from each spot corresponded to a specific protein that was deemed the predominant protein in the spot ( table 2 and supplemental files 2 and 3 ) . the ms / ms data confirmed the presence of the five protein types that had been initially identified through comparison of spot positions with the proteomic map of butte 86 flour . for spots 2c and 2d , the ms / ms data indicated that the spots contained purinin proteins ( bu_purinin#1 and bu_purinin#2 ) ( supplemental files 2 and 3 and table 2 ) , even though nonpurinin type proteins in those spots had the highest exclusive unique spectrum count . five distinct protein groups were identified as the main targets of the antibody response through comparison of the positions of the reactive proteins with a proteomic map of butte 86 flour : serpins , purinins , -amylase / protease inhibitors , globulins , and farinins . numbers refer to the specific immunoreactive protein groups , as marked in figure 3a . number of sera found to have igg and/or iga antibody reactivity to spots in each protein type group , as described in the materials and methods section . protein in each spot for which the highest number of exclusive unique spectra were obtained . protein sequences derived from the translation of butte 86 ests or contigs are indicated by protein sequences derived from the translation of contigs from harvest 1.14 est assembly [ http://harvest.ucr.edu/ ] are indicated by sequences of all identified proteins in the table are provided in supplemental file 4 . number of sera found to have igg and/or iga antibody reactivity to each protein spot , as described in the materials and methods section . ms / ms data indicate that the spot also contains a purinin , bu_purinin # 1 ( supplemental files 2 and 3 ) . ms / ms data indicate that the spot also contains a purinin , bu_purinin # 2 ( supplemental files 2 and 3 ) . in this spot , a similar number of exclusive unique spectra were obtained for globulin-2 , bu_globulin-2_contig_18428 , but a greater number of total spectra were obtained for bu_purinin # 3 ( supplemental files 2 and 3 ) . description of results according to patient group is presented in tables 1 and 2 . patients were most frequently reactive to serpins ( 75% of tested celiac disease and dermatitis herpetiformis patient samples ) , followed by purinins ( 65% ) , -amylase / protease inhibitors ( 60% ) , globulins ( 40% ) , and farinins ( 35% ) . the most frequently reactive individual protein spot was identified as a serpin ( spot 1a in figure 3a ; gi : 224589270 ) ( table 2 ) . fifteen of the 20 patients ( 75% ) and none of the controls exhibited antibody reactivity to this protein spot . within the purinin group , the most reactive spot ( 2a ) was identified as bu_purinin # 3 ; 10 of 20 patients ( 50% ) displayed antibody reactivity to it . frequencies of reactivity to the identified proteins did not appear to be substantially different between the celiac disease and dermatitis herpetiformis groups . in order to further confirm the presence of antibody reactivity to selected nongluten antigens identified in this study , we generated proteins by recombinant expression based on the available amino acid sequences for the serpin in spot 1a and the purinin in spot 2a . immunoblotting with the recombinant proteins confirmed the presence of igg and/or iga antibody reactivity to the specific serpin and purinin proteins in patients with celiac disease or dermatitis herpetiformis who had been found to be positive for antibodies to spot 1a and/or spot 2a ( figure 4 ) . healthy controls did not display any reactivity at the serum dilutions and image acquisition exposures used . serum antibody reactivity to a recombinant serpin , representing spot 1a ( gi : 224589270 ) , and a recombinant purinin representing spot 2a ( bu_purinin # 3 ) . ( a ) sds - page profile of the purified recombinant serpin ( lane 1 ) and purinin ( lane 2 ) proteins , following coomassie staining . ( b e ) immunoblotting with the recombinant proteins , demonstrating iga ( b , d ) and igg ( c , e ) antibody reactivity to serpin ( b , c ) and purinin ( d , e ) . lanes 1 and 2 correspond to sera from two representative patients , while lanes 3 and 4 represent sera from two healthy controls . molecular weight markers are shown to the left of each panel ( in kda ) . heightened adaptive immune response to ingested gluten proteins of wheat and related cereals is a hallmark of celiac disease . a few earlier studies , carried out on small numbers of celiac disease patients , had attempted to assess immune reactivity to nongluten proteins of wheat . however , the results were inconsistent , and the purity of the antigenic mixture in the crude nongluten protein extracts used for detecting immune reactivity was later questioned . here , we have demonstrated the presence of a robust humoral response to specific nongluten proteins of wheat in patients with celiac disease and dermatitis herpetiformis . the detailed analysis of antibody reactivity through two - dimensional immunoblotting and ms / ms identification of target proteins provides unequivocal confirmation that the main targets of the antibody response are , in fact , specific proteins that have not been categorized as gluten or previously recognized as triggers of humoral response in celiac disease . wheat serpin proteins belong to the large family of serine protease inhibitors that are present in many organisms , most of them acting as suicide substrate inhibitors of chymotrypsin - like proteases . other nongluten target antigens of wheat included purinin , -amylase / protease inhibitor , globulin , and farinin proteins . the mechanism responsible for generating antibodies against proteins that are generally thought of as nontoxic in the context of celiac disease is not clear . the elevated antibody reactivity to nongluten proteins may be driven by the enhanced inflammatory environment in the gut , brought on by the villous damage and ensuing epithelial barrier dysfunction that are triggered by gluten in celiac disease . however , it should be noted that we did not detect antibodies against a broad array of nongluten proteins . for example , no antibody reactivity to triticin proteins , which are present at similar or higher concentration in wheat flour as serpins , globulins , farinins , and purinins , was found . also , serpins make up a substantially lower percentage of total protein content of butte 86 flour than the -amylase / protease inhibitors but were a more frequent target of patient antibody response . therefore , the elevated antibody reactivity to nongluten proteins appears to be directed at a rather specific set of antigens . another possibility is that the peripheral antibody response to gluten proteins in celiac disease may cross - react with specific nongluten antigens that contain similar epitopes , thus contributing to the detected levels of antibodies against nongluten proteins . a homology analysis indicates that the newly named purinin proteins are close in sequence to -gliadins . in addition , there are short sequences in the identified -amylase / protease inhibitors and another newly characterized group of proteins , the farinins , which are similar to those in certain -gliadin and low molecular weight glutenin proteins . in addition , the reactive centers of some of the identified serpin antigens resemble the glutamine - rich repeats in gluten proteins . whether these particular shared sequences are actually immunogenic and capable of contributing to the presence of cross - reactive antibodies the observed increase in iga and igg antibody responses to specific nongluten proteins brings up the obvious question of whether the identified immune response might be relevant to the pathogenic mechanism of celiac disease . similar to gluten proteins , serpins and -amylase / protease inhibitors are resistant to proteolytic digestion and are therefore likely to be present in the form of long stretches of incompletely digested , and potentially immunogenic , sequences in the small intestine . the observed iga , along with igg , reactivity to the identified nongluten proteins points to a mucosal origin for the immune - triggering event . however , it remains to be seen whether the mucosal b cell response is accompanied by a corresponding intestinal cd4 t cell reactivity to the nongluten proteins . presence of lamina propria cd4 t cells with specificity for sequences of the identified nongluten proteins may contribute to the celiac disease - associated pathways in the gut , for example , by providing additional help to the autoreactive cd8 intraepithelial cytotoxic t cells that drive epithelial cell damage . it is also possible that the detected antibodies to nongluten proteins would themselves contribute to the mucosal lesion . similar to the effect shown for antigluten antibodies , the antibodies against the nongluten proteins may be involved in inducing local complement activation and mucosal damage . they may also contribute to recruitment of various leukocytes that lead to antibody - dependent cell - mediated cytotoxicity , a process that has been previously demonstrated for antibodies to gluten . however , the observed immune response to nongluten proteins , as was mentioned earlier , may be the result and a bystander marker of intestinal barrier damage and inflammation , without playing a role in the pathogenic mechanism of celiac disease . in conclusion , the results of this study clearly demonstrate that the humoral response to wheat in celiac disease is not limited to gluten antigens but is also directed at specific nongluten proteins . while direct conclusions can not be drawn about the pathogenic effects of the identified nongluten proteins , these findings should prompt further research into their potential role in contributing to the inflammatory processes that result in mucosal damage in patients with celiac disease . the possibility of such a role for these proteins is worthy of attention , especially as therapies other than gluten exclusion from the diet are under development . for example , proteolytic enzymes with specificity for the toxic fragments of gluten , may be insufficiently active against other immunogenic proteins . in addition , further investigation of the utility of the identified antibodies as potential biomarkers in celiac disease or other gluten - related disorders may be warranted .

while the antigenic specificity and pathogenic relevance of immunologic reactivity to gluten in celiac disease have been extensively researched , the immune response to nongluten proteins of wheat has not been characterized . we aimed to investigate the level and molecular specificity of antibody response to wheat nongluten proteins in celiac disease . serum samples from patients and controls were screened for igg and iga antibody reactivity to a nongluten protein extract from the wheat cultivar triticum aestivum butte 86 . antibodies were further analyzed for reactivity to specific nongluten proteins by two - dimensional gel electrophoresis and immunoblotting . immunoreactive molecules were identified by tandem mass spectrometry . compared with healthy controls , patients exhibited significantly higher levels of antibody reactivity to nongluten proteins . the main immunoreactive nongluten antibody target proteins were identified as serpins , purinins , -amylase / protease inhibitors , globulins , and farinins . assessment of reactivity toward purified recombinant proteins further confirmed the presence of antibody response to specific antigens . the results demonstrate that , in addition to the well - recognized immune reaction to gluten , celiac disease is associated with a robust humoral response directed at a specific subset of the nongluten proteins of wheat .

Introduction Materials and Methods Results Discussion

in addition to intestinal symptoms , celiac disease may involve extra - intestinal complications . willem karel dicke was the first to recognize the importance of the removal of wheat and related cereals from diets of celiac disease patients in 1950 . gliadin proteins were found to trigger b and t cell - mediated immune responses , which were thought to play a significant role in the inflammatory cascade in celiac disease . antibodies against gliadin proteins were demonstrated to be closely associated with celiac disease and were widely utilized as serologic markers of the condition prior to the discovery of anti - tg2 autoantibodies . in addition to the gliadins , glutenin proteins of wheat , initially thought to be harmless , were later reported to trigger antibody and t cell immune responses in celiac disease patients . the nongluten proteins comprise the remainder of the wheat proteome and include several minor storage proteins , -amylase / protease inhibitors , and a variety of other enzymes . several nongluten proteins , including -amylase / protease inhibitor , thiol reductase , serine protease inhibitor ( serpin ) , and -amylase have been identified as potent allergens in ige - mediated wheat allergy and/or baker s asthma . however , the nongluten proteins of wheat and related cereals generally have been considered to be nontoxic and to lack immunogenic potential in the context of celiac disease . the earliest of these investigated antibody reactivities in 24 untreated celiac disease patients ( but no healthy controls ) by an immunodiffusion assay , reporting antibodies to gliadin in six and to a crude pbs extract of wheat flour in seven patients . a subsequently published article reported elevated igg reactivity to both gliadin and albumin / globulin protein extracts in 15 untreated celiac disease patients when compared with three normal controls , although the patients exhibited significantly lower titers of antibody to the albumin / globulin extract than to gliadin . a study of six children with celiac disease found increased antibodies to albumin and globulin fractions in comparison to six unaffected controls , although information about the relative purity of the antigenic mixtures used in the immunoassays was not provided . in contrast , the latest of these studies with 10 celiac disease patients found no significant cellular or humoral response to the total extracts of albumins or globulins in comparison to 10 unaffected controls . a recently published report suggested that the nongluten -amylase / protease inhibitor proteins play a role in triggering the inflammatory response associated with celiac disease as innate immune activators through the engagement of the tlr4-md2-cd14 complex , but probably without concomitant b or t cell involvement . apart from these specific studies , the nongluten proteins of wheat and related cereals have not been examined in the context of celiac disease , and their potential for immunogenicity remains unclear . in this study , we use high - resolution two - dimensional gel electrophoresis , immunoblotting , proteome mapping , and tandem mass spectrometry ( ms / ms ) to show that specific nongluten proteins of wheat trigger a robust humoral immune response in patients with celiac disease or dermatitis herpetiformis . serum samples were from 120 individuals , including 50 patients with celiac disease ( 18 male , 46 white race , mean [ sd ] age 44.6 [ 17.5 ] years ) , 20 patients with dermatitis herpetiformis ( 11 male , 20 white race , mean age 43.1 [ 20.8 ] years ) , and 50 unaffected controls ( 20 male , 46 white race , mean age 37.1 [ 11.3 ] years ) . serum samples were obtained from patients and healthy controls under institutional review board - approved protocols at columbia university and at university of utah . all patients and controls were tested for the currently recommended full panel of the most sensitive and specific serologic markers of celiac disease , including iga antibody to tg2 , igg antibody to deamidated gliadin , and iga antibody to deamidated gliadin . igg and iga antibody reactivities to deamidated gliadin , as represented by a previously described glutamine glutamate substituted trimer of a fusion peptide containing the sequences plqpeqpfp and peqlpqfee , were measured by separate elisas , according to the manufacturer s protocols ( euroimmun ag ) . serum igg and iga antibodies to the gluten and nongluten protein extracts were measured separately by elisa as previously described , with some modifications . a 2 mg / ml stock solution of the gluten extract in 70% ethanol or the nongluten protein extract in pbs was prepared . serum samples were diluted at 1:200 for iga and at 1:800 for igg measurement , added at 50 l / well in duplicate , and incubated for 1 h. each plate contained a positive control sample from a patient with biopsy - proven celiac disease and elevated igg and iga antibodies to each protein extract . the kcl - soluble proteins were separated by two - dimensional electrophoresis as previously described . incubation with patient and control serum specimens ( 1:2000 for iga and 1:4000 for igg determination in dilution buffer containing 10% blocking solution and 10% fetal bovine serum in tbst ) was done for 1 h. serum samples from celiac disease ( n = 14 ) and dermatitis herpetiformis ( n = 6 ) patients with elevated iga and/or igg antibody reactivity to nongluten proteins , in addition to 5 healthy controls , were included . proteins in the two - dimensional electrophoresis spots that were the main targets of the antibody response were identified initially by comparison to a previously generated proteomic map of butte 86 flour . proteins in each sample - well were reduced , alkylated , and then digested with trypsin using a digestpro instrument ( intavis , koeln , germany ) according to the manufacturer s instructions . the protein with the greatest number of exclusive unique spectra sequence coverages determined by scaffold were adjusted for the presence of the signal peptides as predicted by signalp 4.1 [ http://www.cbs.dtu.dk/services/signalp/ ] . antibody reactivity to the generated recombinant serpin and purinin proteins was assessed by immunoblotting . iga antibody to tg2 and igg / iga antibodies to deamidated gliadin , which are considered to be highly specific and sensitive for celiac disease , were measured in all patients and controls . celiac disease and dermatitis herpetiformis groups displayed significantly higher mean levels of iga anti - tg2 antibody ( figure 1a ) , iga antideamidated gliadin antibody ( figure 1b ) , and igg antideamidated gliadin antibody ( figure 1b ) than those of healthy controls ( p < 0.0001 for all comparisons ) . mean levels of antibody to ( a ) human tg2 ( iga ) and ( b ) deamidated gliadin fusion peptide ( iga and igg ) in patients with celiac disease and dermatitis herpetiformis in comparison with unaffected controls , as determined by elisa . levels of iga and igg class antibodies to the extracted gluten and nongluten protein fractions were measured in all patients and controls . compared with healthy controls , the two patient groups displayed significantly higher mean levels of iga ( p < 0.0001 for celiac disease ; p < 0.001 for dermatitis herpetiformis ) and igg ( p < 0.0001 for both ) antibody to gluten proteins ( figure 2b ) . in addition , compared with healthy controls , the celiac disease and dermatitis herpetiformis patient cohorts exhibited significantly increased serum iga ( p < 0.0001 for both ) and igg ( p < 0.0001 for both ) antibody reactivity to the proteins of the nongluten extract ( figure 2c ) . ( a ) gel electrophoresis profile of the butte 86 protein preparations used for the antibody assays : ( 1 ) gluten extract ( 5 g ) and ( 2 ) nongluten protein extract ( 5 g ) . ( b , c ) comparison of mean levels of iga and igg antibodies to gluten ( b ) and nongluten ( c ) proteins in patients with celiac disease or dermatitis herpetiformis in comparison with unaffected controls , as determined by elisa . in order to identify the molecular targets of the detected increased antibody response to nongluten proteins in celiac disease and dermatitis herpetiformis patients , a randomly selected subset of antibody - positive sera was further analyzed . antibody reactivity was characterized by immunoblotting following two - dimensional separation of the nongluten proteins of the wheat cultivar butte 86 . the two - dimensional electrophoresis pattern of the nongluten proteins following coomassie staining is shown in figure 3a . the immunoblotting analyses demonstrated antibody reactivity to specific nongluten proteins of wheat in 19 of 20 analyzed patient sera . ( a ) pattern of proteins of the nongluten extract after separation by two - dimensional gel electrophoresis and coomassie staining . proteins in five groups , shown in boxes , were found to be the main nongluten antigen targets of the antibody response : serpins , purinins , -amylase / protease inhibitors , globulins , and farinins . ( b e ) immunoblots showing serum antibody reactivity to the two - dimensionally separated proteins in representative patients and healthy controls : ( b ) igg reactivity to serpins , purinins , -amylase / protease inhibitors , globulins , and farinins in a patient with celiac disease ; ( c ) igg reactivity to serpins , purinins , and -amylase / protease inhibitors in a patient with celiac disease ; ( d ) igg reactivity to serpins and globulins in a patient with dermatitis herpetiformis ; ( e ) iga reactivity to serpins and farinins in a patient with dermatitis herpetiformis ; ( f ) igg and ( g ) iga reactivity in healthy controls , indicating lack of binding to nongluten proteins . comparison of the positions of the reactive proteins with a proteomic map of butte 86 flour suggested that the reactive proteins belonged to the following distinct protein groups : serpins , purinins , -amylase / protease inhibitors , globulins , and farinins ( figure 3 and table 1 ) . however , the majority of the spectra obtained from each spot corresponded to a specific protein that was deemed the predominant protein in the spot ( table 2 and supplemental files 2 and 3 ) . the ms / ms data confirmed the presence of the five protein types that had been initially identified through comparison of spot positions with the proteomic map of butte 86 flour . five distinct protein groups were identified as the main targets of the antibody response through comparison of the positions of the reactive proteins with a proteomic map of butte 86 flour : serpins , purinins , -amylase / protease inhibitors , globulins , and farinins . patients were most frequently reactive to serpins ( 75% of tested celiac disease and dermatitis herpetiformis patient samples ) , followed by purinins ( 65% ) , -amylase / protease inhibitors ( 60% ) , globulins ( 40% ) , and farinins ( 35% ) . within the purinin group , the most reactive spot ( 2a ) was identified as bu_purinin # 3 ; 10 of 20 patients ( 50% ) displayed antibody reactivity to it . frequencies of reactivity to the identified proteins did not appear to be substantially different between the celiac disease and dermatitis herpetiformis groups . in order to further confirm the presence of antibody reactivity to selected nongluten antigens identified in this study , we generated proteins by recombinant expression based on the available amino acid sequences for the serpin in spot 1a and the purinin in spot 2a . immunoblotting with the recombinant proteins confirmed the presence of igg and/or iga antibody reactivity to the specific serpin and purinin proteins in patients with celiac disease or dermatitis herpetiformis who had been found to be positive for antibodies to spot 1a and/or spot 2a ( figure 4 ) . serum antibody reactivity to a recombinant serpin , representing spot 1a ( gi : 224589270 ) , and a recombinant purinin representing spot 2a ( bu_purinin # 3 ) . heightened adaptive immune response to ingested gluten proteins of wheat and related cereals is a hallmark of celiac disease . a few earlier studies , carried out on small numbers of celiac disease patients , had attempted to assess immune reactivity to nongluten proteins of wheat . however , the results were inconsistent , and the purity of the antigenic mixture in the crude nongluten protein extracts used for detecting immune reactivity was later questioned . here , we have demonstrated the presence of a robust humoral response to specific nongluten proteins of wheat in patients with celiac disease and dermatitis herpetiformis . the detailed analysis of antibody reactivity through two - dimensional immunoblotting and ms / ms identification of target proteins provides unequivocal confirmation that the main targets of the antibody response are , in fact , specific proteins that have not been categorized as gluten or previously recognized as triggers of humoral response in celiac disease . other nongluten target antigens of wheat included purinin , -amylase / protease inhibitor , globulin , and farinin proteins . the elevated antibody reactivity to nongluten proteins may be driven by the enhanced inflammatory environment in the gut , brought on by the villous damage and ensuing epithelial barrier dysfunction that are triggered by gluten in celiac disease . for example , no antibody reactivity to triticin proteins , which are present at similar or higher concentration in wheat flour as serpins , globulins , farinins , and purinins , was found . also , serpins make up a substantially lower percentage of total protein content of butte 86 flour than the -amylase / protease inhibitors but were a more frequent target of patient antibody response . therefore , the elevated antibody reactivity to nongluten proteins appears to be directed at a rather specific set of antigens . another possibility is that the peripheral antibody response to gluten proteins in celiac disease may cross - react with specific nongluten antigens that contain similar epitopes , thus contributing to the detected levels of antibodies against nongluten proteins . in addition , there are short sequences in the identified -amylase / protease inhibitors and another newly characterized group of proteins , the farinins , which are similar to those in certain -gliadin and low molecular weight glutenin proteins . whether these particular shared sequences are actually immunogenic and capable of contributing to the presence of cross - reactive antibodies the observed increase in iga and igg antibody responses to specific nongluten proteins brings up the obvious question of whether the identified immune response might be relevant to the pathogenic mechanism of celiac disease . similar to gluten proteins , serpins and -amylase / protease inhibitors are resistant to proteolytic digestion and are therefore likely to be present in the form of long stretches of incompletely digested , and potentially immunogenic , sequences in the small intestine . the observed iga , along with igg , reactivity to the identified nongluten proteins points to a mucosal origin for the immune - triggering event . however , it remains to be seen whether the mucosal b cell response is accompanied by a corresponding intestinal cd4 t cell reactivity to the nongluten proteins . presence of lamina propria cd4 t cells with specificity for sequences of the identified nongluten proteins may contribute to the celiac disease - associated pathways in the gut , for example , by providing additional help to the autoreactive cd8 intraepithelial cytotoxic t cells that drive epithelial cell damage . it is also possible that the detected antibodies to nongluten proteins would themselves contribute to the mucosal lesion . similar to the effect shown for antigluten antibodies , the antibodies against the nongluten proteins may be involved in inducing local complement activation and mucosal damage . however , the observed immune response to nongluten proteins , as was mentioned earlier , may be the result and a bystander marker of intestinal barrier damage and inflammation , without playing a role in the pathogenic mechanism of celiac disease . in conclusion , the results of this study clearly demonstrate that the humoral response to wheat in celiac disease is not limited to gluten antigens but is also directed at specific nongluten proteins . while direct conclusions can not be drawn about the pathogenic effects of the identified nongluten proteins , these findings should prompt further research into their potential role in contributing to the inflammatory processes that result in mucosal damage in patients with celiac disease . in addition , further investigation of the utility of the identified antibodies as potential biomarkers in celiac disease or other gluten - related disorders may be warranted .

celiac disease is a prevalent autoimmune disorder , with documented presence in populations of north and south america , europe , north africa , south and west asia , and australia . the ensuing innate and adaptive immune responses to the ingested proteins are responsible for inflammation , villous atrophy , and crypt hyperplasia in the small intestine , as well as the production of autoantibodies against the transglutaminase 2 ( tg2 ) enzyme . dermatitis herpetiformis is the skin manifestation of celiac disease , affecting about 1020% of celiac disease patients and is characterized by papulovesicular lesions and presence of granular deposits of iga in the dermal papillae . willem karel dicke was the first to recognize the importance of the removal of wheat and related cereals from diets of celiac disease patients in 1950 . shortly after that , the alcohol - soluble subfraction containing the gliadin proteins was determined to contain the main toxic factor in the offending grains . gliadin proteins were found to trigger b and t cell - mediated immune responses , which were thought to play a significant role in the inflammatory cascade in celiac disease . antibodies against gliadin proteins were demonstrated to be closely associated with celiac disease and were widely utilized as serologic markers of the condition prior to the discovery of anti - tg2 autoantibodies . in addition to the gliadins , glutenin proteins of wheat , initially thought to be harmless , were later reported to trigger antibody and t cell immune responses in celiac disease patients . the nongluten proteins comprise the remainder of the wheat proteome and include several minor storage proteins , -amylase / protease inhibitors , and a variety of other enzymes . several nongluten proteins , including -amylase / protease inhibitor , thiol reductase , serine protease inhibitor ( serpin ) , and -amylase have been identified as potent allergens in ige - mediated wheat allergy and/or baker s asthma . however , the nongluten proteins of wheat and related cereals generally have been considered to be nontoxic and to lack immunogenic potential in the context of celiac disease . the earliest of these investigated antibody reactivities in 24 untreated celiac disease patients ( but no healthy controls ) by an immunodiffusion assay , reporting antibodies to gliadin in six and to a crude pbs extract of wheat flour in seven patients . a subsequently published article reported elevated igg reactivity to both gliadin and albumin / globulin protein extracts in 15 untreated celiac disease patients when compared with three normal controls , although the patients exhibited significantly lower titers of antibody to the albumin / globulin extract than to gliadin . a study of six children with celiac disease found increased antibodies to albumin and globulin fractions in comparison to six unaffected controls , although information about the relative purity of the antigenic mixtures used in the immunoassays was not provided . in contrast , the latest of these studies with 10 celiac disease patients found no significant cellular or humoral response to the total extracts of albumins or globulins in comparison to 10 unaffected controls . a recently published report suggested that the nongluten -amylase / protease inhibitor proteins play a role in triggering the inflammatory response associated with celiac disease as innate immune activators through the engagement of the tlr4-md2-cd14 complex , but probably without concomitant b or t cell involvement . apart from these specific studies , the nongluten proteins of wheat and related cereals have not been examined in the context of celiac disease , and their potential for immunogenicity remains unclear . in this study , we use high - resolution two - dimensional gel electrophoresis , immunoblotting , proteome mapping , and tandem mass spectrometry ( ms / ms ) to show that specific nongluten proteins of wheat trigger a robust humoral immune response in patients with celiac disease or dermatitis herpetiformis . serum samples were from 120 individuals , including 50 patients with celiac disease ( 18 male , 46 white race , mean [ sd ] age 44.6 [ 17.5 ] years ) , 20 patients with dermatitis herpetiformis ( 11 male , 20 white race , mean age 43.1 [ 20.8 ] years ) , and 50 unaffected controls ( 20 male , 46 white race , mean age 37.1 [ 11.3 ] years ) . all patients and controls were tested for the currently recommended full panel of the most sensitive and specific serologic markers of celiac disease , including iga antibody to tg2 , igg antibody to deamidated gliadin , and iga antibody to deamidated gliadin . igg and iga antibody reactivities to deamidated gliadin , as represented by a previously described glutamine glutamate substituted trimer of a fusion peptide containing the sequences plqpeqpfp and peqlpqfee , were measured by separate elisas , according to the manufacturer s protocols ( euroimmun ag ) . prior to the elisa analyses , the protein profile of each extract was assessed by sds - page , using the xcell surelock mini - cell electrophoresis system , 412% nupage bis - tris precast gels , and 2-(n - morpholino)ethanesulfonic acid ( mes ) buffer ( life technologies , carlsbad , calif . ) mg / ml in a solution containing 9 m urea , 4% nonidet p-40 , 1% dithiothreitol , and 2% servalyt 3 - 10 iso - dalt the first dimension capillary tube gels contained 9.2 m urea , 4% ( total monomer ) acrylamide / bis , 2% nonidet p-40 , 2% servalyt 3 - 10 iso - dalt , 0.015% ammonium persulfate , and 0.125% temed . eighteen micrograms of protein was loaded for gels that were to be coomassie - stained for visualization of the separated proteins , while 3.6 g of protein was loaded for gels that were to be used for subsequent transfer and immunoblotting . incubation with patient and control serum specimens ( 1:2000 for iga and 1:4000 for igg determination in dilution buffer containing 10% blocking solution and 10% fetal bovine serum in tbst ) was done for 1 h. serum samples from celiac disease ( n = 14 ) and dermatitis herpetiformis ( n = 6 ) patients with elevated iga and/or igg antibody reactivity to nongluten proteins , in addition to 5 healthy controls , were included . , washed with 20 l of solvent a to remove salts , then switched in - line with the analytical column ( 75 m 10 cm of 3 m , 120 , reprosil - pur c18 aq packing ) ( new objective ) and eluted with a gradient of 100% solvent a to 30% solvent b over 30 min into the orbitrap elite mass spectrometer . criteria for protein acceptance in the second pass search was set in the scaffold validation software package to a protein probability of 99% and a requirement for four matching peptides having a parent peptide mass accuracy of 2 ppm and a calculated 95% probability . the protein with the greatest number of exclusive unique spectra sequence coverages determined by scaffold were adjusted for the presence of the signal peptides as predicted by signalp 4.1 [ http://www.cbs.dtu.dk/services/signalp/ ] . to visualize the instrument raw files , the cdnas encoding the identified serpin protein in spot 1a ( gi : 224589270 ) and the purinin protein in spot 2a ( bu_purinin#3 ) ( table 2 ) were synthesized ( after codon optimization for an e. coli expression system ) based on amino acid sequences shown in supplemental file 4 . the synthesized genes were inserted between the restriction sites ndei and hindiii of the tag - free vector e3 to create the e. coli expression vectors e3 - 224589270 and e3-bu_purinin#3 . celiac disease and dermatitis herpetiformis groups displayed significantly higher mean levels of iga anti - tg2 antibody ( figure 1a ) , iga antideamidated gliadin antibody ( figure 1b ) , and igg antideamidated gliadin antibody ( figure 1b ) than those of healthy controls ( p < 0.0001 for all comparisons ) . mean levels of antibody to ( a ) human tg2 ( iga ) and ( b ) deamidated gliadin fusion peptide ( iga and igg ) in patients with celiac disease and dermatitis herpetiformis in comparison with unaffected controls , as determined by elisa . compared with healthy controls , the two patient groups displayed significantly higher mean levels of iga ( p < 0.0001 for celiac disease ; p < 0.001 for dermatitis herpetiformis ) and igg ( p < 0.0001 for both ) antibody to gluten proteins ( figure 2b ) . in addition , compared with healthy controls , the celiac disease and dermatitis herpetiformis patient cohorts exhibited significantly increased serum iga ( p < 0.0001 for both ) and igg ( p < 0.0001 for both ) antibody reactivity to the proteins of the nongluten extract ( figure 2c ) . ( b , c ) comparison of mean levels of iga and igg antibodies to gluten ( b ) and nongluten ( c ) proteins in patients with celiac disease or dermatitis herpetiformis in comparison with unaffected controls , as determined by elisa . in order to identify the molecular targets of the detected increased antibody response to nongluten proteins in celiac disease and dermatitis herpetiformis patients , a randomly selected subset of antibody - positive sera was further analyzed . these included 10 of 11 celiac disease and 2 of 3 dermatitis herpetiformis samples tested for iga reactivity , and 9 of 9 celiac disease and 6 of 6 dermatitis herpetiformis samples tested for igg reactivity . proteins in five groups , shown in boxes , were found to be the main nongluten antigen targets of the antibody response : serpins , purinins , -amylase / protease inhibitors , globulins , and farinins . ( b e ) immunoblots showing serum antibody reactivity to the two - dimensionally separated proteins in representative patients and healthy controls : ( b ) igg reactivity to serpins , purinins , -amylase / protease inhibitors , globulins , and farinins in a patient with celiac disease ; ( c ) igg reactivity to serpins , purinins , and -amylase / protease inhibitors in a patient with celiac disease ; ( d ) igg reactivity to serpins and globulins in a patient with dermatitis herpetiformis ; ( e ) iga reactivity to serpins and farinins in a patient with dermatitis herpetiformis ; ( f ) igg and ( g ) iga reactivity in healthy controls , indicating lack of binding to nongluten proteins . comparison of the positions of the reactive proteins with a proteomic map of butte 86 flour suggested that the reactive proteins belonged to the following distinct protein groups : serpins , purinins , -amylase / protease inhibitors , globulins , and farinins ( figure 3 and table 1 ) . however , the majority of the spectra obtained from each spot corresponded to a specific protein that was deemed the predominant protein in the spot ( table 2 and supplemental files 2 and 3 ) . the ms / ms data confirmed the presence of the five protein types that had been initially identified through comparison of spot positions with the proteomic map of butte 86 flour . for spots 2c and 2d , the ms / ms data indicated that the spots contained purinin proteins ( bu_purinin#1 and bu_purinin#2 ) ( supplemental files 2 and 3 and table 2 ) , even though nonpurinin type proteins in those spots had the highest exclusive unique spectrum count . five distinct protein groups were identified as the main targets of the antibody response through comparison of the positions of the reactive proteins with a proteomic map of butte 86 flour : serpins , purinins , -amylase / protease inhibitors , globulins , and farinins . protein sequences derived from the translation of butte 86 ests or contigs are indicated by protein sequences derived from the translation of contigs from harvest 1.14 est assembly [ http://harvest.ucr.edu/ ] are indicated by sequences of all identified proteins in the table are provided in supplemental file 4 . in this spot , a similar number of exclusive unique spectra were obtained for globulin-2 , bu_globulin-2_contig_18428 , but a greater number of total spectra were obtained for bu_purinin # 3 ( supplemental files 2 and 3 ) . patients were most frequently reactive to serpins ( 75% of tested celiac disease and dermatitis herpetiformis patient samples ) , followed by purinins ( 65% ) , -amylase / protease inhibitors ( 60% ) , globulins ( 40% ) , and farinins ( 35% ) . in order to further confirm the presence of antibody reactivity to selected nongluten antigens identified in this study , we generated proteins by recombinant expression based on the available amino acid sequences for the serpin in spot 1a and the purinin in spot 2a . immunoblotting with the recombinant proteins confirmed the presence of igg and/or iga antibody reactivity to the specific serpin and purinin proteins in patients with celiac disease or dermatitis herpetiformis who had been found to be positive for antibodies to spot 1a and/or spot 2a ( figure 4 ) . ( b e ) immunoblotting with the recombinant proteins , demonstrating iga ( b , d ) and igg ( c , e ) antibody reactivity to serpin ( b , c ) and purinin ( d , e ) . however , the results were inconsistent , and the purity of the antigenic mixture in the crude nongluten protein extracts used for detecting immune reactivity was later questioned . here , we have demonstrated the presence of a robust humoral response to specific nongluten proteins of wheat in patients with celiac disease and dermatitis herpetiformis . the detailed analysis of antibody reactivity through two - dimensional immunoblotting and ms / ms identification of target proteins provides unequivocal confirmation that the main targets of the antibody response are , in fact , specific proteins that have not been categorized as gluten or previously recognized as triggers of humoral response in celiac disease . wheat serpin proteins belong to the large family of serine protease inhibitors that are present in many organisms , most of them acting as suicide substrate inhibitors of chymotrypsin - like proteases . the elevated antibody reactivity to nongluten proteins may be driven by the enhanced inflammatory environment in the gut , brought on by the villous damage and ensuing epithelial barrier dysfunction that are triggered by gluten in celiac disease . another possibility is that the peripheral antibody response to gluten proteins in celiac disease may cross - react with specific nongluten antigens that contain similar epitopes , thus contributing to the detected levels of antibodies against nongluten proteins . in addition , there are short sequences in the identified -amylase / protease inhibitors and another newly characterized group of proteins , the farinins , which are similar to those in certain -gliadin and low molecular weight glutenin proteins . whether these particular shared sequences are actually immunogenic and capable of contributing to the presence of cross - reactive antibodies the observed increase in iga and igg antibody responses to specific nongluten proteins brings up the obvious question of whether the identified immune response might be relevant to the pathogenic mechanism of celiac disease . similar to gluten proteins , serpins and -amylase / protease inhibitors are resistant to proteolytic digestion and are therefore likely to be present in the form of long stretches of incompletely digested , and potentially immunogenic , sequences in the small intestine . presence of lamina propria cd4 t cells with specificity for sequences of the identified nongluten proteins may contribute to the celiac disease - associated pathways in the gut , for example , by providing additional help to the autoreactive cd8 intraepithelial cytotoxic t cells that drive epithelial cell damage . however , the observed immune response to nongluten proteins , as was mentioned earlier , may be the result and a bystander marker of intestinal barrier damage and inflammation , without playing a role in the pathogenic mechanism of celiac disease . in conclusion , the results of this study clearly demonstrate that the humoral response to wheat in celiac disease is not limited to gluten antigens but is also directed at specific nongluten proteins . while direct conclusions can not be drawn about the pathogenic effects of the identified nongluten proteins , these findings should prompt further research into their potential role in contributing to the inflammatory processes that result in mucosal damage in patients with celiac disease .

according to the world health organization , resistance to antibiotics is the biggest challenge in drug development . there is a need not for one new antibiotic substance but several , so these can be combined and strengthen the human position in the fight against bacterial resistance ( kaplan and laing 2004 ) . bacterial pathogens have a variety of mechanisms that contribute to their ability to colonize and cause disease in human hosts ( staskawicz et al . it is well known that the virulence of a microbe , i.e. its ability to infect , is correlated with its ability to adhere to tissue , and , in particular , to its capability to withstand forces from rinsing flows ( ofek et al . some bacteria mediate adhesion by use of long attachment organelles , also referred to as fimbriae or pili . p pili , a prototype helix - like pili , are produced by uropathogenic escherichia coli ( upec ) strains and are predominantly associated with kidney infection ( pyelonephritis ) ( sauer et al . 2000 ) . the rod of p pili consists of more than a thousand papa subunits , assembled into a helical right - handed structure with 3.3 subunits per turn ( gong and makowski 1992 ; bullitt and makowski 1995 ) , ending in a linear tip structure that anchors the papg adhesin at the distal end ( jacob - dubuisson et al . 1993 ; kuehn et al . as is further alluded to below , the ability of such attached bacteria to withstand forces caused by rinsing flows is not only given by the properties of the adhesin receptor interactions , but is also strongly affected by the biomechanical properties of these organelles ( duncan et al . it has been found that the compliance of helix - like pili ( i.e. their response to force ) , and thus their ability to assist in the adhesion process , depends strongly on the quaternary structure of the pilus . these pili have an intricate and extraordinary force versus elongation response that consists of a combination of a constant elongation force ( in the so - called region ii ) , originating from sequential uncoiling of the helix - like structure , and a sigmoidal pseudo - elastic response ( region iii ) , caused by a conformational change of the head - to - tail interaction between the subunits of pili that takes place in a random order ( jass et al . 2007 , 2008 ; bjrnham et al . 2008 ; axner et al . 2009 ) . these extraordinary biomechanical properties , primarily those of region ii , which enable significant elongation of pili length [ more than five times the original length for p pili ( jass et al . 2004 ) ] under a constant force , and the fact that the uncoiling is fully reversible ( fllman et al . 2005 ) , have been regarded as being of special importance to the ability of a piliated bacterium to remain attached to the host tissue ( despite being exposed to a substantial external force ) by redistributing an external shear force among a multitude of pili ( bjrnham et al . 2008 ) . presumably , they are a major reason why the ability of attached bacteria to withstand forces caused by urine flow is strongly affected by the properties of the pili shaft per se ( duncan et al . this implies that the pilus rod and the tip - associated adhesin are both important in the ability of bacteria to withstand strong rinsing forces ( bjrnham and axner 2009 ) . during recent decades for example , fimbrial tip - mediated and shear - dependent adhesion ( le trong et al . 2010 ) and a shear - enhanced adhesion mechanism ( whitfield et al . inhibitors targeting the adhesion process , i.e. carbohydrate derived competitive inhibitors for adhesin receptor interactions , have been developed ( wellens et al . 2008 ; salminen et al . 2007 ; ohlsson et al . 2002 ; krogfelt et al . 1990 ) . in addition to this ligand - based approach , several bicyclic 2-pyridones , referred to as pilicides , have been rationally designed to prevent pili formation . it has been suggested that pilicides inhibit the chaperone usher interaction , thus preventing the essential event in which the n - terminus of the usher ( a membrane protein involved in the pili assembly ) recognizes and binds to incoming chaperone subunit complexes ( pinkner et al . these pilicides can efficiently inhibit pili biogenesis without affecting bacterial growth ( berg et al . 2006 ; pinkner et al . 2006 ; cegelski et al . 2009 ; chorell et al . 2010 ) . in contrast , so far , the pili rod has not been used as a target for antibiotics . in this work we investigated whether it is possible to affect the biomechanical properties of helix - like pili by using a substance that can bind to an elongated pilus at the single organelle level , primarily compromising the uncoiling recoiling process . we show , by use of force - measuring optical tweezers ( fmot ) , that the biomechanical compliance of p pili can be impaired without breaking the backbone of the pilus . thus , the pili rod can be regarded as a new target for novel drug candidates combating uropathogenic bacterial infections . the escherichia coli ( e. coli ) strain used was hb101/ppap5 , which is a clone carrying the wild type pap gene cluster from upec strain j96 ( lindberg et al . 1a . because it has previously been found that study of fewer pili per bacterium enhances the ability to capture a single organelle for force measurements , a pilicide , a substituted ring - fused 2-pyridone ( bibc10 , fig . 1b ) , referred to as pilicide 1 by berg and almqvist ( 2007 ) , was used to chemically manipulate the number of pili expressed per bacterial cell . for pilicide concentrations below that required for total inhibition of pili , the bacteria express fewer pili , in a concentration - dependent manner , although with the same biomechanical properties as for untreated bacteria ( berg et al . bacteria were therefore cultured on trypticase soy agar ( tsa ; becton dickinson , nj , usa ) , supplemented with 50 g / ml carbenicillin ( duchefa biochemie , limhamn , sweden ) in the presence of 2.2 mm pilicide at 37c overnight . to remove pilicide possibly present on the surface of the bacteria , the bacteria were washed three times by dilution ( 20,000-fold ) with dulbecco s phosphate - buffered saline ( dpbs , 10 mm phosphate , 130 mm nacl , ph 7.0 , sigma - aldrich , schnelldorf , germany ) just before use in force measurements . expression of pili was confirmed by atomic - force microscopy ( afm ) imaging as described elsewhere ( berg and almqvist 2007 ) . a typical bacterium cultured on a plate containing 2.2 mm pilicide 1 is shown in the afm micrograph in fig . 1afm micrographs of hb101/ppap5 cells expressing p pili : a bacterium cultured under normal conditions , b chemical structure of pilicide 1 , c bacterium cultured on a plate containing 2.2 mm pilicide afm micrographs of hb101/ppap5 cells expressing p pili : a bacterium cultured under normal conditions , b chemical structure of pilicide 1 , c bacterium cultured on a plate containing 2.2 mm pilicide papd and fimc , periplasmic chaperones from e. coli , were expressed and purified as described elsewhere ( lindberg et al . bovine serum albumin ( bsa ) and insulin - like growth factor binding protein-5 ( igfbp-5 ; both from sigma - aldrich ) were used as controls of the papd specific inhibitory effect . these proteins were separated by spinning for 12 min at 14,000g at room temperature using a microcon centrifugal filter device ( millipore , solna , sweden ) in a hermle z200 m / h centrifuge . polyacrylamide gel electrophoresis ( sds - page ; acrylamide - bis ( 50:1 ) ; biorad laboratories , hercules , ca , usa ) , stained by pageblue protein staining solution ( fermentas international , helsingborg , sweden ) . polymeric microspheres ( 9.7 m , duke scientific , palo alto , ca , usa ) were immobilized by heating a droplet of suspension at 60c for 1 h on microscope cover slips ( vwr , stockholm , sweden ) and functionalized with 0.01% poly - l - lysine ( sigma - aldrich , stockholm , sweden ) . on top of these spheres , samples containing bacteria , papd ( 1.4 , 2.8 and 11.3 m ) , or control proteins ( 2.8 m ) , and 3.0-m polymeric microspheres ( duke scientific ) were applied in a total volume of 10 l . a string of high - vacuum grease ( dow corning , midland , mi , usa ) around the sample and a second cover slip were used to protect the sample against evaporation . force measurements were performed by use of optical tweezers , as described elsewhere ( fllman et al . briefly , a free - floating bacterium was trapped at low power by use of a focused laser beam and attached to a poly - l - lysine - coated bead fixed on the cover slip . a small bead ( 3.0 m ) was then captured and the trap was calibrated by use of the power spectrum method in order to determine the stiffness of the trap ( fllman et al . thereafter , the trapped bead was moved back and forth close to the bacterium until pili attached to it . data acquisition was started and the piezo - stage automatically set in motion , at an elongation speed of 0.1 m / s , in order to separate the bacterium from the small bead . if several pili were initially attached to the trapped bead , the separation was repeated so pili detached from the bead until a single pilus remained . the escherichia coli ( e. coli ) strain used was hb101/ppap5 , which is a clone carrying the wild type pap gene cluster from upec strain j96 ( lindberg et al . 1a . because it has previously been found that study of fewer pili per bacterium enhances the ability to capture a single organelle for force measurements , a pilicide , a substituted ring - fused 2-pyridone ( bibc10 , fig . 1b ) , referred to as pilicide 1 by berg and almqvist ( 2007 ) , was used to chemically manipulate the number of pili expressed per bacterial cell . for pilicide concentrations below that required for total inhibition of pili , the bacteria express fewer pili , in a concentration - dependent manner , although with the same biomechanical properties as for untreated bacteria ( berg et al . bacteria were therefore cultured on trypticase soy agar ( tsa ; becton dickinson , nj , usa ) , supplemented with 50 g / ml carbenicillin ( duchefa biochemie , limhamn , sweden ) in the presence of 2.2 mm pilicide at 37c overnight . to remove pilicide possibly present on the surface of the bacteria , the bacteria were washed three times by dilution ( 20,000-fold ) with dulbecco s phosphate - buffered saline ( dpbs , 10 mm phosphate , 130 mm nacl , ph 7.0 , sigma - aldrich , schnelldorf , germany ) just before use in force measurements . expression of pili was confirmed by atomic - force microscopy ( afm ) imaging as described elsewhere ( berg and almqvist 2007 ) . a typical bacterium cultured on a plate containing 2.2 mm pilicide 1 is shown in the afm micrograph in fig . 1afm micrographs of hb101/ppap5 cells expressing p pili : a bacterium cultured under normal conditions , b chemical structure of pilicide 1 , c bacterium cultured on a plate containing 2.2 mm pilicide afm micrographs of hb101/ppap5 cells expressing p pili : a bacterium cultured under normal conditions , b chemical structure of pilicide 1 , c bacterium cultured on a plate containing 2.2 mm pilicide papd and fimc , periplasmic chaperones from e. coli , were expressed and purified as described elsewhere ( lindberg et al . bovine serum albumin ( bsa ) and insulin - like growth factor binding protein-5 ( igfbp-5 ; both from sigma - aldrich ) were used as controls of the papd specific inhibitory effect . these proteins were separated by spinning for 12 min at 14,000g at room temperature using a microcon centrifugal filter device ( millipore , solna , sweden ) in a hermle z200 m / h centrifuge . polyacrylamide gel electrophoresis ( sds - page ; acrylamide - bis ( 50:1 ) ; biorad laboratories , hercules , ca , usa ) , stained by pageblue protein staining solution ( fermentas international , helsingborg , sweden ) . polymeric microspheres ( 9.7 m , duke scientific , palo alto , ca , usa ) were immobilized by heating a droplet of suspension at 60c for 1 h on microscope cover slips ( vwr , stockholm , sweden ) and functionalized with 0.01% poly - l - lysine ( sigma - aldrich , stockholm , sweden ) . on top of these spheres , samples containing bacteria , papd ( 1.4 , 2.8 and 11.3 m ) , or control proteins ( 2.8 m ) , and 3.0-m polymeric microspheres ( duke scientific ) were applied in a total volume of 10 l . a string of high - vacuum grease ( dow corning , midland , mi , usa ) around the sample and a second cover slip were used to protect the sample against evaporation . force measurements were performed by use of optical tweezers , as described elsewhere ( fllman et al . briefly , a free - floating bacterium was trapped at low power by use of a focused laser beam and attached to a poly - l - lysine - coated bead fixed on the cover slip . a small bead ( 3.0 m ) was then captured and the trap was calibrated by use of the power spectrum method in order to determine the stiffness of the trap ( fllman et al . thereafter , the trapped bead was moved back and forth close to the bacterium until pili attached to it . data acquisition was started and the piezo - stage automatically set in motion , at an elongation speed of 0.1 m / s , in order to separate the bacterium from the small bead . if several pili were initially attached to the trapped bead , the separation was repeated so pili detached from the bead until a single pilus remained . fmot have previously been used for characterization of the biomechanical properties of several different types of pili ( andersson et al . 2011 ) , so the compliance of various types of pili is well known . a typical force versus elongation curve for p the data show that a helix - like pilus has a force versus elongation response that can be seen as composed of three regions ( andersson et al . 2force versus elongation response of a single p pilus elongated at a speed of 0.1 m / s . the characteristic elongation regions i , ii , and iii are marked force versus elongation response of a single p pilus elongated at a speed of 0.1 m / s . the characteristic elongation regions i , ii , and iii are marked region i is a short region , of little relevance to this study , in which the entire pilus is elongated elastically with no conformational change . in region ii , elongation originating from sequential breaking of the turn - to - turn [ tt ; previously referred to as layer - to - layer ( andersson et al . 2006b ) ] interactions of the quaternary structure of the pilus occurs at constant force ; this is often referred to as an uncoiling of the pilus rod . the breaking of interactions in this region is sequential , caused by the fact that each turn of the quaternary structure of pili consists of ~3 subunits , each of which interacts with a corresponding subunit in the neighboring turn . hence , each turn interacts with the subsequent turn via ~3 subunits , each of which experiences a fraction ( ~1/3 ) of the force to which the pilus is exposed . the interaction connecting the outermost unit in the folded part of the rod , on the other hand , experiences the entire force . this implies that , during elongation , breaking of the tt interactions occurs sequentially , which gives rise to a force plateau . for sufficiently low elongation velocities , p pili have been found to uncoil at a force of 28 pn ( jass et al . 2004 ; fllman et al . 2005 ) . for higher velocities , above the so called corner frequency , the uncoiling force increases logarithmically with elongation velocity . moreover , inasmuch as each tt interaction can open and close a number of times , uncoiling of the helix - like structure has been found to be fully reversible , which , in turn , implies that the pili can rapidly and automatically contract after being unfolded by an external force ( fllman et al . 2005 ) . as was alluded to above , these unique biomechanical properties [ together with the fact that the uncoiling process of the rod is assumed to reduce the persistence length of the pili substantially , from being similar to the length of the pili , as demonstrated for type 3 pili of klebsiella pneumoniae ( chen et al . 2011 ) , to a fraction of it ] provide bacteria with extraordinary ability to redistribute an external shear force along several attachment organelles in such a way that no single pilus will experience a substantially greater force than the others ; this is beneficial for the ability of a bacterium to sustain substantial rinsing flows . to investigate the possibility of compromising the biomechanical properties of p pili , primarily by reducing the force within region ii of the force versus elongation curve , we used papd , the chaperone involved in the biogenesis of p pili in vivo , as a test substance . the mechanism of assembly of different types of pili has been well studied ( vetsch et al . 2006 ; sauer et al . 1999 ; remaut et al . 2006 ; rose et al . 2008 ) , and it is known that papd is responsible for folding of the subunits and for their transport to the usher for assembly of p pili ( barnhart et al . thus , in the formed pili the binding site of papd to pili subunits , in what is known from the donor strand exchange mechanism , is already occupied by the neighboring subunit . however , the crystal structure of a protein complex consisting of two papa molecules interacting with each other via donor strand complementation suggests that the papd molecule can still interact with this type of complex ( verger et al . 3overall 3d structure of the papd / papa / papa complex ( pdb i d 2uy6 ) : papd ( blue ) , donor strand complemented ( dsc ) papa ( purple ) , and donor strand exchanged ( dce ) papa ( green ) are shown in ribbon representation with -stands as arrows and -helixes indicated as cylinders . the f--stand of papd ( amino acids 107113 ) is indicated by an arrow , as is the c - terminal -stand of dscpapa ( amino acids 152162 ) overall 3d structure of the papd / papa / papa complex ( pdb i d 2uy6 ) : papd ( blue ) , donor strand complemented ( dsc ) papa ( purple ) , and donor strand exchanged ( dce ) papa ( green ) are shown in ribbon representation with -stands as arrows and -helixes indicated as cylinders . the f--stand of papd ( amino acids 107113 ) is indicated by an arrow , as is the c - terminal -stand of dscpapa ( amino acids 152162 ) the donor strand complemented papa subunit is drawn in purple and referred to as dscpapa . the nte of this unit is inserted into the donor strand exchanged papa subunit , which , according to the donor strand complementation mechanism , is referred to as dsepapa , which is drawn in green . beside the well - documented donor strand complementation between the two papa subunits , an additional presumptive interaction has been observed in the papa / papa / papd complex . the c - terminal -strand of dscpapa and the f--strand of the papd [ amino acids 107113 ( holmgren and branden 1989 ) ] are located in close proximity and can probably interact with each other ( indicated by black arrows in fig . possible interaction with papa subunits in the pili rod led to the choice of papd as the test molecule for this proof - of - principle demonstration of compromise of the biomechanical properties of the pili rod in this work . 2 , helix - like pili have a unique and complex force versus elongation response . normally , a single pilus has a characteristic force plateau in the force versus elongation response ( region ii ) that is flat and repeatable ( which originates from sequential uncoiling of the tt interactions in the helix ) , whereas region iii has a sigmoidal shape ( which originates from entropy - driven transitions of the head - to - tail bonds opening and closing in random order ) ( jass et al . 2004 ; fllman et al . during retraction , and for sufficiently low elongation and retraction speeds , the response is fully reversible [ with the exception of a dip in the transition from region iii to ii , which is considered to be caused by the lack of a nucleation kernel for formation of the first turn of the quaternary structure during contraction ( axner et al . 2006a , 2009 ; lugmaier et al . 2008 ) ] . to assess the effect of papd on the compliance of the pilus rod , force versus elongation data were collected from eight series of seven replicate elongations and retractions of p pili in the presence of different concentrations of papd and control proteins . it was found that in the presence of papd , the force versus elongation data and the contraction response of p pili were altered markedly . as can be seen in fig . 4 , which shows a typical force versus contraction response after the first elongation in the presence of papd at three different concentrations , the recoiling forces of p pili at 1.4 m ( violet line ) and 2.8 m ( green line ) of papd were slightly lower than when no papd was present ( red line ) and contained a number of recurring dips.fig . 4transition from region iii to ii during retraction of a single p pilus in the presence of papd at different concentrations . in samples with no or a low concentration of papd , contraction from an elongation of approximately 2.7 m takes place in region ii , indicating recoiling , whereas in the sample with 11.3 m papd , the force decreases towards zero monotonically ( which is reached for an elongation of approximately 1.6 m ) , indicating no recoiling transition from region iii to ii during retraction of a single p pilus in the presence of papd at different concentrations . in samples with no or a low concentration of papd , contraction from an elongation of approximately 2.7 m takes place in region ii , indicating recoiling , whereas in the sample with 11.3 m papd , the force decreases towards zero monotonically ( which is reached for an elongation of approximately 1.6 m ) , indicating no recoiling when a higher concentration of papd was used ( 5.6 m , black line , and 11.3 m , blue line ) the recoiling force dropped drastically . at 11.3 m papd the force went monotonically to zero with no characteristic plateau . at 5.6 m papd did not completely impair the recoiling of p pili , but the recoiling force was lower than for additions of 1.4 and 2.8 m papd . numerical analysis of the recoiling process during the first half of the plateau in region ii ( from 2.7 to 1.2 m in fig . 4 ) resulted in recoiling forces of 29 0.7 , 25 0.8 , 23 1.3 , 17 1.4 , and 5.5 2.2 pn for 0 , 1.4 , 2.8 , 5.6 , and 11.3 m papd , respectively ( expressed as the mean standard error of the mean ( sem ) , n = 8) . this indicates that papd significantly affects the retraction properties of the rod , suggesting that papd interacts with papa subunits in the uncoiled pili by inhibiting the ability of the latter to form tt interactions , thus preventing the pili from recoiling and contracting . this implies that in the presence of the highest papd concentration the pilus lost virtually all of its compliance . also the uncoiling force for subsequent uncoilings was found to be affected by the presence of papd . for the lowest papd concentrations , the uncoiling force decreased with the number of elongations . figure 5a shows seven repeated uncoilings of the same pilus in the presence of 2.8 m papd . in addition , from the characteristic appearance ( with a plateau ) during the first uncoiling ( red curve ) , for repeated elongations , the uncoiling curve gradually deteriorated ; the plateau became shorter and the force curve transformed to a sigmoidal shape , as is illustrated by the blue curve ( the seventh recoiling).fig . 5a seven successive elongations of a single p pilus in the presence of 2.8 m papd . whereas the response from the first elongation has the conventional plateau , indicative of sequential uncoiling , a sigmoid like response was observed for subsequent elongations , indicating the presence of entropy , which in turn originates from stochastic ( non - sequential ) opening of the tt - bonds . d force versus elongation curves for a single p pilus in the presence of 2.8 m bsa , fimc , and igfbp-5 respectively . elongation of the pilus was stopped when the pilus had become fully uncoiled and the elongation had proceeded into region iii ; this was followed by retraction , giving rise to a recoiling of the pilus . this procedure was repeated seven times on the same pilus a seven successive elongations of a single p pilus in the presence of 2.8 m papd . whereas the response from the first elongation has the conventional plateau , indicative of sequential uncoiling , a sigmoid like response was observed for subsequent elongations , indicating the presence of entropy , which in turn originates from stochastic ( non - sequential ) opening of the tt - bonds . b d force versus elongation curves for a single p pilus in the presence of 2.8 m bsa , fimc , and igfbp-5 respectively . elongation of the pilus was stopped when the pilus had become fully uncoiled and the elongation had proceeded into region iii ; this was followed by retraction , giving rise to a recoiling of the pilus . this procedure was repeated seven times on the same pilus the results from analysis of the uncoiling force ( the force plateau ) in the presence of papd and control proteins and the shortening of the plateau are summarized in table 1 , in which each entry represents the average of eight series of seven uncoiling cycles . because pili length can vary , the lengths of the force plateaus were normalized to that of the first elongation in each set of measurements . any normalized length below unity thus indicates shortening of region ii.table 1uncoiling forces in the presence of 2.8 m papd and control proteins during seven repeated elongations , and the relative lengths of the force plateau compared with the length of the plateau during the first elongation cycle ( mean sem , n = 8)cycleforceplateau , pnrelative length of the plateaupapdcontrolpapdcontrol128 1.428 0.311228 1.830 0.50.83 0.061327 1.828 0.50.61 0.051426 1.828 0.40.56 0.031522 1.127 0.30.44 0.041621 1.426 0.30.44 0.041720 1.427 0.40.39 0.061 uncoiling forces in the presence of 2.8 m papd and control proteins during seven repeated elongations , and the relative lengths of the force plateau compared with the length of the plateau during the first elongation cycle ( mean sem , n = 8) the sigmoidal shape signifies the occurrence of entropic processes , which indicates that the tt interactions can open and close in a random order . this suggests that consecutive turns in the helical structure of the pilus rod no longer interact with each other via multiple tt interactions per turn , which is the underlying cause of the sequential unfolding of the quaternary structure ; it reveals that the turns , instead , interact by a single tt interaction per turn , which then enables opening of the helix - like coil in a random order , caused by blocking of the tt interactions by papd . 5a , the form of the elongation curves is not fully sigmoidal ; instead they often have a plateau ( or a slightly sloping plateau ) in their center . this originates from sequential opening of tt interactions from the part of the pilus in which the consecutive turns still bind by several interactions . the relative length of the plateau , given in table 1 , thus represents how large a fraction of all the turns of the pili can still bind by multiple tt interactions . conversely , the complementary length , i.e. one minus the relative length , indicates how large a fraction of all the turns bind solely by a single tt interaction . table 1 indicates that the number of turns that bind solely by a single tt interaction increased as a function of the number of elongation retraction cycles the pilus was exposed to . after three elongation retraction cycles ( i.e. for the 4th elongation ) under the prevailing conditions , approximately half of the turns interacted with their neighboring turns by one tt interaction only . after six elongation retraction cycles ( i.e. for the 7th elongation ) most , approximately 60% , of the turns bound solely by one tt interaction . this is in agreement with the assumption that the binding of papd is cumulative . a similar change in the shape of the response with the number of elongations was observed during the recoiling process ( data not shown ) . moreover , the number of dips in the force versus contraction plot was found to increase with the number of elongations . spontaneous dips in the recoiling curves have been observed previously and attributed to spontaneous miscoilings ( andersson et al . in addition , the effect of repeated elongations for low papd concentrations was comparable that of a single elongation at a higher papd concentration . this implies that the effect of papd increased cumulatively with both the concentration of papd and the number of elongations ( the latter is illustrated by the data presented in table 1 ) . furthermore , it was found that papd only affected linearized ( stretched or uncoiled ) pili ; the first uncoiling curve in a series of elongations was always unaffected by its presence , even after an hour of exposure of unstretched ( coiled ) pili to papd . the fact that papd only bound to papa units when the rod had been uncoiled indicates that it exclusively binds to the inner parts of the helix - like rod , e.g. as was indicated by fig . in addition , the cumulative effect indicates that the interaction between papd and uncoiled p pili is strong enough for papd to remain bound to the pili during repeated elongation and retraction cycles . to verify the recoiling inhibiting effect of papd , bsa , igfbp-5 , and fimc the molecular weight of igfbp-5 is similar to that of papd but with a secondary structure that is dissimilar to that of papd . bsa has another secondary structure and a molecular weight that is almost twice that of papd ( hung et al . the third control protein , fimc , is a chaperone that is involved in the biogenesis of type 1 pili in e. coli in the same way as papd mediates the biogenesis of p pili . fimc has 50% amino acid sequence homology with papd , and the two proteins have similar 3d structures and molecular weights ( jones et al . 1993 ) . as can be seen by comparison of a single pilus in the presence and absence of 2.8 m control proteins ( fig . 2 ) , it was found that subsequent elongation of p pili was only very slightly affected by the presence of control proteins . it was , moreover , found that , in the presence of these control proteins , the force versus extension / contraction response of p pili remained virtually unchanged after the first elongation in a series of seven repeated elongations ( table 1 ) , the number of dips in the recoiling curves was found to be similar during the first and the last extension cycles indicating that the effect of control proteins is weaker than that of papd and not cumulative . a much higher concentration of control proteins ( 11.3 m ) resulted in detectable disturbances of the recoiling process occasional dips in the recoiling curves resulting in a temporary reduction in uncoiling force . however , the uncoiling force reverted to the normal level during repeated elongations ( data not shown ) . a schematic illustration of the recoiling inhibitor mechanism from a single bacterium that bind by two helix - like pili is shown in fig . 6 . to mimic the effect of mechanical stress of urine flow in vivo the six horizontal panel pairs ( 16 ) represent a series of consecutive flow conditions : no flow ( panel pair 1 ) , high flow ( 2 ) , low flow ( 3 ) , and high flow ( illustrated by three panel pairs , 46 ) . the high and low flow conditions represent cases where the force to which the bacterium is exposed , ftot , is slightly larger than twice the uncoiling force ( fuc ) and slightly smaller than the uncoiling force , i.e. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f_{\text{tot } } > 2f_{\text{uc } } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f_{\text{tot } } < f_{\text{uc } } $ $ \end{document } , respectively . the low flow condition ( 3 ) can even represent a case with no flow . the first and the second columns of the panels represent pili in the absence and presence of recoiling inhibitor , respectively.fig . 6schematic illustration of the recoiling inhibitor mechanism on helix - like pili . a bacterium is attached to the host tissue by two identical pili adhering by specific adhesin the helix - like pili are depicted as springs , with their length approximately representing their degree of elongation . the arrows indicate the force each pilus sustains schematic illustration of the recoiling inhibitor mechanism on helix - like pili . a bacterium is attached to the host tissue by two identical pili adhering by specific adhesin the helix - like pili are depicted as springs , with their length approximately representing their degree of elongation . the arrows indicate the force each pilus sustains in the absence of flow , the bacterium is initially attached by both pili , neither of them elongated ( panels a1 and b1 ) . when the bacterium is exposed to a shear flow , it will rotate ( as is indicated in the subsequent panels ) , so the uppermost pili will be elongated the most . for high flow conditions ( panels a2 and b2 ) as long as they do so , the two pili will take up the same force , half of the force to which they are exposed , i.e. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f_{\text{upper } } = f_{\text{lower } } = f_{\text{tot } } /2 $ $ \end{document } , which is a force that the specific adhesin receptor interaction is presumed to be able to withstand for a substantial time ( longer than the force exposure time ) , so the bacterium will remain attached . in the absence of a recoiling inhibitor ( the first column ) , when the flow decreases ( panel a3 ) the two pili will retract until both have contracted into region i. during subsequent elongation ( panels a4a6 ) , the two pili will again elongate into region ii in a manner similar to that in panel a2 , i.e. by sharing the force equally , i.e. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f_{\text{upper } } = f_{\text{lower } } = f_{\text{tot } } /2 $ $ \end{document } , which is a force the specific adhesin receptor interaction can withstand for the duration of the force exposure , so the bacterium will stay attached . when a recoiling inhibitor is present ( the second column ) , on the other hand , the compliance of the pili will be compromised as a result of linearization of the pilus ( panel b2 ) . however , there will be no indication of this during the first elongation cycle ( because the recoiling inhibitor only attaches to the uncoiled part of the pilus ) ; the reduced compliance will affect the force response solely during subsequent refolding , and more so for the pilus that has been extended the most , which here is assumed to be the upper one ( because this has enabled binding of more of the recoiling inhibitor ) . under the simplified assumption that the upper pilus loses most of its compliance , as was the case for the highest concentrations of papd in fig . 4 , whereas the lower one is affected less , the upper pilus will take significantly less force than the lower ( in the limiting case when it has lost all of its compliance , it will take no force , whereas the lower one will take all , i.e. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f_{\text{upper } } \approx 0 $ $ \end{document } whereas \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f_{\text{lower } } \approx f_{\text{tot } } $ $ \end{document } ) and so will retract less than the lower pilus during the subsequent low flow exposure ( panel b3 ) . under the condition that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f_{\text{tot } } < f_{\text{uc } } $ $ \end{document } , the lower pilus will retract all the way to region i. when the bacterium is again exposed to a higher flow , the upper pilus will take virtually no force ( because of its compromised compliance ) , whereby the lower one will sustain virtually all of the force ( panel b4 ) . this implies that the adhesin receptor bond of the lower pilus will be exposed to a significantly larger force , close to ftot ( > 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f_{\text{uc } } $ $ \end{document } ) , which is a force at which the adhesin receptor bond will rapidly break ( panel b5 ) . when this pilus has detached , the upper pilus will be exposed to the entire force , and this hence , in the presence of a coilicide , the bacterium becomes detached from the host tissue . taken together , our findings suggest that the presence of a substance that can bind to the inner parts of an elongated helix - like pilus as a coilicide should also affect the attachment lifetime of piliated bacteria exposed to urinary flows . as is schematically indicated in fig . ( by the six pairs of panels that illustrate different consecutive flow conditions ) , for a bacterium that becomes attached to a host surface with multiple pili ( in this case illustrated by two pili ) , a force that is slightly larger than twice the pili uncoiling force will , in the absence of any coilicide ( depicted by the left column of panels ) , be distributed fairly evenly among the two pili ( exactly evenly as long as both pili elongate slowly in region ii ) , also for a multitude of subsequent elongations and retraction cycles ( a2a4 ) . as has been discussed previously ( bjrnham and axner 2009 ) , this provides the bacteria with a capability to distribute an external force among a mulitude of pili , which implies they can withstand strong rinsing flows , which , in turn , facilitates adherence and colonization processes critical for virulence . however , as can be seen by a comparison of the two columns of fig . 6 , in which the second column illustrates the response of a bacterium in the presence of a coilicide , the situation changes drastically when the bacterium is exposed to a coilicide . although the first elongation ( panel b2 ) is identical to that without papd present ( panel a2 ) , subsequent recoiling will be compromised ( as is schematically indicated for one of the two pili in panel b3 ) . when a coilicide binds permanently to the inner parts of the helix - like rod , subsequent elongation will be affected . because one of the pili has lost its compliance , this pilus can no longer withstand substantial force , the other ( less compromised , or non - compromised ) pilus will be exposed to most of the force ( panel b4 ) . this implies that adhesin at the tip of this pilus will be exposed to a significantly larger force , so it will rapidly become detached from its receptor [ under the condition it is attached by a slip bond ( bjrnham and axner 2009 ) ] , which in turn quickly leads to detachment of the entire bacterium ( panels b5b6 ) . in this study , we have investigated whether it is possible to compromise the compliance of helix - like pili by the use of a so - called coilicide . the test substance in this work was papd , because it has been found to bind to the inner parts of a linearized helix - form p pili rod . the experiments showed that although the first uncoiling of a given pilus was not affected by the substance investigated , subsequent uncoiling and recoiling ( i.e. after the first elongation - and - contraction cycle of the pili ) were affected by the presence of papd . moreover , use of control substances showed that this effect was obtained specifically in the presence of papd ( and not in the presence of the different tested control proteins ) . at the highest papd concentration used the recoiling force dropped to zero , implying the pilus lost all ability to contract , which in turn affected subsequent elongations . at lower papd concentrations , the plateau during elongations that constitutes region ii was transformed into a sigmoidal response , similar to the response of region iii , which indicates that elongation in region ii was predominantly governed by entropic processes , indicative of a bond opening / closure process that takes place in a randomized order . this behavior suggests that the consecutive turns of subunits no longer form a multitude of parallel interactions . our results also show that papd can affect the pilus response even at low concentrations ( a single pilus consists of ~1,000 papa subunits and , for the papd concentrations used in this work the estimated number of papd molecules per single pilus is only a few ) . all this indicates that the presence of papd can significantly affect the compliance of p pili and that it can do so even at low concentrations . it has thus been demonstrated that the biomechanical properties of helix - like pili can be compromised by a coilicide . it is our expectation that this can be demonstrated also with other types of substance than papd and that coilicides can be found for other types of pili also ; because different kinds of helix - like pili have a similar uncoiling mechanism , it is therefore feasible that one can affect many other types pili in a similar manner . the results of this study thus provide a new strategy in the search for novel antibacterial drugs small synthetic molecules that can compromise the biomechanical properties of bacterial pili . in contrast to previously known pilicides that inhibit the chaperone usher interaction during pili biogenesis , it is proposed the coilicides inhibit the uncoiling / recoiling process of already assembled and surface - located pili . if the ability of bacteria to redistribute external shear forces is compromised , they will no longer be able to resist urine flow , and they will , therefore , presumably be swiftly removed from the host . it is possible that a substance with pili - compliance - compromising ability either can serve as an alternative to existing antibiotics in the future or can be part of a broader strategy , also including substances that can compromise other parts of the adhesion process . this study also demonstrates that fmot is not only a technique useful for redistribution of micrometer - sized objects and force measurements , but also for active manipulation of individual macromolecules , serving as an important tool in the search for novel anti - virulence targets , for example the inner parts of helix - like pili .

gram - negative bacteria often initiate their colonization by use of extended attachment organelles , so called pili . when exposed to force , the rod of helix - like pili has been found to be highly extendable , mainly attributed to uncoiling and recoiling of its quaternary structure . this provides the bacteria with the ability to redistribute an external force among a multitude of pili , which enables them to withstand strong rinsing flows , which , in turn , facilitates adherence and colonization processes critical to virulence . thus , pili fibers are possible targets for novel antibacterial agents . by use of a substance that compromises compliance of the pili , the ability of bacteria to redistribute external forces can be impaired , so they will no longer be able to resist strong urine flow and thus be removed from the host . it is possible such a substance can serve as an alternative to existing antibiotics in the future or be a part of a multi - drug . in this work we investigated whether it is possible to achieve this by targeting the recoiling process . the test substance was purified papd . the effect of papd on the compliance of p pili was assessed at the single organelle level by use of force - measuring optical tweezers . we showed that the recoiling process , and thus the biomechanical compliance , in particular the recoiling process , can be impaired by the presence of papd . this leads to a new concept in the search for novel drug candidates combating uropathogenic bacterial infectionscoilicides , targeting the subunits of which the pilus rod is composed .

Introduction Materials and methods Culture of bacteria PapD and control proteins Force-measurement procedure Results and discussion Conclusions

its ability to infect , is correlated with its ability to adhere to tissue , and , in particular , to its capability to withstand forces from rinsing flows ( ofek et al . p pili , a prototype helix - like pili , are produced by uropathogenic escherichia coli ( upec ) strains and are predominantly associated with kidney infection ( pyelonephritis ) ( sauer et al . as is further alluded to below , the ability of such attached bacteria to withstand forces caused by rinsing flows is not only given by the properties of the adhesin receptor interactions , but is also strongly affected by the biomechanical properties of these organelles ( duncan et al . it has been found that the compliance of helix - like pili ( i.e. their response to force ) , and thus their ability to assist in the adhesion process , depends strongly on the quaternary structure of the pilus . these pili have an intricate and extraordinary force versus elongation response that consists of a combination of a constant elongation force ( in the so - called region ii ) , originating from sequential uncoiling of the helix - like structure , and a sigmoidal pseudo - elastic response ( region iii ) , caused by a conformational change of the head - to - tail interaction between the subunits of pili that takes place in a random order ( jass et al . 2005 ) , have been regarded as being of special importance to the ability of a piliated bacterium to remain attached to the host tissue ( despite being exposed to a substantial external force ) by redistributing an external shear force among a multitude of pili ( bjrnham et al . presumably , they are a major reason why the ability of attached bacteria to withstand forces caused by urine flow is strongly affected by the properties of the pili shaft per se ( duncan et al . this implies that the pilus rod and the tip - associated adhesin are both important in the ability of bacteria to withstand strong rinsing forces ( bjrnham and axner 2009 ) . in this work we investigated whether it is possible to affect the biomechanical properties of helix - like pili by using a substance that can bind to an elongated pilus at the single organelle level , primarily compromising the uncoiling recoiling process . we show , by use of force - measuring optical tweezers ( fmot ) , that the biomechanical compliance of p pili can be impaired without breaking the backbone of the pilus . thus , the pili rod can be regarded as a new target for novel drug candidates combating uropathogenic bacterial infections . for pilicide concentrations below that required for total inhibition of pili , the bacteria express fewer pili , in a concentration - dependent manner , although with the same biomechanical properties as for untreated bacteria ( berg et al . briefly , a free - floating bacterium was trapped at low power by use of a focused laser beam and attached to a poly - l - lysine - coated bead fixed on the cover slip . for pilicide concentrations below that required for total inhibition of pili , the bacteria express fewer pili , in a concentration - dependent manner , although with the same biomechanical properties as for untreated bacteria ( berg et al . briefly , a free - floating bacterium was trapped at low power by use of a focused laser beam and attached to a poly - l - lysine - coated bead fixed on the cover slip . 2006b ) ] interactions of the quaternary structure of the pilus occurs at constant force ; this is often referred to as an uncoiling of the pilus rod . the breaking of interactions in this region is sequential , caused by the fact that each turn of the quaternary structure of pili consists of ~3 subunits , each of which interacts with a corresponding subunit in the neighboring turn . moreover , inasmuch as each tt interaction can open and close a number of times , uncoiling of the helix - like structure has been found to be fully reversible , which , in turn , implies that the pili can rapidly and automatically contract after being unfolded by an external force ( fllman et al . as was alluded to above , these unique biomechanical properties [ together with the fact that the uncoiling process of the rod is assumed to reduce the persistence length of the pili substantially , from being similar to the length of the pili , as demonstrated for type 3 pili of klebsiella pneumoniae ( chen et al . 2011 ) , to a fraction of it ] provide bacteria with extraordinary ability to redistribute an external shear force along several attachment organelles in such a way that no single pilus will experience a substantially greater force than the others ; this is beneficial for the ability of a bacterium to sustain substantial rinsing flows . to investigate the possibility of compromising the biomechanical properties of p pili , primarily by reducing the force within region ii of the force versus elongation curve , we used papd , the chaperone involved in the biogenesis of p pili in vivo , as a test substance . 2008 ) , and it is known that papd is responsible for folding of the subunits and for their transport to the usher for assembly of p pili ( barnhart et al . thus , in the formed pili the binding site of papd to pili subunits , in what is known from the donor strand exchange mechanism , is already occupied by the neighboring subunit . possible interaction with papa subunits in the pili rod led to the choice of papd as the test molecule for this proof - of - principle demonstration of compromise of the biomechanical properties of the pili rod in this work . during retraction , and for sufficiently low elongation and retraction speeds , the response is fully reversible [ with the exception of a dip in the transition from region iii to ii , which is considered to be caused by the lack of a nucleation kernel for formation of the first turn of the quaternary structure during contraction ( axner et al . to assess the effect of papd on the compliance of the pilus rod , force versus elongation data were collected from eight series of seven replicate elongations and retractions of p pili in the presence of different concentrations of papd and control proteins . it was found that in the presence of papd , the force versus elongation data and the contraction response of p pili were altered markedly . 4 , which shows a typical force versus contraction response after the first elongation in the presence of papd at three different concentrations , the recoiling forces of p pili at 1.4 m ( violet line ) and 2.8 m ( green line ) of papd were slightly lower than when no papd was present ( red line ) and contained a number of recurring dips.fig . in samples with no or a low concentration of papd , contraction from an elongation of approximately 2.7 m takes place in region ii , indicating recoiling , whereas in the sample with 11.3 m papd , the force decreases towards zero monotonically ( which is reached for an elongation of approximately 1.6 m ) , indicating no recoiling transition from region iii to ii during retraction of a single p pilus in the presence of papd at different concentrations . in samples with no or a low concentration of papd , contraction from an elongation of approximately 2.7 m takes place in region ii , indicating recoiling , whereas in the sample with 11.3 m papd , the force decreases towards zero monotonically ( which is reached for an elongation of approximately 1.6 m ) , indicating no recoiling when a higher concentration of papd was used ( 5.6 m , black line , and 11.3 m , blue line ) the recoiling force dropped drastically . this indicates that papd significantly affects the retraction properties of the rod , suggesting that papd interacts with papa subunits in the uncoiled pili by inhibiting the ability of the latter to form tt interactions , thus preventing the pili from recoiling and contracting . this implies that in the presence of the highest papd concentration the pilus lost virtually all of its compliance . also the uncoiling force for subsequent uncoilings was found to be affected by the presence of papd . whereas the response from the first elongation has the conventional plateau , indicative of sequential uncoiling , a sigmoid like response was observed for subsequent elongations , indicating the presence of entropy , which in turn originates from stochastic ( non - sequential ) opening of the tt - bonds . this procedure was repeated seven times on the same pilus a seven successive elongations of a single p pilus in the presence of 2.8 m papd . whereas the response from the first elongation has the conventional plateau , indicative of sequential uncoiling , a sigmoid like response was observed for subsequent elongations , indicating the presence of entropy , which in turn originates from stochastic ( non - sequential ) opening of the tt - bonds . this procedure was repeated seven times on the same pilus the results from analysis of the uncoiling force ( the force plateau ) in the presence of papd and control proteins and the shortening of the plateau are summarized in table 1 , in which each entry represents the average of eight series of seven uncoiling cycles . any normalized length below unity thus indicates shortening of region ii.table 1uncoiling forces in the presence of 2.8 m papd and control proteins during seven repeated elongations , and the relative lengths of the force plateau compared with the length of the plateau during the first elongation cycle ( mean sem , n = 8)cycleforceplateau , pnrelative length of the plateaupapdcontrolpapdcontrol128 1.428 0.311228 1.830 0.50.83 0.061327 1.828 0.50.61 0.051426 1.828 0.40.56 0.031522 1.127 0.30.44 0.041621 1.426 0.30.44 0.041720 1.427 0.40.39 0.061 uncoiling forces in the presence of 2.8 m papd and control proteins during seven repeated elongations , and the relative lengths of the force plateau compared with the length of the plateau during the first elongation cycle ( mean sem , n = 8) the sigmoidal shape signifies the occurrence of entropic processes , which indicates that the tt interactions can open and close in a random order . this suggests that consecutive turns in the helical structure of the pilus rod no longer interact with each other via multiple tt interactions per turn , which is the underlying cause of the sequential unfolding of the quaternary structure ; it reveals that the turns , instead , interact by a single tt interaction per turn , which then enables opening of the helix - like coil in a random order , caused by blocking of the tt interactions by papd . 2 ) , it was found that subsequent elongation of p pili was only very slightly affected by the presence of control proteins . it was , moreover , found that , in the presence of these control proteins , the force versus extension / contraction response of p pili remained virtually unchanged after the first elongation in a series of seven repeated elongations ( table 1 ) , the number of dips in the recoiling curves was found to be similar during the first and the last extension cycles indicating that the effect of control proteins is weaker than that of papd and not cumulative . a schematic illustration of the recoiling inhibitor mechanism from a single bacterium that bind by two helix - like pili is shown in fig . 6schematic illustration of the recoiling inhibitor mechanism on helix - like pili . the arrows indicate the force each pilus sustains schematic illustration of the recoiling inhibitor mechanism on helix - like pili . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f_{\text{upper } } = f_{\text{lower } } = f_{\text{tot } } /2 $ $ \end{document } , which is a force that the specific adhesin receptor interaction is presumed to be able to withstand for a substantial time ( longer than the force exposure time ) , so the bacterium will remain attached . when a recoiling inhibitor is present ( the second column ) , on the other hand , the compliance of the pili will be compromised as a result of linearization of the pilus ( panel b2 ) . however , there will be no indication of this during the first elongation cycle ( because the recoiling inhibitor only attaches to the uncoiled part of the pilus ) ; the reduced compliance will affect the force response solely during subsequent refolding , and more so for the pilus that has been extended the most , which here is assumed to be the upper one ( because this has enabled binding of more of the recoiling inhibitor ) . this implies that the adhesin receptor bond of the lower pilus will be exposed to a significantly larger force , close to ftot ( > 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f_{\text{uc } } $ $ \end{document } ) , which is a force at which the adhesin receptor bond will rapidly break ( panel b5 ) . when this pilus has detached , the upper pilus will be exposed to the entire force , and this hence , in the presence of a coilicide , the bacterium becomes detached from the host tissue . taken together , our findings suggest that the presence of a substance that can bind to the inner parts of an elongated helix - like pilus as a coilicide should also affect the attachment lifetime of piliated bacteria exposed to urinary flows . ( by the six pairs of panels that illustrate different consecutive flow conditions ) , for a bacterium that becomes attached to a host surface with multiple pili ( in this case illustrated by two pili ) , a force that is slightly larger than twice the pili uncoiling force will , in the absence of any coilicide ( depicted by the left column of panels ) , be distributed fairly evenly among the two pili ( exactly evenly as long as both pili elongate slowly in region ii ) , also for a multitude of subsequent elongations and retraction cycles ( a2a4 ) . as has been discussed previously ( bjrnham and axner 2009 ) , this provides the bacteria with a capability to distribute an external force among a mulitude of pili , which implies they can withstand strong rinsing flows , which , in turn , facilitates adherence and colonization processes critical for virulence . 6 , in which the second column illustrates the response of a bacterium in the presence of a coilicide , the situation changes drastically when the bacterium is exposed to a coilicide . because one of the pili has lost its compliance , this pilus can no longer withstand substantial force , the other ( less compromised , or non - compromised ) pilus will be exposed to most of the force ( panel b4 ) . this implies that adhesin at the tip of this pilus will be exposed to a significantly larger force , so it will rapidly become detached from its receptor [ under the condition it is attached by a slip bond ( bjrnham and axner 2009 ) ] , which in turn quickly leads to detachment of the entire bacterium ( panels b5b6 ) . in this study , we have investigated whether it is possible to compromise the compliance of helix - like pili by the use of a so - called coilicide . the test substance in this work was papd , because it has been found to bind to the inner parts of a linearized helix - form p pili rod . the experiments showed that although the first uncoiling of a given pilus was not affected by the substance investigated , subsequent uncoiling and recoiling ( i.e. after the first elongation - and - contraction cycle of the pili ) were affected by the presence of papd . moreover , use of control substances showed that this effect was obtained specifically in the presence of papd ( and not in the presence of the different tested control proteins ) . at the highest papd concentration used the recoiling force dropped to zero , implying the pilus lost all ability to contract , which in turn affected subsequent elongations . all this indicates that the presence of papd can significantly affect the compliance of p pili and that it can do so even at low concentrations . it has thus been demonstrated that the biomechanical properties of helix - like pili can be compromised by a coilicide . it is our expectation that this can be demonstrated also with other types of substance than papd and that coilicides can be found for other types of pili also ; because different kinds of helix - like pili have a similar uncoiling mechanism , it is therefore feasible that one can affect many other types pili in a similar manner . the results of this study thus provide a new strategy in the search for novel antibacterial drugs small synthetic molecules that can compromise the biomechanical properties of bacterial pili . if the ability of bacteria to redistribute external shear forces is compromised , they will no longer be able to resist urine flow , and they will , therefore , presumably be swiftly removed from the host . it is possible that a substance with pili - compliance - compromising ability either can serve as an alternative to existing antibiotics in the future or can be part of a broader strategy , also including substances that can compromise other parts of the adhesion process . this study also demonstrates that fmot is not only a technique useful for redistribution of micrometer - sized objects and force measurements , but also for active manipulation of individual macromolecules , serving as an important tool in the search for novel anti - virulence targets , for example the inner parts of helix - like pili .

the rod of p pili consists of more than a thousand papa subunits , assembled into a helical right - handed structure with 3.3 subunits per turn ( gong and makowski 1992 ; bullitt and makowski 1995 ) , ending in a linear tip structure that anchors the papg adhesin at the distal end ( jacob - dubuisson et al . as is further alluded to below , the ability of such attached bacteria to withstand forces caused by rinsing flows is not only given by the properties of the adhesin receptor interactions , but is also strongly affected by the biomechanical properties of these organelles ( duncan et al . these pili have an intricate and extraordinary force versus elongation response that consists of a combination of a constant elongation force ( in the so - called region ii ) , originating from sequential uncoiling of the helix - like structure , and a sigmoidal pseudo - elastic response ( region iii ) , caused by a conformational change of the head - to - tail interaction between the subunits of pili that takes place in a random order ( jass et al . 2005 ) , have been regarded as being of special importance to the ability of a piliated bacterium to remain attached to the host tissue ( despite being exposed to a substantial external force ) by redistributing an external shear force among a multitude of pili ( bjrnham et al . this implies that the pilus rod and the tip - associated adhesin are both important in the ability of bacteria to withstand strong rinsing forces ( bjrnham and axner 2009 ) . it has been suggested that pilicides inhibit the chaperone usher interaction , thus preventing the essential event in which the n - terminus of the usher ( a membrane protein involved in the pili assembly ) recognizes and binds to incoming chaperone subunit complexes ( pinkner et al . in this work we investigated whether it is possible to affect the biomechanical properties of helix - like pili by using a substance that can bind to an elongated pilus at the single organelle level , primarily compromising the uncoiling recoiling process . for pilicide concentrations below that required for total inhibition of pili , the bacteria express fewer pili , in a concentration - dependent manner , although with the same biomechanical properties as for untreated bacteria ( berg et al . to remove pilicide possibly present on the surface of the bacteria , the bacteria were washed three times by dilution ( 20,000-fold ) with dulbecco s phosphate - buffered saline ( dpbs , 10 mm phosphate , 130 mm nacl , ph 7.0 , sigma - aldrich , schnelldorf , germany ) just before use in force measurements . 1afm micrographs of hb101/ppap5 cells expressing p pili : a bacterium cultured under normal conditions , b chemical structure of pilicide 1 , c bacterium cultured on a plate containing 2.2 mm pilicide afm micrographs of hb101/ppap5 cells expressing p pili : a bacterium cultured under normal conditions , b chemical structure of pilicide 1 , c bacterium cultured on a plate containing 2.2 mm pilicide papd and fimc , periplasmic chaperones from e. coli , were expressed and purified as described elsewhere ( lindberg et al . for pilicide concentrations below that required for total inhibition of pili , the bacteria express fewer pili , in a concentration - dependent manner , although with the same biomechanical properties as for untreated bacteria ( berg et al . to remove pilicide possibly present on the surface of the bacteria , the bacteria were washed three times by dilution ( 20,000-fold ) with dulbecco s phosphate - buffered saline ( dpbs , 10 mm phosphate , 130 mm nacl , ph 7.0 , sigma - aldrich , schnelldorf , germany ) just before use in force measurements . 1afm micrographs of hb101/ppap5 cells expressing p pili : a bacterium cultured under normal conditions , b chemical structure of pilicide 1 , c bacterium cultured on a plate containing 2.2 mm pilicide afm micrographs of hb101/ppap5 cells expressing p pili : a bacterium cultured under normal conditions , b chemical structure of pilicide 1 , c bacterium cultured on a plate containing 2.2 mm pilicide papd and fimc , periplasmic chaperones from e. coli , were expressed and purified as described elsewhere ( lindberg et al . moreover , inasmuch as each tt interaction can open and close a number of times , uncoiling of the helix - like structure has been found to be fully reversible , which , in turn , implies that the pili can rapidly and automatically contract after being unfolded by an external force ( fllman et al . as was alluded to above , these unique biomechanical properties [ together with the fact that the uncoiling process of the rod is assumed to reduce the persistence length of the pili substantially , from being similar to the length of the pili , as demonstrated for type 3 pili of klebsiella pneumoniae ( chen et al . 2011 ) , to a fraction of it ] provide bacteria with extraordinary ability to redistribute an external shear force along several attachment organelles in such a way that no single pilus will experience a substantially greater force than the others ; this is beneficial for the ability of a bacterium to sustain substantial rinsing flows . to investigate the possibility of compromising the biomechanical properties of p pili , primarily by reducing the force within region ii of the force versus elongation curve , we used papd , the chaperone involved in the biogenesis of p pili in vivo , as a test substance . thus , in the formed pili the binding site of papd to pili subunits , in what is known from the donor strand exchange mechanism , is already occupied by the neighboring subunit . however , the crystal structure of a protein complex consisting of two papa molecules interacting with each other via donor strand complementation suggests that the papd molecule can still interact with this type of complex ( verger et al . 3overall 3d structure of the papd / papa / papa complex ( pdb i d 2uy6 ) : papd ( blue ) , donor strand complemented ( dsc ) papa ( purple ) , and donor strand exchanged ( dce ) papa ( green ) are shown in ribbon representation with -stands as arrows and -helixes indicated as cylinders . the f--stand of papd ( amino acids 107113 ) is indicated by an arrow , as is the c - terminal -stand of dscpapa ( amino acids 152162 ) overall 3d structure of the papd / papa / papa complex ( pdb i d 2uy6 ) : papd ( blue ) , donor strand complemented ( dsc ) papa ( purple ) , and donor strand exchanged ( dce ) papa ( green ) are shown in ribbon representation with -stands as arrows and -helixes indicated as cylinders . the f--stand of papd ( amino acids 107113 ) is indicated by an arrow , as is the c - terminal -stand of dscpapa ( amino acids 152162 ) the donor strand complemented papa subunit is drawn in purple and referred to as dscpapa . the c - terminal -strand of dscpapa and the f--strand of the papd [ amino acids 107113 ( holmgren and branden 1989 ) ] are located in close proximity and can probably interact with each other ( indicated by black arrows in fig . possible interaction with papa subunits in the pili rod led to the choice of papd as the test molecule for this proof - of - principle demonstration of compromise of the biomechanical properties of the pili rod in this work . normally , a single pilus has a characteristic force plateau in the force versus elongation response ( region ii ) that is flat and repeatable ( which originates from sequential uncoiling of the tt interactions in the helix ) , whereas region iii has a sigmoidal shape ( which originates from entropy - driven transitions of the head - to - tail bonds opening and closing in random order ) ( jass et al . during retraction , and for sufficiently low elongation and retraction speeds , the response is fully reversible [ with the exception of a dip in the transition from region iii to ii , which is considered to be caused by the lack of a nucleation kernel for formation of the first turn of the quaternary structure during contraction ( axner et al . to assess the effect of papd on the compliance of the pilus rod , force versus elongation data were collected from eight series of seven replicate elongations and retractions of p pili in the presence of different concentrations of papd and control proteins . 4 , which shows a typical force versus contraction response after the first elongation in the presence of papd at three different concentrations , the recoiling forces of p pili at 1.4 m ( violet line ) and 2.8 m ( green line ) of papd were slightly lower than when no papd was present ( red line ) and contained a number of recurring dips.fig . in samples with no or a low concentration of papd , contraction from an elongation of approximately 2.7 m takes place in region ii , indicating recoiling , whereas in the sample with 11.3 m papd , the force decreases towards zero monotonically ( which is reached for an elongation of approximately 1.6 m ) , indicating no recoiling transition from region iii to ii during retraction of a single p pilus in the presence of papd at different concentrations . in samples with no or a low concentration of papd , contraction from an elongation of approximately 2.7 m takes place in region ii , indicating recoiling , whereas in the sample with 11.3 m papd , the force decreases towards zero monotonically ( which is reached for an elongation of approximately 1.6 m ) , indicating no recoiling when a higher concentration of papd was used ( 5.6 m , black line , and 11.3 m , blue line ) the recoiling force dropped drastically . 4 ) resulted in recoiling forces of 29 0.7 , 25 0.8 , 23 1.3 , 17 1.4 , and 5.5 2.2 pn for 0 , 1.4 , 2.8 , 5.6 , and 11.3 m papd , respectively ( expressed as the mean standard error of the mean ( sem ) , n = 8) . this indicates that papd significantly affects the retraction properties of the rod , suggesting that papd interacts with papa subunits in the uncoiled pili by inhibiting the ability of the latter to form tt interactions , thus preventing the pili from recoiling and contracting . in addition , from the characteristic appearance ( with a plateau ) during the first uncoiling ( red curve ) , for repeated elongations , the uncoiling curve gradually deteriorated ; the plateau became shorter and the force curve transformed to a sigmoidal shape , as is illustrated by the blue curve ( the seventh recoiling).fig . whereas the response from the first elongation has the conventional plateau , indicative of sequential uncoiling , a sigmoid like response was observed for subsequent elongations , indicating the presence of entropy , which in turn originates from stochastic ( non - sequential ) opening of the tt - bonds . whereas the response from the first elongation has the conventional plateau , indicative of sequential uncoiling , a sigmoid like response was observed for subsequent elongations , indicating the presence of entropy , which in turn originates from stochastic ( non - sequential ) opening of the tt - bonds . this procedure was repeated seven times on the same pilus the results from analysis of the uncoiling force ( the force plateau ) in the presence of papd and control proteins and the shortening of the plateau are summarized in table 1 , in which each entry represents the average of eight series of seven uncoiling cycles . any normalized length below unity thus indicates shortening of region ii.table 1uncoiling forces in the presence of 2.8 m papd and control proteins during seven repeated elongations , and the relative lengths of the force plateau compared with the length of the plateau during the first elongation cycle ( mean sem , n = 8)cycleforceplateau , pnrelative length of the plateaupapdcontrolpapdcontrol128 1.428 0.311228 1.830 0.50.83 0.061327 1.828 0.50.61 0.051426 1.828 0.40.56 0.031522 1.127 0.30.44 0.041621 1.426 0.30.44 0.041720 1.427 0.40.39 0.061 uncoiling forces in the presence of 2.8 m papd and control proteins during seven repeated elongations , and the relative lengths of the force plateau compared with the length of the plateau during the first elongation cycle ( mean sem , n = 8) the sigmoidal shape signifies the occurrence of entropic processes , which indicates that the tt interactions can open and close in a random order . this suggests that consecutive turns in the helical structure of the pilus rod no longer interact with each other via multiple tt interactions per turn , which is the underlying cause of the sequential unfolding of the quaternary structure ; it reveals that the turns , instead , interact by a single tt interaction per turn , which then enables opening of the helix - like coil in a random order , caused by blocking of the tt interactions by papd . it was , moreover , found that , in the presence of these control proteins , the force versus extension / contraction response of p pili remained virtually unchanged after the first elongation in a series of seven repeated elongations ( table 1 ) , the number of dips in the recoiling curves was found to be similar during the first and the last extension cycles indicating that the effect of control proteins is weaker than that of papd and not cumulative . to mimic the effect of mechanical stress of urine flow in vivo the six horizontal panel pairs ( 16 ) represent a series of consecutive flow conditions : no flow ( panel pair 1 ) , high flow ( 2 ) , low flow ( 3 ) , and high flow ( illustrated by three panel pairs , 46 ) . in the absence of a recoiling inhibitor ( the first column ) , when the flow decreases ( panel a3 ) the two pili will retract until both have contracted into region i. during subsequent elongation ( panels a4a6 ) , the two pili will again elongate into region ii in a manner similar to that in panel a2 , i.e. however , there will be no indication of this during the first elongation cycle ( because the recoiling inhibitor only attaches to the uncoiled part of the pilus ) ; the reduced compliance will affect the force response solely during subsequent refolding , and more so for the pilus that has been extended the most , which here is assumed to be the upper one ( because this has enabled binding of more of the recoiling inhibitor ) . this implies that the adhesin receptor bond of the lower pilus will be exposed to a significantly larger force , close to ftot ( > 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f_{\text{uc } } $ $ \end{document } ) , which is a force at which the adhesin receptor bond will rapidly break ( panel b5 ) . taken together , our findings suggest that the presence of a substance that can bind to the inner parts of an elongated helix - like pilus as a coilicide should also affect the attachment lifetime of piliated bacteria exposed to urinary flows . ( by the six pairs of panels that illustrate different consecutive flow conditions ) , for a bacterium that becomes attached to a host surface with multiple pili ( in this case illustrated by two pili ) , a force that is slightly larger than twice the pili uncoiling force will , in the absence of any coilicide ( depicted by the left column of panels ) , be distributed fairly evenly among the two pili ( exactly evenly as long as both pili elongate slowly in region ii ) , also for a multitude of subsequent elongations and retraction cycles ( a2a4 ) . this implies that adhesin at the tip of this pilus will be exposed to a significantly larger force , so it will rapidly become detached from its receptor [ under the condition it is attached by a slip bond ( bjrnham and axner 2009 ) ] , which in turn quickly leads to detachment of the entire bacterium ( panels b5b6 ) . at lower papd concentrations , the plateau during elongations that constitutes region ii was transformed into a sigmoidal response , similar to the response of region iii , which indicates that elongation in region ii was predominantly governed by entropic processes , indicative of a bond opening / closure process that takes place in a randomized order . our results also show that papd can affect the pilus response even at low concentrations ( a single pilus consists of ~1,000 papa subunits and , for the papd concentrations used in this work the estimated number of papd molecules per single pilus is only a few ) . it is our expectation that this can be demonstrated also with other types of substance than papd and that coilicides can be found for other types of pili also ; because different kinds of helix - like pili have a similar uncoiling mechanism , it is therefore feasible that one can affect many other types pili in a similar manner . this study also demonstrates that fmot is not only a technique useful for redistribution of micrometer - sized objects and force measurements , but also for active manipulation of individual macromolecules , serving as an important tool in the search for novel anti - virulence targets , for example the inner parts of helix - like pili .

globally , diarrhoea is the second greatest cause of mortality in children aged < 5 years after pneumonia . children in low- to middle - income countries are at risk of frequent diarrhoea episodes ; the 2011 national survey in ethiopia estimated that 134% of children in the < 5 years age group had suffered an episode of diarrhoea in the preceding 2 weeks , although prevalence was > 20% in some regions . numerous infectious agents cause diarrhoea ; many of which are zoonotic , with campylobacter being one of the main bacterial causes , especially in children aged < 2 years [ 46 ] . while the organism is also frequently isolated from healthy children and adults , and morbidity appears to decline with age , adults with hiv infection are also at increased risk of campylobacter - associated diarrhoea and bacteraemia . different routes by which humans are exposed to campylobacter include ingestion of contaminated food and water , direct contact with infected animals or carcasses , from environments contaminated with animal faeces or sewage , or direct person - to - person transmission [ 4 , 7 ] . in countries with a highly developed intensive poultry industry , there is considerable epidemiological evidence that chickens are the main source of human campylobacter infection [ 810 ] . in developing countries , far less is known about the epidemiology of campylobacter , but it has been found at high levels in retail poultry meat , and strains isolated from chickens have been observed to correlate with human isolates in terms of biotype and serotype . around 83% of ethiopia 's population , currently estimated at 96 million , chicken production is an integral part of most rural families livelihoods ( an estimated two - thirds of ethiopian villagers keep poultry ) and birds are commonly kept at night on perches within the family dwelling , frequently in the kitchen [ 14 , 15 ] . in ethiopia , it has been reported that chickens and poultry meat have a higher campylobacter prevalence compared to other farm animal and meat products [ 16 , 17 ] , and exposure to pets and livestock , including chickens , has been associated with increased odds of campylobacter isolation in diarrhoeal children aged < 5 years . furthermore , in a study from egypt , campylobacter diarrhoea was more common in households where animals were present in food preparation areas . it might , therefore , be anticipated that an important route by which rural families are exposed to campylobacter is from the faeces of infected chickens , which may contaminate the shared living environment . the national poultry population in ethiopia is estimated at 493 million , and is constituted of about 97% indigenous chicken ecotypes . these birds are predominantly maintained under a traditional free - range , scavenging or semi - scavenging system with minimal inputs for housing , feeding or healthcare . despite their low production performances in terms of egg and meat output , indigenous chickens are well adapted to the local environmental conditions and fulfil a variety of social and cultural functions , in addition to being both nutritional and economic assets . in the last 20 years , poultry production in ethiopia has started to be seen as a profitable venture , and more families in urban and peri - urban areas have started to keep small- to medium - sized flocks ( about 501000 birds ) under semi - intensive management . entrepreneurs have also started to invest in the poultry industry by setting up larger flocks of exotic breeds kept under semi - intensive / intensive management , particularly in the cities of dukem , debre zeit and nazeret , close to the capital , addis ababa . efforts have also been made by the government of ethiopia since the early 1990s to boost the productivity of indigenous birds kept by poor rural families through its genetic improvement programme , based on the introduction and distribution of exotic breeds by poultry multiplication centres throughout the country . several non - governmental organizations are also involved in the distribution of intensively reared chickens to smallholder farmers . although still accounting for a small proportion of ethiopia 's poultry , this on - going shift to more intensive production using commercial breeds may alter the dynamics of zoonotic infections such as campylobacteriosis . while housing chickens may limit their exposure to environmental pathogens and reduce direct and indirect contacts between people and chickens , campylobacter is known to spread very rapidly in housed flocks and interventions to limit exposure and transmission are likely to be ineffective , impractical or unaffordable in village poultry production settings . stresses , such as heat stress , or food and water restriction , is known to be detrimental to the chicken 's immune function , altering tissue invasion of intestinal bacteria [ 24 , 25 ] . however , different types or lines of chickens show variation in both the effect of heat stress on their immune response and their ability to regulate the immune response to campylobacter . the links between management system , chicken welfare , stress and the biology of campylobacter infections remain poorly understood , but have raised concerns about how farming systems may impact on public health risk [ 2830 ] . as yet in ethiopia there has been little research examining how the changing dynamics of chicken production may have consequences for public health . the aim of this study was to investigate the impact of production settings and systems in ethiopia on the epidemiology of campylobacter , and to consider the potential implications of this evolving production system for human health in the country . therefore , the main objectives were ( i ) to determine the environmental prevalence of campylobacter in different geographical areas , including rural and peri - urban areas ; and ( ii ) to investigate whether campylobacter prevalence varied in chickens from the same area reared under different production systems . to investigate differences in campylobacter prevalence in village chickens between geographical areas , a cross - sectional study was conducted between october 2012 and april 2013 in three woredas ( administrative regions ) in the oromia region of ethiopia . woredas are further subdivided into kebeles , which are the lowest administrative unit in ethiopia , and may contain several villages or sub - areas . woredas were purposively selected based on known characteristics of their poultry production systems and bird ecotypes and , for the two rural sites , knowledge that there had been no recent poultry development programmes in the areas . horro woreda is located about 310 km west of the capital , addis ababa , and jarso is about 560 km east . over 95% of the population horro is considered an area of high agricultural potential and produces an annual food surplus , whereas , in jarso , agricultural land is limited and the area tends to have a food deficit every year , with the poor reliant on food aid to cover the food gap . debre zeit ( also called bishoftu ) is an urban centre located 47 km east of addis ababa within the ada'a woreda in the east shewa zone . sampling was conducted in four kebeles in each of the two rural districts , where over 70% of households were estimated to own chickens , but where there were no known semi - intensive or intensive poultry farms . twenty households were recruited to participate from each kebele ; names of potential participants were randomly selected from a list of farmers in each kebele , provided by local development agents . development agents are employees of the local agricultural extension service , whose role is to provide farmers with access to training , research and technologies ( http://www.moa.gov.et/policies-and-strategies ) . households were excluded from the study if they did not own at least two chickens : 30% extra additional names of potential participants were included in the random selection to allow for exclusions or non - participation . in the peri - urban area of debre zeit , five kebeles were selected for inclusion in the study , all of which contained at least one intensive chicken farm . as chicken ownership was believed to be markedly lower in this woreda , random selection of households was not considered feasible ; instead , convenience sampling of households was undertaken based on transect walks through the kebeles and identification of households which kept free - ranging scavenging chickens . in addition , in order to investigate differences in campylobacter prevalence between different production systems , 20 intensive / semi - intensive farms within the same debre zeit kebeles were purposively selected for sampling during the same time period as the cross - sectional study . sample sizes were determined based on available logistic and laboratory resources ( i.e. dna extraction kits ) between the three study sites . the unit of sampling was defined as either a backyard chicken - producing household ( i.e. a household that kept at least two chickens under free - ranging scavenging conditions , with or without feed supplementation ) or an intensive poultry farm . for the purposes of this study , an intensive farm was defined as one which kept at least 50 birds under confined conditions and provided all feed . on the day of sampling , flock information such as flock size and type was collected by interviewing the householder / farmer . flock type was broadly grouped into four categories : indigenous ecotypes ; rhode island red ( rir ) , an established multi - purpose breed , or rir hybrids ( normally rir crossed with local ecotypes , which are difficult to distinguish morphologically from pure rir birds ) ; cobb 500 , a commercial fast - growing broiler breed ; and mixed flocks , which contained both local ecotypes and pure or hybrid rir birds . for each unit , a single sample was collected from the ground by walking through the household environment where chicken faeces were concentrated in the case of the extensive system or inside the chicken shed in the more intensive system , using one disposable fabric overshoe ( boot sock ) worn over the footwear . boot socks were pre - moistened with sterile physiological saline before use , in order to allow maximum uptake of campylobacter . to avoid cross - contamination between different flocks , for each sampling a clean disposable plastic overshoe was worn to separate the footwear from the fabric boot sock . after collection , each boot - sock sample was carefully placed into a clean ziplock sterile plastic bag and kept under refrigerated conditions until processing . samples were processed within 6 h to 30 days depending on the district of collection . boot socks were processed individually in the microbiology laboratory of addis ababa university college of veterinary medicine and agriculture . each boot sock was placed in a sterile plastic stomacher bag with 200 ml sterile saline water , and the mixture was treated in a stomacher at medium speed for 1 min to release detached matter . the boot sock was then left for about 10 min at room temperature to allow the solid material to settle . without disturbing the sediment on the bottom , 1 ml of the suspension the suspension was centrifuged for 7 min at ~12 000 g. the supernatant was discarded and the bacterial dna was extracted from the pellet using a commercial dna extraction kit ( promega wizard genomic dna purification kit , usa ) . after dna extraction , 50 l of the supernatant , containing the suspended dna , was added to 450 l sterile distilled water , and stored at 4 c . samples were then exported under license to the university of liverpool , where they were analysed with a pcr assay specific for the genus campylobacter on the basis of 16s rrna gene sequences ( 16s rdna ; table 1 ) [ 35 , 36 ] . table 1.primers used in the 16s rrna pcr assay for identification of the genus campylobacter ( product size 857-bp dna)primersequence 53c412 fgga tga cac ttt tcg gag ccampr2ggc ttc atg ctc tcg agt t primers used in the 16s rrna pcr assay for identification of the genus campylobacter ( product size 857-bp dna ) isolates were confirmed and identified to species level ( c. jejuni or c. coli ) using a multiplex pcr as described by klena et al . based on differences in the lpxa gene ( table 2 ) . all primers used in this work were obtained from eurofins mwg operon ( germany ) and all pcr constituents were supplied by thermo scientific ( uk ) . table 2.primers used in the lpx gene pcr assay for campylobacter isolates speciation ( product size : c. coli 391 bp and c. jejuni 331 bp)primersequence 53lpxac . jejuniacaacttggtgacgatgttgtalpxarkk2 mcaatcatgdgcdatatgasaatahgccat primers used in the lpx gene pcr assay for campylobacter isolates speciation ( product size : c. coli 391 bp and c. jejuni 331 bp ) basic descriptive statistics , graphs and multilevel logistic regression modelling were performed using r software , using the core functions , the plotrix library , the mgcv library and the lme4 library ( r foundation for statistical computing , austria ) . a multilevel multivariable model was constructed to investigate whether area , production system or breed kept had any effect on the odds of a flock being detected as campylobacter positive . as cobb 500 chickens were only kept under intensive systems , and all other types were only kept under backyard scavenging systems , the variables breed and production system were combined to create a variable called type of flock , with four categories : intensive - cobb 500 , scavenging - rir , scavenging - mixed , and scavenging - indigenous. the functional form of the relationship between flock size ( the only continuous explanatory variable ) and the outcome was assessed using generalized additive models and suggested inclusion of a linear relationship was appropriate . backward stepwise model selection was used to identify fixed effects to be retained in the model . the full model included the presence or absence of campylobacter ( at the genus level , as detected by pcr ) as the outcome variable and the type of area ( urban or rural ) , the type of flock and the flock size variables as fixed effects . variables with a likelihood ratio test statistic p value 005 were retained in the model . the authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the helsinki declaration of 1975 , as revised in 2008 . a total of 239 boot - sock samples were collected from the three districts : 79 from horro , 80 from jarso and 80 from debre zeit . the overall campylobacter prevalence detected across the three geographical areas using pcr diagnostic techniques was 184% . pcr assay for assignment of campylobacter species was performed on all 44 campylobacter - positive samples . in total , only 16 ( 364% ) samples gave positive results using this multiplex pcr assay , and for all 16 samples a positive result was obtained for c. jejuni . in the rural horro region , all flocks sampled consisted purely of indigenous bird ecotypes , while in jarso , despite a reported absence of current distribution programmes for exotic birds in this rural region , 12 ( 15% ) of the 80 flocks sampled were made up either partially ( n = 9 ) , or entirely ( n = 3 ) of rir or rir hybrids . in the peri - urban kebeles around debre zeit , 28% of the scavenging flocks consisted partly ( n = 10 ) , or exclusively ( n = 7 ) of rir or rir hybrids . higher campylobacter prevalence was found in the peri - urban ( 463% ) compared to the rural ( 44% ) environments ( fig . the prevalence was still significantly higher in the peri - urban flocks ( 417% ) , even when the intensive flocks were excluded ( = 455 , p < 0001 ) . of the two rural areas , horro had the higher prevalence ( 89% ) of positive flocks , while no flocks were detected as positive by pcr in the jarso region . 1.proportion of flocks tested positive by pcr for campylobacter , showing location , bird and farm type . proportion of flocks tested positive by pcr for campylobacter , showing location , bird and farm type . points are scaled relative to the number of flocks of each type tested . the final multi - level multivariable model ( table 3 ) suggested that flocks in the peri - urban area were at considerably greater odds of being detected campylobacter positive [ odds ratio ( or ) 219 , 95% confidence interval ( ci ) 202350 ] , even after controlling for the type of flock . after controlling for type of area , there was insufficient evidence to reject the null hypothesis of no difference in risk between mixed exotic / indigenous and indigenous - only backyard flocks . by contrast , backyard flocks which consisted only of rir or rir hybrids and housed flocks of cobb 500 chickens were at increased risk compared to the backyard mixed flocks ( or 181 , 95% ci 181839 and or 65 , 95% ci 11387 , respectively ) . inclusion of flock size or any two - way interaction terms did not significantly improve the model ( likelihood ratio test statistic p values > 005 ) ; hence these were not included in the final model . table 3.final multi - level multivariable logistic regression model of factors associated with detection of campylobacter by pcr from environmental samples collected from chicken production sites in three regions of ethiopiavariablelevelsparameter estimates.e.or95% cip valuefixed effectsintercept4511type of arearuralreference004*peri - urban3112219202350001type of flockbackyard : mixedreference003*backyard : indigenous1108290615202backyard : rir / hybrid2912181181839001housed : cobb 50019096511387004random effectvariance estimatekebele08s.e . , standard error ; or , odds ratio ; ci , confidence interval ; rir , rhode island red.*likelihood ratio statistic p value for overall variable . final multi - level multivariable logistic regression model of factors associated with detection of campylobacter by pcr from environmental samples collected from chicken production sites in three regions of ethiopia s.e . , standard error ; or , odds ratio ; ci , confidence interval ; rir , rhode island red . the greatest risk factor identified in this study for detection of campylobacter in the environment of the sampled chicken flocks was location in the peri - urban area , where many farms are starting to intensify their production systems . campylobacter is highly prevalent in intensive production systems in developed countries , with birds often having high levels of infection , posing a public health risk . while it is therefore perhaps unsurprising that the more intensively farmed flocks in the peri - urban areas were at increased risk , it is of particular concern that the greater risk in the peri - urban area persisted despite controlling for the type of flock , suggesting increased risk in all flock types in the peri - urban area , irrespective of breed and production type . although the high levels of campylobacter in intensive and semi - intensive flocks are clearly of concern for farm and abattoir workers and for public exposure through contaminated meat ( as is the case in more economically developed countries ) , this study suggests that attention should also be paid to other potential routes of transmission in situations where scavenging poultry remain commonplace . the backyard flocks , which live closely with people , can potentially contaminate the human living and food preparation environment , and infections may be shared with livestock and other animals ( including peri - domestic pests ) in the household . further work is needed to identify the reasons for the increased detection of campylobacter in flocks in the peri - urban area , compared to the rural areas . key questions to be addressed should include the impact of the close proximity of backyard production to large intensive farms , the potential effect of the distribution of exotic or improved birds from poultry multiplication centres to smallholder producers , the effect of movement of birds through trade , and other factors which may be associated with differences in the ecology of campylobacter between peri - urban and rural areas . furthermore , where there is close inter - species contact , such as was evident in many households in this study , the potential role of transmission from humans or other peri - domestic animals and livestock to chickens should also be considered . an alternative explanation for the higher prevalence of detection in the peri - urban area , independent of the type of flock , is that there is some other local factor in this region which increases the risk of detection of campylobacter in this area , such as a more favourable environment , climate or increased presence of wild bird , insect or rodent vectors . one of the features of debre zeit is the presence of numerous permanent and seasonal lakes , which provide habitat for birds and insects . the positive flocks in horro were also located in kebeles where bodies of water are a feature , whereas jarso , where no positive flocks were identified , has a much more arid climate and no substantial water bodies were present in the kebeles where sampling was carried out . only a single peri - urban region was sampled in this study , therefore these results may not be generalizable to other areas . however , it is of note that other major urban centres of poultry production and distribution in ethiopia , such as awassa and bahir dar , are also located close to large permanent lakes . of the backyard flocks , the odds of campylobacter detection in the environmental samples was greatest for those flocks consisting of only rir birds , or their hybrids . the cause of this is unknown , but factors underpinning this finding may include a genetic susceptibility of this breed , that this breed type is subjected to particular stress when kept under backyard management , or differences in the campylobacter - exposure risk between these and indigenous flocks , such as bird sources , contact patterns or behaviours . it has previously been reported that survival of rir - type birds in backyard conditions is poor , compared to indigenous breeds , and they are poorly adapted to scavenging and highly susceptible to disease . it should be considered that such stress might have implications beyond the health and welfare of the bird , if it alters carriage of zoonotic pathogens such as campylobacter . a potential limitation of this study was that samples collected from the two rural areas had , of necessity , a longer gap between collection and processing than samples collected in the peri - urban area , which was in close proximity to the laboratory facility . it has not been possible , in this study , to analyse whether this time lag had any impact on the detection rate of campylobacter from the study samples . while viable campylobacter decline rapidly in stored samples , especially at temperatures > 4 c , it has been shown that dna - based pcr methods continue to detect campylobacter in manure for at least 30 days even in samples stored at temperatures up to 52 c . however , this also highlights that the pcr - based method used in this study detected campylobacter dna , not viable bacteria , so further work is required to better evaluate the public health risk of these findings . only just over one - third of campylobacter - positive samples could be identified to the species level . this is not surprising , as the multiplex pcr was not designed for speciation on dna extracted from boot - sock samples and , given the amount of dna present in such samples , sensitivity may be an issue . that all 16 samples identified to the species level were found to be c. however , the possibility that other species are present should not be discounted , particularly as these may include some species less pathogenic to people . further investigation with more sensitive methods is needed in order to clarify the relative importance of different campylobacter spp . and molecular epidemiological approaches are required to determine the extent to which the campylobacter identified in each area and flock - type are similar . there is currently much interest in ethiopia in developing the poultry industry and enhancing the productivity of backyard chickens , and current programmes often advocate the adoption of high - producing breeds and changing from scavenging to semi - intensive management . this can be readily observed in the peri - urban areas , such as debre zeit , where exotic chickens such as the rir have been incorporated into the scavenging flocks , with the aim of enhancing egg production . there is also ongoing research to retain the desirable characteristics of the indigenous poultry types while raising the output of eggs and meat from the scavenging system by selective and cross - breeding of the local ecotypes with exotic breeds . since this study was performed , improved indigenous birds selected over seven generations for increased productivity traits ( body weight at age 16 weeks and cumulative egg number at week 45 of lay ) in debre zeit have been placed on farms in the horro and debre zeit regions to evaluate their performance under semi - intensive conditions . while higher productivity is desirable to increase food availability , it is important that measures to enhance food security are also evaluated in terms of their ability to produce safe food . backyard chickens are an important component of families livelihoods , and likely to remain so for many years to come , even in the face of increasing urbanization . while chicken production can contribute to improved livelihoods and nutrition , careful consideration is required of all impacts of programmes to develop the chicken production sector if negative human health effects may also occur . here , we identify a greater risk of environmental detection of campylobacter in a major peri - urban production area in ethiopia , compared to rural areas . further work is needed to determine whether other zoonotic pathogens may be similarly affected . if chicken production in urban / peri - urban areas increases the risk of environmental contamination with campylobacter ( and , potentially , other zoonotic pathogens ) , then managing these risks will be a vital component of veterinary public health services in these areas . furthermore , agricultural development programmes aimed at improving chicken production in rural areas , especially where this involves distribution of intensively reared birds or major changes to the management system , need to carefully consider the potential for perturbation to the ecology of campylobacter in these areas .

summarycampylobacter is a leading cause of diarrhoea , and its presence in chickens is a significant risk for zoonotic infection . poultry production is becoming increasingly intensive in ethiopia and is incorporating more high - producing breeds into traditionally managed smallholdings , especially in peri - urban areas . this cross - sectional study sampled 219 household environments in one peri - urban and two rural areas of ethiopia , and an additional 20 semi - intensive farms in the peri - urban district . campylobacter was detected by polymerase chain reaction ( pcr)-specific assays in 44 samples ; 16 of which could be identified as c. jejuni . flocks in the peri - urban area were at significantly greater odds of detection , including those which only kept indigenous birds under a scavenging system . it was also noted that scavenging flocks of exotic high - production birds ( rhode island red ) were at slightly greater risk , perhaps as exotic birds are under more stress when kept under traditional management systems . we suggest that changes to the system of chicken production may alter the ecology and epidemiology of campylobacter in the environment , chickens and people , which may drive emergence of new epidemiological patterns of disease . further research is needed to determine the extent to which the current management intensification and the distribution programmes of exotic and/or improved indigenous birds may alter campylobacter epidemiology , ecology and public health risk , before their widespread adoption .

INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION

children in low- to middle - income countries are at risk of frequent diarrhoea episodes ; the 2011 national survey in ethiopia estimated that 134% of children in the < 5 years age group had suffered an episode of diarrhoea in the preceding 2 weeks , although prevalence was > 20% in some regions . numerous infectious agents cause diarrhoea ; many of which are zoonotic , with campylobacter being one of the main bacterial causes , especially in children aged < 2 years [ 46 ] . while the organism is also frequently isolated from healthy children and adults , and morbidity appears to decline with age , adults with hiv infection are also at increased risk of campylobacter - associated diarrhoea and bacteraemia . in developing countries , far less is known about the epidemiology of campylobacter , but it has been found at high levels in retail poultry meat , and strains isolated from chickens have been observed to correlate with human isolates in terms of biotype and serotype . around 83% of ethiopia 's population , currently estimated at 96 million , chicken production is an integral part of most rural families livelihoods ( an estimated two - thirds of ethiopian villagers keep poultry ) and birds are commonly kept at night on perches within the family dwelling , frequently in the kitchen [ 14 , 15 ] . in ethiopia , it has been reported that chickens and poultry meat have a higher campylobacter prevalence compared to other farm animal and meat products [ 16 , 17 ] , and exposure to pets and livestock , including chickens , has been associated with increased odds of campylobacter isolation in diarrhoeal children aged < 5 years . it might , therefore , be anticipated that an important route by which rural families are exposed to campylobacter is from the faeces of infected chickens , which may contaminate the shared living environment . the national poultry population in ethiopia is estimated at 493 million , and is constituted of about 97% indigenous chicken ecotypes . these birds are predominantly maintained under a traditional free - range , scavenging or semi - scavenging system with minimal inputs for housing , feeding or healthcare . in the last 20 years , poultry production in ethiopia has started to be seen as a profitable venture , and more families in urban and peri - urban areas have started to keep small- to medium - sized flocks ( about 501000 birds ) under semi - intensive management . entrepreneurs have also started to invest in the poultry industry by setting up larger flocks of exotic breeds kept under semi - intensive / intensive management , particularly in the cities of dukem , debre zeit and nazeret , close to the capital , addis ababa . efforts have also been made by the government of ethiopia since the early 1990s to boost the productivity of indigenous birds kept by poor rural families through its genetic improvement programme , based on the introduction and distribution of exotic breeds by poultry multiplication centres throughout the country . several non - governmental organizations are also involved in the distribution of intensively reared chickens to smallholder farmers . although still accounting for a small proportion of ethiopia 's poultry , this on - going shift to more intensive production using commercial breeds may alter the dynamics of zoonotic infections such as campylobacteriosis . while housing chickens may limit their exposure to environmental pathogens and reduce direct and indirect contacts between people and chickens , campylobacter is known to spread very rapidly in housed flocks and interventions to limit exposure and transmission are likely to be ineffective , impractical or unaffordable in village poultry production settings . the links between management system , chicken welfare , stress and the biology of campylobacter infections remain poorly understood , but have raised concerns about how farming systems may impact on public health risk [ 2830 ] . as yet in ethiopia there has been little research examining how the changing dynamics of chicken production may have consequences for public health . the aim of this study was to investigate the impact of production settings and systems in ethiopia on the epidemiology of campylobacter , and to consider the potential implications of this evolving production system for human health in the country . therefore , the main objectives were ( i ) to determine the environmental prevalence of campylobacter in different geographical areas , including rural and peri - urban areas ; and ( ii ) to investigate whether campylobacter prevalence varied in chickens from the same area reared under different production systems . to investigate differences in campylobacter prevalence in village chickens between geographical areas , a cross - sectional study was conducted between october 2012 and april 2013 in three woredas ( administrative regions ) in the oromia region of ethiopia . woredas are further subdivided into kebeles , which are the lowest administrative unit in ethiopia , and may contain several villages or sub - areas . woredas were purposively selected based on known characteristics of their poultry production systems and bird ecotypes and , for the two rural sites , knowledge that there had been no recent poultry development programmes in the areas . over 95% of the population horro is considered an area of high agricultural potential and produces an annual food surplus , whereas , in jarso , agricultural land is limited and the area tends to have a food deficit every year , with the poor reliant on food aid to cover the food gap . sampling was conducted in four kebeles in each of the two rural districts , where over 70% of households were estimated to own chickens , but where there were no known semi - intensive or intensive poultry farms . households were excluded from the study if they did not own at least two chickens : 30% extra additional names of potential participants were included in the random selection to allow for exclusions or non - participation . in the peri - urban area of debre zeit , five kebeles were selected for inclusion in the study , all of which contained at least one intensive chicken farm . in addition , in order to investigate differences in campylobacter prevalence between different production systems , 20 intensive / semi - intensive farms within the same debre zeit kebeles were purposively selected for sampling during the same time period as the cross - sectional study . the unit of sampling was defined as either a backyard chicken - producing household ( i.e. for the purposes of this study , an intensive farm was defined as one which kept at least 50 birds under confined conditions and provided all feed . flock type was broadly grouped into four categories : indigenous ecotypes ; rhode island red ( rir ) , an established multi - purpose breed , or rir hybrids ( normally rir crossed with local ecotypes , which are difficult to distinguish morphologically from pure rir birds ) ; cobb 500 , a commercial fast - growing broiler breed ; and mixed flocks , which contained both local ecotypes and pure or hybrid rir birds . for each unit , a single sample was collected from the ground by walking through the household environment where chicken faeces were concentrated in the case of the extensive system or inside the chicken shed in the more intensive system , using one disposable fabric overshoe ( boot sock ) worn over the footwear . to avoid cross - contamination between different flocks , for each sampling a clean disposable plastic overshoe was worn to separate the footwear from the fabric boot sock . after collection , each boot - sock sample was carefully placed into a clean ziplock sterile plastic bag and kept under refrigerated conditions until processing . each boot sock was placed in a sterile plastic stomacher bag with 200 ml sterile saline water , and the mixture was treated in a stomacher at medium speed for 1 min to release detached matter . after dna extraction , 50 l of the supernatant , containing the suspended dna , was added to 450 l sterile distilled water , and stored at 4 c . table 1.primers used in the 16s rrna pcr assay for identification of the genus campylobacter ( product size 857-bp dna)primersequence 53c412 fgga tga cac ttt tcg gag ccampr2ggc ttc atg ctc tcg agt t primers used in the 16s rrna pcr assay for identification of the genus campylobacter ( product size 857-bp dna ) isolates were confirmed and identified to species level ( c. jejuni or c. coli ) using a multiplex pcr as described by klena et al . table 2.primers used in the lpx gene pcr assay for campylobacter isolates speciation ( product size : c. coli 391 bp and c. jejuni 331 bp)primersequence 53lpxac . jejuniacaacttggtgacgatgttgtalpxarkk2 mcaatcatgdgcdatatgasaatahgccat primers used in the lpx gene pcr assay for campylobacter isolates speciation ( product size : c. coli 391 bp and c. jejuni 331 bp ) basic descriptive statistics , graphs and multilevel logistic regression modelling were performed using r software , using the core functions , the plotrix library , the mgcv library and the lme4 library ( r foundation for statistical computing , austria ) . as cobb 500 chickens were only kept under intensive systems , and all other types were only kept under backyard scavenging systems , the variables breed and production system were combined to create a variable called type of flock , with four categories : intensive - cobb 500 , scavenging - rir , scavenging - mixed , and scavenging - indigenous. the full model included the presence or absence of campylobacter ( at the genus level , as detected by pcr ) as the outcome variable and the type of area ( urban or rural ) , the type of flock and the flock size variables as fixed effects . in total , only 16 ( 364% ) samples gave positive results using this multiplex pcr assay , and for all 16 samples a positive result was obtained for c. jejuni . in the rural horro region , all flocks sampled consisted purely of indigenous bird ecotypes , while in jarso , despite a reported absence of current distribution programmes for exotic birds in this rural region , 12 ( 15% ) of the 80 flocks sampled were made up either partially ( n = 9 ) , or entirely ( n = 3 ) of rir or rir hybrids . in the peri - urban kebeles around debre zeit , 28% of the scavenging flocks consisted partly ( n = 10 ) , or exclusively ( n = 7 ) of rir or rir hybrids . higher campylobacter prevalence was found in the peri - urban ( 463% ) compared to the rural ( 44% ) environments ( fig . the prevalence was still significantly higher in the peri - urban flocks ( 417% ) , even when the intensive flocks were excluded ( = 455 , p < 0001 ) . of the two rural areas , horro had the higher prevalence ( 89% ) of positive flocks , while no flocks were detected as positive by pcr in the jarso region . points are scaled relative to the number of flocks of each type tested . the final multi - level multivariable model ( table 3 ) suggested that flocks in the peri - urban area were at considerably greater odds of being detected campylobacter positive [ odds ratio ( or ) 219 , 95% confidence interval ( ci ) 202350 ] , even after controlling for the type of flock . by contrast , backyard flocks which consisted only of rir or rir hybrids and housed flocks of cobb 500 chickens were at increased risk compared to the backyard mixed flocks ( or 181 , 95% ci 181839 and or 65 , 95% ci 11387 , respectively ) . inclusion of flock size or any two - way interaction terms did not significantly improve the model ( likelihood ratio test statistic p values > 005 ) ; hence these were not included in the final model . table 3.final multi - level multivariable logistic regression model of factors associated with detection of campylobacter by pcr from environmental samples collected from chicken production sites in three regions of ethiopiavariablelevelsparameter estimates.e.or95% cip valuefixed effectsintercept4511type of arearuralreference004*peri - urban3112219202350001type of flockbackyard : mixedreference003*backyard : indigenous1108290615202backyard : rir / hybrid2912181181839001housed : cobb 50019096511387004random effectvariance estimatekebele08s.e . , standard error ; or , odds ratio ; ci , confidence interval ; rir , rhode island red. final multi - level multivariable logistic regression model of factors associated with detection of campylobacter by pcr from environmental samples collected from chicken production sites in three regions of ethiopia s.e . , standard error ; or , odds ratio ; ci , confidence interval ; rir , rhode island red . the greatest risk factor identified in this study for detection of campylobacter in the environment of the sampled chicken flocks was location in the peri - urban area , where many farms are starting to intensify their production systems . campylobacter is highly prevalent in intensive production systems in developed countries , with birds often having high levels of infection , posing a public health risk . while it is therefore perhaps unsurprising that the more intensively farmed flocks in the peri - urban areas were at increased risk , it is of particular concern that the greater risk in the peri - urban area persisted despite controlling for the type of flock , suggesting increased risk in all flock types in the peri - urban area , irrespective of breed and production type . although the high levels of campylobacter in intensive and semi - intensive flocks are clearly of concern for farm and abattoir workers and for public exposure through contaminated meat ( as is the case in more economically developed countries ) , this study suggests that attention should also be paid to other potential routes of transmission in situations where scavenging poultry remain commonplace . the backyard flocks , which live closely with people , can potentially contaminate the human living and food preparation environment , and infections may be shared with livestock and other animals ( including peri - domestic pests ) in the household . further work is needed to identify the reasons for the increased detection of campylobacter in flocks in the peri - urban area , compared to the rural areas . key questions to be addressed should include the impact of the close proximity of backyard production to large intensive farms , the potential effect of the distribution of exotic or improved birds from poultry multiplication centres to smallholder producers , the effect of movement of birds through trade , and other factors which may be associated with differences in the ecology of campylobacter between peri - urban and rural areas . furthermore , where there is close inter - species contact , such as was evident in many households in this study , the potential role of transmission from humans or other peri - domestic animals and livestock to chickens should also be considered . an alternative explanation for the higher prevalence of detection in the peri - urban area , independent of the type of flock , is that there is some other local factor in this region which increases the risk of detection of campylobacter in this area , such as a more favourable environment , climate or increased presence of wild bird , insect or rodent vectors . the positive flocks in horro were also located in kebeles where bodies of water are a feature , whereas jarso , where no positive flocks were identified , has a much more arid climate and no substantial water bodies were present in the kebeles where sampling was carried out . only a single peri - urban region was sampled in this study , therefore these results may not be generalizable to other areas . however , it is of note that other major urban centres of poultry production and distribution in ethiopia , such as awassa and bahir dar , are also located close to large permanent lakes . of the backyard flocks , the odds of campylobacter detection in the environmental samples was greatest for those flocks consisting of only rir birds , or their hybrids . the cause of this is unknown , but factors underpinning this finding may include a genetic susceptibility of this breed , that this breed type is subjected to particular stress when kept under backyard management , or differences in the campylobacter - exposure risk between these and indigenous flocks , such as bird sources , contact patterns or behaviours . it has previously been reported that survival of rir - type birds in backyard conditions is poor , compared to indigenous breeds , and they are poorly adapted to scavenging and highly susceptible to disease . a potential limitation of this study was that samples collected from the two rural areas had , of necessity , a longer gap between collection and processing than samples collected in the peri - urban area , which was in close proximity to the laboratory facility . while viable campylobacter decline rapidly in stored samples , especially at temperatures > 4 c , it has been shown that dna - based pcr methods continue to detect campylobacter in manure for at least 30 days even in samples stored at temperatures up to 52 c . however , this also highlights that the pcr - based method used in this study detected campylobacter dna , not viable bacteria , so further work is required to better evaluate the public health risk of these findings . only just over one - third of campylobacter - positive samples could be identified to the species level . and molecular epidemiological approaches are required to determine the extent to which the campylobacter identified in each area and flock - type are similar . there is currently much interest in ethiopia in developing the poultry industry and enhancing the productivity of backyard chickens , and current programmes often advocate the adoption of high - producing breeds and changing from scavenging to semi - intensive management . this can be readily observed in the peri - urban areas , such as debre zeit , where exotic chickens such as the rir have been incorporated into the scavenging flocks , with the aim of enhancing egg production . there is also ongoing research to retain the desirable characteristics of the indigenous poultry types while raising the output of eggs and meat from the scavenging system by selective and cross - breeding of the local ecotypes with exotic breeds . since this study was performed , improved indigenous birds selected over seven generations for increased productivity traits ( body weight at age 16 weeks and cumulative egg number at week 45 of lay ) in debre zeit have been placed on farms in the horro and debre zeit regions to evaluate their performance under semi - intensive conditions . backyard chickens are an important component of families livelihoods , and likely to remain so for many years to come , even in the face of increasing urbanization . here , we identify a greater risk of environmental detection of campylobacter in a major peri - urban production area in ethiopia , compared to rural areas . further work is needed to determine whether other zoonotic pathogens may be similarly affected . if chicken production in urban / peri - urban areas increases the risk of environmental contamination with campylobacter ( and , potentially , other zoonotic pathogens ) , then managing these risks will be a vital component of veterinary public health services in these areas . furthermore , agricultural development programmes aimed at improving chicken production in rural areas , especially where this involves distribution of intensively reared birds or major changes to the management system , need to carefully consider the potential for perturbation to the ecology of campylobacter in these areas .

children in low- to middle - income countries are at risk of frequent diarrhoea episodes ; the 2011 national survey in ethiopia estimated that 134% of children in the < 5 years age group had suffered an episode of diarrhoea in the preceding 2 weeks , although prevalence was > 20% in some regions . numerous infectious agents cause diarrhoea ; many of which are zoonotic , with campylobacter being one of the main bacterial causes , especially in children aged < 2 years [ 46 ] . while the organism is also frequently isolated from healthy children and adults , and morbidity appears to decline with age , adults with hiv infection are also at increased risk of campylobacter - associated diarrhoea and bacteraemia . different routes by which humans are exposed to campylobacter include ingestion of contaminated food and water , direct contact with infected animals or carcasses , from environments contaminated with animal faeces or sewage , or direct person - to - person transmission [ 4 , 7 ] . in countries with a highly developed intensive poultry industry , there is considerable epidemiological evidence that chickens are the main source of human campylobacter infection [ 810 ] . in developing countries , far less is known about the epidemiology of campylobacter , but it has been found at high levels in retail poultry meat , and strains isolated from chickens have been observed to correlate with human isolates in terms of biotype and serotype . around 83% of ethiopia 's population , currently estimated at 96 million , chicken production is an integral part of most rural families livelihoods ( an estimated two - thirds of ethiopian villagers keep poultry ) and birds are commonly kept at night on perches within the family dwelling , frequently in the kitchen [ 14 , 15 ] . in ethiopia , it has been reported that chickens and poultry meat have a higher campylobacter prevalence compared to other farm animal and meat products [ 16 , 17 ] , and exposure to pets and livestock , including chickens , has been associated with increased odds of campylobacter isolation in diarrhoeal children aged < 5 years . it might , therefore , be anticipated that an important route by which rural families are exposed to campylobacter is from the faeces of infected chickens , which may contaminate the shared living environment . despite their low production performances in terms of egg and meat output , indigenous chickens are well adapted to the local environmental conditions and fulfil a variety of social and cultural functions , in addition to being both nutritional and economic assets . in the last 20 years , poultry production in ethiopia has started to be seen as a profitable venture , and more families in urban and peri - urban areas have started to keep small- to medium - sized flocks ( about 501000 birds ) under semi - intensive management . entrepreneurs have also started to invest in the poultry industry by setting up larger flocks of exotic breeds kept under semi - intensive / intensive management , particularly in the cities of dukem , debre zeit and nazeret , close to the capital , addis ababa . efforts have also been made by the government of ethiopia since the early 1990s to boost the productivity of indigenous birds kept by poor rural families through its genetic improvement programme , based on the introduction and distribution of exotic breeds by poultry multiplication centres throughout the country . several non - governmental organizations are also involved in the distribution of intensively reared chickens to smallholder farmers . although still accounting for a small proportion of ethiopia 's poultry , this on - going shift to more intensive production using commercial breeds may alter the dynamics of zoonotic infections such as campylobacteriosis . while housing chickens may limit their exposure to environmental pathogens and reduce direct and indirect contacts between people and chickens , campylobacter is known to spread very rapidly in housed flocks and interventions to limit exposure and transmission are likely to be ineffective , impractical or unaffordable in village poultry production settings . stresses , such as heat stress , or food and water restriction , is known to be detrimental to the chicken 's immune function , altering tissue invasion of intestinal bacteria [ 24 , 25 ] . however , different types or lines of chickens show variation in both the effect of heat stress on their immune response and their ability to regulate the immune response to campylobacter . the links between management system , chicken welfare , stress and the biology of campylobacter infections remain poorly understood , but have raised concerns about how farming systems may impact on public health risk [ 2830 ] . as yet in ethiopia there has been little research examining how the changing dynamics of chicken production may have consequences for public health . the aim of this study was to investigate the impact of production settings and systems in ethiopia on the epidemiology of campylobacter , and to consider the potential implications of this evolving production system for human health in the country . therefore , the main objectives were ( i ) to determine the environmental prevalence of campylobacter in different geographical areas , including rural and peri - urban areas ; and ( ii ) to investigate whether campylobacter prevalence varied in chickens from the same area reared under different production systems . to investigate differences in campylobacter prevalence in village chickens between geographical areas , a cross - sectional study was conducted between october 2012 and april 2013 in three woredas ( administrative regions ) in the oromia region of ethiopia . woredas were purposively selected based on known characteristics of their poultry production systems and bird ecotypes and , for the two rural sites , knowledge that there had been no recent poultry development programmes in the areas . over 95% of the population horro is considered an area of high agricultural potential and produces an annual food surplus , whereas , in jarso , agricultural land is limited and the area tends to have a food deficit every year , with the poor reliant on food aid to cover the food gap . development agents are employees of the local agricultural extension service , whose role is to provide farmers with access to training , research and technologies ( http://www.moa.gov.et/policies-and-strategies ) . in the peri - urban area of debre zeit , five kebeles were selected for inclusion in the study , all of which contained at least one intensive chicken farm . as chicken ownership was believed to be markedly lower in this woreda , random selection of households was not considered feasible ; instead , convenience sampling of households was undertaken based on transect walks through the kebeles and identification of households which kept free - ranging scavenging chickens . in addition , in order to investigate differences in campylobacter prevalence between different production systems , 20 intensive / semi - intensive farms within the same debre zeit kebeles were purposively selected for sampling during the same time period as the cross - sectional study . flock type was broadly grouped into four categories : indigenous ecotypes ; rhode island red ( rir ) , an established multi - purpose breed , or rir hybrids ( normally rir crossed with local ecotypes , which are difficult to distinguish morphologically from pure rir birds ) ; cobb 500 , a commercial fast - growing broiler breed ; and mixed flocks , which contained both local ecotypes and pure or hybrid rir birds . for each unit , a single sample was collected from the ground by walking through the household environment where chicken faeces were concentrated in the case of the extensive system or inside the chicken shed in the more intensive system , using one disposable fabric overshoe ( boot sock ) worn over the footwear . boot socks were pre - moistened with sterile physiological saline before use , in order to allow maximum uptake of campylobacter . without disturbing the sediment on the bottom , 1 ml of the suspension the suspension was centrifuged for 7 min at ~12 000 g. the supernatant was discarded and the bacterial dna was extracted from the pellet using a commercial dna extraction kit ( promega wizard genomic dna purification kit , usa ) . samples were then exported under license to the university of liverpool , where they were analysed with a pcr assay specific for the genus campylobacter on the basis of 16s rrna gene sequences ( 16s rdna ; table 1 ) [ 35 , 36 ] . table 1.primers used in the 16s rrna pcr assay for identification of the genus campylobacter ( product size 857-bp dna)primersequence 53c412 fgga tga cac ttt tcg gag ccampr2ggc ttc atg ctc tcg agt t primers used in the 16s rrna pcr assay for identification of the genus campylobacter ( product size 857-bp dna ) isolates were confirmed and identified to species level ( c. jejuni or c. coli ) using a multiplex pcr as described by klena et al . table 2.primers used in the lpx gene pcr assay for campylobacter isolates speciation ( product size : c. coli 391 bp and c. jejuni 331 bp)primersequence 53lpxac . jejuniacaacttggtgacgatgttgtalpxarkk2 mcaatcatgdgcdatatgasaatahgccat primers used in the lpx gene pcr assay for campylobacter isolates speciation ( product size : c. coli 391 bp and c. jejuni 331 bp ) basic descriptive statistics , graphs and multilevel logistic regression modelling were performed using r software , using the core functions , the plotrix library , the mgcv library and the lme4 library ( r foundation for statistical computing , austria ) . a multilevel multivariable model was constructed to investigate whether area , production system or breed kept had any effect on the odds of a flock being detected as campylobacter positive . as cobb 500 chickens were only kept under intensive systems , and all other types were only kept under backyard scavenging systems , the variables breed and production system were combined to create a variable called type of flock , with four categories : intensive - cobb 500 , scavenging - rir , scavenging - mixed , and scavenging - indigenous. the functional form of the relationship between flock size ( the only continuous explanatory variable ) and the outcome was assessed using generalized additive models and suggested inclusion of a linear relationship was appropriate . the full model included the presence or absence of campylobacter ( at the genus level , as detected by pcr ) as the outcome variable and the type of area ( urban or rural ) , the type of flock and the flock size variables as fixed effects . in the rural horro region , all flocks sampled consisted purely of indigenous bird ecotypes , while in jarso , despite a reported absence of current distribution programmes for exotic birds in this rural region , 12 ( 15% ) of the 80 flocks sampled were made up either partially ( n = 9 ) , or entirely ( n = 3 ) of rir or rir hybrids . in the peri - urban kebeles around debre zeit , 28% of the scavenging flocks consisted partly ( n = 10 ) , or exclusively ( n = 7 ) of rir or rir hybrids . higher campylobacter prevalence was found in the peri - urban ( 463% ) compared to the rural ( 44% ) environments ( fig . the prevalence was still significantly higher in the peri - urban flocks ( 417% ) , even when the intensive flocks were excluded ( = 455 , p < 0001 ) . of the two rural areas , horro had the higher prevalence ( 89% ) of positive flocks , while no flocks were detected as positive by pcr in the jarso region . the final multi - level multivariable model ( table 3 ) suggested that flocks in the peri - urban area were at considerably greater odds of being detected campylobacter positive [ odds ratio ( or ) 219 , 95% confidence interval ( ci ) 202350 ] , even after controlling for the type of flock . after controlling for type of area , there was insufficient evidence to reject the null hypothesis of no difference in risk between mixed exotic / indigenous and indigenous - only backyard flocks . by contrast , backyard flocks which consisted only of rir or rir hybrids and housed flocks of cobb 500 chickens were at increased risk compared to the backyard mixed flocks ( or 181 , 95% ci 181839 and or 65 , 95% ci 11387 , respectively ) . table 3.final multi - level multivariable logistic regression model of factors associated with detection of campylobacter by pcr from environmental samples collected from chicken production sites in three regions of ethiopiavariablelevelsparameter estimates.e.or95% cip valuefixed effectsintercept4511type of arearuralreference004*peri - urban3112219202350001type of flockbackyard : mixedreference003*backyard : indigenous1108290615202backyard : rir / hybrid2912181181839001housed : cobb 50019096511387004random effectvariance estimatekebele08s.e . final multi - level multivariable logistic regression model of factors associated with detection of campylobacter by pcr from environmental samples collected from chicken production sites in three regions of ethiopia s.e . the greatest risk factor identified in this study for detection of campylobacter in the environment of the sampled chicken flocks was location in the peri - urban area , where many farms are starting to intensify their production systems . while it is therefore perhaps unsurprising that the more intensively farmed flocks in the peri - urban areas were at increased risk , it is of particular concern that the greater risk in the peri - urban area persisted despite controlling for the type of flock , suggesting increased risk in all flock types in the peri - urban area , irrespective of breed and production type . although the high levels of campylobacter in intensive and semi - intensive flocks are clearly of concern for farm and abattoir workers and for public exposure through contaminated meat ( as is the case in more economically developed countries ) , this study suggests that attention should also be paid to other potential routes of transmission in situations where scavenging poultry remain commonplace . the backyard flocks , which live closely with people , can potentially contaminate the human living and food preparation environment , and infections may be shared with livestock and other animals ( including peri - domestic pests ) in the household . further work is needed to identify the reasons for the increased detection of campylobacter in flocks in the peri - urban area , compared to the rural areas . key questions to be addressed should include the impact of the close proximity of backyard production to large intensive farms , the potential effect of the distribution of exotic or improved birds from poultry multiplication centres to smallholder producers , the effect of movement of birds through trade , and other factors which may be associated with differences in the ecology of campylobacter between peri - urban and rural areas . furthermore , where there is close inter - species contact , such as was evident in many households in this study , the potential role of transmission from humans or other peri - domestic animals and livestock to chickens should also be considered . an alternative explanation for the higher prevalence of detection in the peri - urban area , independent of the type of flock , is that there is some other local factor in this region which increases the risk of detection of campylobacter in this area , such as a more favourable environment , climate or increased presence of wild bird , insect or rodent vectors . one of the features of debre zeit is the presence of numerous permanent and seasonal lakes , which provide habitat for birds and insects . the positive flocks in horro were also located in kebeles where bodies of water are a feature , whereas jarso , where no positive flocks were identified , has a much more arid climate and no substantial water bodies were present in the kebeles where sampling was carried out . of the backyard flocks , the odds of campylobacter detection in the environmental samples was greatest for those flocks consisting of only rir birds , or their hybrids . the cause of this is unknown , but factors underpinning this finding may include a genetic susceptibility of this breed , that this breed type is subjected to particular stress when kept under backyard management , or differences in the campylobacter - exposure risk between these and indigenous flocks , such as bird sources , contact patterns or behaviours . it has previously been reported that survival of rir - type birds in backyard conditions is poor , compared to indigenous breeds , and they are poorly adapted to scavenging and highly susceptible to disease . a potential limitation of this study was that samples collected from the two rural areas had , of necessity , a longer gap between collection and processing than samples collected in the peri - urban area , which was in close proximity to the laboratory facility . while viable campylobacter decline rapidly in stored samples , especially at temperatures > 4 c , it has been shown that dna - based pcr methods continue to detect campylobacter in manure for at least 30 days even in samples stored at temperatures up to 52 c . however , this also highlights that the pcr - based method used in this study detected campylobacter dna , not viable bacteria , so further work is required to better evaluate the public health risk of these findings . this is not surprising , as the multiplex pcr was not designed for speciation on dna extracted from boot - sock samples and , given the amount of dna present in such samples , sensitivity may be an issue . that all 16 samples identified to the species level were found to be c. however , the possibility that other species are present should not be discounted , particularly as these may include some species less pathogenic to people . there is currently much interest in ethiopia in developing the poultry industry and enhancing the productivity of backyard chickens , and current programmes often advocate the adoption of high - producing breeds and changing from scavenging to semi - intensive management . this can be readily observed in the peri - urban areas , such as debre zeit , where exotic chickens such as the rir have been incorporated into the scavenging flocks , with the aim of enhancing egg production . there is also ongoing research to retain the desirable characteristics of the indigenous poultry types while raising the output of eggs and meat from the scavenging system by selective and cross - breeding of the local ecotypes with exotic breeds . since this study was performed , improved indigenous birds selected over seven generations for increased productivity traits ( body weight at age 16 weeks and cumulative egg number at week 45 of lay ) in debre zeit have been placed on farms in the horro and debre zeit regions to evaluate their performance under semi - intensive conditions . here , we identify a greater risk of environmental detection of campylobacter in a major peri - urban production area in ethiopia , compared to rural areas . if chicken production in urban / peri - urban areas increases the risk of environmental contamination with campylobacter ( and , potentially , other zoonotic pathogens ) , then managing these risks will be a vital component of veterinary public health services in these areas . furthermore , agricultural development programmes aimed at improving chicken production in rural areas , especially where this involves distribution of intensively reared birds or major changes to the management system , need to carefully consider the potential for perturbation to the ecology of campylobacter in these areas .

osteonecrosis or avascular osteonecrosis ( avn ) of the femoral head is a devastating multifactorial disease that affects 20 000 persons each year in the united states . although the pathophysiology of avn has not been completely elucidated , this progressive clinical condition is characterized by bone death and reduced local blood flow . as bone repair occurs , the imbalance of osteoclast - medicated resorption and delayed new bone formation result in mechanically weak bone that collapses under the load of weight . after collapse , due to extreme pain and loss of hip function , most patients require standard total hip arthroplasty ( tha ) . however , because of the young age of many of these patients , a hip replacement can not be expected to last the patient s lifetime ; therefore , when feasible , attempts should be made to save the femoral head prior to collapse with use of less invasive treatment modalities [ 79 ] . there is currently no uniformly accepted pharmacologic treatment that retards avn progression and prevents bone collapse . in contrast to other drugs , bisphosphonates ( bps ) are potent anti - reabsorptive agents that act by inhibiting the action of mature osteoclasts in the bone , which theoretically normalizes the uncoupled bone remodeling , contributing to femoral head collapse . in the last decade , many studies have investigated the application of bps in the treatment of avn [ 1019 ] . nevertheless , the lack of controlled and long - term results , the substantial heterogeneities of genres in bps , the unclear indication of various stages of avn , and the combination of adults and juveniles , complicate the interpretation of recent systematic reviews and necessitate new evidence . of different bps , in addition , application of bps in children raises great concern due to its potential harmful effects on the growing skeleton of juveniles . therefore , we performed a systematic review restricted to alendronate therapy for adult avn . by summarizing recent randomized controlled trials and long - term follow - up studies , the purpose of this systematic review was to determine the efficacy and safety of alendronate for adult avn during short- and long - term follow - up . our hypothesis was that alendronate therapy could be well - tolerated and : 1 ) improve clinical function and pain , 2 ) retard the progression of femoral head collapse and/or reduce the incidence of tha , and 3 ) be influenced by stage of the disease . four electronic databases ( pubmed , embase , the cochrane central register of controlled trials , and china national knowledge infrastructure ) were searched using a search strategy combining the terms in boolean logic : ( alendronate or fosamax ) and ( avascular necrosis or aseptic necrosis or osteonecrosis ) and ( femoral head ) . trials were included if they were randomized or nonrandomized clinical trials , cohort , case - control studies , and series of cases in which alendronate was used for treatment of avn of the femoral head in adults , with adequately reported data on diminishment of pain , or improvement of articular function , or retardation of bone collapse progression , or need for arthroplasty . we also manually searched reference lists of review articles , and included studies to identify other potentially eligible studies . after exclusion of duplicates , 1 reviewer ( lrb ) performed an initial title and abstract screening of articles to discard those that were clearly ineligible , then 2 reviewers ( lt and zhm ) independently examined the full article to assess the trials for eligibility for inclusion , with disagreements resolved by discussion . citations were excluded if they were animal studies or targeted adolescences or used alendronate in combination with any other treatments . if necessary , we attempted to contact the author of the original report to obtain further details . from each article we extracted the following details by using standardized forms : authors , year of publication , geographical location of study , study design / level of evidence , study population ( hips / patients ) , patient sex and age , follow - up duration , interventions , outcomes , and adverse events . the level of evidence of each study was rated on basis of oxford centre for evidence - based medicine levels of evidence ( march 2009 ) . the outcome of interest included clinic function and hip pain improvement of avn patients after alendronate treatment . clinical failure was defined as the need for tha . for the radiographic evaluation , although various classification systems were used among the studies , they shared fundamental similarities ; therefore , radiographic failure here were defined as any lesions progressed to a higher stage from baseline stage . due to the included avn comprising both pre - collapse and early - collapse stage , collapse rates were separated and considered as a new occurrence of collapse or an increased collapse of greater than 2 mm . data could not be analyzed using a meta - analysis due to the methodological heterogeneity and limited number of the available controlled studies . of these , we included 8 articles with 788 hips in this systematic review [ 1219 ] . all the studies targeted adult avn patients within stage iii classified by x - ray , magnetic resonance imaging ( mri ) , both according to ficat and arlet ( 3 studies ) , association research circulation osseous ( arco , 2 studies ) , or steinberg ( university of pennsylvania system , 2 studies ) . four studies were restricted to non - traumatic avn patients and the other 4 articles from the same group did not specify the etiology . the doses and duration of alendronate administration differed among the studies . in 5 of the included studies full weight - bearing was only permitted in 1 study and 4 studies only allowed partial or no weight - bearing . six studies reported short - term results of alendronate treatment on avn ( < 4 years ) [ 12,1519 ] , and the other 2 reported long - term results ( 4 years ) . only 3 studies contained a control group , 2 of which were randomized controlled trials that reported the details of randomization and blinding . most of the included studies were prospective non - controlled studies and 4 of them were from the same institution reporting on the same group during different follow - up period . as presented in table 1 , the level of evidence for the studies ranged from 1b to 3b . the current evidence is level 3a , which is limited to the small number with small sample size and the majority of non - controlled studies . table 2 showed the outcomes reported from studies evaluating alendronate use in avascular osteonecrosis of the femoral head . studied 16 patients with avn of the femoral head most of them secondary to the use of corticosteroids . they used a regimen of alendronate 10 mg / day + calcium 1 g / day + vitamin d supplement , and the mean duration of therapy with alendronate was 24.7 weeks . patients that used alendronate had a significant improvement in pain as early as 12 weeks , with a reduction in the need for analgesics and improvement in functional capacity in all patients , and this improvement was maintained for 24 weeks . the observation was extended to a total of 60 patients ( 100 hips ) , with an average follow - up of 37 months . alendronate was used in daily doses of 10 mg or weekly doses of 70 mg ; these authors confirmed these findings and further suggested that alendronate would retard the progression of avn and avoid the early indication of surgery in mid - term follow - up . another study with short - term follow - up was performed by chen et al . , which included 83 patients with non - traumatic avn of the femoral head 33 of the patients were arco i and the rest were arco ii . they were given oral alendronate 70 mg weekly , and evaluated with harris criteria at baseline and 3 months after treatment . in the patients with arco i avn , the scores of pain and function were improved after treatment ( p<0.01 ) . similarly , in the patients with arco ii avn the scores of pain and function were both improved after treatment ( p<0.01 ) . and the score of activity was also enhanced obviously , which was not observed in arco i avn patients . they concluded that alendronate is effective in treatment of early - stage adult non - traumatic avn of the femoral head , especially for arco ii patients . the first rct with short - term results evaluating the treatment of alendronate on avn of the femoral head was by lai et al . . they studied 40 patients with steinberg stage ii or iii c non - traumatic avn of the femoral head . the patients were divided into 2 groups , half of them received alendronate 70 mg / week orally and the other half did not receive this medication . the patients were monitored radiologically every 10 weeks and observed for a minimum of 24 months . at the end of the study , the mean hhs was 49.29.2 points in the control group and 74.47.8 points in the alendronate group . it was also demonstrated that only 2/29 femoral heads with an ( 0/17 in stage ii , 2/12 in stage iii ) collapsed in the group that received alendronate 70 mg / week , whereas in the group that was randomized to not receive this medication , collapse occurred in 19/25 ( 9/13 in stage ii , 10/12 in stage iii ) femoral heads ( p<0.001 ) . one hip in the alendronate group underwent tha , whereas 16 hips in the control group underwent tha ( p<0.001 ) . thus , they concluded that alendronate appeared to prevent early collapse of the femoral head in the hips with steinberg stage ii or iii c non - traumatic avn . in a study by nishii et al . , 14 patients ( 20 hips ) with arco i iii avn received alendronate 5 mg / day and were compared in a nonrandomized manner with a group of 8 patients ( 13 hips ) that did not receive alendronate . all the patients received periodical radiologic evaluation at 3 , 6 , and 12 months . at the end of follow - up , the group of patients receiving alendronate had less pain and a lower frequency of femoral head collapse when compared with the control group . specifically , they found progressive collapse occurred only in hips with extensive necrosis ( greater than the medial 2/3 of the weight - bearing area of the femoral head with / without involvement of acetabula edge , termed type c2 and c1 , respectively ) in both groups , which had a much higher incidence in the control groups . in total , collapse occurred in 6/13 of articulations in the control group and in only 1/20 in the alendronate group ( p=0.008 ) . moreover , 2 of 13 hips in the control group needed tha , but none of patients needed surgery in the alendronate group . therefore they suggested alendronate had the potential to prevent collapse of the femoral head , even with extensive necrosis , within 1 year . chen et al . recently performed a 2-year , multicenter , prospective , randomized , double - blind study involving a total of 52 patients ( 65 hips ) . twenty - six patients ( 32 hips ) were assigned to a scheme of alendronate 70 mg / week for 104 weeks and were compared with a group of 26 patients ( 33 hips ) that received placebo . at the end of the study , mri evaluation revealed that 21 of the 32 hips in the alendronate group and 20 of the 33 hips in the placebo group had progressed ( p=0.636 ) . four of 32 hips in the alendronate treatment group underwent tha , and 5 of 33 hips in the placebo group had tha ( p=0.837 ) . no differences were noted in hhs , or short form 36 scores between the 2 groups . thus , the extensive lesion of necrotic area might impair the prognosis of the avn when alendronate was used . the long - term data was from a study by agarwala et al . , who presented a clinic radiological analysis of 395 hips with a mean follow - up of 4 years and then further extended the follow - up of 40 patients ( 53 hips ) with avn to 10 years ; those patients were treated with oral alendronate for 3 years , and the data demonstrated although clinical functions showed a general trend of worsening after discontinuation , patients tolerated the decline well , as suggested by the reduction in pain of ficat and arlet stage i iii patients 10 years after onset of alendronate treatment . more importantly , the rates of radiologic progression and femoral head collapse were markedly reduced even at 10 years as compared to the historical data available for natural history of hips with untreated avn . at 10 years , 46 ( 87% ) of the 53 hips survived , that is , had a satisfactory clinical result . hip loss to arthroplasty occurred in 1 each of stage i ( 7% ) and stage ii ( 5% ) hips and in 5 ( 26% ) of stage iii hips . of the 34 hips that were in pre - collapse stages at the onset of the study , at 10 years 10 had collapsed , indicating a collapse rate of 29% for a period of 10 years . they thereafter indicated that the benefit is particularly marked if the treatment is begun in the pre - collapse stages of the disease ( stage i or ii ) . even in stage iii hips some benefit was obtained from treatment with alendronate by at least a delay in the need for total hip replacement . the most common adverse effects across the studies were gastric dyspepsia mentioned in 3 studies and dizziness mentioned in 2 studies , both of which occurred after treatment initiation and were self - limiting . no osteonecrosis of the jaw were seen irrespective of the dose or duration of alendronate . the most studied is core decompression , which works by reduction of intramedullary pressure inside the femoral head by making a drill hole , thus improving blood flow to bone . osteotomy could relocate the necrotic area of bone from the weight - loading area of the acetabulum , so as to redistribute the weight loading to articular cartilage , which is supported by healthy bone . the increasingly applied method is bone graft , aiming to provide mechanical support to subchondral bone or cartilage . there are various types of bone grafting , some combined with osteotomy , osteochondral grafts , muscle pedicle bone grafts , and some are vascularized grafts to improve blood flow of the bone by achieving revascularization . it has been used to replace the necrotic bone segment to prevent collapse in steinberg stage i iii femoral avn . however , the efficacy and safety of the above procedures are still controversial [ 7,2729 ] . due to the reported efficacy of total hip arthroplasty and the typical age of patients with osteonecrosis , it has recently been questioned whether these invasive procedures are appropriate , given the potential difficulty of later conversion to a hip replacement . conservative treatment that helps improve function and delays femoral head deformity could be valuable time - buying strategy for some patients . some of the pharmacologic agents that have been used to treat osteonecrosis of the hip are statins , anticoagulants , prostacyclin , and bps [ 1019 ] . the theoretical benefit of statins is based on the association of increased fat cell size with an increased risk of development of hip osteonecrosis . anticoagulants may inhibit the aggregation of platelets and enhance blood flow to ischemic areas of bone . however , collapse of the femoral head appears to be a consequence of the non - coupling of bone reabsorption and bone regeneration rates . in this context , collapse could be prevented if the reabsorptive activity of the necrotic bone during the repair phase was inhibited or slowed until the formation of sufficient new bone . the efficacy of alendronate therapy of femoral head deformity was indicated by several experimental studies consisting of both adult rat and rabbit models induced by femoral head ischemia . in 2 closely related studies , alendronate given subcutaneously ( 200 mg / kg / d ) was able to preserve femoral head structure in mature rats during a 6-week follow - up . in another study , 3-week alendronate therapy reduced degeneration of articular cartilage and improved subchondral bone volume and mineral density in adult rabbits at 12 months , which therefore might be the reason that alendronate treatment could preserve the shape of the femoral head affected by avn . regarding alendronate for the treatment of avn in clinical trials , in the present review , only 8 articles were published . our literature search found were 2 recent systematic reviews evaluating bps for avn , 1 of which only included 3 observational short - term studies in juveniles , and the other with 6 small short - term trials that reported substantial heterogeneities across studies in patient group ( adults and adolescents ) and treatments ( other bps and combined therapy ) . in contrast to previous reviews , the inclusion criteria of the current review were restricted to articles that studied alendronate treatment for adult avn . due to ongoing debate on long - term effects of alendronate on the growing skeleton , we limited the studied population to only adults . the inclusion of most recent longer - term results and rct would also help to update the previous evidence . furthermore , after determining an overall profile of clinic outcome and the rates of radiographic and clinical failure , the collected data were further stratified by radiographic stage and by duration of follow - up to determine whether any of these factors influenced the results . however , generally speaking , the studies included in the current review still present various limitations most used observational non - controlled methods ; small numbers of patients ; different avn stages of patients when treatment was initiated ; and lack of uniformity in dose and time of alendronate use . these articles , in addition , had various durations of follow - up and were composed of various subgroups of patient populations . bearing in mind the above - mentioned limitations , most studies suggested a positive short - term and middle - term efficacy in pain reduction , improvement of articular function , slowing of bone collapse progression , and delaying the need for arthroplasty in adult avn patients with the use of alendronate treatment . this is of great clinical significance , as most of the included patients are young or active patients who are likely to require a revision at some point in the future and the effective slowing of femoral head deformity by alendronate could help to avoid early tha . favorable long - term results were also presented by agarwala s 10-year study in treated patients even after alendronate discontinuation . in addition , there were no severe adverse effects associated with alendronate treatment observed during short- or long - term follow - up . another finding of the current review is that although patients in all stages appeared to have potential benefit from alendronate treatment , the application in early avn with small size lesion was suggested by most of the included studies . specially , as shown in chen s study , when extensive osteonecrosis ( stage ii c and iii c ) were radiographically presented , alendronate did not have any benefits . thus , the efficacy of alendronate for avn with large necrotic legions should be considered more carefully . agarwala et al . studied 16 patients with avn of the femoral head most of them secondary to the use of corticosteroids . they used a regimen of alendronate 10 mg / day + calcium 1 g / day + vitamin d supplement , and the mean duration of therapy with alendronate was 24.7 weeks . patients that used alendronate had a significant improvement in pain as early as 12 weeks , with a reduction in the need for analgesics and improvement in functional capacity in all patients , and this improvement was maintained for 24 weeks . the observation was extended to a total of 60 patients ( 100 hips ) , with an average follow - up of 37 months . alendronate was used in daily doses of 10 mg or weekly doses of 70 mg ; these authors confirmed these findings and further suggested that alendronate would retard the progression of avn and avoid the early indication of surgery in mid - term follow - up . another study with short - term follow - up was performed by chen et al . , which included 83 patients with non - traumatic avn of the femoral head 33 of the patients were arco i and the rest were arco ii . they were given oral alendronate 70 mg weekly , and evaluated with harris criteria at baseline and 3 months after treatment . in the patients with arco i avn , the scores of pain and function were improved after treatment ( p<0.01 ) . similarly , in the patients with arco ii avn the scores of pain and function were both improved after treatment ( p<0.01 ) . and the score of activity was also enhanced obviously , which was not observed in arco i avn patients . they concluded that alendronate is effective in treatment of early - stage adult non - traumatic avn of the femoral head , especially for arco ii patients . the first rct with short - term results evaluating the treatment of alendronate on avn of the femoral head was by lai et al . . they studied 40 patients with steinberg stage ii or iii c non - traumatic avn of the femoral head . the patients were divided into 2 groups , half of them received alendronate 70 mg / week orally and the other half did not receive this medication . the patients were monitored radiologically every 10 weeks and observed for a minimum of 24 months . at the end of the study , the mean hhs was 49.29.2 points in the control group and 74.47.8 points in the alendronate group . it was also demonstrated that only 2/29 femoral heads with an ( 0/17 in stage ii , 2/12 in stage iii ) collapsed in the group that received alendronate 70 mg / week , whereas in the group that was randomized to not receive this medication , collapse occurred in 19/25 ( 9/13 in stage ii , 10/12 in stage iii ) femoral heads ( p<0.001 ) . one hip in the alendronate group underwent tha , whereas 16 hips in the control group underwent tha ( p<0.001 ) . thus , they concluded that alendronate appeared to prevent early collapse of the femoral head in the hips with steinberg stage ii or iii c non - traumatic avn . in a study by nishii et al . , 14 patients ( 20 hips ) with arco i iii avn received alendronate 5 mg / day and were compared in a nonrandomized manner with a group of 8 patients ( 13 hips ) that did not receive alendronate . all the patients received periodical radiologic evaluation at 3 , 6 , and 12 months . at the end of follow - up , the group of patients receiving alendronate had less pain and a lower frequency of femoral head collapse when compared with the control group . specifically , they found progressive collapse occurred only in hips with extensive necrosis ( greater than the medial 2/3 of the weight - bearing area of the femoral head with / without involvement of acetabula edge , termed type c2 and c1 , respectively ) in both groups , which had a much higher incidence in the control groups . in total , collapse occurred in 6/13 of articulations in the control group and in only 1/20 in the alendronate group ( p=0.008 ) . moreover , 2 of 13 hips in the control group needed tha , but none of patients needed surgery in the alendronate group . therefore they suggested alendronate had the potential to prevent collapse of the femoral head , even with extensive necrosis , within 1 year . chen et al . recently performed a 2-year , multicenter , prospective , randomized , double - blind study involving a total of 52 patients ( 65 hips ) . twenty - six patients ( 32 hips ) were assigned to a scheme of alendronate 70 mg / week for 104 weeks and were compared with a group of 26 patients ( 33 hips ) that received placebo . at the end of the study , mri evaluation revealed that 21 of the 32 hips in the alendronate group and 20 of the 33 hips in the placebo group had progressed ( p=0.636 ) . four of 32 hips in the alendronate treatment group underwent tha , and 5 of 33 hips in the placebo group had tha ( p=0.837 ) . no differences were noted in hhs , or short form 36 scores between the 2 groups . thus , the extensive lesion of necrotic area might impair the prognosis of the avn when alendronate was used . who presented a clinic radiological analysis of 395 hips with a mean follow - up of 4 years and then further extended the follow - up of 40 patients ( 53 hips ) with avn to 10 years ; those patients were treated with oral alendronate for 3 years , and the data demonstrated although clinical functions showed a general trend of worsening after discontinuation , patients tolerated the decline well , as suggested by the reduction in pain of ficat and arlet stage i iii patients 10 years after onset of alendronate treatment . more importantly , the rates of radiologic progression and femoral head collapse were markedly reduced even at 10 years as compared to the historical data available for natural history of hips with untreated avn . at 10 years , 46 ( 87% ) of the 53 hips survived , that is , had a satisfactory clinical result . hip loss to arthroplasty occurred in 1 each of stage i ( 7% ) and stage ii ( 5% ) hips and in 5 ( 26% ) of stage iii hips . of the 34 hips that were in pre - collapse stages at the onset of the study , at 10 years 10 had collapsed , indicating a collapse rate of 29% for a period of 10 years . mean time to collapse was 4.2 years . they thereafter indicated that the benefit is particularly marked if the treatment is begun in the pre - collapse stages of the disease ( stage i or ii ) . even in stage iii hips some benefit was obtained from treatment with alendronate by at least a delay in the need for total hip replacement . the most common adverse effects across the studies were gastric dyspepsia mentioned in 3 studies and dizziness mentioned in 2 studies , both of which occurred after treatment initiation and were self - limiting . no osteonecrosis of the jaw were seen irrespective of the dose or duration of alendronate . the most studied is core decompression , which works by reduction of intramedullary pressure inside the femoral head by making a drill hole , thus improving blood flow to bone . osteotomy could relocate the necrotic area of bone from the weight - loading area of the acetabulum , so as to redistribute the weight loading to articular cartilage , which is supported by healthy bone . the increasingly applied method is bone graft , aiming to provide mechanical support to subchondral bone or cartilage . there are various types of bone grafting , some combined with osteotomy , osteochondral grafts , muscle pedicle bone grafts , and some are vascularized grafts to improve blood flow of the bone by achieving revascularization . it has been used to replace the necrotic bone segment to prevent collapse in steinberg stage i iii femoral avn . however , the efficacy and safety of the above procedures are still controversial [ 7,2729 ] . due to the reported efficacy of total hip arthroplasty and the typical age of patients with osteonecrosis , it has recently been questioned whether these invasive procedures are appropriate , given the potential difficulty of later conversion to a hip replacement . conservative treatment that helps improve function and delays femoral head deformity could be valuable time - buying strategy for some patients . some of the pharmacologic agents that have been used to treat osteonecrosis of the hip are statins , anticoagulants , prostacyclin , and bps [ 1019 ] . the theoretical benefit of statins is based on the association of increased fat cell size with an increased risk of development of hip osteonecrosis . anticoagulants may inhibit the aggregation of platelets and enhance blood flow to ischemic areas of bone . however , collapse of the femoral head appears to be a consequence of the non - coupling of bone reabsorption and bone regeneration rates . in this context , collapse could be prevented if the reabsorptive activity of the necrotic bone during the repair phase was inhibited or slowed until the formation of sufficient new bone . the efficacy of alendronate therapy of femoral head deformity was indicated by several experimental studies consisting of both adult rat and rabbit models induced by femoral head ischemia . in 2 closely related studies , alendronate given subcutaneously ( 200 mg / kg / d ) was able to preserve femoral head structure in mature rats during a 6-week follow - up . in another study , 3-week alendronate therapy reduced degeneration of articular cartilage and improved subchondral bone volume and mineral density in adult rabbits at 12 months , which therefore might be the reason that alendronate treatment could preserve the shape of the femoral head affected by avn . regarding alendronate for the treatment of avn in clinical trials , in the present review , our literature search found were 2 recent systematic reviews evaluating bps for avn , 1 of which only included 3 observational short - term studies in juveniles , and the other with 6 small short - term trials that reported substantial heterogeneities across studies in patient group ( adults and adolescents ) and treatments ( other bps and combined therapy ) . in contrast to previous reviews , the inclusion criteria of the current review were restricted to articles that studied alendronate treatment for adult avn . due to ongoing debate on long - term effects of alendronate on the growing skeleton , we limited the studied population to only adults . the inclusion of most recent longer - term results and rct would also help to update the previous evidence . furthermore , after determining an overall profile of clinic outcome and the rates of radiographic and clinical failure , the collected data were further stratified by radiographic stage and by duration of follow - up to determine whether any of these factors influenced the results . however , generally speaking , the studies included in the current review still present various limitations most used observational non - controlled methods ; small numbers of patients ; different avn stages of patients when treatment was initiated ; and lack of uniformity in dose and time of alendronate use . these articles , in addition , had various durations of follow - up and were composed of various subgroups of patient populations . bearing in mind the above - mentioned limitations , most studies suggested a positive short - term and middle - term efficacy in pain reduction , improvement of articular function , slowing of bone collapse progression , and delaying the need for arthroplasty in adult avn patients with the use of alendronate treatment . this is of great clinical significance , as most of the included patients are young or active patients who are likely to require a revision at some point in the future and the effective slowing of femoral head deformity by alendronate could help to avoid early tha . favorable long - term results were also presented by agarwala s 10-year study in treated patients even after alendronate discontinuation . in addition , there were no severe adverse effects associated with alendronate treatment observed during short- or long - term follow - up . another finding of the current review is that although patients in all stages appeared to have potential benefit from alendronate treatment , the application in early avn with small size lesion was suggested by most of the included studies . specially , as shown in chen s study , when extensive osteonecrosis ( stage ii c and iii c ) were radiographically presented , alendronate did not have any benefits . thus , the efficacy of alendronate for avn with large necrotic legions should be considered more carefully . our findings support consideration of alendronate use for avn in adults because short - term and long - term favorable results could be expected , particularly with the early stage and with small necrotic size . nevertheless , the lack of large - scale , randomized , and double - blind studies should be noted , and future studies should be developed to demonstrate the following aspects . 1 ) the detailed indication of avn for alendronate treatment should be further clarified ; for example , what type of avn , traumatic or non - traumatic , which stage of avn , including what size and what location of the necrotic lesion should be preferentially indicated . 2 ) there are a number of patient - specific factors that must be considered , including age , comorbidities , life expectancy , health , and activity level . 3 ) we also need to optimize the strategy of treatment , including timing of treatment initiation and alendronate therapy dose and duration .

osteonecrosis or avascular osteonecrosis ( avn ) of the femoral head is a devastating multifactorial disease that affects 20 000 persons each year in the united states . the purpose of this systematic review was to determine the efficacy and safety of alendronate for adult avn during short- and long - term follow - up . electronic databases were searched for randomized or nonrandomized clinical trials , cohort , case - control studies , and series of cases in which alendronate was used for treatment of adult avn of the femoral head . relevant articles with adequate data on reduction of pain , improvement of articular function , slowing of bone collapse progression , or need for total hip arthroplasty ( tha ) were included after applying inclusion and exclusion criteria . eight articles involving 788 hips with evidence level 1b to 3b were included in this systematic review . most studies suggested a positive short - term efficacy of alendronate treatment in reducing pain , improving articular function , slowing of bone collapse progression , and delaying the need for tha for adult avn patients . the favorable long - term results were also presented in those treated patients after 10-year follow - up . in addition , there were no severe adverse effects associated with alendronate treatment observed during short- and long - term follow - up , and most of the included studies suggested use of alendronate in early avn with small necrotic lesion to achieve better outcomes . the findings support consideration of alendronate use for adult avn , particularly with early stage and small necrotic size . the lack of large - scale , randomized , and double - blind studies justifies new studies to demonstrate the detailed indication and the optimized strategy of alendronate treatment . level of evidence : level 3a .

Background Short-term outcomes analysis Long-term outcomes analysis Adverse events analysis Conclusions

osteonecrosis or avascular osteonecrosis ( avn ) of the femoral head is a devastating multifactorial disease that affects 20 000 persons each year in the united states . after collapse , due to extreme pain and loss of hip function , most patients require standard total hip arthroplasty ( tha ) . however , because of the young age of many of these patients , a hip replacement can not be expected to last the patient s lifetime ; therefore , when feasible , attempts should be made to save the femoral head prior to collapse with use of less invasive treatment modalities [ 79 ] . nevertheless , the lack of controlled and long - term results , the substantial heterogeneities of genres in bps , the unclear indication of various stages of avn , and the combination of adults and juveniles , complicate the interpretation of recent systematic reviews and necessitate new evidence . therefore , we performed a systematic review restricted to alendronate therapy for adult avn . by summarizing recent randomized controlled trials and long - term follow - up studies , the purpose of this systematic review was to determine the efficacy and safety of alendronate for adult avn during short- and long - term follow - up . our hypothesis was that alendronate therapy could be well - tolerated and : 1 ) improve clinical function and pain , 2 ) retard the progression of femoral head collapse and/or reduce the incidence of tha , and 3 ) be influenced by stage of the disease . four electronic databases ( pubmed , embase , the cochrane central register of controlled trials , and china national knowledge infrastructure ) were searched using a search strategy combining the terms in boolean logic : ( alendronate or fosamax ) and ( avascular necrosis or aseptic necrosis or osteonecrosis ) and ( femoral head ) . trials were included if they were randomized or nonrandomized clinical trials , cohort , case - control studies , and series of cases in which alendronate was used for treatment of avn of the femoral head in adults , with adequately reported data on diminishment of pain , or improvement of articular function , or retardation of bone collapse progression , or need for arthroplasty . we also manually searched reference lists of review articles , and included studies to identify other potentially eligible studies . from each article we extracted the following details by using standardized forms : authors , year of publication , geographical location of study , study design / level of evidence , study population ( hips / patients ) , patient sex and age , follow - up duration , interventions , outcomes , and adverse events . the outcome of interest included clinic function and hip pain improvement of avn patients after alendronate treatment . clinical failure was defined as the need for tha . of these , we included 8 articles with 788 hips in this systematic review [ 1219 ] . all the studies targeted adult avn patients within stage iii classified by x - ray , magnetic resonance imaging ( mri ) , both according to ficat and arlet ( 3 studies ) , association research circulation osseous ( arco , 2 studies ) , or steinberg ( university of pennsylvania system , 2 studies ) . in 5 of the included studies full weight - bearing was only permitted in 1 study and 4 studies only allowed partial or no weight - bearing . six studies reported short - term results of alendronate treatment on avn ( < 4 years ) [ 12,1519 ] , and the other 2 reported long - term results ( 4 years ) . most of the included studies were prospective non - controlled studies and 4 of them were from the same institution reporting on the same group during different follow - up period . as presented in table 1 , the level of evidence for the studies ranged from 1b to 3b . table 2 showed the outcomes reported from studies evaluating alendronate use in avascular osteonecrosis of the femoral head . studied 16 patients with avn of the femoral head most of them secondary to the use of corticosteroids . they used a regimen of alendronate 10 mg / day + calcium 1 g / day + vitamin d supplement , and the mean duration of therapy with alendronate was 24.7 weeks . patients that used alendronate had a significant improvement in pain as early as 12 weeks , with a reduction in the need for analgesics and improvement in functional capacity in all patients , and this improvement was maintained for 24 weeks . alendronate was used in daily doses of 10 mg or weekly doses of 70 mg ; these authors confirmed these findings and further suggested that alendronate would retard the progression of avn and avoid the early indication of surgery in mid - term follow - up . another study with short - term follow - up was performed by chen et al . , which included 83 patients with non - traumatic avn of the femoral head 33 of the patients were arco i and the rest were arco ii . they concluded that alendronate is effective in treatment of early - stage adult non - traumatic avn of the femoral head , especially for arco ii patients . the first rct with short - term results evaluating the treatment of alendronate on avn of the femoral head was by lai et al . they studied 40 patients with steinberg stage ii or iii c non - traumatic avn of the femoral head . thus , they concluded that alendronate appeared to prevent early collapse of the femoral head in the hips with steinberg stage ii or iii c non - traumatic avn . at the end of follow - up , the group of patients receiving alendronate had less pain and a lower frequency of femoral head collapse when compared with the control group . specifically , they found progressive collapse occurred only in hips with extensive necrosis ( greater than the medial 2/3 of the weight - bearing area of the femoral head with / without involvement of acetabula edge , termed type c2 and c1 , respectively ) in both groups , which had a much higher incidence in the control groups . therefore they suggested alendronate had the potential to prevent collapse of the femoral head , even with extensive necrosis , within 1 year . recently performed a 2-year , multicenter , prospective , randomized , double - blind study involving a total of 52 patients ( 65 hips ) . four of 32 hips in the alendronate treatment group underwent tha , and 5 of 33 hips in the placebo group had tha ( p=0.837 ) . thus , the extensive lesion of necrotic area might impair the prognosis of the avn when alendronate was used . , who presented a clinic radiological analysis of 395 hips with a mean follow - up of 4 years and then further extended the follow - up of 40 patients ( 53 hips ) with avn to 10 years ; those patients were treated with oral alendronate for 3 years , and the data demonstrated although clinical functions showed a general trend of worsening after discontinuation , patients tolerated the decline well , as suggested by the reduction in pain of ficat and arlet stage i iii patients 10 years after onset of alendronate treatment . even in stage iii hips some benefit was obtained from treatment with alendronate by at least a delay in the need for total hip replacement . the most studied is core decompression , which works by reduction of intramedullary pressure inside the femoral head by making a drill hole , thus improving blood flow to bone . there are various types of bone grafting , some combined with osteotomy , osteochondral grafts , muscle pedicle bone grafts , and some are vascularized grafts to improve blood flow of the bone by achieving revascularization . however , the efficacy and safety of the above procedures are still controversial [ 7,2729 ] . due to the reported efficacy of total hip arthroplasty and the typical age of patients with osteonecrosis , it has recently been questioned whether these invasive procedures are appropriate , given the potential difficulty of later conversion to a hip replacement . however , collapse of the femoral head appears to be a consequence of the non - coupling of bone reabsorption and bone regeneration rates . the efficacy of alendronate therapy of femoral head deformity was indicated by several experimental studies consisting of both adult rat and rabbit models induced by femoral head ischemia . in 2 closely related studies , alendronate given subcutaneously ( 200 mg / kg / d ) was able to preserve femoral head structure in mature rats during a 6-week follow - up . in another study , 3-week alendronate therapy reduced degeneration of articular cartilage and improved subchondral bone volume and mineral density in adult rabbits at 12 months , which therefore might be the reason that alendronate treatment could preserve the shape of the femoral head affected by avn . regarding alendronate for the treatment of avn in clinical trials , in the present review , only 8 articles were published . our literature search found were 2 recent systematic reviews evaluating bps for avn , 1 of which only included 3 observational short - term studies in juveniles , and the other with 6 small short - term trials that reported substantial heterogeneities across studies in patient group ( adults and adolescents ) and treatments ( other bps and combined therapy ) . in contrast to previous reviews , the inclusion criteria of the current review were restricted to articles that studied alendronate treatment for adult avn . due to ongoing debate on long - term effects of alendronate on the growing skeleton , we limited the studied population to only adults . furthermore , after determining an overall profile of clinic outcome and the rates of radiographic and clinical failure , the collected data were further stratified by radiographic stage and by duration of follow - up to determine whether any of these factors influenced the results . however , generally speaking , the studies included in the current review still present various limitations most used observational non - controlled methods ; small numbers of patients ; different avn stages of patients when treatment was initiated ; and lack of uniformity in dose and time of alendronate use . these articles , in addition , had various durations of follow - up and were composed of various subgroups of patient populations . bearing in mind the above - mentioned limitations , most studies suggested a positive short - term and middle - term efficacy in pain reduction , improvement of articular function , slowing of bone collapse progression , and delaying the need for arthroplasty in adult avn patients with the use of alendronate treatment . this is of great clinical significance , as most of the included patients are young or active patients who are likely to require a revision at some point in the future and the effective slowing of femoral head deformity by alendronate could help to avoid early tha . favorable long - term results were also presented by agarwala s 10-year study in treated patients even after alendronate discontinuation . in addition , there were no severe adverse effects associated with alendronate treatment observed during short- or long - term follow - up . another finding of the current review is that although patients in all stages appeared to have potential benefit from alendronate treatment , the application in early avn with small size lesion was suggested by most of the included studies . thus , the efficacy of alendronate for avn with large necrotic legions should be considered more carefully . studied 16 patients with avn of the femoral head most of them secondary to the use of corticosteroids . they used a regimen of alendronate 10 mg / day + calcium 1 g / day + vitamin d supplement , and the mean duration of therapy with alendronate was 24.7 weeks . patients that used alendronate had a significant improvement in pain as early as 12 weeks , with a reduction in the need for analgesics and improvement in functional capacity in all patients , and this improvement was maintained for 24 weeks . alendronate was used in daily doses of 10 mg or weekly doses of 70 mg ; these authors confirmed these findings and further suggested that alendronate would retard the progression of avn and avoid the early indication of surgery in mid - term follow - up . another study with short - term follow - up was performed by chen et al . , which included 83 patients with non - traumatic avn of the femoral head 33 of the patients were arco i and the rest were arco ii . in the patients with arco i avn , the scores of pain and function were improved after treatment ( p<0.01 ) . they concluded that alendronate is effective in treatment of early - stage adult non - traumatic avn of the femoral head , especially for arco ii patients . the first rct with short - term results evaluating the treatment of alendronate on avn of the femoral head was by lai et al . they studied 40 patients with steinberg stage ii or iii c non - traumatic avn of the femoral head . thus , they concluded that alendronate appeared to prevent early collapse of the femoral head in the hips with steinberg stage ii or iii c non - traumatic avn . at the end of follow - up , the group of patients receiving alendronate had less pain and a lower frequency of femoral head collapse when compared with the control group . specifically , they found progressive collapse occurred only in hips with extensive necrosis ( greater than the medial 2/3 of the weight - bearing area of the femoral head with / without involvement of acetabula edge , termed type c2 and c1 , respectively ) in both groups , which had a much higher incidence in the control groups . therefore they suggested alendronate had the potential to prevent collapse of the femoral head , even with extensive necrosis , within 1 year . recently performed a 2-year , multicenter , prospective , randomized , double - blind study involving a total of 52 patients ( 65 hips ) . four of 32 hips in the alendronate treatment group underwent tha , and 5 of 33 hips in the placebo group had tha ( p=0.837 ) . thus , the extensive lesion of necrotic area might impair the prognosis of the avn when alendronate was used . who presented a clinic radiological analysis of 395 hips with a mean follow - up of 4 years and then further extended the follow - up of 40 patients ( 53 hips ) with avn to 10 years ; those patients were treated with oral alendronate for 3 years , and the data demonstrated although clinical functions showed a general trend of worsening after discontinuation , patients tolerated the decline well , as suggested by the reduction in pain of ficat and arlet stage i iii patients 10 years after onset of alendronate treatment . even in stage iii hips some benefit was obtained from treatment with alendronate by at least a delay in the need for total hip replacement . the most studied is core decompression , which works by reduction of intramedullary pressure inside the femoral head by making a drill hole , thus improving blood flow to bone . there are various types of bone grafting , some combined with osteotomy , osteochondral grafts , muscle pedicle bone grafts , and some are vascularized grafts to improve blood flow of the bone by achieving revascularization . however , the efficacy and safety of the above procedures are still controversial [ 7,2729 ] . due to the reported efficacy of total hip arthroplasty and the typical age of patients with osteonecrosis , it has recently been questioned whether these invasive procedures are appropriate , given the potential difficulty of later conversion to a hip replacement . however , collapse of the femoral head appears to be a consequence of the non - coupling of bone reabsorption and bone regeneration rates . the efficacy of alendronate therapy of femoral head deformity was indicated by several experimental studies consisting of both adult rat and rabbit models induced by femoral head ischemia . in 2 closely related studies , alendronate given subcutaneously ( 200 mg / kg / d ) was able to preserve femoral head structure in mature rats during a 6-week follow - up . in another study , 3-week alendronate therapy reduced degeneration of articular cartilage and improved subchondral bone volume and mineral density in adult rabbits at 12 months , which therefore might be the reason that alendronate treatment could preserve the shape of the femoral head affected by avn . regarding alendronate for the treatment of avn in clinical trials , in the present review , our literature search found were 2 recent systematic reviews evaluating bps for avn , 1 of which only included 3 observational short - term studies in juveniles , and the other with 6 small short - term trials that reported substantial heterogeneities across studies in patient group ( adults and adolescents ) and treatments ( other bps and combined therapy ) . in contrast to previous reviews , the inclusion criteria of the current review were restricted to articles that studied alendronate treatment for adult avn . due to ongoing debate on long - term effects of alendronate on the growing skeleton , we limited the studied population to only adults . furthermore , after determining an overall profile of clinic outcome and the rates of radiographic and clinical failure , the collected data were further stratified by radiographic stage and by duration of follow - up to determine whether any of these factors influenced the results . however , generally speaking , the studies included in the current review still present various limitations most used observational non - controlled methods ; small numbers of patients ; different avn stages of patients when treatment was initiated ; and lack of uniformity in dose and time of alendronate use . these articles , in addition , had various durations of follow - up and were composed of various subgroups of patient populations . bearing in mind the above - mentioned limitations , most studies suggested a positive short - term and middle - term efficacy in pain reduction , improvement of articular function , slowing of bone collapse progression , and delaying the need for arthroplasty in adult avn patients with the use of alendronate treatment . this is of great clinical significance , as most of the included patients are young or active patients who are likely to require a revision at some point in the future and the effective slowing of femoral head deformity by alendronate could help to avoid early tha . favorable long - term results were also presented by agarwala s 10-year study in treated patients even after alendronate discontinuation . in addition , there were no severe adverse effects associated with alendronate treatment observed during short- or long - term follow - up . another finding of the current review is that although patients in all stages appeared to have potential benefit from alendronate treatment , the application in early avn with small size lesion was suggested by most of the included studies . thus , the efficacy of alendronate for avn with large necrotic legions should be considered more carefully . our findings support consideration of alendronate use for avn in adults because short - term and long - term favorable results could be expected , particularly with the early stage and with small necrotic size . nevertheless , the lack of large - scale , randomized , and double - blind studies should be noted , and future studies should be developed to demonstrate the following aspects . 1 ) the detailed indication of avn for alendronate treatment should be further clarified ; for example , what type of avn , traumatic or non - traumatic , which stage of avn , including what size and what location of the necrotic lesion should be preferentially indicated .

as bone repair occurs , the imbalance of osteoclast - medicated resorption and delayed new bone formation result in mechanically weak bone that collapses under the load of weight . however , because of the young age of many of these patients , a hip replacement can not be expected to last the patient s lifetime ; therefore , when feasible , attempts should be made to save the femoral head prior to collapse with use of less invasive treatment modalities [ 79 ] . in contrast to other drugs , bisphosphonates ( bps ) are potent anti - reabsorptive agents that act by inhibiting the action of mature osteoclasts in the bone , which theoretically normalizes the uncoupled bone remodeling , contributing to femoral head collapse . in the last decade , many studies have investigated the application of bps in the treatment of avn [ 1019 ] . nevertheless , the lack of controlled and long - term results , the substantial heterogeneities of genres in bps , the unclear indication of various stages of avn , and the combination of adults and juveniles , complicate the interpretation of recent systematic reviews and necessitate new evidence . of different bps , in addition , application of bps in children raises great concern due to its potential harmful effects on the growing skeleton of juveniles . by summarizing recent randomized controlled trials and long - term follow - up studies , the purpose of this systematic review was to determine the efficacy and safety of alendronate for adult avn during short- and long - term follow - up . our hypothesis was that alendronate therapy could be well - tolerated and : 1 ) improve clinical function and pain , 2 ) retard the progression of femoral head collapse and/or reduce the incidence of tha , and 3 ) be influenced by stage of the disease . four electronic databases ( pubmed , embase , the cochrane central register of controlled trials , and china national knowledge infrastructure ) were searched using a search strategy combining the terms in boolean logic : ( alendronate or fosamax ) and ( avascular necrosis or aseptic necrosis or osteonecrosis ) and ( femoral head ) . trials were included if they were randomized or nonrandomized clinical trials , cohort , case - control studies , and series of cases in which alendronate was used for treatment of avn of the femoral head in adults , with adequately reported data on diminishment of pain , or improvement of articular function , or retardation of bone collapse progression , or need for arthroplasty . after exclusion of duplicates , 1 reviewer ( lrb ) performed an initial title and abstract screening of articles to discard those that were clearly ineligible , then 2 reviewers ( lt and zhm ) independently examined the full article to assess the trials for eligibility for inclusion , with disagreements resolved by discussion . from each article we extracted the following details by using standardized forms : authors , year of publication , geographical location of study , study design / level of evidence , study population ( hips / patients ) , patient sex and age , follow - up duration , interventions , outcomes , and adverse events . for the radiographic evaluation , although various classification systems were used among the studies , they shared fundamental similarities ; therefore , radiographic failure here were defined as any lesions progressed to a higher stage from baseline stage . due to the included avn comprising both pre - collapse and early - collapse stage , collapse rates were separated and considered as a new occurrence of collapse or an increased collapse of greater than 2 mm . all the studies targeted adult avn patients within stage iii classified by x - ray , magnetic resonance imaging ( mri ) , both according to ficat and arlet ( 3 studies ) , association research circulation osseous ( arco , 2 studies ) , or steinberg ( university of pennsylvania system , 2 studies ) . four studies were restricted to non - traumatic avn patients and the other 4 articles from the same group did not specify the etiology . most of the included studies were prospective non - controlled studies and 4 of them were from the same institution reporting on the same group during different follow - up period . patients that used alendronate had a significant improvement in pain as early as 12 weeks , with a reduction in the need for analgesics and improvement in functional capacity in all patients , and this improvement was maintained for 24 weeks . alendronate was used in daily doses of 10 mg or weekly doses of 70 mg ; these authors confirmed these findings and further suggested that alendronate would retard the progression of avn and avoid the early indication of surgery in mid - term follow - up . , which included 83 patients with non - traumatic avn of the femoral head 33 of the patients were arco i and the rest were arco ii . in the patients with arco i avn , the scores of pain and function were improved after treatment ( p<0.01 ) . similarly , in the patients with arco ii avn the scores of pain and function were both improved after treatment ( p<0.01 ) . they concluded that alendronate is effective in treatment of early - stage adult non - traumatic avn of the femoral head , especially for arco ii patients . at the end of the study , the mean hhs was 49.29.2 points in the control group and 74.47.8 points in the alendronate group . it was also demonstrated that only 2/29 femoral heads with an ( 0/17 in stage ii , 2/12 in stage iii ) collapsed in the group that received alendronate 70 mg / week , whereas in the group that was randomized to not receive this medication , collapse occurred in 19/25 ( 9/13 in stage ii , 10/12 in stage iii ) femoral heads ( p<0.001 ) . one hip in the alendronate group underwent tha , whereas 16 hips in the control group underwent tha ( p<0.001 ) . thus , they concluded that alendronate appeared to prevent early collapse of the femoral head in the hips with steinberg stage ii or iii c non - traumatic avn . , 14 patients ( 20 hips ) with arco i iii avn received alendronate 5 mg / day and were compared in a nonrandomized manner with a group of 8 patients ( 13 hips ) that did not receive alendronate . at the end of follow - up , the group of patients receiving alendronate had less pain and a lower frequency of femoral head collapse when compared with the control group . specifically , they found progressive collapse occurred only in hips with extensive necrosis ( greater than the medial 2/3 of the weight - bearing area of the femoral head with / without involvement of acetabula edge , termed type c2 and c1 , respectively ) in both groups , which had a much higher incidence in the control groups . in total , collapse occurred in 6/13 of articulations in the control group and in only 1/20 in the alendronate group ( p=0.008 ) . moreover , 2 of 13 hips in the control group needed tha , but none of patients needed surgery in the alendronate group . twenty - six patients ( 32 hips ) were assigned to a scheme of alendronate 70 mg / week for 104 weeks and were compared with a group of 26 patients ( 33 hips ) that received placebo . at the end of the study , mri evaluation revealed that 21 of the 32 hips in the alendronate group and 20 of the 33 hips in the placebo group had progressed ( p=0.636 ) . four of 32 hips in the alendronate treatment group underwent tha , and 5 of 33 hips in the placebo group had tha ( p=0.837 ) . , who presented a clinic radiological analysis of 395 hips with a mean follow - up of 4 years and then further extended the follow - up of 40 patients ( 53 hips ) with avn to 10 years ; those patients were treated with oral alendronate for 3 years , and the data demonstrated although clinical functions showed a general trend of worsening after discontinuation , patients tolerated the decline well , as suggested by the reduction in pain of ficat and arlet stage i iii patients 10 years after onset of alendronate treatment . more importantly , the rates of radiologic progression and femoral head collapse were markedly reduced even at 10 years as compared to the historical data available for natural history of hips with untreated avn . hip loss to arthroplasty occurred in 1 each of stage i ( 7% ) and stage ii ( 5% ) hips and in 5 ( 26% ) of stage iii hips . of the 34 hips that were in pre - collapse stages at the onset of the study , at 10 years 10 had collapsed , indicating a collapse rate of 29% for a period of 10 years . the most common adverse effects across the studies were gastric dyspepsia mentioned in 3 studies and dizziness mentioned in 2 studies , both of which occurred after treatment initiation and were self - limiting . osteotomy could relocate the necrotic area of bone from the weight - loading area of the acetabulum , so as to redistribute the weight loading to articular cartilage , which is supported by healthy bone . there are various types of bone grafting , some combined with osteotomy , osteochondral grafts , muscle pedicle bone grafts , and some are vascularized grafts to improve blood flow of the bone by achieving revascularization . due to the reported efficacy of total hip arthroplasty and the typical age of patients with osteonecrosis , it has recently been questioned whether these invasive procedures are appropriate , given the potential difficulty of later conversion to a hip replacement . the theoretical benefit of statins is based on the association of increased fat cell size with an increased risk of development of hip osteonecrosis . however , collapse of the femoral head appears to be a consequence of the non - coupling of bone reabsorption and bone regeneration rates . in another study , 3-week alendronate therapy reduced degeneration of articular cartilage and improved subchondral bone volume and mineral density in adult rabbits at 12 months , which therefore might be the reason that alendronate treatment could preserve the shape of the femoral head affected by avn . regarding alendronate for the treatment of avn in clinical trials , in the present review , only 8 articles were published . our literature search found were 2 recent systematic reviews evaluating bps for avn , 1 of which only included 3 observational short - term studies in juveniles , and the other with 6 small short - term trials that reported substantial heterogeneities across studies in patient group ( adults and adolescents ) and treatments ( other bps and combined therapy ) . in contrast to previous reviews , the inclusion criteria of the current review were restricted to articles that studied alendronate treatment for adult avn . furthermore , after determining an overall profile of clinic outcome and the rates of radiographic and clinical failure , the collected data were further stratified by radiographic stage and by duration of follow - up to determine whether any of these factors influenced the results . however , generally speaking , the studies included in the current review still present various limitations most used observational non - controlled methods ; small numbers of patients ; different avn stages of patients when treatment was initiated ; and lack of uniformity in dose and time of alendronate use . bearing in mind the above - mentioned limitations , most studies suggested a positive short - term and middle - term efficacy in pain reduction , improvement of articular function , slowing of bone collapse progression , and delaying the need for arthroplasty in adult avn patients with the use of alendronate treatment . this is of great clinical significance , as most of the included patients are young or active patients who are likely to require a revision at some point in the future and the effective slowing of femoral head deformity by alendronate could help to avoid early tha . another finding of the current review is that although patients in all stages appeared to have potential benefit from alendronate treatment , the application in early avn with small size lesion was suggested by most of the included studies . patients that used alendronate had a significant improvement in pain as early as 12 weeks , with a reduction in the need for analgesics and improvement in functional capacity in all patients , and this improvement was maintained for 24 weeks . , which included 83 patients with non - traumatic avn of the femoral head 33 of the patients were arco i and the rest were arco ii . in the patients with arco i avn , the scores of pain and function were improved after treatment ( p<0.01 ) . similarly , in the patients with arco ii avn the scores of pain and function were both improved after treatment ( p<0.01 ) . they concluded that alendronate is effective in treatment of early - stage adult non - traumatic avn of the femoral head , especially for arco ii patients . at the end of the study , the mean hhs was 49.29.2 points in the control group and 74.47.8 points in the alendronate group . it was also demonstrated that only 2/29 femoral heads with an ( 0/17 in stage ii , 2/12 in stage iii ) collapsed in the group that received alendronate 70 mg / week , whereas in the group that was randomized to not receive this medication , collapse occurred in 19/25 ( 9/13 in stage ii , 10/12 in stage iii ) femoral heads ( p<0.001 ) . one hip in the alendronate group underwent tha , whereas 16 hips in the control group underwent tha ( p<0.001 ) . , 14 patients ( 20 hips ) with arco i iii avn received alendronate 5 mg / day and were compared in a nonrandomized manner with a group of 8 patients ( 13 hips ) that did not receive alendronate . at the end of follow - up , the group of patients receiving alendronate had less pain and a lower frequency of femoral head collapse when compared with the control group . specifically , they found progressive collapse occurred only in hips with extensive necrosis ( greater than the medial 2/3 of the weight - bearing area of the femoral head with / without involvement of acetabula edge , termed type c2 and c1 , respectively ) in both groups , which had a much higher incidence in the control groups . in total , collapse occurred in 6/13 of articulations in the control group and in only 1/20 in the alendronate group ( p=0.008 ) . moreover , 2 of 13 hips in the control group needed tha , but none of patients needed surgery in the alendronate group . twenty - six patients ( 32 hips ) were assigned to a scheme of alendronate 70 mg / week for 104 weeks and were compared with a group of 26 patients ( 33 hips ) that received placebo . at the end of the study , mri evaluation revealed that 21 of the 32 hips in the alendronate group and 20 of the 33 hips in the placebo group had progressed ( p=0.636 ) . four of 32 hips in the alendronate treatment group underwent tha , and 5 of 33 hips in the placebo group had tha ( p=0.837 ) . who presented a clinic radiological analysis of 395 hips with a mean follow - up of 4 years and then further extended the follow - up of 40 patients ( 53 hips ) with avn to 10 years ; those patients were treated with oral alendronate for 3 years , and the data demonstrated although clinical functions showed a general trend of worsening after discontinuation , patients tolerated the decline well , as suggested by the reduction in pain of ficat and arlet stage i iii patients 10 years after onset of alendronate treatment . more importantly , the rates of radiologic progression and femoral head collapse were markedly reduced even at 10 years as compared to the historical data available for natural history of hips with untreated avn . of the 34 hips that were in pre - collapse stages at the onset of the study , at 10 years 10 had collapsed , indicating a collapse rate of 29% for a period of 10 years . the most common adverse effects across the studies were gastric dyspepsia mentioned in 3 studies and dizziness mentioned in 2 studies , both of which occurred after treatment initiation and were self - limiting . osteotomy could relocate the necrotic area of bone from the weight - loading area of the acetabulum , so as to redistribute the weight loading to articular cartilage , which is supported by healthy bone . there are various types of bone grafting , some combined with osteotomy , osteochondral grafts , muscle pedicle bone grafts , and some are vascularized grafts to improve blood flow of the bone by achieving revascularization . due to the reported efficacy of total hip arthroplasty and the typical age of patients with osteonecrosis , it has recently been questioned whether these invasive procedures are appropriate , given the potential difficulty of later conversion to a hip replacement . however , collapse of the femoral head appears to be a consequence of the non - coupling of bone reabsorption and bone regeneration rates . in another study , 3-week alendronate therapy reduced degeneration of articular cartilage and improved subchondral bone volume and mineral density in adult rabbits at 12 months , which therefore might be the reason that alendronate treatment could preserve the shape of the femoral head affected by avn . regarding alendronate for the treatment of avn in clinical trials , in the present review , our literature search found were 2 recent systematic reviews evaluating bps for avn , 1 of which only included 3 observational short - term studies in juveniles , and the other with 6 small short - term trials that reported substantial heterogeneities across studies in patient group ( adults and adolescents ) and treatments ( other bps and combined therapy ) . furthermore , after determining an overall profile of clinic outcome and the rates of radiographic and clinical failure , the collected data were further stratified by radiographic stage and by duration of follow - up to determine whether any of these factors influenced the results . however , generally speaking , the studies included in the current review still present various limitations most used observational non - controlled methods ; small numbers of patients ; different avn stages of patients when treatment was initiated ; and lack of uniformity in dose and time of alendronate use . bearing in mind the above - mentioned limitations , most studies suggested a positive short - term and middle - term efficacy in pain reduction , improvement of articular function , slowing of bone collapse progression , and delaying the need for arthroplasty in adult avn patients with the use of alendronate treatment . this is of great clinical significance , as most of the included patients are young or active patients who are likely to require a revision at some point in the future and the effective slowing of femoral head deformity by alendronate could help to avoid early tha . another finding of the current review is that although patients in all stages appeared to have potential benefit from alendronate treatment , the application in early avn with small size lesion was suggested by most of the included studies . our findings support consideration of alendronate use for avn in adults because short - term and long - term favorable results could be expected , particularly with the early stage and with small necrotic size . 1 ) the detailed indication of avn for alendronate treatment should be further clarified ; for example , what type of avn , traumatic or non - traumatic , which stage of avn , including what size and what location of the necrotic lesion should be preferentially indicated .

cesarean sections have been a global public health concern for decades.13 as a key component of comprehensive emergency obstetric care ( emoc ) , the adequate provision of cesarean sections during intrapartum care is a life - saving procedure.2,3 this is particularly vital for mothers living in low and middle income countries , where access to emoc is low and resources to provide quality care are limited.35 notwithstanding the well - recognized life - saving role of cesarean sections , international comparisons show that rates of cesarean sections vary greatly across countries and high coverage does not necessarily lead to good health outcomes.1,2 elective cesareans can increase the risk of maternal morbidity , neonatal death , and neonatal admission to an intensive care unit.1 the overuse of cesareans is therefore a real public health concern , not only because cesareans are costly , incurring a financial burden for the families and society , but also because they may entail a health risk for women and children s health . the threshold above which cesarean sections are in excess of need is not well known , but the world health organization suggests a maximum threshold of around 15%.6 recent global estimates consistently show that rates of cesareans have been rising dramatically over the recommended threshold , particularly in latin american and some asian countries.7,8 the debate on why rates of cesareans rise quickly continues , and both supply and demand side factors are thought to play a role.1,9 the preponderance of obstetricians in providing intrapartum care and the perverse financial incentives associated with cesareans are the main factors considered from the supply side , whilst for the demand side , discussions center around women and families preferences . to date no consensus has been reached on the main factors driving the cesarean epidemic . as the largest developing country , the people s republic of china bears a substantial burden of maternal and child mortality worldwide , and was included as one of the 68 countdown countries to achieve the two millennium development goals ( mdg ) related to maternal and child health ( mdg 4 and 5).10,11 to address the high burden of maternal mortality , a universal hospital delivery strategy was introduced by the chinese government in the mid-1990s.12 in 1995 , the law on maternal and infant health care was passed to guarantee each woman s right to a hospital for quality intrapartum care . both supply and demand side interventions have been put forward since then . infrastructure has been strengthened , medical staff were trained and supervised in township and lower level hospitals , and referral channels to tertiary hospitals were reinforced.12 in 2003 , the government launched the new cooperative medical scheme ( ncms ) , which provides health insurance coverage for the majority of the rural chinese population for medical care , including hospital delivery . by 2007 , health insurance had achieved near universal coverage in the people s republic of china , greatly increasing the financial accessibility to facility - based intrapartum care for the chinese population.13 consistent national data show that the people s republic of china s hospital delivery strategy has been successful in reducing maternal and neonatal mortality.10,11 in 1988 , less than half of all women gave birth in a hospital , yet 20 years later hospital births have become nearly universal and socioeconomic inequalities in hospital births have nearly disappeared.13 over the same period , maternal mortality declined by 47%86% and neonatal mortality by 48%70% . as a result , the people s republic of china is now quoted as a successful example in the developing world in achieving mdg 4 and 5.10,11 at the same time however , rates of cesarean sections have been rising dramatically . in 20072008 , the national rate was 46.2% and 40% of cesareans were reported to have no clinical indications.14 in this paper , we systematically reviewed peer - reviewed articles in both chinese and international journals on the theme of cesarean sections in the people s republic of china . from a multidisciplinary perspective , particularly health policy and systems research , we discuss the current knowledge on the prevalence , reasons , and consequences regarding cesarean sections in the people s republic of china . we add up the literature not only by describing the specific phenomenon from the people s republic of china , but also by contributing to the international debate on why cesareans rise and how it might be related to a country s health system development . we systematically searched pubmed for peer - reviewed articles . since only a limited amount of chinese journals were indexed by pubmed , we also comprehensively searched the following chinese databases : the china national knowledge infrastructure , wanfang , and the vip information . the databases were searched from inception to september 1 , 2013 . to identify further relevant studies , reference lists of included articles were hand - searched and reviewed . when searching pubmed , we included studies that reported caesarean or the following study types were included : clinical trial , meta - analysis , randomized controlled trials , and all types of observational studies , including cohort studies , case control studies , cross - sectional studies , and case reports . however , since most of the chinese journals , which are not indexed by pubmed , are not peer - reviewed and the majority of these papers are not original research , we screened the title and abstract and intentionally chose important and relevant articles for this review . finally , we got 97 results on pubmed . full texts of these potentially suitable articles were obtained and reviewed . with 17 articles picked out from chinese databases and five additional papers hand - searched , we finally arrived at 82 papers for this analysis ( figure 1 ) . amongst these articles , 30 reported the prevalence and trends in cesareans , 48 reported the reasons for performing cesareans , and 30 reported the consequences of cesareans , where 23 articles reported both the prevalence and the reasons , 1 reported both the prevalence and the consequences , and one reported all three themes . the following search formula was used for pubmed : ( humans[mesh terms ] and ( clinical trial[all fields ] or meta - analysis[all fields ] or randomized controlled trial[all fields ] or case reports[all fields ] or classical article[all fields ] or clinical trial , phase i[all fields ] or clinical trial , phase ii[all fields ] or clinical trial , phase iii[all fields ] or clinical trial , phase iv[all fields ] or comparative study[all fields ] or controlled clinical trial[all fields ] or corrected and republished article[all fields ] or english abstract[all fields ] or evaluation studies[all fields ] or historical article[all fields ] or journal article[all fields ] or multicenter study[all fields ] or twin study[all fields ] ) ) and ( caesarean or caesarean section or cesarean or cesarean section ) and ( china [ title ] or chinese [ title ] ) and ( china [ all fields ] not veterinary[mesh subheading ] ) . there are two major types of studies estimating the prevalence of and trends in cesarean sections in the people s republic of china : hospital - based studies and population surveys ( table 1 ) . the earliest attempts used hospital records to report trends in cesarean sections in specific hospitals , categorizing cesareans by clinical indications . a typical example was zheng et al.15 using clinical records from one of the best tertiary hospitals in beijing , they reported that the proportion of cesarean births was low in 1960 ( 2.0% ) , increasing dramatically to 50.0% in 1994 . the authors categorized clinical decisions into five groups : 1 ) maternal factors , including pregnancy - induced hypertension and medical complications during pregnancy ; 2 ) fetal factors , including fetal distress and placental position abnormality ; 3 ) cephalopelvic disproportion ; 4 ) social factors ; and 5 ) other factors . interestingly , a category of social factors was included , increasing from 0% of cesareans in 1960 to 10.7% in 1994 . for example , zhao et al16 reported that cesareans increased from 25.4% of births in 1990 to 39.6% in 2002 in a secondary level hospital , and feng and yue17 reported a rise from 2.6% in 1955 to 64.7% in 2004 in a tertiary hospital in middle china . such types of studies also appeared in english peer - reviewed journals , for example han et al18 and ma et al.19 in summary , these analyses consistently reported a substantial rise in the proportion of social factors , which has become the leading reason affecting clinical decisions to perform cesarean sections in recent years . in the second type of studies health facility surveys information is also based on hospital records ; each hospital itself was required to calculate the proportion of cesarean deliveries . for example , wang et al20 surveyed 887 health facilities for six types of birth attendance procedures taken in the year 2002 . they reported a national rate of cesarean section at 38.0% and extremely low utilization of sericeps and forceps in birth attendance . while the strength of this type of research is that it could yield robust national rates , the weakness is that trends could not be arrived at due to the hardship in organizing facility surveys from academic institutions . the above two study designs based on hospital records could yield robust estimates of trends and proportions of cesarean section for births in hospitals . however , as illustrated by a systematic review based on other countries,21 information extracted from clinical records may yield non - trivial misclassifications for clinical indications on the categorization of cesarean sections . furthermore , such types of studies ignore births that are out of health facilities , resulting in an overestimate of the rates of cesarean section at national or regional level . hospital records also lack information on the socioeconomic characteristics of the population level , limiting the scope for further policy analysis . in 1998 , cai et al22 conducted a population - based survey in a district in shanghai , one of the largest metropolitan cities in the people s republic of china . using information on cesarean sections collected from women s recall , the authors reported , for the first time , a rise of cesarean sections from 4.7% from 19601979 to 22.5% from 19881993 at the population level . cai et al also analyzed whether cesarean sections were associated with clinical , demographic , and socioeconomic factors . they found that medical insurance coverage , older maternal age , high birth weight , and self - reported complications were factors affecting the utilization of cesareans . following cai et al , population - based surveys became the design of choice to estimate rates of cesarean section and their determinants ( 15 studies as shown in table 2 ) . for example , sufang et al23 reported trends in cesareans in the people s republic of china between 1993 and 2002 using a national cross - sectional survey and lu et al24 reported trends in southern china between 1994 and 2005 . repeated cross - sectional surveys were also used for the estimation of longer time trends . using the people s republic of china s national health service surveys , tang et al25 reported trends in cesareans in urban china from 19902002 , while klemetti et al26 reported such trends in rural china from 19912002 . with the same national data , feng et al27 did a comprehensive analysis on the trends in cesarean section rates in the people s republic of china from 1988 to 2008 . using the four national surveys conducted in the years 1993 , 1998 , 2003 , and 2008 , the study estimated the people s republic of china s national cesarean section rates at 3.4% in 1988 , increasing ten - fold to 39.3% in 2008 . the most extraordinary increase was witnessed in urban settings , where 64.1% of the women gave birth by cesarean section in 2008 ; while even in the least developed rural region , the rate rose from 0% in 1988 to 11.3% in 2008 . both clinical factors and nonclinical factors were investigated to explain the epidemic of cesarean sections in the people s republic of china . while studies on clinical factors for choosing cesareans are relatively rare and disagreement is seldom made , the debates on nonclinical factors are quite lively and in - depth , particularly on whether the rise in cesareans in the people s republic of china are fueled by the women s own choice or the obstetricians induced demand . a comprehensive list of studies addressing the factors that affect the use of cesarean sections in the people s republic of china is shown in table 2 . in summary , there are 14 studies focusing on clinical factors and 34 studies on nonclinical factors . as early as 1989 , following an observation that rising cesareans were not associated with a reduction in perinatal mortality , a national academic conference was organized in the obstetricians society to discuss clinical indications for cesarean sections.28 among the clinical indications , precious child was increasingly reported , alongside other more conventional clinical factors . there was no clear definition of what was meant by precious child , and the terminology evolved to a broader but equally undefined category of social factors.15,17 clinical records from various hospitals in the people s republic of china consistently show a rise in the proportion of cesareans performed for social factors and social factors have recently become the leading reason in some hospitals.1517 there are 11 studies that reported on the women s choice for performing cesarean sections . from in - depth interviews based on relatively small sizes , qualitative evidence shows that cesareans may in part be due to women s own choices ; where convenience , perceived safety , painless birth , and choice of birth date are the main reasons for cesarean preferences . some women even believe that a baby delivered by cesarean section may be cleverer because the baby was not squeezed in the birth canal.2931 over time , various authors have introduced a new terminology in their analyses : a cesarean delivery on maternal request ( cdmr ) . for example , zhang et al proposed a number of criteria to define cdmr.32 the five criteria that are required to qualify as cdmr are : singleton birth , cesarean delivery based on woman s request , the fetus is alive prior to delivery , gestational age of 38 weeks or more , and cesarean section before onset of spontaneous or induced labor . the following eight criteria disqualify women from cdmr : previous cesarean delivery , malpresentation , placenta previa , antepartum placenta abruption , antepartum severe preeclampsia , abnormal fetal heart rate before labor , suspected fetal growth restriction , and suspected large - for - gestational - age fetus . using this definition , the authors found that while cesarean section rates rose from 22% in 1994 to 60% in 2003 in southeast china , the proportion that was accounted for by cdmr rose from less than 1/20 to more than 1/3 over the decade . since good quality clinical information is required to define a cdmr , it can not be identified through population surveys . long et al33 proposed a simple definition of nonemergency cesareans to be used in survey instruments . emergency cesareans were identified by asking the woman whether she felt that her child s or her own condition were in danger such that a cesarean was necessary . with population survey data from five rural counties , the authors found that half of the nonemergency cesareans were suggested by a doctor and half by the women s own choices . first , since obstetricians are powerful in clinical decisions , the power imbalance between providers and patients during labor and delivery may mask the real decision - making , and interpretation of cdmr from clinical records therefore warrants careful attention . second , for the emergency classification approach , women s specific expectations and experiences of labor and delivery may introduce substantial information bias . how this affects the results is uncertain , but caution is required in the interpretation of preferences . unfortunately , more robust classification such as that proposed by robson34 to identify unnecessary cesareans has never been done , leaving a substantial literature gap . there are 29 studies on structural factors fueling rising cesarean section in the people s republic of china . using information on cesarean sections collected from women s recollections , for the first time , the authors reported a rise of cesarean sections from 4.7% in 19601979 to 22.5% in 19881993 at the population level.22 using a population - based survey , the authors analyzed the reproductive history of 1,957 married women in 1993 . information on delivery mode was collected for a 30-year period , and the association of cesareans with clinical , demographic , and socioeconomic factors was analyzed . the study reported that form of medical payment was associated with use of cesarean section . they found that women who were covered by the government insurance scheme had a 6 times higher odds of cesarean birth compared with those covered by cooperative medical insurance . since the government insurance offered higher cost reimbursement , the authors argued that it was the distorted incentives within the insurance arrangements that contributed to the rise in cesarean births . they further suggested that the fee - for - service payment arrangement to hospitals may exaggerate the perverse incentives introduced for doctors , since doctors would have strong motivations for providing cesareans to make more money . with similar designs as cai et al,22 tang et al25 analyzed factors affecting rising cesarean delivery rates in urban china . using three national cross - sectional survey databases , the authors analyzed 3,559 primiparous women s reproductive information , representing urban china in 1993 , 1998 , and 2003 . the authors found that rates of cesarean section tripled in urban china from 1990 to 2003 , and women living in large cities had 2.4 times the odds of having a cesarean delivery compared to women living in small cities . women s educational achievement was one of the main determinants of cesarean section in urban china , with 4.5 times higher odds of cesarean section for those who had college or higher education compared with the illiterate / primary school group . while insurance coverage seemed to be positively associated with cesarean delivery , the role of income was not statistically significant . tang et al attributed the association between higher level of education and cesarean birth to women s own choices . they argued that women may fear pain , or want to give birth on a specific date or at a time believed to be auspicious , they may want to protect the baby s brain , and may also be concerned for their sexual life following a vaginal delivery , and this would be more pronounced amongst those who are well - educated . the authors also discussed the distorted providers incentives as a potential determinant of rising cesareans , however no clear evidence was provided in support of this hypothesis . klemetti et al26 analyzed the factors affecting rising cesarean section in rural china , using the same data sources as tang et al25 used . however , unlike tang et al s findings for urban china , the study found that household income was a strong predictor of cesarean section in rural china , where an odds ratio ( or ) of 2.4 was found comparing the highest with the lowest quintiles . while the effect of education was less strong than in the urban settings , with an or of 2.6 , the authors reported that higher level hospitals were more likely to provide cesareans than lower level ones ( or 4.0 ) . more interestingly , the authors found that the strongest predictor for cesarean delivery in rural china was the uptake of antenatal care , where women who had any antenatal care had 8.7 times the odds for a cesarean section compared to those who did not use such services . with this evidence , the authors argued that the increased cesarean rates can not be explained solely by medical reasons , and there might be an overuse of unnecessary , expensive services for chinese health providers , though the underlying mechanisms were not deliberately discussed . using the other national data source from the united nations population fund , sufang et al23 confirmed that higher level hospitals had higher rates of cesareans than lower level ones , but the rates in lower level hospitals were rising substantially , nearing those in the large hospitals . based on a natural experiment design , bogg et al35 observed rising trends of cesarean sections in five rural counties from 20012007 and found that revenue from cesareans made up 72%85% of total delivery fee revenue . they suggested that the rapid increase in cesareans and the associated expenditures in rural china may be attributed to the introduction of the ncms , which offers more generous cost reimbursement for cesarean delivery . the authors argued that the incentives of chinese health providers are distorted due to the fee - forservice payment system , which has introduced distorted behaviors for providers , such as over - prescription of drugs and antibiotics , overuse of high - technological diagnostic tests , and a treatment pattern of expensive curative rather than basic and preventive primary care . the concerns about whether health providers distorted behavior affects the rise in cesareans in the people s republic of china have generated a lively debate . those who support such a hypothesis justify it largely on the basis that higher income households and those who have insurance coverage are more likely to have cesarean sections , since doctors can make more money from these groups.3638 but there is also some evidence refuting this hypothesis . for example , comparing women who were covered by the ncms and those who were not , xiao et al39 found that there was no overuse of delivery services , including cesareans , among the women reimbursed by the ncms . similarly , hong,40 separating clinically indicated and nonclinically indicated cesarean sections in 32 tertiary hospitals in beijing , found that nonmedically indicated cesarean sections were not associated with health insurance in urban settings . long et al,33 on the other hand , found an association between insurance and nonemergency cesarean delivery ; however , only when cesarean rates were at a modest level ( 13%82% ) . the choice of such thresholds was somewhat arbitrary and since the insurance coverage for intrapartum care is already universal in the people s republic of china , it is hard to believe that the comparison between women who are insured and those who are not insured are robust to substantial selection bias . the most robust evidence relating cesarean sections to financial incentives is based on randomly allocated interventions in the context of a trial . using a community - based cluster randomized controlled trial in two provinces in the people s republic of china , hemminki et al41 found that financial interventions covered part of women s costs for prenatal and postnatal care , but they were associated with only a moderate increase in cesarean sections ( or 1.17 ; 95% confidence interval [ 0.961.43 ] ) . feng et al27 performed a trends analysis on the factors affecting rising cesarean sections in the people s republic of china . since doctors would expect higher revenue from the families with a higher ability to pay , the authors hypothesized that , if the cesarean epidemic in the people s republic of china is fuelled by distorted financial incentives , then women who are richer and insured would have experienced a faster rise in cesarean births over recent decades . however , using data from four national cross - sectional surveys between 1988 and 2008 , the authors found that although cesareans were more common among the richer , well - educated , and insured women , the rate increased alarmingly in all socioeconomic groups and had a faster rise amongst the groups with lower income , no health insurance coverage , and less educational achievement . furthermore , this study found that the socio - economic region of residence was a more important determinant for cesarean sections than the women s individual socioeconomic characteristics , questioning the link between women s personal preference , and cesarean births . hence , supply side factors may be more important predictors for cesarean sections than ability to pay or education . the strong regional variation in the cesarean section rate , as found in this study , points to structural factors related to the supply of services as an important driver of the increase . as an interpretation , the authors argued that it may be due to the people s republic of china s health development approach focusing on specialized care that contributes to the epidemic of cesarean sections in this country . the people s republic of china has marginalized midwifery and midwifery education was discontinued in 1993 . as a result , the density of nurses and midwives is lower than in other countries with similar income levels , and the number of doctors trained in western medicine doubled in the past 20 years . hospitals in urban and wealthier regions are better equipped and have more qualified staff than those in more remote rural regions , making cesarean section more accessible . the contribution of specialized obstetric care to rising cesarean sections , as proposed by feng et al27 is supported by several intervention studies initiated from the clinical and midwifery disciplines . for example , runmei et al42 conducted an intervention study in a tertiary referral hospital in south - western china . with the auditing of surgeons practices as the key intervention , cesarean section rates in this hospital decreased by 20% per year between 2005 and 2011 . cheung et al43 organized an innovative midwife - led normal birth unit in one tertiary hospital in 2008 , aiming to facilitate normal birth and enhance midwifery practice . based on a retrospective cohort design , the authors reported a 33% reduction in the rates of cesarean section for women who used midwife - led care . as a recommendation , the authors proposed a separation of obstetric care from maternity care and a revisit of midwife care as an independent discipline . notwithstanding these findings , midwifery and normal delivery propositions seems to be out of chinese health policy makers perspective and evidence on this topic is still insufficient to influence current policy making . as a summary for nonclinical factors affecting rising cesareans in the people s republic of china , there is a lively debate on whether women s preferences or providers distorted financial incentives affected the rise in cesarean sections . notwithstanding , recent evidence suggests that it might be the people s republic of china s health development approach focusing on specialized care and marginalizing primary care that is playing a role , though further work is warranted to strengthen the causal link . there are three types of designs used to investigate pure clinical factors affecting cesarean sections in the people s republic of china : hospital records analysis ( 6 studies ) , population surveys ( 2 studies ) , and case control studies ( 4 studies ) . by describing the proportions of various clinical indications for performing cesareans , the first type of design , hospital records analysis , generally shows that cephalopelvic disproportion , fetal distress , breech presentation , abnormal pelvis , macrosomia , dystocia , hypertension , multiple birth , premature rupture of membranes , oligohydramnios , late preterm births , and older maternal age are the most commonly reported clinical indications.1517,44,45 however , the limitation of this type of research is obvious since only proportions of each clinical indication are described , while no relative risks would be arrived at for comparisons between factors . to further strengthen the association of various risk factors with the use of cesarean section , some authors sampled out cases of deliveries from hospital records , completed them with some demographic factors , and did cross - sectional analyses on the various clinical factors affecting the utilization of cesarean section . using such an approach , ma et al19 reported a positive association of multiple births , large for gestational age , pregnancy complications , and older maternal age with cesarean births . to accommodate wider factors , some authors generalized such an approach to the population level . for example , huang et al46 investigated the association between times of antenatal ultrasound utilization and the use of cesarean section based on population surveys and found an or of 1.36 , which means that one additional antenatal ultrasound scan would increase the possibility of performing a cesarean section around 1.36 times . to strengthen the causal relationship , some authors build up this approach by using case control or nested case control designs . for example , cheng et al47 compared 6,421 vaginal and 7,650 cesarean births in 48 hospitals from three metropolitan cities in the people s republic of china and found that birth weight larger than 4,000 g , maternal obesity , cephalopelvic disproportion , fetal distress , dystocia , and placenta previa were the main determinants for having a cesarean section . zhou et al48 conducted a similar analysis for a rural chinese population and identified hypertension , breech presentation , dystocia , preterm delivery , and older age as predictors of cesarean births . furthermore , there are some case control studies examining whether a specific disease is association with cesarean section . lu et al49 compared mothers who were hepatitis b surface antigen carriers with those who were not based on hospital records , and reported that such a factor is not statistically associated with cesarean procedures . using a similar design , zhang et al50 reported that mothers with uterine anomalies have a higher probability of cesarean birth . research on the consequences of cesareans are relatively rare and out of focus , and in general the evidence ranked weakly . of the 30 articles identified , 13 studied clinical outcomes for cesarean birth , 9 studied child health outcomes , 7 studied maternal health outcomes , and only 1 studied health system level outcomes ( table 3 ) . in the one study addressing structural consequences , long et al37 using repeated cross - sectional survey data found that the expenditure for facility - based delivery greatly increased in rural china over 19982007 because of greater use of more cesarean deliveries . on average , the out of pocket payment for cesarean section constituted 31.8% of a poor rural household s annual income . two studies were based on a case control design and the other eleven were cross - sectional studies . for the two case control studies , one found that cesarean sections were associated with lower cord blood plasmin activity and lower umbilical blood igg , igm , c3 , and c4 compared to vaginal delivery.51 the second case control study , making the same comparisons , found no association between cesarean birth and intraventricular hemorrhage.52 the eleven cross - sectional studies consistently found that cesarean sections were associated with higher risks of venous thromboembolism , surgical site infection , and postpartum infection . however , evidence on whether women who had a cesarean section are more likely to have postpartum hemorrhage is contradictory between the two studies reporting such outcomes.53,54 for example , cai53 found that performing cesareans is associated with higher risk of postpartum hemorrhage , while jiang et al54 found no associations that were statistically significant . cesarean births seem to have higher risks of neonatal injury as reported by cai.53 and repeated cesareans were associated with higher hysterectomy.55 however , the associations of cesarean section with other clinical outcomes were not profound ; for example , overactive bladder , lower urinary tract storage symptoms , incontinence , vertical transmission of chlamydia trachomatis , intraventricular hemorrhage in preterm babies , intrapartum fetal death , neonatal respiratory distress syndrome , and low neonatal apgar scores . studies examining the consequences of cesarean section on child health examined breastfeeding behaviors , child obesity , and physiological development . of the 8 articles identified , only 3 were published in peer - reviewed journals , including 2 retrospective cohort studies56,57 and 1 case control design.58 in general , the findings are consistent in showing that cesarean section on maternal request is a risk factor for child obesity , which is consistent with international evidence.59 however , the link from cesareans to child physiological development is not robust . there are 5 papers published in peer - review journals on the maternal health consequences after performing a cesarean section . one was based on cross - sectional data , where no associations of breast milk zinc concentration and self - reported postpartum health with cesarean births were found.60 the other 4 were based on prospective cohort study designs , where one cohort reported that women performing cesarean births were less likely to provide exclusive breastfeeding and formula feeding was more likely to be offered to the infant,61 while another found a contrary result.62 based on a 2-week follow - up of 534 women delivering babies in two hospitals , xie et al63 found that cesarean section is associated with increased risk of postpartum depression with an adjusted relative risk near 2 . notwithstanding , the actual or perceived safety of cesarean section in the people s republic of china is pretty rare . with a national rate near 40% and a rapid rise in the country over the past decade , there is no doubt that the rising rate of cesarean sections has become a national epidemic in the people s republic of china , warranting careful policy considerations . however , no consensus has been made on the leverage factors that drive the increase in the use of cesarean section , particularly for those nonclinical factors . while the main debate is around whether the rise in cesarean sections is fueled by women s own preferences or providers distorted financial incentives , recent evidence suggest that it might be the more macro level structural factors that are playing a part , ie , the people s republic of china s health development approach focusing on specialized care and marginalizing primary care , though further research is warranted to understand the underlying mechanisms . compared to the lively debate analyzing the trends and factors that affect the rising number of cesarean sections , knowledge of the consequences of cesareans is quite limited in the people s republic of china . the only robust evidence is that children of cesarean births are more likely to be obese . however , research on how the uptake of cesarean section affects women s health is underdeveloped .

objectiveto review the current knowledge on the prevalence , reasons , and consequences of cesarean sections in the people s republic of china.methodspeer-reviewed articles were systematically searched on pubmed . the following chinese databases were comprehensively searched : the china national knowledge infrastructure , wanfang , and the vip information . the databases were searched from inception to september 1 , 2013 . two reviewers independently screened the titles and abstracts for eligibility . full texts of eligible papers were reviewed , where relevant references were hand - searched and reviewed.findingssixty articles were included from pubmed , 17 articles were intentionally picked out from chinese journals , and five additional articles were added , for a total of 82 articles for the analysis . with a current national rate near 40% , the literature consistently reported a rapid rise of cesarean sections in the people s republic of china in the past decades , irrespective of where people lived or their socioeconomic standing . nonclinical factors were considered as the main drivers fueling the rise of cesareans in the people s republic of china . there was a lively debate on whether women s preferences or providers distorted financial incentives affected the rise in cesarean sections . however , recent evidence suggests that it might be the people s republic of china s health development approach focusing on specialized care and marginalizing primary care that is playing a role . although 30 articles were identified studying the consequences of cesareans , the methodologies are in general weak and the themes are out of focus.conclusionthe overuse of cesareans is rising alarmingly in the people s republic of china and has become a real public health problem . no consensus has been made on the leverage factors that drive the cesarean epidemic , particularly for those nonclinical factors . the more macro level structural factors may have played a part , though further research is warranted to understand the mechanisms . knowledge of the consequences of cesareans , particularly for women , is limited in the people s republic of china , leaving a substantial literature gap .

Introduction Search strategy and findings Prevalence and trends in cesareans Hospital-based studies Population-based surveys Reasons for performing cesarean sections Nonclinical factors Womens preferences Structural factors Clinical factors Consequences of cesarean sections Conclusion

cesarean sections have been a global public health concern for decades.13 as a key component of comprehensive emergency obstetric care ( emoc ) , the adequate provision of cesarean sections during intrapartum care is a life - saving procedure.2,3 this is particularly vital for mothers living in low and middle income countries , where access to emoc is low and resources to provide quality care are limited.35 notwithstanding the well - recognized life - saving role of cesarean sections , international comparisons show that rates of cesarean sections vary greatly across countries and high coverage does not necessarily lead to good health outcomes.1,2 elective cesareans can increase the risk of maternal morbidity , neonatal death , and neonatal admission to an intensive care unit.1 the overuse of cesareans is therefore a real public health concern , not only because cesareans are costly , incurring a financial burden for the families and society , but also because they may entail a health risk for women and children s health . the threshold above which cesarean sections are in excess of need is not well known , but the world health organization suggests a maximum threshold of around 15%.6 recent global estimates consistently show that rates of cesareans have been rising dramatically over the recommended threshold , particularly in latin american and some asian countries.7,8 the debate on why rates of cesareans rise quickly continues , and both supply and demand side factors are thought to play a role.1,9 the preponderance of obstetricians in providing intrapartum care and the perverse financial incentives associated with cesareans are the main factors considered from the supply side , whilst for the demand side , discussions center around women and families preferences . to date no consensus has been reached on the main factors driving the cesarean epidemic . as the largest developing country , the people s republic of china bears a substantial burden of maternal and child mortality worldwide , and was included as one of the 68 countdown countries to achieve the two millennium development goals ( mdg ) related to maternal and child health ( mdg 4 and 5).10,11 to address the high burden of maternal mortality , a universal hospital delivery strategy was introduced by the chinese government in the mid-1990s.12 in 1995 , the law on maternal and infant health care was passed to guarantee each woman s right to a hospital for quality intrapartum care . infrastructure has been strengthened , medical staff were trained and supervised in township and lower level hospitals , and referral channels to tertiary hospitals were reinforced.12 in 2003 , the government launched the new cooperative medical scheme ( ncms ) , which provides health insurance coverage for the majority of the rural chinese population for medical care , including hospital delivery . by 2007 , health insurance had achieved near universal coverage in the people s republic of china , greatly increasing the financial accessibility to facility - based intrapartum care for the chinese population.13 consistent national data show that the people s republic of china s hospital delivery strategy has been successful in reducing maternal and neonatal mortality.10,11 in 1988 , less than half of all women gave birth in a hospital , yet 20 years later hospital births have become nearly universal and socioeconomic inequalities in hospital births have nearly disappeared.13 over the same period , maternal mortality declined by 47%86% and neonatal mortality by 48%70% . as a result , the people s republic of china is now quoted as a successful example in the developing world in achieving mdg 4 and 5.10,11 at the same time however , rates of cesarean sections have been rising dramatically . in 20072008 , the national rate was 46.2% and 40% of cesareans were reported to have no clinical indications.14 in this paper , we systematically reviewed peer - reviewed articles in both chinese and international journals on the theme of cesarean sections in the people s republic of china . from a multidisciplinary perspective , particularly health policy and systems research , we discuss the current knowledge on the prevalence , reasons , and consequences regarding cesarean sections in the people s republic of china . we add up the literature not only by describing the specific phenomenon from the people s republic of china , but also by contributing to the international debate on why cesareans rise and how it might be related to a country s health system development . since only a limited amount of chinese journals were indexed by pubmed , we also comprehensively searched the following chinese databases : the china national knowledge infrastructure , wanfang , and the vip information . the databases were searched from inception to september 1 , 2013 . to identify further relevant studies , reference lists of included articles were hand - searched and reviewed . when searching pubmed , we included studies that reported caesarean or the following study types were included : clinical trial , meta - analysis , randomized controlled trials , and all types of observational studies , including cohort studies , case control studies , cross - sectional studies , and case reports . however , since most of the chinese journals , which are not indexed by pubmed , are not peer - reviewed and the majority of these papers are not original research , we screened the title and abstract and intentionally chose important and relevant articles for this review . full texts of these potentially suitable articles were obtained and reviewed . with 17 articles picked out from chinese databases and five additional papers hand - searched , we finally arrived at 82 papers for this analysis ( figure 1 ) . amongst these articles , 30 reported the prevalence and trends in cesareans , 48 reported the reasons for performing cesareans , and 30 reported the consequences of cesareans , where 23 articles reported both the prevalence and the reasons , 1 reported both the prevalence and the consequences , and one reported all three themes . there are two major types of studies estimating the prevalence of and trends in cesarean sections in the people s republic of china : hospital - based studies and population surveys ( table 1 ) . such types of studies also appeared in english peer - reviewed journals , for example han et al18 and ma et al.19 in summary , these analyses consistently reported a substantial rise in the proportion of social factors , which has become the leading reason affecting clinical decisions to perform cesarean sections in recent years . however , as illustrated by a systematic review based on other countries,21 information extracted from clinical records may yield non - trivial misclassifications for clinical indications on the categorization of cesarean sections . furthermore , such types of studies ignore births that are out of health facilities , resulting in an overestimate of the rates of cesarean section at national or regional level . in 1998 , cai et al22 conducted a population - based survey in a district in shanghai , one of the largest metropolitan cities in the people s republic of china . using information on cesarean sections collected from women s recall , the authors reported , for the first time , a rise of cesarean sections from 4.7% from 19601979 to 22.5% from 19881993 at the population level . for example , sufang et al23 reported trends in cesareans in the people s republic of china between 1993 and 2002 using a national cross - sectional survey and lu et al24 reported trends in southern china between 1994 and 2005 . using the people s republic of china s national health service surveys , tang et al25 reported trends in cesareans in urban china from 19902002 , while klemetti et al26 reported such trends in rural china from 19912002 . with the same national data , feng et al27 did a comprehensive analysis on the trends in cesarean section rates in the people s republic of china from 1988 to 2008 . using the four national surveys conducted in the years 1993 , 1998 , 2003 , and 2008 , the study estimated the people s republic of china s national cesarean section rates at 3.4% in 1988 , increasing ten - fold to 39.3% in 2008 . the most extraordinary increase was witnessed in urban settings , where 64.1% of the women gave birth by cesarean section in 2008 ; while even in the least developed rural region , the rate rose from 0% in 1988 to 11.3% in 2008 . both clinical factors and nonclinical factors were investigated to explain the epidemic of cesarean sections in the people s republic of china . while studies on clinical factors for choosing cesareans are relatively rare and disagreement is seldom made , the debates on nonclinical factors are quite lively and in - depth , particularly on whether the rise in cesareans in the people s republic of china are fueled by the women s own choice or the obstetricians induced demand . a comprehensive list of studies addressing the factors that affect the use of cesarean sections in the people s republic of china is shown in table 2 . there was no clear definition of what was meant by precious child , and the terminology evolved to a broader but equally undefined category of social factors.15,17 clinical records from various hospitals in the people s republic of china consistently show a rise in the proportion of cesareans performed for social factors and social factors have recently become the leading reason in some hospitals.1517 there are 11 studies that reported on the women s choice for performing cesarean sections . unfortunately , more robust classification such as that proposed by robson34 to identify unnecessary cesareans has never been done , leaving a substantial literature gap . there are 29 studies on structural factors fueling rising cesarean section in the people s republic of china . using information on cesarean sections collected from women s recollections , for the first time , the authors reported a rise of cesarean sections from 4.7% in 19601979 to 22.5% in 19881993 at the population level.22 using a population - based survey , the authors analyzed the reproductive history of 1,957 married women in 1993 . information on delivery mode was collected for a 30-year period , and the association of cesareans with clinical , demographic , and socioeconomic factors was analyzed . since the government insurance offered higher cost reimbursement , the authors argued that it was the distorted incentives within the insurance arrangements that contributed to the rise in cesarean births . using three national cross - sectional survey databases , the authors analyzed 3,559 primiparous women s reproductive information , representing urban china in 1993 , 1998 , and 2003 . women s educational achievement was one of the main determinants of cesarean section in urban china , with 4.5 times higher odds of cesarean section for those who had college or higher education compared with the illiterate / primary school group . however , unlike tang et al s findings for urban china , the study found that household income was a strong predictor of cesarean section in rural china , where an odds ratio ( or ) of 2.4 was found comparing the highest with the lowest quintiles . with this evidence , the authors argued that the increased cesarean rates can not be explained solely by medical reasons , and there might be an overuse of unnecessary , expensive services for chinese health providers , though the underlying mechanisms were not deliberately discussed . based on a natural experiment design , bogg et al35 observed rising trends of cesarean sections in five rural counties from 20012007 and found that revenue from cesareans made up 72%85% of total delivery fee revenue . the concerns about whether health providers distorted behavior affects the rise in cesareans in the people s republic of china have generated a lively debate . the choice of such thresholds was somewhat arbitrary and since the insurance coverage for intrapartum care is already universal in the people s republic of china , it is hard to believe that the comparison between women who are insured and those who are not insured are robust to substantial selection bias . the most robust evidence relating cesarean sections to financial incentives is based on randomly allocated interventions in the context of a trial . using a community - based cluster randomized controlled trial in two provinces in the people s republic of china , hemminki et al41 found that financial interventions covered part of women s costs for prenatal and postnatal care , but they were associated with only a moderate increase in cesarean sections ( or 1.17 ; 95% confidence interval [ 0.961.43 ] ) . feng et al27 performed a trends analysis on the factors affecting rising cesarean sections in the people s republic of china . since doctors would expect higher revenue from the families with a higher ability to pay , the authors hypothesized that , if the cesarean epidemic in the people s republic of china is fuelled by distorted financial incentives , then women who are richer and insured would have experienced a faster rise in cesarean births over recent decades . however , using data from four national cross - sectional surveys between 1988 and 2008 , the authors found that although cesareans were more common among the richer , well - educated , and insured women , the rate increased alarmingly in all socioeconomic groups and had a faster rise amongst the groups with lower income , no health insurance coverage , and less educational achievement . furthermore , this study found that the socio - economic region of residence was a more important determinant for cesarean sections than the women s individual socioeconomic characteristics , questioning the link between women s personal preference , and cesarean births . the strong regional variation in the cesarean section rate , as found in this study , points to structural factors related to the supply of services as an important driver of the increase . as an interpretation , the authors argued that it may be due to the people s republic of china s health development approach focusing on specialized care that contributes to the epidemic of cesarean sections in this country . the people s republic of china has marginalized midwifery and midwifery education was discontinued in 1993 . as a result , the density of nurses and midwives is lower than in other countries with similar income levels , and the number of doctors trained in western medicine doubled in the past 20 years . based on a retrospective cohort design , the authors reported a 33% reduction in the rates of cesarean section for women who used midwife - led care . as a summary for nonclinical factors affecting rising cesareans in the people s republic of china , there is a lively debate on whether women s preferences or providers distorted financial incentives affected the rise in cesarean sections . notwithstanding , recent evidence suggests that it might be the people s republic of china s health development approach focusing on specialized care and marginalizing primary care that is playing a role , though further work is warranted to strengthen the causal link . there are three types of designs used to investigate pure clinical factors affecting cesarean sections in the people s republic of china : hospital records analysis ( 6 studies ) , population surveys ( 2 studies ) , and case control studies ( 4 studies ) . by describing the proportions of various clinical indications for performing cesareans , the first type of design , hospital records analysis , generally shows that cephalopelvic disproportion , fetal distress , breech presentation , abnormal pelvis , macrosomia , dystocia , hypertension , multiple birth , premature rupture of membranes , oligohydramnios , late preterm births , and older maternal age are the most commonly reported clinical indications.1517,44,45 however , the limitation of this type of research is obvious since only proportions of each clinical indication are described , while no relative risks would be arrived at for comparisons between factors . for example , cheng et al47 compared 6,421 vaginal and 7,650 cesarean births in 48 hospitals from three metropolitan cities in the people s republic of china and found that birth weight larger than 4,000 g , maternal obesity , cephalopelvic disproportion , fetal distress , dystocia , and placenta previa were the main determinants for having a cesarean section . zhou et al48 conducted a similar analysis for a rural chinese population and identified hypertension , breech presentation , dystocia , preterm delivery , and older age as predictors of cesarean births . research on the consequences of cesareans are relatively rare and out of focus , and in general the evidence ranked weakly . for the two case control studies , one found that cesarean sections were associated with lower cord blood plasmin activity and lower umbilical blood igg , igm , c3 , and c4 compared to vaginal delivery.51 the second case control study , making the same comparisons , found no association between cesarean birth and intraventricular hemorrhage.52 the eleven cross - sectional studies consistently found that cesarean sections were associated with higher risks of venous thromboembolism , surgical site infection , and postpartum infection . however , evidence on whether women who had a cesarean section are more likely to have postpartum hemorrhage is contradictory between the two studies reporting such outcomes.53,54 for example , cai53 found that performing cesareans is associated with higher risk of postpartum hemorrhage , while jiang et al54 found no associations that were statistically significant . cesarean births seem to have higher risks of neonatal injury as reported by cai.53 and repeated cesareans were associated with higher hysterectomy.55 however , the associations of cesarean section with other clinical outcomes were not profound ; for example , overactive bladder , lower urinary tract storage symptoms , incontinence , vertical transmission of chlamydia trachomatis , intraventricular hemorrhage in preterm babies , intrapartum fetal death , neonatal respiratory distress syndrome , and low neonatal apgar scores . studies examining the consequences of cesarean section on child health examined breastfeeding behaviors , child obesity , and physiological development . of the 8 articles identified , only 3 were published in peer - reviewed journals , including 2 retrospective cohort studies56,57 and 1 case control design.58 in general , the findings are consistent in showing that cesarean section on maternal request is a risk factor for child obesity , which is consistent with international evidence.59 however , the link from cesareans to child physiological development is not robust . notwithstanding , the actual or perceived safety of cesarean section in the people s republic of china is pretty rare . with a national rate near 40% and a rapid rise in the country over the past decade , there is no doubt that the rising rate of cesarean sections has become a national epidemic in the people s republic of china , warranting careful policy considerations . however , no consensus has been made on the leverage factors that drive the increase in the use of cesarean section , particularly for those nonclinical factors . while the main debate is around whether the rise in cesarean sections is fueled by women s own preferences or providers distorted financial incentives , recent evidence suggest that it might be the more macro level structural factors that are playing a part , ie , the people s republic of china s health development approach focusing on specialized care and marginalizing primary care , though further research is warranted to understand the underlying mechanisms . compared to the lively debate analyzing the trends and factors that affect the rising number of cesarean sections , knowledge of the consequences of cesareans is quite limited in the people s republic of china . however , research on how the uptake of cesarean section affects women s health is underdeveloped .

cesarean sections have been a global public health concern for decades.13 as a key component of comprehensive emergency obstetric care ( emoc ) , the adequate provision of cesarean sections during intrapartum care is a life - saving procedure.2,3 this is particularly vital for mothers living in low and middle income countries , where access to emoc is low and resources to provide quality care are limited.35 notwithstanding the well - recognized life - saving role of cesarean sections , international comparisons show that rates of cesarean sections vary greatly across countries and high coverage does not necessarily lead to good health outcomes.1,2 elective cesareans can increase the risk of maternal morbidity , neonatal death , and neonatal admission to an intensive care unit.1 the overuse of cesareans is therefore a real public health concern , not only because cesareans are costly , incurring a financial burden for the families and society , but also because they may entail a health risk for women and children s health . the threshold above which cesarean sections are in excess of need is not well known , but the world health organization suggests a maximum threshold of around 15%.6 recent global estimates consistently show that rates of cesareans have been rising dramatically over the recommended threshold , particularly in latin american and some asian countries.7,8 the debate on why rates of cesareans rise quickly continues , and both supply and demand side factors are thought to play a role.1,9 the preponderance of obstetricians in providing intrapartum care and the perverse financial incentives associated with cesareans are the main factors considered from the supply side , whilst for the demand side , discussions center around women and families preferences . as the largest developing country , the people s republic of china bears a substantial burden of maternal and child mortality worldwide , and was included as one of the 68 countdown countries to achieve the two millennium development goals ( mdg ) related to maternal and child health ( mdg 4 and 5).10,11 to address the high burden of maternal mortality , a universal hospital delivery strategy was introduced by the chinese government in the mid-1990s.12 in 1995 , the law on maternal and infant health care was passed to guarantee each woman s right to a hospital for quality intrapartum care . infrastructure has been strengthened , medical staff were trained and supervised in township and lower level hospitals , and referral channels to tertiary hospitals were reinforced.12 in 2003 , the government launched the new cooperative medical scheme ( ncms ) , which provides health insurance coverage for the majority of the rural chinese population for medical care , including hospital delivery . by 2007 , health insurance had achieved near universal coverage in the people s republic of china , greatly increasing the financial accessibility to facility - based intrapartum care for the chinese population.13 consistent national data show that the people s republic of china s hospital delivery strategy has been successful in reducing maternal and neonatal mortality.10,11 in 1988 , less than half of all women gave birth in a hospital , yet 20 years later hospital births have become nearly universal and socioeconomic inequalities in hospital births have nearly disappeared.13 over the same period , maternal mortality declined by 47%86% and neonatal mortality by 48%70% . in 20072008 , the national rate was 46.2% and 40% of cesareans were reported to have no clinical indications.14 in this paper , we systematically reviewed peer - reviewed articles in both chinese and international journals on the theme of cesarean sections in the people s republic of china . from a multidisciplinary perspective , particularly health policy and systems research , we discuss the current knowledge on the prevalence , reasons , and consequences regarding cesarean sections in the people s republic of china . we add up the literature not only by describing the specific phenomenon from the people s republic of china , but also by contributing to the international debate on why cesareans rise and how it might be related to a country s health system development . when searching pubmed , we included studies that reported caesarean or the following study types were included : clinical trial , meta - analysis , randomized controlled trials , and all types of observational studies , including cohort studies , case control studies , cross - sectional studies , and case reports . however , since most of the chinese journals , which are not indexed by pubmed , are not peer - reviewed and the majority of these papers are not original research , we screened the title and abstract and intentionally chose important and relevant articles for this review . amongst these articles , 30 reported the prevalence and trends in cesareans , 48 reported the reasons for performing cesareans , and 30 reported the consequences of cesareans , where 23 articles reported both the prevalence and the reasons , 1 reported both the prevalence and the consequences , and one reported all three themes . there are two major types of studies estimating the prevalence of and trends in cesarean sections in the people s republic of china : hospital - based studies and population surveys ( table 1 ) . for example , zhao et al16 reported that cesareans increased from 25.4% of births in 1990 to 39.6% in 2002 in a secondary level hospital , and feng and yue17 reported a rise from 2.6% in 1955 to 64.7% in 2004 in a tertiary hospital in middle china . such types of studies also appeared in english peer - reviewed journals , for example han et al18 and ma et al.19 in summary , these analyses consistently reported a substantial rise in the proportion of social factors , which has become the leading reason affecting clinical decisions to perform cesarean sections in recent years . using the four national surveys conducted in the years 1993 , 1998 , 2003 , and 2008 , the study estimated the people s republic of china s national cesarean section rates at 3.4% in 1988 , increasing ten - fold to 39.3% in 2008 . the most extraordinary increase was witnessed in urban settings , where 64.1% of the women gave birth by cesarean section in 2008 ; while even in the least developed rural region , the rate rose from 0% in 1988 to 11.3% in 2008 . while studies on clinical factors for choosing cesareans are relatively rare and disagreement is seldom made , the debates on nonclinical factors are quite lively and in - depth , particularly on whether the rise in cesareans in the people s republic of china are fueled by the women s own choice or the obstetricians induced demand . a comprehensive list of studies addressing the factors that affect the use of cesarean sections in the people s republic of china is shown in table 2 . as early as 1989 , following an observation that rising cesareans were not associated with a reduction in perinatal mortality , a national academic conference was organized in the obstetricians society to discuss clinical indications for cesarean sections.28 among the clinical indications , precious child was increasingly reported , alongside other more conventional clinical factors . there was no clear definition of what was meant by precious child , and the terminology evolved to a broader but equally undefined category of social factors.15,17 clinical records from various hospitals in the people s republic of china consistently show a rise in the proportion of cesareans performed for social factors and social factors have recently become the leading reason in some hospitals.1517 there are 11 studies that reported on the women s choice for performing cesarean sections . from in - depth interviews based on relatively small sizes , qualitative evidence shows that cesareans may in part be due to women s own choices ; where convenience , perceived safety , painless birth , and choice of birth date are the main reasons for cesarean preferences . for example , zhang et al proposed a number of criteria to define cdmr.32 the five criteria that are required to qualify as cdmr are : singleton birth , cesarean delivery based on woman s request , the fetus is alive prior to delivery , gestational age of 38 weeks or more , and cesarean section before onset of spontaneous or induced labor . the following eight criteria disqualify women from cdmr : previous cesarean delivery , malpresentation , placenta previa , antepartum placenta abruption , antepartum severe preeclampsia , abnormal fetal heart rate before labor , suspected fetal growth restriction , and suspected large - for - gestational - age fetus . using this definition , the authors found that while cesarean section rates rose from 22% in 1994 to 60% in 2003 in southeast china , the proportion that was accounted for by cdmr rose from less than 1/20 to more than 1/3 over the decade . first , since obstetricians are powerful in clinical decisions , the power imbalance between providers and patients during labor and delivery may mask the real decision - making , and interpretation of cdmr from clinical records therefore warrants careful attention . using information on cesarean sections collected from women s recollections , for the first time , the authors reported a rise of cesarean sections from 4.7% in 19601979 to 22.5% in 19881993 at the population level.22 using a population - based survey , the authors analyzed the reproductive history of 1,957 married women in 1993 . women s educational achievement was one of the main determinants of cesarean section in urban china , with 4.5 times higher odds of cesarean section for those who had college or higher education compared with the illiterate / primary school group . they argued that women may fear pain , or want to give birth on a specific date or at a time believed to be auspicious , they may want to protect the baby s brain , and may also be concerned for their sexual life following a vaginal delivery , and this would be more pronounced amongst those who are well - educated . however , unlike tang et al s findings for urban china , the study found that household income was a strong predictor of cesarean section in rural china , where an odds ratio ( or ) of 2.4 was found comparing the highest with the lowest quintiles . while the effect of education was less strong than in the urban settings , with an or of 2.6 , the authors reported that higher level hospitals were more likely to provide cesareans than lower level ones ( or 4.0 ) . more interestingly , the authors found that the strongest predictor for cesarean delivery in rural china was the uptake of antenatal care , where women who had any antenatal care had 8.7 times the odds for a cesarean section compared to those who did not use such services . with this evidence , the authors argued that the increased cesarean rates can not be explained solely by medical reasons , and there might be an overuse of unnecessary , expensive services for chinese health providers , though the underlying mechanisms were not deliberately discussed . using the other national data source from the united nations population fund , sufang et al23 confirmed that higher level hospitals had higher rates of cesareans than lower level ones , but the rates in lower level hospitals were rising substantially , nearing those in the large hospitals . they suggested that the rapid increase in cesareans and the associated expenditures in rural china may be attributed to the introduction of the ncms , which offers more generous cost reimbursement for cesarean delivery . the authors argued that the incentives of chinese health providers are distorted due to the fee - forservice payment system , which has introduced distorted behaviors for providers , such as over - prescription of drugs and antibiotics , overuse of high - technological diagnostic tests , and a treatment pattern of expensive curative rather than basic and preventive primary care . the choice of such thresholds was somewhat arbitrary and since the insurance coverage for intrapartum care is already universal in the people s republic of china , it is hard to believe that the comparison between women who are insured and those who are not insured are robust to substantial selection bias . using a community - based cluster randomized controlled trial in two provinces in the people s republic of china , hemminki et al41 found that financial interventions covered part of women s costs for prenatal and postnatal care , but they were associated with only a moderate increase in cesarean sections ( or 1.17 ; 95% confidence interval [ 0.961.43 ] ) . since doctors would expect higher revenue from the families with a higher ability to pay , the authors hypothesized that , if the cesarean epidemic in the people s republic of china is fuelled by distorted financial incentives , then women who are richer and insured would have experienced a faster rise in cesarean births over recent decades . however , using data from four national cross - sectional surveys between 1988 and 2008 , the authors found that although cesareans were more common among the richer , well - educated , and insured women , the rate increased alarmingly in all socioeconomic groups and had a faster rise amongst the groups with lower income , no health insurance coverage , and less educational achievement . furthermore , this study found that the socio - economic region of residence was a more important determinant for cesarean sections than the women s individual socioeconomic characteristics , questioning the link between women s personal preference , and cesarean births . the strong regional variation in the cesarean section rate , as found in this study , points to structural factors related to the supply of services as an important driver of the increase . as a summary for nonclinical factors affecting rising cesareans in the people s republic of china , there is a lively debate on whether women s preferences or providers distorted financial incentives affected the rise in cesarean sections . notwithstanding , recent evidence suggests that it might be the people s republic of china s health development approach focusing on specialized care and marginalizing primary care that is playing a role , though further work is warranted to strengthen the causal link . there are three types of designs used to investigate pure clinical factors affecting cesarean sections in the people s republic of china : hospital records analysis ( 6 studies ) , population surveys ( 2 studies ) , and case control studies ( 4 studies ) . by describing the proportions of various clinical indications for performing cesareans , the first type of design , hospital records analysis , generally shows that cephalopelvic disproportion , fetal distress , breech presentation , abnormal pelvis , macrosomia , dystocia , hypertension , multiple birth , premature rupture of membranes , oligohydramnios , late preterm births , and older maternal age are the most commonly reported clinical indications.1517,44,45 however , the limitation of this type of research is obvious since only proportions of each clinical indication are described , while no relative risks would be arrived at for comparisons between factors . to further strengthen the association of various risk factors with the use of cesarean section , some authors sampled out cases of deliveries from hospital records , completed them with some demographic factors , and did cross - sectional analyses on the various clinical factors affecting the utilization of cesarean section . for example , huang et al46 investigated the association between times of antenatal ultrasound utilization and the use of cesarean section based on population surveys and found an or of 1.36 , which means that one additional antenatal ultrasound scan would increase the possibility of performing a cesarean section around 1.36 times . for example , cheng et al47 compared 6,421 vaginal and 7,650 cesarean births in 48 hospitals from three metropolitan cities in the people s republic of china and found that birth weight larger than 4,000 g , maternal obesity , cephalopelvic disproportion , fetal distress , dystocia , and placenta previa were the main determinants for having a cesarean section . of the 30 articles identified , 13 studied clinical outcomes for cesarean birth , 9 studied child health outcomes , 7 studied maternal health outcomes , and only 1 studied health system level outcomes ( table 3 ) . in the one study addressing structural consequences , long et al37 using repeated cross - sectional survey data found that the expenditure for facility - based delivery greatly increased in rural china over 19982007 because of greater use of more cesarean deliveries . for the two case control studies , one found that cesarean sections were associated with lower cord blood plasmin activity and lower umbilical blood igg , igm , c3 , and c4 compared to vaginal delivery.51 the second case control study , making the same comparisons , found no association between cesarean birth and intraventricular hemorrhage.52 the eleven cross - sectional studies consistently found that cesarean sections were associated with higher risks of venous thromboembolism , surgical site infection , and postpartum infection . however , evidence on whether women who had a cesarean section are more likely to have postpartum hemorrhage is contradictory between the two studies reporting such outcomes.53,54 for example , cai53 found that performing cesareans is associated with higher risk of postpartum hemorrhage , while jiang et al54 found no associations that were statistically significant . cesarean births seem to have higher risks of neonatal injury as reported by cai.53 and repeated cesareans were associated with higher hysterectomy.55 however , the associations of cesarean section with other clinical outcomes were not profound ; for example , overactive bladder , lower urinary tract storage symptoms , incontinence , vertical transmission of chlamydia trachomatis , intraventricular hemorrhage in preterm babies , intrapartum fetal death , neonatal respiratory distress syndrome , and low neonatal apgar scores . of the 8 articles identified , only 3 were published in peer - reviewed journals , including 2 retrospective cohort studies56,57 and 1 case control design.58 in general , the findings are consistent in showing that cesarean section on maternal request is a risk factor for child obesity , which is consistent with international evidence.59 however , the link from cesareans to child physiological development is not robust . one was based on cross - sectional data , where no associations of breast milk zinc concentration and self - reported postpartum health with cesarean births were found.60 the other 4 were based on prospective cohort study designs , where one cohort reported that women performing cesarean births were less likely to provide exclusive breastfeeding and formula feeding was more likely to be offered to the infant,61 while another found a contrary result.62 based on a 2-week follow - up of 534 women delivering babies in two hospitals , xie et al63 found that cesarean section is associated with increased risk of postpartum depression with an adjusted relative risk near 2 . with a national rate near 40% and a rapid rise in the country over the past decade , there is no doubt that the rising rate of cesarean sections has become a national epidemic in the people s republic of china , warranting careful policy considerations . while the main debate is around whether the rise in cesarean sections is fueled by women s own preferences or providers distorted financial incentives , recent evidence suggest that it might be the more macro level structural factors that are playing a part , ie , the people s republic of china s health development approach focusing on specialized care and marginalizing primary care , though further research is warranted to understand the underlying mechanisms . compared to the lively debate analyzing the trends and factors that affect the rising number of cesarean sections , knowledge of the consequences of cesareans is quite limited in the people s republic of china .

it is now evident that adipose tissue not only stores excess triacylglycerols , but functions as an endocrine organ by releasing adipokines , which have important roles in the regulation of appetite , glucose and lipid metabolism , inflammation , and insulin resistance.1,2 such adipokines include adiponectin , leptin , resistin , tumor necrosis factor ( tnf)- , plasminogen activator inhibitor-1 ( pai-1 ) , and interleukin ( il)-6 . tnf- , pai-1 , and il-6 are all proinflammatory cytokines , although il-6 can further exert an anti - inflammatory action.1 it has also been observed that leptin , resistin , and tnf- impair insulin sensitivity and trigger atherogenesis.1 these adipokines usually increase with adiposity and can have a detrimental role on an individual s health . however , adiponectin , unlike other adipokines produced by adipose tissue , possesses antiatherogenic properties and is found to decrease with increased adiposity.1,2 several studies provide evidence of improvement in adipokine profiles with weight loss.36 however , it remains unclear as to whether changes in the circulating adipokines investigated to date contribute significantly to the beneficial effects on health associated with weight loss.7 the extent of weight loss required to elicit such changes in adipokine levels remains unclear . in addition , the method by which the weight loss is achieved may also influence the adipokine response . varady et al8 suggest that a minimum weight loss of 5% is required to have an effect on levels of key adipokines ( adiponectin , leptin , and resistin ) , but this differs from other studies which report the need for a 10% weight loss for modification in levels of some adipokines ( eg , adiponectin ) and that this varies with the degree of obesity.9 further studies are needed to clarify the relationship between changes in adipokine levels and the health benefits of weight loss , and whether specific dietary manipulations have differential effects on such changes . we hypothesized that the extent of weight loss rather than the macronutrient intake will determine the degree of change in adipokine levels . we aimed to investigate the effect of weight loss on adipokine levels in individuals receiving a low carbohydrate / high protein ( lchp ) diet compared with a very low calorie diet ( lighterlife [ ll ] ) diet . the present analysis is an ancillary study to that previously presented by rolland et al10 where the methods were described . dropouts were not included in this analysis because the goal was to evaluate the effect of diet on adipokine levels and not an intention - to - treat clinical trial . in brief , patients referred to a specialist obesity clinic were entered into a randomized controlled clinical trial of differing dietary interventions in the management of obesity . men and women older than 18 years of age and a body mass index ( bmi ) 35 kg / m were included . patients with a history of hepatic or renal disease , cancer , current pregnancy / lactation , on antidepressant or antiobesity medication , or eating disorders were excluded . the study included a three - month screening period where patients were assigned to a 600 calorie - deficient diet ( cdd ) aiming to achieve a 5% weight loss . this was done to select patients who would not respond to a low - fat , reduced - energy approach and randomly assign them to a lchp or a ll diet in the form of a continuous method approach . those who lost > 5% of their body weight were maintained on this approach for an additional three months . if weight loss was > 10% at this time , the cdd was continued for an additional six months . completer data for this group were limited ( n = 4 ) and therefore were not included in this paper . those patients who failed to achieve the weight loss targets were randomly allocated to either a lchp or ll diet , and continued on the assigned diet for an additional nine months ( figure 1 ) . patients on cdd who achieved a 5% weight loss at screening but did not achieve the 10% weight loss three months following screening were randomized to the ll or the lchp diet but their data were omitted from this analysis . the study was approved by the north of scotland research ethics service . using the schofield and the harris benedict equations , the amount of energy allowed was broken down into portions from the different food groups.10 portion sizes were explained to the patients and written information was also provided . patients were reviewed at weeks 2 , 4 , 8 , and 12 when they were weighed , their weight loss progress was discussed , and they were provided with dietary advice . patients randomized to the low lchp diet were restricted to 40 g of carbohydrate per day . the energy intake was 8001500 kcal where an 800 kcal diet was composed of 20% carbohydrate , 40% protein , and 40% fat . patients were given a booklet with information about which foods to eat and which to avoid . the diet was supplemented with multivitamins and minerals ( forceval , alliance pharmaceuticals , chippenham , uk ) . the ll diet used in this study is administered in the form of soups , shakes , and bars to replace conventional food and provides a daily average of 550 kcal ( 36% carbohydrate , 36% protein , 28% fat , and at least 100% of the recommended daily allowance for all micronutrients ) . ll has two distinctive stages , ie , weight loss and food reintroduction . during each stage , patients attend weekly single - sex group meetings of 712 people for behavior change therapy based on cognitive behavior therapy and transactional analysis methodology delivered by a trained ll advisor . patients were required to remain on the weight loss phase for a minimum of three months , after which they were given a choice to continue for up to another six months or were assigned to the food reintroduction phase . on average , patients who completed the study remained on the diet for 6.9 ( range 49 ) months . for the food reintroduction phase , solid foods were reintroduced over a 12-week period where patients were slowly weaned off food packs while still receiving advice and support . all patients came monthly to the trial center to be weighed for the first three months and then every alternate month after screening , resulting in six visits in nine months . fat mass and fat free mass was estimated using bioelectrical impedance ( tanita bc-418 ma , [ tanita corporation , arlington heights , il ] ) . body composition and waist circumference were measured prescreening , at screening , and at months 3 and 9 after screening . blood samples were obtained after an overnight fast prescreening , at screening , and at months 3 and 9 after screening to measure fasting plasma glucose , insulin , and adipokines , including leptin , resistin , adiponectin , pai-1 ( active ) , il-6 , and tnf-. serum samples for adipokines and insulin were measured in duplicate with commercially available immunoassay kits from millipore using the lincoplex system ( st charles , mo ) . the protocol for measurement of insulin and all the adipokines was carried out as described by the manufacturer . high - density lipoprotein cholesterol ( hdl ) , triacylglycerol , and fasting glucose were analyzed in the department of clinical biochemistry at nhs grampian . insulin resistance using fasting glucose and insulin values was calculated using the homeostasis model of assessment of insulin resistance ( homa - ir ) where homa - ir = [ insulin ] [ glucose]/22.5 . any skewed data were log - transformed and subsequently assessed using parametric tests . for within group analysis , a paired t - test was used . for between - group analysis of changes , the pearson correlation was used to assess the relationship between changes in adipokines and changes in other continuous measurements . statistical tests were carried out using the spss 15.0 for windows software program ( spss inc , chicago , il ) . because this is an ancillary analysis following on from a previously published study,10 the power for this analysis a p value of < 0.05 ( two - tailed ) was considered statistically significant . based on the mean change ( 31.0 kg ) and standard deviation ( 16.4 kg ) for weight loss observed at nine months,10 14 patients in each group resulted in a > 99% power for weight change . using the schofield and the harris benedict equations , 600 kcal were removed from the patients estimated daily energy intake . the amount of energy allowed was broken down into portions from the different food groups.10 portion sizes were explained to the patients and written information was also provided . patients were reviewed at weeks 2 , 4 , 8 , and 12 when they were weighed , their weight loss progress was discussed , and they were provided with dietary advice . patients randomized to the low lchp diet were restricted to 40 g of carbohydrate per day . the energy intake was 8001500 kcal where an 800 kcal diet was composed of 20% carbohydrate , 40% protein , and 40% fat . patients were given a booklet with information about which foods to eat and which to avoid . the diet was supplemented with multivitamins and minerals ( forceval , alliance pharmaceuticals , chippenham , uk ) . the ll diet used in this study is administered in the form of soups , shakes , and bars to replace conventional food and provides a daily average of 550 kcal ( 36% carbohydrate , 36% protein , 28% fat , and at least 100% of the recommended daily allowance for all micronutrients ) . ll has two distinctive stages , ie , weight loss and food reintroduction . during each stage , patients attend weekly single - sex group meetings of 712 people for behavior change therapy based on cognitive behavior therapy and transactional analysis methodology delivered by a trained ll advisor . patients were required to remain on the weight loss phase for a minimum of three months , after which they were given a choice to continue for up to another six months or were assigned to the food reintroduction phase . on average , patients who completed the study remained on the diet for 6.9 ( range 49 ) months . for the food reintroduction phase , solid foods were reintroduced over a 12-week period where patients were slowly weaned off food packs while still receiving advice and support . all patients came monthly to the trial center to be weighed for the first three months and then every alternate month after screening , resulting in six visits in nine months . fat mass and fat free mass was estimated using bioelectrical impedance ( tanita bc-418 ma , [ tanita corporation , arlington heights , il ] ) . body composition and waist circumference were measured prescreening , at screening , and at months 3 and 9 after screening . blood samples were obtained after an overnight fast prescreening , at screening , and at months 3 and 9 after screening to measure fasting plasma glucose , insulin , and adipokines , including leptin , resistin , adiponectin , pai-1 ( active ) , il-6 , and tnf-. serum samples for adipokines and insulin were measured in duplicate with commercially available immunoassay kits from millipore using the lincoplex system ( st charles , mo ) . the protocol for measurement of insulin and all the adipokines high - density lipoprotein cholesterol ( hdl ) , triacylglycerol , and fasting glucose were analyzed in the department of clinical biochemistry at nhs grampian . insulin resistance using fasting glucose and insulin values was calculated using the homeostasis model of assessment of insulin resistance ( homa - ir ) where homa - ir = [ insulin ] [ glucose]/22.5 . all variables were assessed for normality using the kolmogorov - smirnov test . any skewed data were log - transformed and subsequently assessed using parametric tests . for within group analysis , a paired t - test was used . for between - group analysis of changes , the pearson correlation was used to assess the relationship between changes in adipokines and changes in other continuous measurements . statistical tests were carried out using the spss 15.0 for windows software program ( spss inc , chicago , il ) . because this is an ancillary analysis following on from a previously published study,10 the power for this analysis was carried out based on weight loss . a p value of < 0.05 ( two - tailed ) was considered statistically significant . based on the mean change ( 31.0 kg ) and standard deviation ( 16.4 kg ) for weight loss observed at nine months,10 14 patients in each group resulted in a > 99% power for weight change . data are presented as means standard deviation , and changes are expressed as the mean difference changes from baseline standard deviation . baseline characteristics are listed in table 2 . a total of 31 ( 14 in ll , 17 in lchp ) patients completed the study . there were significantly more men in the ll than the lchp group ( n = 5 and n = 1 , respectively ) . weight , bmi , and fat free mass were significantly greater in the ll group than in the lchp group ( table 2 ) . when investigating the whole group , significant associations were observed between adiponectin , hdl , and pai-1 . leptin was significantly associated with percentage body fat and tnf- , while il-6 was inversely associated with hdl ( table 3 ) . there were no significant gender differences for the adipokines with the exception of leptin , for which the levels were significantly greater in women than in men ( 46.7 15.5 ng / ml versus 28.2 14.6 ng / ml , p = 0.014 ) . however , this was no longer significant after adjusting for percentage body fat ( p = 0.089 ) . at three months , percentage weight loss for the lchp group was 2.9% 4.2% and for ll was 18.4% 5.4% ( p < 0.0001 ) . weight , waist circumference , percentage body fat , fat mass , leptin , pai-1 , fasting glucose , homa - ir , and triacylglycerols had improved significantly in the ll group ( table 4 ) . however , hdl and fat free mass decreased significantly from baseline in the ll group ( table 4 ) . a significant improvement from baseline to three months was observed for weight , percentage body fat , fat mass , insulin , and homa - ir in the lchp group ( table 4 ) . at nine months , the percentage weight change for the lchp group was 1.4% 1.0% and for ll was 23.8% 4.0% ( p in the ll group , weight loss , waist circumference , percentage body fat , fat mass , fat free mass , leptin , pai-1 , fasting glucose , and triacylglycerols were still significantly improved compared with baseline ( table 4 ) . in addition , significant improvements in circulating levels of adiponectin and hdl were observed , but homa - ir was no longer significant ( table 4 ) . changes in adiponectin were inversely associated with changes in weight , bmi , fat mass , percentage body fat , waist circumference , il-6 , and tnf-. changes in leptin were associated with changes in weight , bmi , fat mass , waist circumference , fat free mass , and percentage body fat . changes in tnf- were associated with changes in il-6 , and inversely associated with changes in adiponectin . there were no significant changes from baseline at nine months for the lchp group ( table 4 ) . however , changes in leptin were inversely associated with changes in hdl ( r = 0.573 , p = 0.032 ) . overall , changes at nine months for weight , waist circumference , fat mass , adiponectin , leptin , fasting glucose , and hdl were significantly greater for the ll group than for the lchp group ( table 4 ) . at three months , percentage weight loss for the lchp group was 2.9% 4.2% and for ll was 18.4% 5.4% ( p < 0.0001 ) . weight , waist circumference , percentage body fat , fat mass , leptin , pai-1 , fasting glucose , homa - ir , and triacylglycerols had improved significantly in the ll group ( table 4 ) . however , hdl and fat free mass decreased significantly from baseline in the ll group ( table 4 ) . a significant improvement from baseline to three months was observed for weight , percentage body fat , fat mass , insulin , and homa - ir in the lchp group ( table 4 ) . at nine months , the percentage weight change for the lchp group was 1.4% 1.0% and for ll was 23.8% 4.0% ( p < 0.0001 ) . in the ll group , weight loss , waist circumference , percentage body fat , fat mass , fat free mass , leptin , pai-1 , fasting glucose , and triacylglycerols were , significant improvements in circulating levels of adiponectin and hdl were observed , but homa - ir was no longer significant ( table 4 ) . changes in adiponectin were inversely associated with changes in weight , bmi , fat mass , percentage body fat , waist circumference , il-6 , and tnf-. changes in leptin were associated with changes in weight , bmi , fat mass , waist circumference , fat free mass , and percentage body fat . changes in tnf- were associated with changes in il-6 , and inversely associated with changes in adiponectin . there were no significant changes from baseline at nine months for the lchp group ( table 4 ) . however , changes in leptin were inversely associated with changes in hdl ( r = 0.573 , p = 0.032 ) . overall , changes at nine months for weight , waist circumference , fat mass , adiponectin , leptin , fasting glucose , and hdl were significantly greater for the ll group than for the lchp group ( table 4 ) . in the present study , there was a significant weight loss at three months for patients on both ll and lchp . however , significant weight loss at nine months was only maintained in the ll group . changes in adiponectin and leptin were significantly greater in the ll group than in the lchp group which may be due to greater weight loss and decrease in fat mass . however , changes in tnf- , il-6 , pai-1 , and resistin did not differ significantly between the dietary groups at nine months . at baseline , we observed a trend for adiponectin to be inversely correlated with weight and bmi , but this did not reach statistical significance , possibly due to the small sample size . surprisingly , there was no significant inverse correlation between adiponectin and waist circumference , which was unexpected because other studies have demonstrated that waist circumference is a good correlate for adiponectemia.12,13 although the sample size was small , this may not be the reason for the lack of correlation between adiponectin , bmi , and waist circumference . . there is evidence to suggest that the incidence of metabolic syndrome decreases beyond a bmi of 37.5 kg / m.14 interestingly , our findings support the evidence presented by plaisance et al,15 who reported that baseline adiponectin levels were strongly correlated with mean hdl cholesterol . association of moderately decreased risk of cvd with increased adiponectin is thought to be mediated in part by the effects of adiponectin on hdl , through parallel increases in both . however , how adiponectin affects hdl remains unknown.16 in the ll group , changes in adiponectin were significant at nine months but not at three months . weight loss increased significantly from three to nine months in this group , which would suggest that a weight loss greater than 18.4% is required for statistically significant improvements in circulating adiponectin levels . changes in adiponectin were inversely associated with fat mass , waist circumference , il-6 , and tnf- at nine months , suggesting an improvement in inflammatory status with weight loss and the associated increase in adiponectin . in the present study , leptin was significantly associated with percentage body fat at baseline , which is consistent with previous findings.17 there was no significant correlation found between leptin and waist circumference in the present study at baseline . this suggests that there may be a greater release of leptin from peripheral subcutaneous adipose tissue compared with visceral adipose tissue . studies have shown that there are variations in leptin gene expression in adipose tissue depending on the site where it is deposited , where expression is greater in subcutaneous compared with visceral adipose tissue.18,19 alternatively , the lack of association between leptin and waist circumference may have been due to issues in the measurement of waist circumference in grade iii obese patients , a difficulty which is well recognized . in addition , different protocols yield different results.20 leptin was significantly decreased at three and nine months in the ll group . the changes in leptin were significantly associated with fat mass , percentage body fat , and waist circumference . this is consistent with a number of studies in which leptin is shown to decrease in response to weight loss.2124 there was also an association between leptin change and waist circumference change at nine months , reflecting an overall loss of fat mass . in addition , despite the minimal weight loss for the lchp group , changes in leptin were inversely associated with hdl at nine months . this may suggest that even a small reduction in weight results in a beneficial trend of improvement in leptin levels which is likely to improve cardiovascular risk . levels of pai-1 at baseline were associated with waist circumference , homa - ir , and insulin , suggesting that pai-1 is involved in insulin resistance as previously reported.25 weight loss has been found to reduce the levels of pai-1,2628 indicating the influence of adipose tissue on the levels of this protein . our study data are in support of this evidence , where changes in pai-1 were associated with changes in fat mass for the ll group at nine months . there was no evidence from the present study to support a strong link between resistin levels and weight . of all the adipokines examined in this study , resistin appears to be the most controversial because the evidence appears to be equivocal and inconclusive.2931 further human studies are required to confirm whether there is a relationship between resistin and obesity , including insulin resistance and type 2 diabetes mellitus . similarly , despite the fact that il-6 has been found to be increased in obesity32,33 and reduced in response to weight loss,34 no significant differences at baseline or in response to diet were observed here . subcutaneous adipose tissue is thought to release approximately 30% of systemic il-6 , and visceral adipose tissue is thought to release even more . however , the lack of change may be explained by the fact that only about 10% of total il-6 is produced by fat cells.35 in addition , mean levels of tnf- did not correlate significantly with weight , waist circumference , or percentage body fat at baseline . these results were unexpected , because it is widely reported that tnf- is linked with obesity.36 also , tnf- did not show any significant decrease in response to weight loss or diet . however , in a study by arvidsson et al,37 circulating levels of tnf- did not show a significant difference after a mean weight loss of 7.5% at 10 weeks . these authors concluded that adipose tissue has only a minor effect on the regulation of circulating tnf- levels . thus , tnf- seems to be produced and acts locally in human fat tissue,38 and there is no in situ release from adipose tissue into the blood.35 a number of studies have observed no changes in il-6 or tnf- after significant reductions in weight ( 59 kg ) with dietary and exercise interventions.39,40 however , it was interesting to observe that changes in il-6 and tnf- were significantly correlated at nine months in the ll group . this could be explained by the relationship between il-6 and tnf- whereby il-6 exerts proinflammatory activity itself and increases tnf-.1 this would suggest that , despite weight loss not resulting in significant improvements in circulating levels of il-6 and tnf- , there appears to be an underlying clinically significant response whereby reduction in weight results in a decrease in inflammation . there is evidence that weight loss achieved using a very low calorie diet results in changes in adipokine levels similar to those observed in response to bariatric surgery . mitterberger et al41 compared a group of patients for whom weight loss was achieved by dietary caloric restriction only with a group of patients in whom caloric restriction was induced by bariatric surgery ( three gastric bypasses and eight gastric bands ) . they reported that despite the fact that only a 26% 7% weight loss was achieved in the dietary caloric restriction as compared with 43% 10% in the bariatric surgery group , changes in adipokines were not significantly different between the two groups . this may suggest an important role for the use of a weight loss approach , such as the very low calorie diet instead of bariatric surgery , which is highly invasive , expensive , and can lead to long - term vitamin and mineral deficiencies.42 although it remains unclear as to whether the adipokines investigated to date are responsible for the beneficial effects on health associated with weight loss , it is clear from this study and several others that there are important associations between weight loss , certain adipokines ( such as adiponectin ) , leptin levels , and cardiovascular disease risk . in addition , the extent of weight loss required to elicit the benefits as well as the effects of the method by which weight loss is obtained remains unknown . however , it can be argued that there does seem to be a minimal weight loss required which was not achieved here in the lchp group and a maximal weight loss beyond which further improvements in circulating adipokine levels are no longer observed , as demonstrated in the study by mitterberg et al.41 further research would include determining if there is a ceiling effect for adipokine change in response to weight loss . this would involve directly comparing adipokine changes in response to weight loss achieved using a very low calorie diet as compared with weight loss achieved using surgical approaches . there were significantly more men on ll than on lchp , but this may not have been too limiting because there were no significant gender effects on adipokine when all patients were combined at baseline , except for leptin , but this discrepancy may have resulted in the significant differences between the two groups at baseline for weight , waist circumference , and fat free mass . in addition , although the sample size provided a > 99% power for weight loss , the sample sizes may have been too small to observe associations and changes in adipokines , which would have been expected based on the literature . moreover , use of the multiplexed assay has been criticized in the scientific literature.43 the multiplexed bead immunoassay allows the simultaneous detection of adipokines in small blood samples which may be particularly useful when samples are difficult to obtain . the use of this approach has been validated and found to be useful for leptin , adiponectin , and insulin in the evaluation of changes in obesity markers following weight reduction.43 the relationship between the multiplexed bead immunoassay when compared with the radioimmunoassay or enzyme - linked immunoassay for other adipokines such as resistin , il-6 , and tnf- , were reported to be quite weak . these differences could be explained by differences in antibody pairs and sample diluents , as well as the low effects of low concentrations of these adipokines . in addition , assay sensitivity remains an issue when compared with ultrasensitive enzyme - linked immunoassay methods.43 however , the ability of multiplexed assays to detect adipokines of a broader dynamic range than enzyme - linked immunoassays , as well as its greater cost - effectiveness , time efficiency , requirement of smaller sample volumes , and the removal of interassay variability suggest that this approach is still to be considered a powerful tool , albeit with the above limitations . it would appear that the significant weight loss of 23.8% observed on ll resulted in significant improvements in circulating levels of leptin , pai-1 , and adiponectin . changes in these adipokines possibly resulted in improvements in fasting glucose , triacylglycerols , and hdl . this is most likely due to the overall weight loss achieved rather than macronutrient intake . further research examining the adipokine response to weight loss using a very low calorie diet in comparison with surgical interventions would be beneficial to determine if the adipokine response to weight loss has a ceiling effect .

background : adipose tissue functions as an endocrine organ by releasing adipokines which have important roles in the regulation of inflammation and insulin sensitivity . although there is evidence of improvement in circulating levels of adipokines with weight loss , few studies relate such changes to specific diets . we investigated the effects of weight loss achieved by two different diets on circulating adipokine levels in obese individuals.methods:a total of 120 obese patients ( body mass index 35 kg / m2 ) underwent a three - month screening period on a low - fat , reduced - calorie diet . patients failing to achieve a 5% weight loss using this approach were randomly allocated to either a low carbohydrate / high protein diet ( n = 17 ) or to a commercial very low calorie diet ( lighterlife , n = 14 ) for a period of nine months.results:at nine months , a significant weight loss was only maintained for lighter - life ( 32.3 22.7 kg , p < 0.0001 ) but not on the low carbohydrate / high protein diet . changes in adiponectin ( 15.8 17.1 ng / ml versus 0.8 6.2 ng / ml , p = 0.003 ) and leptin ( 17.6 24.3 ng / ml versus 3.0 9.2 ng / ml , p = 0.049 ) at nine months were significantly greater for lighterlife than for the low carbohydrate / high protein diet , which may reflect greater weight loss and decrease in fat mass . changes in tumor necrosis factor - alpha , interleukin-6 , and plasminogen activator inhibitor type 1 did not differ significantly between the dietary interventions at nine months.conclusion:a significant weight loss of 23.8% from baseline weight was observed using a very low calorie diet and resulted in significant improvements in circulating levels of leptin , plasminogen activator inhibitor type 1 , and adiponectin , which are likely to be due to weight loss and not macronutrient intake .

Introduction Methods Dietary intervention Randomization and diet allocation Data collection Statistical analysis Results Three-month data Nine-month data Discussion Conclusion

it is now evident that adipose tissue not only stores excess triacylglycerols , but functions as an endocrine organ by releasing adipokines , which have important roles in the regulation of appetite , glucose and lipid metabolism , inflammation , and insulin resistance.1,2 such adipokines include adiponectin , leptin , resistin , tumor necrosis factor ( tnf)- , plasminogen activator inhibitor-1 ( pai-1 ) , and interleukin ( il)-6 . tnf- , pai-1 , and il-6 are all proinflammatory cytokines , although il-6 can further exert an anti - inflammatory action.1 it has also been observed that leptin , resistin , and tnf- impair insulin sensitivity and trigger atherogenesis.1 these adipokines usually increase with adiposity and can have a detrimental role on an individual s health . however , adiponectin , unlike other adipokines produced by adipose tissue , possesses antiatherogenic properties and is found to decrease with increased adiposity.1,2 several studies provide evidence of improvement in adipokine profiles with weight loss.36 however , it remains unclear as to whether changes in the circulating adipokines investigated to date contribute significantly to the beneficial effects on health associated with weight loss.7 the extent of weight loss required to elicit such changes in adipokine levels remains unclear . varady et al8 suggest that a minimum weight loss of 5% is required to have an effect on levels of key adipokines ( adiponectin , leptin , and resistin ) , but this differs from other studies which report the need for a 10% weight loss for modification in levels of some adipokines ( eg , adiponectin ) and that this varies with the degree of obesity.9 further studies are needed to clarify the relationship between changes in adipokine levels and the health benefits of weight loss , and whether specific dietary manipulations have differential effects on such changes . we hypothesized that the extent of weight loss rather than the macronutrient intake will determine the degree of change in adipokine levels . we aimed to investigate the effect of weight loss on adipokine levels in individuals receiving a low carbohydrate / high protein ( lchp ) diet compared with a very low calorie diet ( lighterlife [ ll ] ) diet . men and women older than 18 years of age and a body mass index ( bmi ) 35 kg / m were included . the study included a three - month screening period where patients were assigned to a 600 calorie - deficient diet ( cdd ) aiming to achieve a 5% weight loss . this was done to select patients who would not respond to a low - fat , reduced - energy approach and randomly assign them to a lchp or a ll diet in the form of a continuous method approach . completer data for this group were limited ( n = 4 ) and therefore were not included in this paper . those patients who failed to achieve the weight loss targets were randomly allocated to either a lchp or ll diet , and continued on the assigned diet for an additional nine months ( figure 1 ) . patients on cdd who achieved a 5% weight loss at screening but did not achieve the 10% weight loss three months following screening were randomized to the ll or the lchp diet but their data were omitted from this analysis . the ll diet used in this study is administered in the form of soups , shakes , and bars to replace conventional food and provides a daily average of 550 kcal ( 36% carbohydrate , 36% protein , 28% fat , and at least 100% of the recommended daily allowance for all micronutrients ) . patients were required to remain on the weight loss phase for a minimum of three months , after which they were given a choice to continue for up to another six months or were assigned to the food reintroduction phase . all patients came monthly to the trial center to be weighed for the first three months and then every alternate month after screening , resulting in six visits in nine months . blood samples were obtained after an overnight fast prescreening , at screening , and at months 3 and 9 after screening to measure fasting plasma glucose , insulin , and adipokines , including leptin , resistin , adiponectin , pai-1 ( active ) , il-6 , and tnf-. based on the mean change ( 31.0 kg ) and standard deviation ( 16.4 kg ) for weight loss observed at nine months,10 14 patients in each group resulted in a > 99% power for weight change . the ll diet used in this study is administered in the form of soups , shakes , and bars to replace conventional food and provides a daily average of 550 kcal ( 36% carbohydrate , 36% protein , 28% fat , and at least 100% of the recommended daily allowance for all micronutrients ) . patients were required to remain on the weight loss phase for a minimum of three months , after which they were given a choice to continue for up to another six months or were assigned to the food reintroduction phase . all patients came monthly to the trial center to be weighed for the first three months and then every alternate month after screening , resulting in six visits in nine months . blood samples were obtained after an overnight fast prescreening , at screening , and at months 3 and 9 after screening to measure fasting plasma glucose , insulin , and adipokines , including leptin , resistin , adiponectin , pai-1 ( active ) , il-6 , and tnf-. based on the mean change ( 31.0 kg ) and standard deviation ( 16.4 kg ) for weight loss observed at nine months,10 14 patients in each group resulted in a > 99% power for weight change . there were significantly more men in the ll than the lchp group ( n = 5 and n = 1 , respectively ) . weight , bmi , and fat free mass were significantly greater in the ll group than in the lchp group ( table 2 ) . there were no significant gender differences for the adipokines with the exception of leptin , for which the levels were significantly greater in women than in men ( 46.7 15.5 ng / ml versus 28.2 14.6 ng / ml , p = 0.014 ) . at three months , percentage weight loss for the lchp group was 2.9% 4.2% and for ll was 18.4% 5.4% ( p < 0.0001 ) . weight , waist circumference , percentage body fat , fat mass , leptin , pai-1 , fasting glucose , homa - ir , and triacylglycerols had improved significantly in the ll group ( table 4 ) . a significant improvement from baseline to three months was observed for weight , percentage body fat , fat mass , insulin , and homa - ir in the lchp group ( table 4 ) . at nine months , the percentage weight change for the lchp group was 1.4% 1.0% and for ll was 23.8% 4.0% ( p in the ll group , weight loss , waist circumference , percentage body fat , fat mass , fat free mass , leptin , pai-1 , fasting glucose , and triacylglycerols were still significantly improved compared with baseline ( table 4 ) . in addition , significant improvements in circulating levels of adiponectin and hdl were observed , but homa - ir was no longer significant ( table 4 ) . changes in adiponectin were inversely associated with changes in weight , bmi , fat mass , percentage body fat , waist circumference , il-6 , and tnf-. changes in leptin were associated with changes in weight , bmi , fat mass , waist circumference , fat free mass , and percentage body fat . changes in tnf- were associated with changes in il-6 , and inversely associated with changes in adiponectin . there were no significant changes from baseline at nine months for the lchp group ( table 4 ) . however , changes in leptin were inversely associated with changes in hdl ( r = 0.573 , p = 0.032 ) . overall , changes at nine months for weight , waist circumference , fat mass , adiponectin , leptin , fasting glucose , and hdl were significantly greater for the ll group than for the lchp group ( table 4 ) . at three months , percentage weight loss for the lchp group was 2.9% 4.2% and for ll was 18.4% 5.4% ( p < 0.0001 ) . weight , waist circumference , percentage body fat , fat mass , leptin , pai-1 , fasting glucose , homa - ir , and triacylglycerols had improved significantly in the ll group ( table 4 ) . a significant improvement from baseline to three months was observed for weight , percentage body fat , fat mass , insulin , and homa - ir in the lchp group ( table 4 ) . at nine months , the percentage weight change for the lchp group was 1.4% 1.0% and for ll was 23.8% 4.0% ( p < 0.0001 ) . in the ll group , weight loss , waist circumference , percentage body fat , fat mass , fat free mass , leptin , pai-1 , fasting glucose , and triacylglycerols were , significant improvements in circulating levels of adiponectin and hdl were observed , but homa - ir was no longer significant ( table 4 ) . changes in adiponectin were inversely associated with changes in weight , bmi , fat mass , percentage body fat , waist circumference , il-6 , and tnf-. changes in leptin were associated with changes in weight , bmi , fat mass , waist circumference , fat free mass , and percentage body fat . there were no significant changes from baseline at nine months for the lchp group ( table 4 ) . however , changes in leptin were inversely associated with changes in hdl ( r = 0.573 , p = 0.032 ) . overall , changes at nine months for weight , waist circumference , fat mass , adiponectin , leptin , fasting glucose , and hdl were significantly greater for the ll group than for the lchp group ( table 4 ) . in the present study , there was a significant weight loss at three months for patients on both ll and lchp . however , significant weight loss at nine months was only maintained in the ll group . changes in adiponectin and leptin were significantly greater in the ll group than in the lchp group which may be due to greater weight loss and decrease in fat mass . however , changes in tnf- , il-6 , pai-1 , and resistin did not differ significantly between the dietary groups at nine months . at baseline , we observed a trend for adiponectin to be inversely correlated with weight and bmi , but this did not reach statistical significance , possibly due to the small sample size . surprisingly , there was no significant inverse correlation between adiponectin and waist circumference , which was unexpected because other studies have demonstrated that waist circumference is a good correlate for adiponectemia.12,13 although the sample size was small , this may not be the reason for the lack of correlation between adiponectin , bmi , and waist circumference . there is evidence to suggest that the incidence of metabolic syndrome decreases beyond a bmi of 37.5 kg / m.14 interestingly , our findings support the evidence presented by plaisance et al,15 who reported that baseline adiponectin levels were strongly correlated with mean hdl cholesterol . association of moderately decreased risk of cvd with increased adiponectin is thought to be mediated in part by the effects of adiponectin on hdl , through parallel increases in both . however , how adiponectin affects hdl remains unknown.16 in the ll group , changes in adiponectin were significant at nine months but not at three months . weight loss increased significantly from three to nine months in this group , which would suggest that a weight loss greater than 18.4% is required for statistically significant improvements in circulating adiponectin levels . changes in adiponectin were inversely associated with fat mass , waist circumference , il-6 , and tnf- at nine months , suggesting an improvement in inflammatory status with weight loss and the associated increase in adiponectin . studies have shown that there are variations in leptin gene expression in adipose tissue depending on the site where it is deposited , where expression is greater in subcutaneous compared with visceral adipose tissue.18,19 alternatively , the lack of association between leptin and waist circumference may have been due to issues in the measurement of waist circumference in grade iii obese patients , a difficulty which is well recognized . the changes in leptin were significantly associated with fat mass , percentage body fat , and waist circumference . this is consistent with a number of studies in which leptin is shown to decrease in response to weight loss.2124 there was also an association between leptin change and waist circumference change at nine months , reflecting an overall loss of fat mass . in addition , despite the minimal weight loss for the lchp group , changes in leptin were inversely associated with hdl at nine months . this may suggest that even a small reduction in weight results in a beneficial trend of improvement in leptin levels which is likely to improve cardiovascular risk . levels of pai-1 at baseline were associated with waist circumference , homa - ir , and insulin , suggesting that pai-1 is involved in insulin resistance as previously reported.25 weight loss has been found to reduce the levels of pai-1,2628 indicating the influence of adipose tissue on the levels of this protein . our study data are in support of this evidence , where changes in pai-1 were associated with changes in fat mass for the ll group at nine months . however , the lack of change may be explained by the fact that only about 10% of total il-6 is produced by fat cells.35 in addition , mean levels of tnf- did not correlate significantly with weight , waist circumference , or percentage body fat at baseline . these results were unexpected , because it is widely reported that tnf- is linked with obesity.36 also , tnf- did not show any significant decrease in response to weight loss or diet . however , in a study by arvidsson et al,37 circulating levels of tnf- did not show a significant difference after a mean weight loss of 7.5% at 10 weeks . these authors concluded that adipose tissue has only a minor effect on the regulation of circulating tnf- levels . thus , tnf- seems to be produced and acts locally in human fat tissue,38 and there is no in situ release from adipose tissue into the blood.35 a number of studies have observed no changes in il-6 or tnf- after significant reductions in weight ( 59 kg ) with dietary and exercise interventions.39,40 however , it was interesting to observe that changes in il-6 and tnf- were significantly correlated at nine months in the ll group . this could be explained by the relationship between il-6 and tnf- whereby il-6 exerts proinflammatory activity itself and increases tnf-.1 this would suggest that , despite weight loss not resulting in significant improvements in circulating levels of il-6 and tnf- , there appears to be an underlying clinically significant response whereby reduction in weight results in a decrease in inflammation . there is evidence that weight loss achieved using a very low calorie diet results in changes in adipokine levels similar to those observed in response to bariatric surgery . mitterberger et al41 compared a group of patients for whom weight loss was achieved by dietary caloric restriction only with a group of patients in whom caloric restriction was induced by bariatric surgery ( three gastric bypasses and eight gastric bands ) . they reported that despite the fact that only a 26% 7% weight loss was achieved in the dietary caloric restriction as compared with 43% 10% in the bariatric surgery group , changes in adipokines were not significantly different between the two groups . this may suggest an important role for the use of a weight loss approach , such as the very low calorie diet instead of bariatric surgery , which is highly invasive , expensive , and can lead to long - term vitamin and mineral deficiencies.42 although it remains unclear as to whether the adipokines investigated to date are responsible for the beneficial effects on health associated with weight loss , it is clear from this study and several others that there are important associations between weight loss , certain adipokines ( such as adiponectin ) , leptin levels , and cardiovascular disease risk . in addition , the extent of weight loss required to elicit the benefits as well as the effects of the method by which weight loss is obtained remains unknown . however , it can be argued that there does seem to be a minimal weight loss required which was not achieved here in the lchp group and a maximal weight loss beyond which further improvements in circulating adipokine levels are no longer observed , as demonstrated in the study by mitterberg et al.41 further research would include determining if there is a ceiling effect for adipokine change in response to weight loss . this would involve directly comparing adipokine changes in response to weight loss achieved using a very low calorie diet as compared with weight loss achieved using surgical approaches . there were significantly more men on ll than on lchp , but this may not have been too limiting because there were no significant gender effects on adipokine when all patients were combined at baseline , except for leptin , but this discrepancy may have resulted in the significant differences between the two groups at baseline for weight , waist circumference , and fat free mass . in addition , although the sample size provided a > 99% power for weight loss , the sample sizes may have been too small to observe associations and changes in adipokines , which would have been expected based on the literature . moreover , use of the multiplexed assay has been criticized in the scientific literature.43 the multiplexed bead immunoassay allows the simultaneous detection of adipokines in small blood samples which may be particularly useful when samples are difficult to obtain . the use of this approach has been validated and found to be useful for leptin , adiponectin , and insulin in the evaluation of changes in obesity markers following weight reduction.43 the relationship between the multiplexed bead immunoassay when compared with the radioimmunoassay or enzyme - linked immunoassay for other adipokines such as resistin , il-6 , and tnf- , were reported to be quite weak . in addition , assay sensitivity remains an issue when compared with ultrasensitive enzyme - linked immunoassay methods.43 however , the ability of multiplexed assays to detect adipokines of a broader dynamic range than enzyme - linked immunoassays , as well as its greater cost - effectiveness , time efficiency , requirement of smaller sample volumes , and the removal of interassay variability suggest that this approach is still to be considered a powerful tool , albeit with the above limitations . it would appear that the significant weight loss of 23.8% observed on ll resulted in significant improvements in circulating levels of leptin , pai-1 , and adiponectin . changes in these adipokines possibly resulted in improvements in fasting glucose , triacylglycerols , and hdl . this is most likely due to the overall weight loss achieved rather than macronutrient intake . further research examining the adipokine response to weight loss using a very low calorie diet in comparison with surgical interventions would be beneficial to determine if the adipokine response to weight loss has a ceiling effect .

it is now evident that adipose tissue not only stores excess triacylglycerols , but functions as an endocrine organ by releasing adipokines , which have important roles in the regulation of appetite , glucose and lipid metabolism , inflammation , and insulin resistance.1,2 such adipokines include adiponectin , leptin , resistin , tumor necrosis factor ( tnf)- , plasminogen activator inhibitor-1 ( pai-1 ) , and interleukin ( il)-6 . tnf- , pai-1 , and il-6 are all proinflammatory cytokines , although il-6 can further exert an anti - inflammatory action.1 it has also been observed that leptin , resistin , and tnf- impair insulin sensitivity and trigger atherogenesis.1 these adipokines usually increase with adiposity and can have a detrimental role on an individual s health . however , adiponectin , unlike other adipokines produced by adipose tissue , possesses antiatherogenic properties and is found to decrease with increased adiposity.1,2 several studies provide evidence of improvement in adipokine profiles with weight loss.36 however , it remains unclear as to whether changes in the circulating adipokines investigated to date contribute significantly to the beneficial effects on health associated with weight loss.7 the extent of weight loss required to elicit such changes in adipokine levels remains unclear . varady et al8 suggest that a minimum weight loss of 5% is required to have an effect on levels of key adipokines ( adiponectin , leptin , and resistin ) , but this differs from other studies which report the need for a 10% weight loss for modification in levels of some adipokines ( eg , adiponectin ) and that this varies with the degree of obesity.9 further studies are needed to clarify the relationship between changes in adipokine levels and the health benefits of weight loss , and whether specific dietary manipulations have differential effects on such changes . we hypothesized that the extent of weight loss rather than the macronutrient intake will determine the degree of change in adipokine levels . we aimed to investigate the effect of weight loss on adipokine levels in individuals receiving a low carbohydrate / high protein ( lchp ) diet compared with a very low calorie diet ( lighterlife [ ll ] ) diet . dropouts were not included in this analysis because the goal was to evaluate the effect of diet on adipokine levels and not an intention - to - treat clinical trial . this was done to select patients who would not respond to a low - fat , reduced - energy approach and randomly assign them to a lchp or a ll diet in the form of a continuous method approach . those patients who failed to achieve the weight loss targets were randomly allocated to either a lchp or ll diet , and continued on the assigned diet for an additional nine months ( figure 1 ) . patients on cdd who achieved a 5% weight loss at screening but did not achieve the 10% weight loss three months following screening were randomized to the ll or the lchp diet but their data were omitted from this analysis . using the schofield and the harris benedict equations , the amount of energy allowed was broken down into portions from the different food groups.10 portion sizes were explained to the patients and written information was also provided . the ll diet used in this study is administered in the form of soups , shakes , and bars to replace conventional food and provides a daily average of 550 kcal ( 36% carbohydrate , 36% protein , 28% fat , and at least 100% of the recommended daily allowance for all micronutrients ) . during each stage , patients attend weekly single - sex group meetings of 712 people for behavior change therapy based on cognitive behavior therapy and transactional analysis methodology delivered by a trained ll advisor . patients were required to remain on the weight loss phase for a minimum of three months , after which they were given a choice to continue for up to another six months or were assigned to the food reintroduction phase . all patients came monthly to the trial center to be weighed for the first three months and then every alternate month after screening , resulting in six visits in nine months . blood samples were obtained after an overnight fast prescreening , at screening , and at months 3 and 9 after screening to measure fasting plasma glucose , insulin , and adipokines , including leptin , resistin , adiponectin , pai-1 ( active ) , il-6 , and tnf-. high - density lipoprotein cholesterol ( hdl ) , triacylglycerol , and fasting glucose were analyzed in the department of clinical biochemistry at nhs grampian . insulin resistance using fasting glucose and insulin values was calculated using the homeostasis model of assessment of insulin resistance ( homa - ir ) where homa - ir = [ insulin ] [ glucose]/22.5 . for between - group analysis of changes , the pearson correlation was used to assess the relationship between changes in adipokines and changes in other continuous measurements . based on the mean change ( 31.0 kg ) and standard deviation ( 16.4 kg ) for weight loss observed at nine months,10 14 patients in each group resulted in a > 99% power for weight change . the ll diet used in this study is administered in the form of soups , shakes , and bars to replace conventional food and provides a daily average of 550 kcal ( 36% carbohydrate , 36% protein , 28% fat , and at least 100% of the recommended daily allowance for all micronutrients ) . during each stage , patients attend weekly single - sex group meetings of 712 people for behavior change therapy based on cognitive behavior therapy and transactional analysis methodology delivered by a trained ll advisor . patients were required to remain on the weight loss phase for a minimum of three months , after which they were given a choice to continue for up to another six months or were assigned to the food reintroduction phase . all patients came monthly to the trial center to be weighed for the first three months and then every alternate month after screening , resulting in six visits in nine months . blood samples were obtained after an overnight fast prescreening , at screening , and at months 3 and 9 after screening to measure fasting plasma glucose , insulin , and adipokines , including leptin , resistin , adiponectin , pai-1 ( active ) , il-6 , and tnf-. the protocol for measurement of insulin and all the adipokines high - density lipoprotein cholesterol ( hdl ) , triacylglycerol , and fasting glucose were analyzed in the department of clinical biochemistry at nhs grampian . insulin resistance using fasting glucose and insulin values was calculated using the homeostasis model of assessment of insulin resistance ( homa - ir ) where homa - ir = [ insulin ] [ glucose]/22.5 . for between - group analysis of changes , the pearson correlation was used to assess the relationship between changes in adipokines and changes in other continuous measurements . based on the mean change ( 31.0 kg ) and standard deviation ( 16.4 kg ) for weight loss observed at nine months,10 14 patients in each group resulted in a > 99% power for weight change . there were significantly more men in the ll than the lchp group ( n = 5 and n = 1 , respectively ) . weight , bmi , and fat free mass were significantly greater in the ll group than in the lchp group ( table 2 ) . when investigating the whole group , significant associations were observed between adiponectin , hdl , and pai-1 . leptin was significantly associated with percentage body fat and tnf- , while il-6 was inversely associated with hdl ( table 3 ) . there were no significant gender differences for the adipokines with the exception of leptin , for which the levels were significantly greater in women than in men ( 46.7 15.5 ng / ml versus 28.2 14.6 ng / ml , p = 0.014 ) . at three months , percentage weight loss for the lchp group was 2.9% 4.2% and for ll was 18.4% 5.4% ( p < 0.0001 ) . weight , waist circumference , percentage body fat , fat mass , leptin , pai-1 , fasting glucose , homa - ir , and triacylglycerols had improved significantly in the ll group ( table 4 ) . a significant improvement from baseline to three months was observed for weight , percentage body fat , fat mass , insulin , and homa - ir in the lchp group ( table 4 ) . at nine months , the percentage weight change for the lchp group was 1.4% 1.0% and for ll was 23.8% 4.0% ( p in the ll group , weight loss , waist circumference , percentage body fat , fat mass , fat free mass , leptin , pai-1 , fasting glucose , and triacylglycerols were still significantly improved compared with baseline ( table 4 ) . in addition , significant improvements in circulating levels of adiponectin and hdl were observed , but homa - ir was no longer significant ( table 4 ) . changes in adiponectin were inversely associated with changes in weight , bmi , fat mass , percentage body fat , waist circumference , il-6 , and tnf-. changes in leptin were associated with changes in weight , bmi , fat mass , waist circumference , fat free mass , and percentage body fat . changes in tnf- were associated with changes in il-6 , and inversely associated with changes in adiponectin . overall , changes at nine months for weight , waist circumference , fat mass , adiponectin , leptin , fasting glucose , and hdl were significantly greater for the ll group than for the lchp group ( table 4 ) . at three months , percentage weight loss for the lchp group was 2.9% 4.2% and for ll was 18.4% 5.4% ( p < 0.0001 ) . weight , waist circumference , percentage body fat , fat mass , leptin , pai-1 , fasting glucose , homa - ir , and triacylglycerols had improved significantly in the ll group ( table 4 ) . a significant improvement from baseline to three months was observed for weight , percentage body fat , fat mass , insulin , and homa - ir in the lchp group ( table 4 ) . at nine months , the percentage weight change for the lchp group was 1.4% 1.0% and for ll was 23.8% 4.0% ( p < 0.0001 ) . in the ll group , weight loss , waist circumference , percentage body fat , fat mass , fat free mass , leptin , pai-1 , fasting glucose , and triacylglycerols were , significant improvements in circulating levels of adiponectin and hdl were observed , but homa - ir was no longer significant ( table 4 ) . changes in adiponectin were inversely associated with changes in weight , bmi , fat mass , percentage body fat , waist circumference , il-6 , and tnf-. changes in leptin were associated with changes in weight , bmi , fat mass , waist circumference , fat free mass , and percentage body fat . changes in tnf- were associated with changes in il-6 , and inversely associated with changes in adiponectin . overall , changes at nine months for weight , waist circumference , fat mass , adiponectin , leptin , fasting glucose , and hdl were significantly greater for the ll group than for the lchp group ( table 4 ) . in the present study , there was a significant weight loss at three months for patients on both ll and lchp . changes in adiponectin and leptin were significantly greater in the ll group than in the lchp group which may be due to greater weight loss and decrease in fat mass . at baseline , we observed a trend for adiponectin to be inversely correlated with weight and bmi , but this did not reach statistical significance , possibly due to the small sample size . surprisingly , there was no significant inverse correlation between adiponectin and waist circumference , which was unexpected because other studies have demonstrated that waist circumference is a good correlate for adiponectemia.12,13 although the sample size was small , this may not be the reason for the lack of correlation between adiponectin , bmi , and waist circumference . there is evidence to suggest that the incidence of metabolic syndrome decreases beyond a bmi of 37.5 kg / m.14 interestingly , our findings support the evidence presented by plaisance et al,15 who reported that baseline adiponectin levels were strongly correlated with mean hdl cholesterol . association of moderately decreased risk of cvd with increased adiponectin is thought to be mediated in part by the effects of adiponectin on hdl , through parallel increases in both . however , how adiponectin affects hdl remains unknown.16 in the ll group , changes in adiponectin were significant at nine months but not at three months . weight loss increased significantly from three to nine months in this group , which would suggest that a weight loss greater than 18.4% is required for statistically significant improvements in circulating adiponectin levels . changes in adiponectin were inversely associated with fat mass , waist circumference , il-6 , and tnf- at nine months , suggesting an improvement in inflammatory status with weight loss and the associated increase in adiponectin . in the present study , leptin was significantly associated with percentage body fat at baseline , which is consistent with previous findings.17 there was no significant correlation found between leptin and waist circumference in the present study at baseline . studies have shown that there are variations in leptin gene expression in adipose tissue depending on the site where it is deposited , where expression is greater in subcutaneous compared with visceral adipose tissue.18,19 alternatively , the lack of association between leptin and waist circumference may have been due to issues in the measurement of waist circumference in grade iii obese patients , a difficulty which is well recognized . this is consistent with a number of studies in which leptin is shown to decrease in response to weight loss.2124 there was also an association between leptin change and waist circumference change at nine months , reflecting an overall loss of fat mass . in addition , despite the minimal weight loss for the lchp group , changes in leptin were inversely associated with hdl at nine months . levels of pai-1 at baseline were associated with waist circumference , homa - ir , and insulin , suggesting that pai-1 is involved in insulin resistance as previously reported.25 weight loss has been found to reduce the levels of pai-1,2628 indicating the influence of adipose tissue on the levels of this protein . our study data are in support of this evidence , where changes in pai-1 were associated with changes in fat mass for the ll group at nine months . of all the adipokines examined in this study , resistin appears to be the most controversial because the evidence appears to be equivocal and inconclusive.2931 further human studies are required to confirm whether there is a relationship between resistin and obesity , including insulin resistance and type 2 diabetes mellitus . similarly , despite the fact that il-6 has been found to be increased in obesity32,33 and reduced in response to weight loss,34 no significant differences at baseline or in response to diet were observed here . however , the lack of change may be explained by the fact that only about 10% of total il-6 is produced by fat cells.35 in addition , mean levels of tnf- did not correlate significantly with weight , waist circumference , or percentage body fat at baseline . thus , tnf- seems to be produced and acts locally in human fat tissue,38 and there is no in situ release from adipose tissue into the blood.35 a number of studies have observed no changes in il-6 or tnf- after significant reductions in weight ( 59 kg ) with dietary and exercise interventions.39,40 however , it was interesting to observe that changes in il-6 and tnf- were significantly correlated at nine months in the ll group . this could be explained by the relationship between il-6 and tnf- whereby il-6 exerts proinflammatory activity itself and increases tnf-.1 this would suggest that , despite weight loss not resulting in significant improvements in circulating levels of il-6 and tnf- , there appears to be an underlying clinically significant response whereby reduction in weight results in a decrease in inflammation . mitterberger et al41 compared a group of patients for whom weight loss was achieved by dietary caloric restriction only with a group of patients in whom caloric restriction was induced by bariatric surgery ( three gastric bypasses and eight gastric bands ) . they reported that despite the fact that only a 26% 7% weight loss was achieved in the dietary caloric restriction as compared with 43% 10% in the bariatric surgery group , changes in adipokines were not significantly different between the two groups . this may suggest an important role for the use of a weight loss approach , such as the very low calorie diet instead of bariatric surgery , which is highly invasive , expensive , and can lead to long - term vitamin and mineral deficiencies.42 although it remains unclear as to whether the adipokines investigated to date are responsible for the beneficial effects on health associated with weight loss , it is clear from this study and several others that there are important associations between weight loss , certain adipokines ( such as adiponectin ) , leptin levels , and cardiovascular disease risk . however , it can be argued that there does seem to be a minimal weight loss required which was not achieved here in the lchp group and a maximal weight loss beyond which further improvements in circulating adipokine levels are no longer observed , as demonstrated in the study by mitterberg et al.41 further research would include determining if there is a ceiling effect for adipokine change in response to weight loss . there were significantly more men on ll than on lchp , but this may not have been too limiting because there were no significant gender effects on adipokine when all patients were combined at baseline , except for leptin , but this discrepancy may have resulted in the significant differences between the two groups at baseline for weight , waist circumference , and fat free mass . moreover , use of the multiplexed assay has been criticized in the scientific literature.43 the multiplexed bead immunoassay allows the simultaneous detection of adipokines in small blood samples which may be particularly useful when samples are difficult to obtain . the use of this approach has been validated and found to be useful for leptin , adiponectin , and insulin in the evaluation of changes in obesity markers following weight reduction.43 the relationship between the multiplexed bead immunoassay when compared with the radioimmunoassay or enzyme - linked immunoassay for other adipokines such as resistin , il-6 , and tnf- , were reported to be quite weak . in addition , assay sensitivity remains an issue when compared with ultrasensitive enzyme - linked immunoassay methods.43 however , the ability of multiplexed assays to detect adipokines of a broader dynamic range than enzyme - linked immunoassays , as well as its greater cost - effectiveness , time efficiency , requirement of smaller sample volumes , and the removal of interassay variability suggest that this approach is still to be considered a powerful tool , albeit with the above limitations .

two basic considerations are necessary to identify a profession : its central area of interest and its mission . although not the only things that identify a profession , these are necessary before other prerequisites of a profession are obtainable . others hold that the definition of a profession pertains to its area of knowledge and boundaries of that knowledge , otherwise known as degree of abstraction . as a practical matter , identity of a profession helps guide its practitioners on scope of practice issues and informs the public on what the profession has to offer . moreover , without a clear identity , there tends to be confusion about the profession . moreover , the formation of a professional identity goes beyond the acquisition of certain skill sets , as it is formed mainly in social and relationship contexts . having an identity that satisfies these descriptions still may not be satisfactory if that identity is not unique , separate , and distinct from those of other professions . one obstacle to achieving a clear and unified chiropractic identity has been that of oppression , as some in the nursing profession have observed . in the chiropractic context medical regulation was used against chiropractors , and many were arrested for practicing medicine without a license . the actions of the brave chiropractic pioneers , who went to jail for chiropractic to protest the charge ( and who were practicing chiropractic , not medicine ) , helped bring about licensure in the united states . during the tumultuous days of harassment by organized medicine another important aspect in professional identity is obtaining trust from the public that the profession serves . this trust requires periodic renewal of a profession s social contract with the public . without such renewal , the public will wrest the contract from the profession s control and shift it to the default model of the market . when 2 professions have identities that are the same or overlap substantially ( eg , both have back pain as their area of focus ) , then 1 of the professions is duplicative and therefore unnecessary . although back pain may not be the main area of interest in all medical practices , it certainly is included as 1 of their areas of interest . when chiropractors identify themselves as back pain specialists , or even spine specialists , they compete not only with medical practitioners , but also with osteopaths , physical therapists , massage therapists , and medicine cabinets in the homes of millions of potential patients . thus , for the chiropractic profession , if its identity is based on neck and back pain , it is lost in the presence of much larger professions ( eg , medicine ) that also treat back and neck pain . moreover , when the osteopathic profession merged into orthodox medicine in the late 1950s , it ceased to offer a unique service and was essentially swallowed up by the medical profession . the purpose of this paper is to discuss various statements related to chiropractic identity from d. d. palmer ; selected chiropractic organizations ( associations ) and colleges ; and attitudes and perceptions of chiropractic from chiropractic students , practitioners , and patients . the article concludes by encouraging the profession to base itself on its original intent , which is improving neurologic function by adjustment of vertebral subluxation . for the purpose of this commentary , identity of chiropractic is considered synonymous with definition or explanation of what chiropractic is . definitions of chiropractic , or statements that at least resembled a definition , were obtained from palmer s famous 1910 book , the science , art , and philosophy of chiropractic ; websites of national and international chiropractic organizations ; websites of chiropractic colleges listed in english by the association of chiropractic colleges ; and internet ( bing , pubmed , and google scholar ) searches for reported attitudes of chiropractic students , practitioners , and patients . the associations comprised the association of chiropractic colleges ( acc ) , american chiropractic association , international chiropractors association , international federation of chiropractors and organizations ( ifco ) , and world federation of chiropractic ( wfc ) . throughout palmer s 1910 textbook , he essentially stated that the main area of interest and therefore identity in chiropractic was analysis and adjustment of vertebral subluxation . among a sample of 5 major chiropractic organizations , 2 specifically mention that chiropractic s focus is subluxation ( acc ) or vertebral subluxation ( ifco ) . another organization s statement is similar in that it mentions spinal adjustment , nervous system function , and general health , without specifically using the term subluxation ( wfc ) ( table 1 ) . a search of the internet was performed in february 2016 on how chiropractic colleges represent themselves and chiropractic identity to the public . among the chiropractic colleges , 3 specifically mention subluxation in their description of chiropractic : life university , life chiropractic college west , and sherman college of chiropractic . other colleges , such as cleveland chiropractic college , new zealand college of chiropractic , and palmer college of chiropractic , describe subluxation with statements related to chiropractic focusing on the spine and its relationship to the nervous system . a number of other colleges mention that chiropractic pertains to general health and/or musculoskeletal conditions ( table 2 ) . survey research by gallup and palmer college indicates that the public generally considers that chiropractic pertains to neck and back pain . however , 31% of those surveyed indicated that they would like to use chiropractic care even if they were asymptomatic . this suggests that about a third of potential patients are open to a wellness approach , which is a component of the vertebral subluxation model ( eg , maintenance care ) . survey research by gliedt et al indicates that a majority ( 61.4% ) of chiropractic students think that the emphasis of chiropractic intervention is to eliminate vertebral subluxations / vertebral subluxation complexes . survey research by mcdonald et al indicated that 88.1% of practicing chiropractors in north america who responded to the survey answered yes to the question , should chiropractic retain the term vertebral subluxation complex ? currently , a consensus viewpoint regarding the identity of the chiropractic profession is lacking , evidenced by the various opinions among its associations and colleges . it is unclear how these varying viewpoints came to be , considering the stated intent and identity of chiropractic as laid down by the founder of the profession . it is possible that these differing views came about simply as a preference that was different from palmer s original intent . academic freedom allows for various viewpoints , even with respect to a profession s identity . however , that freedom may come with a price , in the way of unintended consequences ; the public may be confused as to what chiropractic is when faced with the variability of viewpoints . moreover , a profession s identity that does not make distinctions between its identity and the identities of other professions seems to invite further confusion . to this point , the wfc has recommended that the identity of the profession should be similar in all countries . i feel that this is a worthy recommendation ; however , the brand that wfc suggests may be too vague , as it has a relatively high degree of abstraction when it states that chiropractors are spinal health care experts in the health care system . the quasi - vagueness here might be considered to be at odds with wfc s call that chiropractic identity should be clear and concise . nonetheless , common ground between the wfc and those within the subluxation model are suggested in its qualifying statements for chiropractic care:ability to improve function in the neuromusculoskeletal system and overall health , wellbeing , and quality of life;specialized approach to examination , diagnosis , and treatment , based on best available research and clinical evidence with particular emphasis on the relationship between the spine and the nervous system;tradition of effectiveness and patient satisfaction;without use of drugs and surgery , enabling patients to avoid these where possible;expertly qualified providers of spinal adjustment , manipulation and other manual treatments , exercise instruction , and patient education;collaboration with other health professionals ; anda patient - centered and biopsychosocial approach , emphasizing the mind / body relationship in health , the self - healing powers of the individual , and individual responsibility for health and encouraging patient independence . ability to improve function in the neuromusculoskeletal system and overall health , wellbeing , and quality of life ; specialized approach to examination , diagnosis , and treatment , based on best available research and clinical evidence with particular emphasis on the relationship between the spine and the nervous system ; tradition of effectiveness and patient satisfaction ; without use of drugs and surgery , enabling patients to avoid these where possible ; expertly qualified providers of spinal adjustment , manipulation and other manual treatments , exercise instruction , and patient education ; collaboration with other health professionals ; and a patient - centered and biopsychosocial approach , emphasizing the mind / body relationship in health , the self - healing powers of the individual , and individual responsibility for health and encouraging patient independence . i suggest that the chiropractic profession s identity should be based on what makes it unique and what the founder of chiropractic envisioned it to be , which is adjustment of vertebral subluxations to improve neurologic function in the patient . although some in the profession have concerns with the subluxation model , there is interest in advancing the profession by addressing these issues.19 , 20 for others , the problem may be with the use of the term subluxation . for example , a former president of the national university of health sciences states that the purpose of chiropractic manual care is to correct a functional articular lesion to produce ( a ) beneficial neurologic effect . labeling subluxation as a functional articular lesion may not necessarily affect the qualities of the entity , if that label carries the same definition as subluxation . still , it may reduce confusion if consistent terminology were used to describe this unique chiropractic area of interest . nonetheless , commonality between different factions within the chiropractic profession may begin to merge perhaps on unifying expressions such as spinal adjustment and functional neurology or improving function of the patient s nervous system through spinal adjustment . to this end , in moving the argument forward , table 3 is a compilation of terms and concepts from across the profession that have at least some semblance to the subluxation model . a perceived problem with the subluxation model is that there are so many definitions for subluxation . for example , gonstead has its protocol on how to analyze for subluxation , activator methods has its own operational definition , and so on . however , there may be general agreement on the concept definition for vertebral subluxation , which essentially is a minor biomechanical dysfunction in the spine that results in a neurologic disturbance . the various operational definitions highlight the need to include them in papers on subluxation , particularly in research papers , so that other researchers who may wish to verify findings of such studies can do so given enough detail on the methods used . it simply does not suffice to say in a research paper that the spine was examined and adjusted . another issue with the subluxation model is the validity and reliability of the methods used to identify it . this challenge though is not unique to the subluxation model , as validity of methods is a challenge in non - subluxation models in chiropractic . for example , the back pain model and the subluxation model may both use motion palpation as a diagnostic method , yet palpation has its own set of challenges regarding its validity ( eg , some studies report low reliability ) . the remedy for such challenges is at the same time an opportunity for different factions of the profession to come together to focus on a common area to advance the profession . as president abraham lincoln said , a house divided against itself can not stand . an example from the history of chiropractic that unity on subluxation research is possible was the study conducted at the university of colorado in the 1970s by sharpless . one last example of a challenge with the subluxation model is whether subluxation has any adverse effect on health . because there may be only some evidence along these lines ( eg , the bakris et al study on upper cervical adjustment and hypertension ) , it would be disingenuous to insinuate that there is none at all . indeed , even some of the harshest critics of the subluxation model , to their credit , state that it is a legitimate , potentially testable , theoretical construct . settling on any particular identity for the chiropractic profession will likely be met with resistance by some , given the plethora of opinions among chiropractic professionals as to what the identity of the chiropractic profession should be . common ground between the different factions within the chiropractic profession might be found in a unifying expression such as spinal adjustment and functional neurology . a limitation to this commentary is that the colleges selected were mostly from one region , north america , the exception being the new zealand college of chiropractic . the international flavor of what chiropractic is can be viewed by the paper s inclusion of international associations ( eg , wfc and ifco ) . throughout palmer s 1910 textbook , he essentially stated that the main area of interest and therefore identity in chiropractic was analysis and adjustment of vertebral subluxation . among a sample of 5 major chiropractic organizations , 2 specifically mention that chiropractic s focus is subluxation ( acc ) or vertebral subluxation ( ifco ) . another organization s statement is similar in that it mentions spinal adjustment , nervous system function , and general health , without specifically using the term subluxation ( wfc ) ( table 1 ) . a search of the internet was performed in february 2016 on how chiropractic colleges represent themselves and chiropractic identity to the public . among the chiropractic colleges , 3 specifically mention subluxation in their description of chiropractic : life university , life chiropractic college west , and sherman college of chiropractic . other colleges , such as cleveland chiropractic college , new zealand college of chiropractic , and palmer college of chiropractic , describe subluxation with statements related to chiropractic focusing on the spine and its relationship to the nervous system . a number of other colleges mention that chiropractic pertains to general health and/or musculoskeletal conditions ( table 2 ) . survey research by gallup and palmer college indicates that the public generally considers that chiropractic pertains to neck and back pain . however , 31% of those surveyed indicated that they would like to use chiropractic care even if they were asymptomatic . this suggests that about a third of potential patients are open to a wellness approach , which is a component of the vertebral subluxation model ( eg , maintenance care ) . survey research by gliedt et al indicates that a majority ( 61.4% ) of chiropractic students think that the emphasis of chiropractic intervention is to eliminate vertebral subluxations / vertebral subluxation complexes . survey research by mcdonald et al indicated that 88.1% of practicing chiropractors in north america who responded to the survey answered yes to the question , should chiropractic retain the term vertebral subluxation complex ? currently , a consensus viewpoint regarding the identity of the chiropractic profession is lacking , evidenced by the various opinions among its associations and colleges . it is unclear how these varying viewpoints came to be , considering the stated intent and identity of chiropractic as laid down by the founder of the profession . it is possible that these differing views came about simply as a preference that was different from palmer s original intent . academic freedom allows for various viewpoints , even with respect to a profession s identity . however , that freedom may come with a price , in the way of unintended consequences ; the public may be confused as to what chiropractic is when faced with the variability of viewpoints . moreover , a profession s identity that does not make distinctions between its identity and the identities of other professions seems to invite further confusion . to this point , the wfc has recommended that the identity of the profession should be similar in all countries . i feel that this is a worthy recommendation ; however , the brand that wfc suggests may be too vague , as it has a relatively high degree of abstraction when it states that chiropractors are spinal health care experts in the health care system . the quasi - vagueness here might be considered to be at odds with wfc s call that chiropractic identity should be clear and concise . nonetheless , common ground between the wfc and those within the subluxation model are suggested in its qualifying statements for chiropractic care:ability to improve function in the neuromusculoskeletal system and overall health , wellbeing , and quality of life;specialized approach to examination , diagnosis , and treatment , based on best available research and clinical evidence with particular emphasis on the relationship between the spine and the nervous system;tradition of effectiveness and patient satisfaction;without use of drugs and surgery , enabling patients to avoid these where possible;expertly qualified providers of spinal adjustment , manipulation and other manual treatments , exercise instruction , and patient education;collaboration with other health professionals ; anda patient - centered and biopsychosocial approach , emphasizing the mind / body relationship in health , the self - healing powers of the individual , and individual responsibility for health and encouraging patient independence . ability to improve function in the neuromusculoskeletal system and overall health , wellbeing , and quality of life ; specialized approach to examination , diagnosis , and treatment , based on best available research and clinical evidence with particular emphasis on the relationship between the spine and the nervous system ; tradition of effectiveness and patient satisfaction ; without use of drugs and surgery , enabling patients to avoid these where possible ; expertly qualified providers of spinal adjustment , manipulation and other manual treatments , exercise instruction , and patient education ; collaboration with other health professionals ; and a patient - centered and biopsychosocial approach , emphasizing the mind / body relationship in health , the self - healing powers of the individual , and individual responsibility for health and encouraging patient independence . i suggest that the chiropractic profession s identity should be based on what makes it unique and what the founder of chiropractic envisioned it to be , which is adjustment of vertebral subluxations to improve neurologic function in the patient . although some in the profession have concerns with the subluxation model , there is interest in advancing the profession by addressing these issues.19 , 20 for others , the problem may be with the use of the term subluxation . for example , a former president of the national university of health sciences states that the purpose of chiropractic manual care is to correct a functional articular lesion to produce ( a ) beneficial neurologic effect . labeling subluxation as a functional articular lesion may not necessarily affect the qualities of the entity , if that label carries the same definition as subluxation . still , it may reduce confusion if consistent terminology were used to describe this unique chiropractic area of interest . nonetheless , commonality between different factions within the chiropractic profession may begin to merge perhaps on unifying expressions such as spinal adjustment and functional neurology or improving function of the patient s nervous system through spinal adjustment . to this end , in moving the argument forward , table 3 is a compilation of terms and concepts from across the profession that have at least some semblance to the subluxation model . a perceived problem with the subluxation model is that there are so many definitions for subluxation . for example , gonstead has its protocol on how to analyze for subluxation , activator methods has its own operational definition , and so on . however , there may be general agreement on the concept definition for vertebral subluxation , which essentially is a minor biomechanical dysfunction in the spine that results in a neurologic disturbance . the various operational definitions highlight the need to include them in papers on subluxation , particularly in research papers , so that other researchers who may wish to verify findings of such studies can do so given enough detail on the methods used . it simply does not suffice to say in a research paper that the spine was examined and adjusted . another issue with the subluxation model is the validity and reliability of the methods used to identify it . this challenge though is not unique to the subluxation model , as validity of methods is a challenge in non - subluxation models in chiropractic . for example , the back pain model and the subluxation model may both use motion palpation as a diagnostic method , yet palpation has its own set of challenges regarding its validity ( eg , some studies report low reliability ) . the remedy for such challenges is at the same time an opportunity for different factions of the profession to come together to focus on a common area to advance the profession . as president abraham lincoln said , an example from the history of chiropractic that unity on subluxation research is possible was the study conducted at the university of colorado in the 1970s by sharpless . one last example of a challenge with the subluxation model is whether subluxation has any adverse effect on health . because there may be only some evidence along these lines ( eg , the bakris et al study on upper cervical adjustment and hypertension ) , it would be disingenuous to insinuate that there is none at all . indeed , even some of the harshest critics of the subluxation model , to their credit , state that it is a legitimate , potentially testable , theoretical construct . settling on any particular identity for the chiropractic profession will likely be met with resistance by some , given the plethora of opinions among chiropractic professionals as to what the identity of the chiropractic profession should be . common ground between the different factions within the chiropractic profession might be found in a unifying expression such as spinal adjustment and functional neurology . a limitation to this commentary is that the colleges selected were mostly from one region , north america , the exception being the new zealand college of chiropractic . the international flavor of what chiropractic is can be viewed by the paper s inclusion of international associations ( eg , wfc and ifco ) . when a profession s identity is not clear with respect to its area of interest and mission , then the public may be less inclined to seek its services . identifying the chiropractic profession with a focus on vertebral subluxation would give the profession uniqueness not duplicated by other health care professions and therefore might legitimatize the existence of chiropractic as a health care profession . an identity having a focus on vertebral subluxation would also be consistent with the original intent of the founding of the chiropractic profession . supervision ( provided oversight , responsible for organization and implementation , writing of the manuscript ) : j.h . data collection / processing ( responsible for experiments , patient management , organization , or reporting data ) : j.h . analysis / interpretation ( responsible for statistical analysis , evaluation , and presentation of the results ) : j.h . critical review ( revised manuscript for intellectual content , this does not relate to spelling and grammar checking ) : j.h.practical applicationsthe various identity statements within the profession tend to result in confusion about the purpose of chiropractic care.the profession should self - identify as having a unique identity and purpose.to this end , i propose that the traditional identity of the chiropractic profession should be used , namely , the location , analysis , and adjustment of vertebral subluxation . the various identity statements within the profession tend to result in confusion about the purpose of chiropractic care.the profession should self - identify as having a unique identity and purpose.to this end , i propose that the traditional identity of the chiropractic profession should be used , namely , the location , analysis , and adjustment of vertebral subluxation . the various identity statements within the profession tend to result in confusion about the purpose of chiropractic care . the profession should self - identify as having a unique identity and purpose . to this end , i propose that the traditional identity of the chiropractic profession should be used , namely , the location , analysis , and adjustment of vertebral subluxation . the various identity statements within the profession tend to result in confusion about the purpose of chiropractic care.the profession should self - identify as having a unique identity and purpose.to this end , i propose that the traditional identity of the chiropractic profession should be used , namely , the location , analysis , and adjustment of vertebral subluxation . the various identity statements within the profession tend to result in confusion about the purpose of chiropractic care . the profession should self - identify as having a unique identity and purpose . to this end , i propose that the traditional identity of the chiropractic profession should be used , namely , the location , analysis , and adjustment of vertebral subluxation .

objectivethe purpose of this paper is to discuss various statements related to chiropractic identity from1 d. d. palmer2 ; selected chiropractic organizations , associations , and colleges ; and3 attitudes and perceptions of chiropractic from chiropractic students , practitioners , and patients.discussionfor comparison purposes , identity statements and perceptions from the various chiropractic associations and colleges , as well as from students and patients , were explored . identity statements for chiropractic were searched in various sources such as palmer s 1910 textbook , recent literature on viewpoints from chiropractic students and practitioners , and websites for chiropractic colleges and organizations . palmer taught that the chiropractor s focus was on vertebral subluxation . today , a number of chiropractic colleges and organizations continue to include the vertebral subluxation model in their instruction , with a majority of students and practitioners subscribing to the model . conversely , a number of other colleges and organizations portray chiropractic as being essentially about the treatment of back and neck pain , which is what patients associate with chiropractic . however , settling on any particular identity for the chiropractic profession will likely be met with resistance by some , given the plethora of opinions among chiropractic professionals as to what the identity of the chiropractic profession should be . common ground between the different factions within the chiropractic profession might be found in a unifying expression such as functional neurology.conclusionwhen a profession s identity is not clear with respect to its area of interest and mission , then the public may be less inclined to seek its services . identifying the chiropractic profession with a focus on vertebral subluxation would give the profession uniqueness not duplicated by other health care professions and , therefore , might legitimatize the existence of chiropractic as a health care profession . an identity having a focus on vertebral subluxation would also be consistent with the original intent of the founding of the chiropractic profession .

Introduction Discussion The Founder of Chiropractic Chiropractic Organizations Chiropractic Colleges The Public Chiropractic Students Chiropractors Lack of Consensus Challenges With the Model Limitations Conclusion Funding Sources and Conflicts of Interest Contributorship Information Practical Applications

two basic considerations are necessary to identify a profession : its central area of interest and its mission . others hold that the definition of a profession pertains to its area of knowledge and boundaries of that knowledge , otherwise known as degree of abstraction . as a practical matter , identity of a profession helps guide its practitioners on scope of practice issues and informs the public on what the profession has to offer . having an identity that satisfies these descriptions still may not be satisfactory if that identity is not unique , separate , and distinct from those of other professions . during the tumultuous days of harassment by organized medicine another important aspect in professional identity is obtaining trust from the public that the profession serves . this trust requires periodic renewal of a profession s social contract with the public . without such renewal , the public will wrest the contract from the profession s control and shift it to the default model of the market . when 2 professions have identities that are the same or overlap substantially ( eg , both have back pain as their area of focus ) , then 1 of the professions is duplicative and therefore unnecessary . thus , for the chiropractic profession , if its identity is based on neck and back pain , it is lost in the presence of much larger professions ( eg , medicine ) that also treat back and neck pain . the purpose of this paper is to discuss various statements related to chiropractic identity from d. d. palmer ; selected chiropractic organizations ( associations ) and colleges ; and attitudes and perceptions of chiropractic from chiropractic students , practitioners , and patients . the article concludes by encouraging the profession to base itself on its original intent , which is improving neurologic function by adjustment of vertebral subluxation . for the purpose of this commentary , identity of chiropractic is considered synonymous with definition or explanation of what chiropractic is . definitions of chiropractic , or statements that at least resembled a definition , were obtained from palmer s famous 1910 book , the science , art , and philosophy of chiropractic ; websites of national and international chiropractic organizations ; websites of chiropractic colleges listed in english by the association of chiropractic colleges ; and internet ( bing , pubmed , and google scholar ) searches for reported attitudes of chiropractic students , practitioners , and patients . the associations comprised the association of chiropractic colleges ( acc ) , american chiropractic association , international chiropractors association , international federation of chiropractors and organizations ( ifco ) , and world federation of chiropractic ( wfc ) . throughout palmer s 1910 textbook , he essentially stated that the main area of interest and therefore identity in chiropractic was analysis and adjustment of vertebral subluxation . among a sample of 5 major chiropractic organizations , 2 specifically mention that chiropractic s focus is subluxation ( acc ) or vertebral subluxation ( ifco ) . a search of the internet was performed in february 2016 on how chiropractic colleges represent themselves and chiropractic identity to the public . among the chiropractic colleges , 3 specifically mention subluxation in their description of chiropractic : life university , life chiropractic college west , and sherman college of chiropractic . other colleges , such as cleveland chiropractic college , new zealand college of chiropractic , and palmer college of chiropractic , describe subluxation with statements related to chiropractic focusing on the spine and its relationship to the nervous system . a number of other colleges mention that chiropractic pertains to general health and/or musculoskeletal conditions ( table 2 ) . survey research by gallup and palmer college indicates that the public generally considers that chiropractic pertains to neck and back pain . this suggests that about a third of potential patients are open to a wellness approach , which is a component of the vertebral subluxation model ( eg , maintenance care ) . survey research by gliedt et al indicates that a majority ( 61.4% ) of chiropractic students think that the emphasis of chiropractic intervention is to eliminate vertebral subluxations / vertebral subluxation complexes . survey research by mcdonald et al indicated that 88.1% of practicing chiropractors in north america who responded to the survey answered yes to the question , should chiropractic retain the term vertebral subluxation complex ? currently , a consensus viewpoint regarding the identity of the chiropractic profession is lacking , evidenced by the various opinions among its associations and colleges . it is unclear how these varying viewpoints came to be , considering the stated intent and identity of chiropractic as laid down by the founder of the profession . it is possible that these differing views came about simply as a preference that was different from palmer s original intent . academic freedom allows for various viewpoints , even with respect to a profession s identity . however , that freedom may come with a price , in the way of unintended consequences ; the public may be confused as to what chiropractic is when faced with the variability of viewpoints . moreover , a profession s identity that does not make distinctions between its identity and the identities of other professions seems to invite further confusion . to this point , the wfc has recommended that the identity of the profession should be similar in all countries . i feel that this is a worthy recommendation ; however , the brand that wfc suggests may be too vague , as it has a relatively high degree of abstraction when it states that chiropractors are spinal health care experts in the health care system . the quasi - vagueness here might be considered to be at odds with wfc s call that chiropractic identity should be clear and concise . nonetheless , common ground between the wfc and those within the subluxation model are suggested in its qualifying statements for chiropractic care:ability to improve function in the neuromusculoskeletal system and overall health , wellbeing , and quality of life;specialized approach to examination , diagnosis , and treatment , based on best available research and clinical evidence with particular emphasis on the relationship between the spine and the nervous system;tradition of effectiveness and patient satisfaction;without use of drugs and surgery , enabling patients to avoid these where possible;expertly qualified providers of spinal adjustment , manipulation and other manual treatments , exercise instruction , and patient education;collaboration with other health professionals ; anda patient - centered and biopsychosocial approach , emphasizing the mind / body relationship in health , the self - healing powers of the individual , and individual responsibility for health and encouraging patient independence . ability to improve function in the neuromusculoskeletal system and overall health , wellbeing , and quality of life ; specialized approach to examination , diagnosis , and treatment , based on best available research and clinical evidence with particular emphasis on the relationship between the spine and the nervous system ; tradition of effectiveness and patient satisfaction ; without use of drugs and surgery , enabling patients to avoid these where possible ; expertly qualified providers of spinal adjustment , manipulation and other manual treatments , exercise instruction , and patient education ; collaboration with other health professionals ; and a patient - centered and biopsychosocial approach , emphasizing the mind / body relationship in health , the self - healing powers of the individual , and individual responsibility for health and encouraging patient independence . i suggest that the chiropractic profession s identity should be based on what makes it unique and what the founder of chiropractic envisioned it to be , which is adjustment of vertebral subluxations to improve neurologic function in the patient . although some in the profession have concerns with the subluxation model , there is interest in advancing the profession by addressing these issues.19 , 20 for others , the problem may be with the use of the term subluxation . for example , a former president of the national university of health sciences states that the purpose of chiropractic manual care is to correct a functional articular lesion to produce ( a ) beneficial neurologic effect . still , it may reduce confusion if consistent terminology were used to describe this unique chiropractic area of interest . nonetheless , commonality between different factions within the chiropractic profession may begin to merge perhaps on unifying expressions such as spinal adjustment and functional neurology or improving function of the patient s nervous system through spinal adjustment . to this end , in moving the argument forward , table 3 is a compilation of terms and concepts from across the profession that have at least some semblance to the subluxation model . however , there may be general agreement on the concept definition for vertebral subluxation , which essentially is a minor biomechanical dysfunction in the spine that results in a neurologic disturbance . the various operational definitions highlight the need to include them in papers on subluxation , particularly in research papers , so that other researchers who may wish to verify findings of such studies can do so given enough detail on the methods used . another issue with the subluxation model is the validity and reliability of the methods used to identify it . this challenge though is not unique to the subluxation model , as validity of methods is a challenge in non - subluxation models in chiropractic . the remedy for such challenges is at the same time an opportunity for different factions of the profession to come together to focus on a common area to advance the profession . an example from the history of chiropractic that unity on subluxation research is possible was the study conducted at the university of colorado in the 1970s by sharpless . indeed , even some of the harshest critics of the subluxation model , to their credit , state that it is a legitimate , potentially testable , theoretical construct . settling on any particular identity for the chiropractic profession will likely be met with resistance by some , given the plethora of opinions among chiropractic professionals as to what the identity of the chiropractic profession should be . common ground between the different factions within the chiropractic profession might be found in a unifying expression such as spinal adjustment and functional neurology . a limitation to this commentary is that the colleges selected were mostly from one region , north america , the exception being the new zealand college of chiropractic . throughout palmer s 1910 textbook , he essentially stated that the main area of interest and therefore identity in chiropractic was analysis and adjustment of vertebral subluxation . among a sample of 5 major chiropractic organizations , 2 specifically mention that chiropractic s focus is subluxation ( acc ) or vertebral subluxation ( ifco ) . a search of the internet was performed in february 2016 on how chiropractic colleges represent themselves and chiropractic identity to the public . among the chiropractic colleges , 3 specifically mention subluxation in their description of chiropractic : life university , life chiropractic college west , and sherman college of chiropractic . other colleges , such as cleveland chiropractic college , new zealand college of chiropractic , and palmer college of chiropractic , describe subluxation with statements related to chiropractic focusing on the spine and its relationship to the nervous system . a number of other colleges mention that chiropractic pertains to general health and/or musculoskeletal conditions ( table 2 ) . this suggests that about a third of potential patients are open to a wellness approach , which is a component of the vertebral subluxation model ( eg , maintenance care ) . survey research by gliedt et al indicates that a majority ( 61.4% ) of chiropractic students think that the emphasis of chiropractic intervention is to eliminate vertebral subluxations / vertebral subluxation complexes . survey research by mcdonald et al indicated that 88.1% of practicing chiropractors in north america who responded to the survey answered yes to the question , should chiropractic retain the term vertebral subluxation complex ? currently , a consensus viewpoint regarding the identity of the chiropractic profession is lacking , evidenced by the various opinions among its associations and colleges . it is unclear how these varying viewpoints came to be , considering the stated intent and identity of chiropractic as laid down by the founder of the profession . it is possible that these differing views came about simply as a preference that was different from palmer s original intent . academic freedom allows for various viewpoints , even with respect to a profession s identity . however , that freedom may come with a price , in the way of unintended consequences ; the public may be confused as to what chiropractic is when faced with the variability of viewpoints . moreover , a profession s identity that does not make distinctions between its identity and the identities of other professions seems to invite further confusion . to this point , the wfc has recommended that the identity of the profession should be similar in all countries . i feel that this is a worthy recommendation ; however , the brand that wfc suggests may be too vague , as it has a relatively high degree of abstraction when it states that chiropractors are spinal health care experts in the health care system . the quasi - vagueness here might be considered to be at odds with wfc s call that chiropractic identity should be clear and concise . nonetheless , common ground between the wfc and those within the subluxation model are suggested in its qualifying statements for chiropractic care:ability to improve function in the neuromusculoskeletal system and overall health , wellbeing , and quality of life;specialized approach to examination , diagnosis , and treatment , based on best available research and clinical evidence with particular emphasis on the relationship between the spine and the nervous system;tradition of effectiveness and patient satisfaction;without use of drugs and surgery , enabling patients to avoid these where possible;expertly qualified providers of spinal adjustment , manipulation and other manual treatments , exercise instruction , and patient education;collaboration with other health professionals ; anda patient - centered and biopsychosocial approach , emphasizing the mind / body relationship in health , the self - healing powers of the individual , and individual responsibility for health and encouraging patient independence . ability to improve function in the neuromusculoskeletal system and overall health , wellbeing , and quality of life ; specialized approach to examination , diagnosis , and treatment , based on best available research and clinical evidence with particular emphasis on the relationship between the spine and the nervous system ; tradition of effectiveness and patient satisfaction ; without use of drugs and surgery , enabling patients to avoid these where possible ; expertly qualified providers of spinal adjustment , manipulation and other manual treatments , exercise instruction , and patient education ; collaboration with other health professionals ; and a patient - centered and biopsychosocial approach , emphasizing the mind / body relationship in health , the self - healing powers of the individual , and individual responsibility for health and encouraging patient independence . i suggest that the chiropractic profession s identity should be based on what makes it unique and what the founder of chiropractic envisioned it to be , which is adjustment of vertebral subluxations to improve neurologic function in the patient . although some in the profession have concerns with the subluxation model , there is interest in advancing the profession by addressing these issues.19 , 20 for others , the problem may be with the use of the term subluxation . for example , a former president of the national university of health sciences states that the purpose of chiropractic manual care is to correct a functional articular lesion to produce ( a ) beneficial neurologic effect . nonetheless , commonality between different factions within the chiropractic profession may begin to merge perhaps on unifying expressions such as spinal adjustment and functional neurology or improving function of the patient s nervous system through spinal adjustment . to this end , in moving the argument forward , table 3 is a compilation of terms and concepts from across the profession that have at least some semblance to the subluxation model . however , there may be general agreement on the concept definition for vertebral subluxation , which essentially is a minor biomechanical dysfunction in the spine that results in a neurologic disturbance . the various operational definitions highlight the need to include them in papers on subluxation , particularly in research papers , so that other researchers who may wish to verify findings of such studies can do so given enough detail on the methods used . it simply does not suffice to say in a research paper that the spine was examined and adjusted . another issue with the subluxation model is the validity and reliability of the methods used to identify it . this challenge though is not unique to the subluxation model , as validity of methods is a challenge in non - subluxation models in chiropractic . the remedy for such challenges is at the same time an opportunity for different factions of the profession to come together to focus on a common area to advance the profession . one last example of a challenge with the subluxation model is whether subluxation has any adverse effect on health . settling on any particular identity for the chiropractic profession will likely be met with resistance by some , given the plethora of opinions among chiropractic professionals as to what the identity of the chiropractic profession should be . common ground between the different factions within the chiropractic profession might be found in a unifying expression such as spinal adjustment and functional neurology . a limitation to this commentary is that the colleges selected were mostly from one region , north america , the exception being the new zealand college of chiropractic . when a profession s identity is not clear with respect to its area of interest and mission , then the public may be less inclined to seek its services . identifying the chiropractic profession with a focus on vertebral subluxation would give the profession uniqueness not duplicated by other health care professions and therefore might legitimatize the existence of chiropractic as a health care profession . an identity having a focus on vertebral subluxation would also be consistent with the original intent of the founding of the chiropractic profession . critical review ( revised manuscript for intellectual content , this does not relate to spelling and grammar checking ) : j.h.practical applicationsthe various identity statements within the profession tend to result in confusion about the purpose of chiropractic care.the profession should self - identify as having a unique identity and purpose.to this end , i propose that the traditional identity of the chiropractic profession should be used , namely , the location , analysis , and adjustment of vertebral subluxation . the various identity statements within the profession tend to result in confusion about the purpose of chiropractic care.the profession should self - identify as having a unique identity and purpose.to this end , i propose that the traditional identity of the chiropractic profession should be used , namely , the location , analysis , and adjustment of vertebral subluxation . the various identity statements within the profession tend to result in confusion about the purpose of chiropractic care . the profession should self - identify as having a unique identity and purpose . to this end , i propose that the traditional identity of the chiropractic profession should be used , namely , the location , analysis , and adjustment of vertebral subluxation . the various identity statements within the profession tend to result in confusion about the purpose of chiropractic care.the profession should self - identify as having a unique identity and purpose.to this end , i propose that the traditional identity of the chiropractic profession should be used , namely , the location , analysis , and adjustment of vertebral subluxation . the various identity statements within the profession tend to result in confusion about the purpose of chiropractic care . the profession should self - identify as having a unique identity and purpose . to this end , i propose that the traditional identity of the chiropractic profession should be used , namely , the location , analysis , and adjustment of vertebral subluxation .

others hold that the definition of a profession pertains to its area of knowledge and boundaries of that knowledge , otherwise known as degree of abstraction . as a practical matter , identity of a profession helps guide its practitioners on scope of practice issues and informs the public on what the profession has to offer . moreover , the formation of a professional identity goes beyond the acquisition of certain skill sets , as it is formed mainly in social and relationship contexts . having an identity that satisfies these descriptions still may not be satisfactory if that identity is not unique , separate , and distinct from those of other professions . one obstacle to achieving a clear and unified chiropractic identity has been that of oppression , as some in the nursing profession have observed . in the chiropractic context medical regulation was used against chiropractors , and many were arrested for practicing medicine without a license . the actions of the brave chiropractic pioneers , who went to jail for chiropractic to protest the charge ( and who were practicing chiropractic , not medicine ) , helped bring about licensure in the united states . during the tumultuous days of harassment by organized medicine another important aspect in professional identity is obtaining trust from the public that the profession serves . without such renewal , the public will wrest the contract from the profession s control and shift it to the default model of the market . when 2 professions have identities that are the same or overlap substantially ( eg , both have back pain as their area of focus ) , then 1 of the professions is duplicative and therefore unnecessary . when chiropractors identify themselves as back pain specialists , or even spine specialists , they compete not only with medical practitioners , but also with osteopaths , physical therapists , massage therapists , and medicine cabinets in the homes of millions of potential patients . thus , for the chiropractic profession , if its identity is based on neck and back pain , it is lost in the presence of much larger professions ( eg , medicine ) that also treat back and neck pain . moreover , when the osteopathic profession merged into orthodox medicine in the late 1950s , it ceased to offer a unique service and was essentially swallowed up by the medical profession . the purpose of this paper is to discuss various statements related to chiropractic identity from d. d. palmer ; selected chiropractic organizations ( associations ) and colleges ; and attitudes and perceptions of chiropractic from chiropractic students , practitioners , and patients . the article concludes by encouraging the profession to base itself on its original intent , which is improving neurologic function by adjustment of vertebral subluxation . for the purpose of this commentary , identity of chiropractic is considered synonymous with definition or explanation of what chiropractic is . definitions of chiropractic , or statements that at least resembled a definition , were obtained from palmer s famous 1910 book , the science , art , and philosophy of chiropractic ; websites of national and international chiropractic organizations ; websites of chiropractic colleges listed in english by the association of chiropractic colleges ; and internet ( bing , pubmed , and google scholar ) searches for reported attitudes of chiropractic students , practitioners , and patients . the associations comprised the association of chiropractic colleges ( acc ) , american chiropractic association , international chiropractors association , international federation of chiropractors and organizations ( ifco ) , and world federation of chiropractic ( wfc ) . throughout palmer s 1910 textbook , he essentially stated that the main area of interest and therefore identity in chiropractic was analysis and adjustment of vertebral subluxation . among a sample of 5 major chiropractic organizations , 2 specifically mention that chiropractic s focus is subluxation ( acc ) or vertebral subluxation ( ifco ) . another organization s statement is similar in that it mentions spinal adjustment , nervous system function , and general health , without specifically using the term subluxation ( wfc ) ( table 1 ) . a search of the internet was performed in february 2016 on how chiropractic colleges represent themselves and chiropractic identity to the public . among the chiropractic colleges , 3 specifically mention subluxation in their description of chiropractic : life university , life chiropractic college west , and sherman college of chiropractic . other colleges , such as cleveland chiropractic college , new zealand college of chiropractic , and palmer college of chiropractic , describe subluxation with statements related to chiropractic focusing on the spine and its relationship to the nervous system . a number of other colleges mention that chiropractic pertains to general health and/or musculoskeletal conditions ( table 2 ) . this suggests that about a third of potential patients are open to a wellness approach , which is a component of the vertebral subluxation model ( eg , maintenance care ) . survey research by gliedt et al indicates that a majority ( 61.4% ) of chiropractic students think that the emphasis of chiropractic intervention is to eliminate vertebral subluxations / vertebral subluxation complexes . survey research by mcdonald et al indicated that 88.1% of practicing chiropractors in north america who responded to the survey answered yes to the question , should chiropractic retain the term vertebral subluxation complex ? currently , a consensus viewpoint regarding the identity of the chiropractic profession is lacking , evidenced by the various opinions among its associations and colleges . it is unclear how these varying viewpoints came to be , considering the stated intent and identity of chiropractic as laid down by the founder of the profession . however , that freedom may come with a price , in the way of unintended consequences ; the public may be confused as to what chiropractic is when faced with the variability of viewpoints . to this point , the wfc has recommended that the identity of the profession should be similar in all countries . i feel that this is a worthy recommendation ; however , the brand that wfc suggests may be too vague , as it has a relatively high degree of abstraction when it states that chiropractors are spinal health care experts in the health care system . the quasi - vagueness here might be considered to be at odds with wfc s call that chiropractic identity should be clear and concise . nonetheless , common ground between the wfc and those within the subluxation model are suggested in its qualifying statements for chiropractic care:ability to improve function in the neuromusculoskeletal system and overall health , wellbeing , and quality of life;specialized approach to examination , diagnosis , and treatment , based on best available research and clinical evidence with particular emphasis on the relationship between the spine and the nervous system;tradition of effectiveness and patient satisfaction;without use of drugs and surgery , enabling patients to avoid these where possible;expertly qualified providers of spinal adjustment , manipulation and other manual treatments , exercise instruction , and patient education;collaboration with other health professionals ; anda patient - centered and biopsychosocial approach , emphasizing the mind / body relationship in health , the self - healing powers of the individual , and individual responsibility for health and encouraging patient independence . ability to improve function in the neuromusculoskeletal system and overall health , wellbeing , and quality of life ; specialized approach to examination , diagnosis , and treatment , based on best available research and clinical evidence with particular emphasis on the relationship between the spine and the nervous system ; tradition of effectiveness and patient satisfaction ; without use of drugs and surgery , enabling patients to avoid these where possible ; expertly qualified providers of spinal adjustment , manipulation and other manual treatments , exercise instruction , and patient education ; collaboration with other health professionals ; and a patient - centered and biopsychosocial approach , emphasizing the mind / body relationship in health , the self - healing powers of the individual , and individual responsibility for health and encouraging patient independence . i suggest that the chiropractic profession s identity should be based on what makes it unique and what the founder of chiropractic envisioned it to be , which is adjustment of vertebral subluxations to improve neurologic function in the patient . although some in the profession have concerns with the subluxation model , there is interest in advancing the profession by addressing these issues.19 , 20 for others , the problem may be with the use of the term subluxation . for example , a former president of the national university of health sciences states that the purpose of chiropractic manual care is to correct a functional articular lesion to produce ( a ) beneficial neurologic effect . labeling subluxation as a functional articular lesion may not necessarily affect the qualities of the entity , if that label carries the same definition as subluxation . nonetheless , commonality between different factions within the chiropractic profession may begin to merge perhaps on unifying expressions such as spinal adjustment and functional neurology or improving function of the patient s nervous system through spinal adjustment . to this end , in moving the argument forward , table 3 is a compilation of terms and concepts from across the profession that have at least some semblance to the subluxation model . however , there may be general agreement on the concept definition for vertebral subluxation , which essentially is a minor biomechanical dysfunction in the spine that results in a neurologic disturbance . this challenge though is not unique to the subluxation model , as validity of methods is a challenge in non - subluxation models in chiropractic . for example , the back pain model and the subluxation model may both use motion palpation as a diagnostic method , yet palpation has its own set of challenges regarding its validity ( eg , some studies report low reliability ) . the remedy for such challenges is at the same time an opportunity for different factions of the profession to come together to focus on a common area to advance the profession . an example from the history of chiropractic that unity on subluxation research is possible was the study conducted at the university of colorado in the 1970s by sharpless . indeed , even some of the harshest critics of the subluxation model , to their credit , state that it is a legitimate , potentially testable , theoretical construct . settling on any particular identity for the chiropractic profession will likely be met with resistance by some , given the plethora of opinions among chiropractic professionals as to what the identity of the chiropractic profession should be . common ground between the different factions within the chiropractic profession might be found in a unifying expression such as spinal adjustment and functional neurology . a limitation to this commentary is that the colleges selected were mostly from one region , north america , the exception being the new zealand college of chiropractic . the international flavor of what chiropractic is can be viewed by the paper s inclusion of international associations ( eg , wfc and ifco ) . throughout palmer s 1910 textbook , he essentially stated that the main area of interest and therefore identity in chiropractic was analysis and adjustment of vertebral subluxation . among a sample of 5 major chiropractic organizations , 2 specifically mention that chiropractic s focus is subluxation ( acc ) or vertebral subluxation ( ifco ) . another organization s statement is similar in that it mentions spinal adjustment , nervous system function , and general health , without specifically using the term subluxation ( wfc ) ( table 1 ) . a search of the internet was performed in february 2016 on how chiropractic colleges represent themselves and chiropractic identity to the public . among the chiropractic colleges , 3 specifically mention subluxation in their description of chiropractic : life university , life chiropractic college west , and sherman college of chiropractic . other colleges , such as cleveland chiropractic college , new zealand college of chiropractic , and palmer college of chiropractic , describe subluxation with statements related to chiropractic focusing on the spine and its relationship to the nervous system . a number of other colleges mention that chiropractic pertains to general health and/or musculoskeletal conditions ( table 2 ) . this suggests that about a third of potential patients are open to a wellness approach , which is a component of the vertebral subluxation model ( eg , maintenance care ) . survey research by gliedt et al indicates that a majority ( 61.4% ) of chiropractic students think that the emphasis of chiropractic intervention is to eliminate vertebral subluxations / vertebral subluxation complexes . survey research by mcdonald et al indicated that 88.1% of practicing chiropractors in north america who responded to the survey answered yes to the question , should chiropractic retain the term vertebral subluxation complex ? currently , a consensus viewpoint regarding the identity of the chiropractic profession is lacking , evidenced by the various opinions among its associations and colleges . it is unclear how these varying viewpoints came to be , considering the stated intent and identity of chiropractic as laid down by the founder of the profession . however , that freedom may come with a price , in the way of unintended consequences ; the public may be confused as to what chiropractic is when faced with the variability of viewpoints . i feel that this is a worthy recommendation ; however , the brand that wfc suggests may be too vague , as it has a relatively high degree of abstraction when it states that chiropractors are spinal health care experts in the health care system . nonetheless , common ground between the wfc and those within the subluxation model are suggested in its qualifying statements for chiropractic care:ability to improve function in the neuromusculoskeletal system and overall health , wellbeing , and quality of life;specialized approach to examination , diagnosis , and treatment , based on best available research and clinical evidence with particular emphasis on the relationship between the spine and the nervous system;tradition of effectiveness and patient satisfaction;without use of drugs and surgery , enabling patients to avoid these where possible;expertly qualified providers of spinal adjustment , manipulation and other manual treatments , exercise instruction , and patient education;collaboration with other health professionals ; anda patient - centered and biopsychosocial approach , emphasizing the mind / body relationship in health , the self - healing powers of the individual , and individual responsibility for health and encouraging patient independence . ability to improve function in the neuromusculoskeletal system and overall health , wellbeing , and quality of life ; specialized approach to examination , diagnosis , and treatment , based on best available research and clinical evidence with particular emphasis on the relationship between the spine and the nervous system ; tradition of effectiveness and patient satisfaction ; without use of drugs and surgery , enabling patients to avoid these where possible ; expertly qualified providers of spinal adjustment , manipulation and other manual treatments , exercise instruction , and patient education ; collaboration with other health professionals ; and a patient - centered and biopsychosocial approach , emphasizing the mind / body relationship in health , the self - healing powers of the individual , and individual responsibility for health and encouraging patient independence . i suggest that the chiropractic profession s identity should be based on what makes it unique and what the founder of chiropractic envisioned it to be , which is adjustment of vertebral subluxations to improve neurologic function in the patient . although some in the profession have concerns with the subluxation model , there is interest in advancing the profession by addressing these issues.19 , 20 for others , the problem may be with the use of the term subluxation . for example , a former president of the national university of health sciences states that the purpose of chiropractic manual care is to correct a functional articular lesion to produce ( a ) beneficial neurologic effect . labeling subluxation as a functional articular lesion may not necessarily affect the qualities of the entity , if that label carries the same definition as subluxation . nonetheless , commonality between different factions within the chiropractic profession may begin to merge perhaps on unifying expressions such as spinal adjustment and functional neurology or improving function of the patient s nervous system through spinal adjustment . to this end , in moving the argument forward , table 3 is a compilation of terms and concepts from across the profession that have at least some semblance to the subluxation model . however , there may be general agreement on the concept definition for vertebral subluxation , which essentially is a minor biomechanical dysfunction in the spine that results in a neurologic disturbance . this challenge though is not unique to the subluxation model , as validity of methods is a challenge in non - subluxation models in chiropractic . for example , the back pain model and the subluxation model may both use motion palpation as a diagnostic method , yet palpation has its own set of challenges regarding its validity ( eg , some studies report low reliability ) . the remedy for such challenges is at the same time an opportunity for different factions of the profession to come together to focus on a common area to advance the profession . indeed , even some of the harshest critics of the subluxation model , to their credit , state that it is a legitimate , potentially testable , theoretical construct . settling on any particular identity for the chiropractic profession will likely be met with resistance by some , given the plethora of opinions among chiropractic professionals as to what the identity of the chiropractic profession should be . common ground between the different factions within the chiropractic profession might be found in a unifying expression such as spinal adjustment and functional neurology . the international flavor of what chiropractic is can be viewed by the paper s inclusion of international associations ( eg , wfc and ifco ) . identifying the chiropractic profession with a focus on vertebral subluxation would give the profession uniqueness not duplicated by other health care professions and therefore might legitimatize the existence of chiropractic as a health care profession . an identity having a focus on vertebral subluxation would also be consistent with the original intent of the founding of the chiropractic profession . critical review ( revised manuscript for intellectual content , this does not relate to spelling and grammar checking ) : j.h.practical applicationsthe various identity statements within the profession tend to result in confusion about the purpose of chiropractic care.the profession should self - identify as having a unique identity and purpose.to this end , i propose that the traditional identity of the chiropractic profession should be used , namely , the location , analysis , and adjustment of vertebral subluxation . the various identity statements within the profession tend to result in confusion about the purpose of chiropractic care.the profession should self - identify as having a unique identity and purpose.to this end , i propose that the traditional identity of the chiropractic profession should be used , namely , the location , analysis , and adjustment of vertebral subluxation . the various identity statements within the profession tend to result in confusion about the purpose of chiropractic care . to this end , i propose that the traditional identity of the chiropractic profession should be used , namely , the location , analysis , and adjustment of vertebral subluxation . the various identity statements within the profession tend to result in confusion about the purpose of chiropractic care.the profession should self - identify as having a unique identity and purpose.to this end , i propose that the traditional identity of the chiropractic profession should be used , namely , the location , analysis , and adjustment of vertebral subluxation . the various identity statements within the profession tend to result in confusion about the purpose of chiropractic care . to this end , i propose that the traditional identity of the chiropractic profession should be used , namely , the location , analysis , and adjustment of vertebral subluxation .

human immunodeficiency virus prevalence in the world has levelled - off in most regions in the world . women formed about 50% of the adult population ( 15 years or older ) infected with hiv / aids in the world . in sub - saharan africa , women living with hiv make up 59% of the adult population living with the infection . sub saharan africa accounts for about 70% of new hiv infections . some might be able to resume normal sexual activity others might not . in ghana , the estimated hiv prevalence is 1.5 with about 226,000 people living with hiv / aids . this is a prong of preventing mother to child transmission ( pmtct ) . most hiv infections in children are transmitted through mother - to - child during pregnancy , labor and breastfeeding . condom use among women living with hiv is also important to prevent the transmission of hiv / sti to the partner . even for those who desire to have a child , , the current contraceptive use of any method among married women ( 15- 49 years ) in the general population is 23.5% and modern contraceptive use is 23.0% . condom use among married women aged 15 49 years is 2.4% in the general population in ghana . the sexual behaviour and contraceptive use among women living with hiv are important in reducing the transmission of hiv . it was projected that about 10,300 mothers need prevention of mother - to - child transmission services and there would be 1,100 new hiv infections in children in 2012 in ghana . condoms have been the main contraceptive that has been promoted for people living with hiv / aids because of its dual purpose of preventing the transmission of hiv / sti and preventing pregnancies . it is therefore important to study the contraceptive use and the factors associated with contraceptive uptake in women living with hiv in order to inform programs designed to reduce the transmission of hiv and prevent unintended pregnancies among this sub - population . this was hospital - based cross - sectional descriptive study conducted from the 10th of july 2012 to the 15th of august 2012 . the study was conducted in the kumasi metropolitan area which has a total population of 2,035,064 ; females constitute 52.2% of the population . for health purposes , the metropolis is divided into five sub - metropolitan areas : bantama , asokwa , manhyia north , manhyia south and subin . two public health institutions in the metropolis with hiv / aids clinics , namely the kumasi south hospital in the asokwa sub - metropolitan health area and the suntreso government hospital in the bantama sub - metropolitan health area were used for this study . the hiv / aids clinics at the kumasi south hospital and the suntreso government hospital are the largest hiv / aids clinics of the ghana health service facilities in the metropolis and they have about 4000 and 2000 clients respectively . the hiv / aids clinics offer counselling and testing , care and treatment , and provision of antiretroviral drugs . they also serve as referral centres for other clinics that do not provide antiretroviral drugs in the metropolis . women living with hiv / aids within the reproductive age 18 to 49 years attending clinics at the above mentioned facilities were recruited for the study . the estimated sample size for the study was 300 . to be eligible for the study , newly diagnosed hiv / aids clients were excluded in the study since they are usually emotionally unstable and not sexually active or had lost interest in sex . individuals in the who clinical stage iv and the very sick were also excluded three hundred women living with hiv / aids were approached to be interviewed . two hundred clients were sampled from the kumasi south hospital while one hundred clients were selected from the suntreso government hospital . we aimed to recruit all eligible hiv / aids clients presenting at the clinic within the study period . if the client agreed to participate in the study , a consent form was signed and interviewed conducted . in all two hundred and ninety five women living with hiv agreed and questionnaires were administered to them . the questionnaire covered socio - demographic characteristics , contraceptive use , haart use , reproductive history , partner s characteristics and desire for children , knowledge of pmtct and contraceptive methods discussed at the hiv / aids clinic . hospital records were reviewed to collect information on who clinical stage and date of diagnosis of hiv of the study participant . questions on contraceptive knowledge and use were adopted from the 2008 ghana demographic health survey . interviews were conducted in english or translated into twi ( local dialect ) depending on preference of the participant , and it lasted between 10 to 15 minutes . analysis was performed on 183 study participants who were sexually active ; frequencies , percentages and odds ratio were calculated . multivariate logistic regression model was fitted to simultaneously adjust for the effect of other covariates . statistical significance level was set at an alpha value of 0.05 with 95% confidence interval . for inclusion of variables into the model a significance level of 0.25 was considered using the likelihood ratio test . ethical clearance was obtained from the committee on human research , publications and ethics , school of medical sciences , kwame nkrumah university of science and technology , kumasi , ghana . this was hospital - based cross - sectional descriptive study conducted from the 10th of july 2012 to the 15th of august 2012 . the study was conducted in the kumasi metropolitan area which has a total population of 2,035,064 ; females constitute 52.2% of the population . for health purposes , the metropolis is divided into five sub - metropolitan areas : bantama , asokwa , manhyia north , manhyia south and subin . two public health institutions in the metropolis with hiv / aids clinics , namely the kumasi south hospital in the asokwa sub - metropolitan health area and the suntreso government hospital in the bantama sub - metropolitan health area were used for this study . the hiv / aids clinics at the kumasi south hospital and the suntreso government hospital are the largest hiv / aids clinics of the ghana health service facilities in the metropolis and they have about 4000 and 2000 clients respectively . the hiv / aids clinics offer counselling and testing , care and treatment , and provision of antiretroviral drugs . they also serve as referral centres for other clinics that do not provide antiretroviral drugs in the metropolis . women living with hiv / aids within the reproductive age 18 to 49 years attending clinics at the above mentioned facilities were recruited for the study . the estimated sample size for the study was 300 . to be eligible for the study , newly diagnosed hiv / aids clients were excluded in the study since they are usually emotionally unstable and not sexually active or had lost interest in sex . individuals in the who clinical stage iv and the very sick were also excluded three hundred women living with hiv / aids were approached to be interviewed . two hundred clients were sampled from the kumasi south hospital while one hundred clients were selected from the suntreso government hospital . we aimed to recruit all eligible hiv / aids clients presenting at the clinic within the study period . if the client agreed to participate in the study , a consent form was signed and interviewed conducted . in all two hundred and ninety five women living with hiv agreed and questionnaires were administered to them . the questionnaire covered socio - demographic characteristics , contraceptive use , haart use , reproductive history , partner s characteristics and desire for children , knowledge of pmtct and contraceptive methods discussed at the hiv / aids clinic . hospital records were reviewed to collect information on who clinical stage and date of diagnosis of hiv of the study participant . questions on contraceptive knowledge and use were adopted from the 2008 ghana demographic health survey . interviews were conducted in english or translated into twi ( local dialect ) depending on preference of the participant , and it lasted between 10 to 15 minutes . analysis was performed on 183 study participants who were sexually active ; frequencies , percentages and odds ratio were calculated . multivariate logistic regression model was fitted to simultaneously adjust for the effect of other covariates . statistical significance level was set at an alpha value of 0.05 with 95% confidence interval . for inclusion of variables into the model a significance level of 0.25 was considered using the likelihood ratio test . ethical clearance was obtained from the committee on human research , publications and ethics , school of medical sciences , kwame nkrumah university of science and technology , kumasi , ghana . this was hospital - based cross - sectional descriptive study conducted from the 10th of july 2012 to the 15th of august 2012 . the study was conducted in the kumasi metropolitan area which has a total population of 2,035,064 ; females constitute 52.2% of the population . for health purposes , the metropolis is divided into five sub - metropolitan areas : bantama , asokwa , manhyia north , manhyia south and subin . two public health institutions in the metropolis with hiv / aids clinics , namely the kumasi south hospital in the asokwa sub - metropolitan health area and the suntreso government hospital in the bantama sub - metropolitan health area were used for this study . the hiv / aids clinics at the kumasi south hospital and the suntreso government hospital are the largest hiv / aids clinics of the ghana health service facilities in the metropolis and they have about 4000 and 2000 clients respectively . the hiv / aids clinics offer counselling and testing , care and treatment , and provision of antiretroviral drugs . they also serve as referral centres for other clinics that do not provide antiretroviral drugs in the metropolis . women living with hiv / aids within the reproductive age 18 to 49 years attending clinics at the above mentioned facilities were recruited for the study . the estimated sample size for the study was 300 . to be eligible for the study newly diagnosed hiv / aids clients were excluded in the study since they are usually emotionally unstable and not sexually active or had lost interest in sex . individuals in the who clinical stage iv and the very sick were also excluded three hundred women living with hiv / aids were approached to be interviewed . two hundred clients were sampled from the kumasi south hospital while one hundred clients were selected from the suntreso government hospital . we aimed to recruit all eligible hiv / aids clients presenting at the clinic within the study period . if the client agreed to participate in the study , a consent form was signed and interviewed conducted . in all two hundred and ninety five women living with hiv agreed and questionnaires were administered to them . the questionnaire covered socio - demographic characteristics , contraceptive use , haart use , reproductive history , partner s characteristics and desire for children , knowledge of pmtct and contraceptive methods discussed at the hiv / aids clinic . hospital records were reviewed to collect information on who clinical stage and date of diagnosis of hiv of the study participant . questions on contraceptive knowledge and use were adopted from the 2008 ghana demographic health survey . interviews were conducted in english or translated into twi ( local dialect ) depending on preference of the participant , and it lasted between 10 to 15 minutes . data analysis was done using stata version eleven ( stata corp . , college station , texas : statacorp lp , usa ) . analysis was performed on 183 study participants who were sexually active ; frequencies , percentages and odds ratio were calculated . multivariate logistic regression model was fitted to simultaneously adjust for the effect of other covariates . statistical significance level was set at an alpha value of 0.05 with 95% confidence interval . for inclusion of variables into the model a significance level of 0.25 was considered using the likelihood ratio test . ethical clearance was obtained from the committee on human research , publications and ethics , school of medical sciences , kwame nkrumah university of science and technology , kumasi , ghana . the mean age of the respondents was 34.5 years with standard deviation of 5.2 . about 79% of the respondents were married and of those who were using a contraceptive 82.2% were married . twenty percent of the respondents were unemployed whilst majority ( 70.5% ) were self employed . the average monthly income of most of the respondents who were employed was below us$ 53 ( ghcedi ( ) 1.90 is equivalent to us$ 1.00 ) ( see table 1 ) . socio - demographic and health characteristics of women living with hiv other christian= all christian organisations ( methodist , presbyterian , pentecost , charismatic ) except catholic , basic education = primary high education= middle / junior high school , secondary / senior high school and tertiary , among those who use contraceptive ( n=125 ) and those who do not use contraceptive ( n=21 ) most of the respondents ( 80.3% ) were menstruating regularly and 94% had ever had children . majority of the women and their partners desire to have children and about 85% of the respondents had disclosed their hiv status to their partners . about one third of the respondents did not know the hiv status of their partners . among the women currently using contraceptive 61.2% desire to have children in the future and 59.2% of their partners also desire to have children ( table 2 ) . reproductive characteristics and intentions of women living with hiv by contraceptive use in the univariate analysis , however , women who were cohabitating were less likely to use contraceptives ( unadjusted odds ratio [ or ] = 0.32 ; 95% ci = 0.12 - 0.88 , p = 0.03 ) compared with those who were married . participants who did not have children were significantly less likely to use a contraceptive ( unadjusted 0r = 0.17 ; 95% ci = 0.05 - 0.60 , p = 0.02 ) compared with women with children . the hiv status of the partner was associated with contraceptive use ; partners whose hiv status were unknown compared with those who were hiv positive were less likely to use a contraceptive ( unadjusted or = 0.27 ; 95% ci = 0.08 - 0.86 , p = 0.03 ) . multivariate analysis showed that , disclosure of hiv status to partner was the only variable that significantly predicted contraceptive use , suggesting that women who have not disclosed their status to their partners were less likely to use contraceptive ( aor = 0.25 ; 95% ci = 0.07 - 0.87 , p = 0.03 ) ( table 3 ) . univariate and adjusted analysis of factors associated with contraceptive use among women living with hiv eighty - five percent of the respondents were using a modern contraceptive method . the distribution of the various contraceptive methods are male condom(77.0% ) , injectables(5.5% ) , female sterilisation(1.1% ) , female condom(0.5% ) , implants(0.5 ) and other non modern methods which is the rhythm(1.1% ) . most of the respondents ( 80.3% ) were menstruating regularly and 94% had ever had children . majority of the women and their partners desire to have children and about 85% of the respondents had disclosed their hiv status to their partners . about one third of the respondents did not know the hiv status of their partners . among the women currently using contraceptive 61.2% desire to have children in the future and 59.2% of their partners also desire to have children ( table 2 ) . reproductive characteristics and intentions of women living with hiv by contraceptive use in the univariate analysis , age group and educational status were not associated with the use of contraceptives . however , women who were cohabitating were less likely to use contraceptives ( unadjusted odds ratio [ or ] = 0.32 ; 95% ci = 0.12 - 0.88 , p = 0.03 ) compared with those who were married . participants who did not have children were significantly less likely to use a contraceptive ( unadjusted 0r = 0.17 ; 95% ci = 0.05 - 0.60 , p = 0.02 ) compared with women with children . the hiv status of the partner was associated with contraceptive use ; partners whose hiv status were unknown compared with those who were hiv positive were less likely to use a contraceptive ( unadjusted or = 0.27 ; 95% ci = 0.08 - 0.86 , p = 0.03 ) . multivariate analysis showed that , disclosure of hiv status to partner was the only variable that significantly predicted contraceptive use , suggesting that women who have not disclosed their status to their partners were less likely to use contraceptive ( aor = 0.25 ; 95% ci = 0.07 - 0.87 , p = 0.03 ) ( table 3 ) . the distribution of the various contraceptive methods are male condom(77.0% ) , injectables(5.5% ) , female sterilisation(1.1% ) , female condom(0.5% ) , implants(0.5 ) and other non modern methods which is the rhythm(1.1% ) . this cross - sectional study serves as a pilot study on the contraceptive characteristics of women living with hiv in the kumasi metropolis , ghana . the results showed that disclosure of hiv sero - status to one s partner was a significant predictor of contraceptive use ; women who had not disclosed their status to their partners were significantly less likely to use contraceptive . similarly , a study in zambia also reported disclosure of hiv status as a predictor of contraceptive use . in uganda disclosure of hiv status was a predictor of modern contraceptive use in women living with hiv . disclosure of one s hiv status to partner would ensure that both parties would understand the importance of using the condom and other modern contraceptives to prevent unintended pregnancies . it would be easier for couples who have completed childbearing to decide to go for a permanent contraceptive method as well as use the condom . women living with hiv could therefore be counselled and encouraged to disclose their hiv status to their partners since it may improve their contraceptive use to prevent unintended pregnancies and the transmission of hiv / sti . disclosure of hiv status to partner is a dilemma because of the stigma and discrimination[9 , 10 ] associated with the infection and more so if the woman is the first to know of her status it sometimes presupposes that she is the one who got the infection first . women in many places in africa are expected to be modest and chaste , and be with only one partner . most hiv infection is transmitted through heterosexual intercourse in africa and if a woman is tested positive it presupposes that she is promiscuous that is why she has the infection . interventions that promote hiv status disclosure and issues relating to non - disclosure could be assessed[11 , 12 ] since it may improve uptake of contraceptives . contraceptive use among women living with hiv is generally higher than the general population[8 , 13 ] . this is mainly because of the high use of the condom which has a dual purpose of preventing the transmission of hiv and preventing unintended pregnancies . this compares with a study done in soweto , south africa were 84% women living with hiv were currently using at least one contraceptive method . a cross - sectional study in kenya reported 44.2% of respondents currently using a contraceptive method . a study in uganda , even reported as low as 25.2% of women currently using a contraceptive method and this could be due to the fact that the study was conducted in a post conflict era . another study in uganda conducted at twelve hiv clinics reported current contraceptive use of 61.8% . in lusaka , zambia , 59.2% of women living with hiv reported current use of a modern contraceptive and this is due to the fact that more than half of the respondents had not disclosed their hiv status to their partners . the differences in the level of contraceptive use can be explained by the differences in the characteristics of the populations sampled . in the current study , more than three - out - of - four women were using the male condom . this is mainly because of its dual purpose in preventing the transmission of sti / hiv and pregnancy , and this is the main contraceptive promoted at hiv / aids clinics in kumasi . similarly in other african countries the commonest contraceptive used by women living with hiv is the male condom[7 , 8 , 13 ] . the female condom which has the advantage of being female controlled and can be used if the man is reluctant to use the male condom was however very low ( 0.5% ) . similarly in the general population in ghana female condom use is very low . it can be argued that the female condom has not been well promoted ; it was recently re - launched by the ghana health service in october 2012 with the aim that it will help with its integration into sector wide policies as well as within hiv / aids programming . similarly in uganda , female condom use was low ( 0.8% ) . female condom use in kenya was higher ( 10.5% ) unlike that in kumasi , ghana but this is still low when compared with male condom use . further research into the low use of female condom among women living with hiv will help clarify the barriers to its use and probably improve its uptake . dual contraceptive method use which is the male condom and any other method was low ( 4.4% ) in the sexually active women living with hiv . other studies however reported higher dual contraceptive use[7 , 8 , 13 , 14 ] . this study recorded lower dual contraceptive method use and it may be due to the fact that much attention has not been given to the other modern contraceptive methods among women living with hiv . dual contraceptive method use has the additional advantage of being an effective method of preventing unintended pregnancies and this is important especially for couples who have completed their families or who do not desire children . the integration of family planning services into hiv / aids clinics creates an ideal opportunity to promote dual method use and this will go a long way in the prevention of mother to child transmission and the transmission of hiv . female sterilisation was very low and compares to other african studies[7 , 8 , 13 ] . in the united states however sterilisation was one of the commonest contraceptives ( 44.4% ) used among women living with hiv / aids . it must however be stated that about a third of the women had sterilisation regret . in counselling women on contraceptive methods , care must be taken so as not to coerce women living with hiv to accept permanent contraceptive methods . women living with hiv have reproductive rights to decide freely on the number of children and spacing of their children and counselling on contraceptive methods should be done in a supportive environment . couple counselling is also another method that may improve use of other contraceptive methods in addition to condom use but further research in this area has to be done . in kumasi , about one tenth of sexually active women living with hiv used other family planning methods without condom . this was slightly lower than the 13% reported in a study in uganda . since the condom is the only contraceptive that has the additional benefit of preventing hiv / sti transmission , its continuous promotion at hiv / aids clinics the clients attending the clinic may be compliant clients who adhere to treatment and counselling . since these clinics are specialised , the potential to elicit high response rate for use of condoms is high . women who were very sick or in who clinical stage iv were excluded from the study . this will affect assessing the health status and contraceptive use of all women living with hiv in general . these limitations notwithstanding the findings from this study provide a good source of information for improving the services to women living with hiv . in interpreting the results caution must however be taken . the clients attending the clinic may be compliant clients who adhere to treatment and counselling . since these clinics are specialised , the potential to elicit high response rate for use of condoms is high . women who were very sick or in who clinical stage iv were excluded from the study . this will affect assessing the health status and contraceptive use of all women living with hiv in general . these limitations notwithstanding the findings from this study provide a good source of information for improving the services to women living with hiv . in interpreting the results caution must however be taken . the clients attending the clinic may be compliant clients who adhere to treatment and counselling . since these clinics are specialised , the potential to elicit high response rate for use of condoms is high . women who were very sick or in who clinical stage iv were excluded from the study . this will affect assessing the health status and contraceptive use of all women living with hiv in general . these limitations notwithstanding the findings from this study provide a good source of information for improving the services to women living with hiv . overall , the use of contraceptives among women living with hiv in the kumasi metropolis , ghana is high . condom is the main contraceptive used because of its dual purpose in preventing the transmission of hiv / sti and preventing unintended pregnancy . factors associated with contraceptive use at univariate analysis : are ever had children , desire for a child , hiv status of partner and hiv disclosure to partner . women who have not disclosed their hiv status to their partners were less likely to use contraceptive . women living with hiv could be encouraged to disclose their hiv status to their partners and encourage their partners to also go for hiv testing . issues relating to non - disclosure may be assessed and interventions that promote disclosure of hiv status may be implemented . women who had not disclosed their hiv status could be as a result of stigma and discrimination associated with the infection and they could also be lacking condom negotiation skills . there may be the need of improving the condom negotiation skills of women living with hiv . as part of the integration of family planning services into hiv / aids services in ghana , attention could be given to temporary long term and permanent contraceptives whilst emphasising condom use as well . there is however the need for much larger and longer follow - up studies in this area to be conducted in ghana .

objectives : contraceptive use among women living with hiv is important to prevent the transmission of the infection to their partners , prevent unintended pregnancies and prevent the mother - to - child transmission of the infection . the study sought to determine the contraceptive characteristics of women living with hiv in the kumasi metropolis.methods:a cross - sectional study was conducted from july to august 2012 at two hiv / aids clinics in the kumasi metropolis in the ashanti region , ghana . interviewer- administered questionnaires were used to collect data from two hundred and ninety five women . data from one hundred and eighty three women living with hiv and who were sexually active were analyzed . factors associated with contraceptive use were examined using logistic regression.results:the overall contraceptive use was high ; 84.7% were using a modern contraceptive method . the male condom was the commonest contraceptive method ( 77.0% ) used and this was the main contraceptive method promoted at the hiv / aids clinic . dual method usage was low ( 4.4% ) . multivariate analysis showed that the significant predictor of contraceptive use was hiv status disclosure to partner ( aor = 0.25 ; 95% ci = 0.07 - 0.87 ; p = 0.03).conclusions and public health implications : the integration of family planning and hiv / aids services could stress dual method use and encourage hiv status disclosure to partner .

Introduction Methods None Study Area and Design Study Population and Study Sample Data Statistical Analysis Ethical Consideration Results Reproductive characteristics and intention of respondents Factors associated with contraceptive usage Current Contraceptive use among women living with HIV Discussion None Study Limitations Conclusion and Public Health Implications

women formed about 50% of the adult population ( 15 years or older ) infected with hiv / aids in the world . in sub - saharan africa , women living with hiv make up 59% of the adult population living with the infection . in ghana , the estimated hiv prevalence is 1.5 with about 226,000 people living with hiv / aids . most hiv infections in children are transmitted through mother - to - child during pregnancy , labor and breastfeeding . condom use among women living with hiv is also important to prevent the transmission of hiv / sti to the partner . even for those who desire to have a child , , the current contraceptive use of any method among married women ( 15- 49 years ) in the general population is 23.5% and modern contraceptive use is 23.0% . the sexual behaviour and contraceptive use among women living with hiv are important in reducing the transmission of hiv . it was projected that about 10,300 mothers need prevention of mother - to - child transmission services and there would be 1,100 new hiv infections in children in 2012 in ghana . condoms have been the main contraceptive that has been promoted for people living with hiv / aids because of its dual purpose of preventing the transmission of hiv / sti and preventing pregnancies . it is therefore important to study the contraceptive use and the factors associated with contraceptive uptake in women living with hiv in order to inform programs designed to reduce the transmission of hiv and prevent unintended pregnancies among this sub - population . this was hospital - based cross - sectional descriptive study conducted from the 10th of july 2012 to the 15th of august 2012 . the study was conducted in the kumasi metropolitan area which has a total population of 2,035,064 ; females constitute 52.2% of the population . two public health institutions in the metropolis with hiv / aids clinics , namely the kumasi south hospital in the asokwa sub - metropolitan health area and the suntreso government hospital in the bantama sub - metropolitan health area were used for this study . the hiv / aids clinics at the kumasi south hospital and the suntreso government hospital are the largest hiv / aids clinics of the ghana health service facilities in the metropolis and they have about 4000 and 2000 clients respectively . the hiv / aids clinics offer counselling and testing , care and treatment , and provision of antiretroviral drugs . women living with hiv / aids within the reproductive age 18 to 49 years attending clinics at the above mentioned facilities were recruited for the study . the estimated sample size for the study was 300 . to be eligible for the study , newly diagnosed hiv / aids clients were excluded in the study since they are usually emotionally unstable and not sexually active or had lost interest in sex . individuals in the who clinical stage iv and the very sick were also excluded three hundred women living with hiv / aids were approached to be interviewed . two hundred clients were sampled from the kumasi south hospital while one hundred clients were selected from the suntreso government hospital . we aimed to recruit all eligible hiv / aids clients presenting at the clinic within the study period . in all two hundred and ninety five women living with hiv agreed and questionnaires were administered to them . the questionnaire covered socio - demographic characteristics , contraceptive use , haart use , reproductive history , partner s characteristics and desire for children , knowledge of pmtct and contraceptive methods discussed at the hiv / aids clinic . hospital records were reviewed to collect information on who clinical stage and date of diagnosis of hiv of the study participant . analysis was performed on 183 study participants who were sexually active ; frequencies , percentages and odds ratio were calculated . this was hospital - based cross - sectional descriptive study conducted from the 10th of july 2012 to the 15th of august 2012 . the study was conducted in the kumasi metropolitan area which has a total population of 2,035,064 ; females constitute 52.2% of the population . two public health institutions in the metropolis with hiv / aids clinics , namely the kumasi south hospital in the asokwa sub - metropolitan health area and the suntreso government hospital in the bantama sub - metropolitan health area were used for this study . the hiv / aids clinics at the kumasi south hospital and the suntreso government hospital are the largest hiv / aids clinics of the ghana health service facilities in the metropolis and they have about 4000 and 2000 clients respectively . the hiv / aids clinics offer counselling and testing , care and treatment , and provision of antiretroviral drugs . women living with hiv / aids within the reproductive age 18 to 49 years attending clinics at the above mentioned facilities were recruited for the study . the estimated sample size for the study was 300 . to be eligible for the study , newly diagnosed hiv / aids clients were excluded in the study since they are usually emotionally unstable and not sexually active or had lost interest in sex . individuals in the who clinical stage iv and the very sick were also excluded three hundred women living with hiv / aids were approached to be interviewed . two hundred clients were sampled from the kumasi south hospital while one hundred clients were selected from the suntreso government hospital . we aimed to recruit all eligible hiv / aids clients presenting at the clinic within the study period . if the client agreed to participate in the study , a consent form was signed and interviewed conducted . in all two hundred and ninety five women living with hiv agreed and questionnaires were administered to them . the questionnaire covered socio - demographic characteristics , contraceptive use , haart use , reproductive history , partner s characteristics and desire for children , knowledge of pmtct and contraceptive methods discussed at the hiv / aids clinic . hospital records were reviewed to collect information on who clinical stage and date of diagnosis of hiv of the study participant . analysis was performed on 183 study participants who were sexually active ; frequencies , percentages and odds ratio were calculated . this was hospital - based cross - sectional descriptive study conducted from the 10th of july 2012 to the 15th of august 2012 . the study was conducted in the kumasi metropolitan area which has a total population of 2,035,064 ; females constitute 52.2% of the population . two public health institutions in the metropolis with hiv / aids clinics , namely the kumasi south hospital in the asokwa sub - metropolitan health area and the suntreso government hospital in the bantama sub - metropolitan health area were used for this study . the hiv / aids clinics at the kumasi south hospital and the suntreso government hospital are the largest hiv / aids clinics of the ghana health service facilities in the metropolis and they have about 4000 and 2000 clients respectively . the hiv / aids clinics offer counselling and testing , care and treatment , and provision of antiretroviral drugs . women living with hiv / aids within the reproductive age 18 to 49 years attending clinics at the above mentioned facilities were recruited for the study . to be eligible for the study newly diagnosed hiv / aids clients were excluded in the study since they are usually emotionally unstable and not sexually active or had lost interest in sex . individuals in the who clinical stage iv and the very sick were also excluded three hundred women living with hiv / aids were approached to be interviewed . two hundred clients were sampled from the kumasi south hospital while one hundred clients were selected from the suntreso government hospital . we aimed to recruit all eligible hiv / aids clients presenting at the clinic within the study period . if the client agreed to participate in the study , a consent form was signed and interviewed conducted . in all two hundred and ninety five women living with hiv agreed and questionnaires were administered to them . the questionnaire covered socio - demographic characteristics , contraceptive use , haart use , reproductive history , partner s characteristics and desire for children , knowledge of pmtct and contraceptive methods discussed at the hiv / aids clinic . hospital records were reviewed to collect information on who clinical stage and date of diagnosis of hiv of the study participant . analysis was performed on 183 study participants who were sexually active ; frequencies , percentages and odds ratio were calculated . about 79% of the respondents were married and of those who were using a contraceptive 82.2% were married . the average monthly income of most of the respondents who were employed was below us$ 53 ( ghcedi ( ) 1.90 is equivalent to us$ 1.00 ) ( see table 1 ) . socio - demographic and health characteristics of women living with hiv other christian= all christian organisations ( methodist , presbyterian , pentecost , charismatic ) except catholic , basic education = primary high education= middle / junior high school , secondary / senior high school and tertiary , among those who use contraceptive ( n=125 ) and those who do not use contraceptive ( n=21 ) most of the respondents ( 80.3% ) were menstruating regularly and 94% had ever had children . majority of the women and their partners desire to have children and about 85% of the respondents had disclosed their hiv status to their partners . about one third of the respondents did not know the hiv status of their partners . among the women currently using contraceptive 61.2% desire to have children in the future and 59.2% of their partners also desire to have children ( table 2 ) . reproductive characteristics and intentions of women living with hiv by contraceptive use in the univariate analysis , however , women who were cohabitating were less likely to use contraceptives ( unadjusted odds ratio [ or ] = 0.32 ; 95% ci = 0.12 - 0.88 , p = 0.03 ) compared with those who were married . participants who did not have children were significantly less likely to use a contraceptive ( unadjusted 0r = 0.17 ; 95% ci = 0.05 - 0.60 , p = 0.02 ) compared with women with children . the hiv status of the partner was associated with contraceptive use ; partners whose hiv status were unknown compared with those who were hiv positive were less likely to use a contraceptive ( unadjusted or = 0.27 ; 95% ci = 0.08 - 0.86 , p = 0.03 ) . multivariate analysis showed that , disclosure of hiv status to partner was the only variable that significantly predicted contraceptive use , suggesting that women who have not disclosed their status to their partners were less likely to use contraceptive ( aor = 0.25 ; 95% ci = 0.07 - 0.87 , p = 0.03 ) ( table 3 ) . univariate and adjusted analysis of factors associated with contraceptive use among women living with hiv eighty - five percent of the respondents were using a modern contraceptive method . majority of the women and their partners desire to have children and about 85% of the respondents had disclosed their hiv status to their partners . about one third of the respondents did not know the hiv status of their partners . reproductive characteristics and intentions of women living with hiv by contraceptive use in the univariate analysis , age group and educational status were not associated with the use of contraceptives . however , women who were cohabitating were less likely to use contraceptives ( unadjusted odds ratio [ or ] = 0.32 ; 95% ci = 0.12 - 0.88 , p = 0.03 ) compared with those who were married . participants who did not have children were significantly less likely to use a contraceptive ( unadjusted 0r = 0.17 ; 95% ci = 0.05 - 0.60 , p = 0.02 ) compared with women with children . the hiv status of the partner was associated with contraceptive use ; partners whose hiv status were unknown compared with those who were hiv positive were less likely to use a contraceptive ( unadjusted or = 0.27 ; 95% ci = 0.08 - 0.86 , p = 0.03 ) . multivariate analysis showed that , disclosure of hiv status to partner was the only variable that significantly predicted contraceptive use , suggesting that women who have not disclosed their status to their partners were less likely to use contraceptive ( aor = 0.25 ; 95% ci = 0.07 - 0.87 , p = 0.03 ) ( table 3 ) . the distribution of the various contraceptive methods are male condom(77.0% ) , injectables(5.5% ) , female sterilisation(1.1% ) , female condom(0.5% ) , implants(0.5 ) and other non modern methods which is the rhythm(1.1% ) . this cross - sectional study serves as a pilot study on the contraceptive characteristics of women living with hiv in the kumasi metropolis , ghana . the results showed that disclosure of hiv sero - status to one s partner was a significant predictor of contraceptive use ; women who had not disclosed their status to their partners were significantly less likely to use contraceptive . similarly , a study in zambia also reported disclosure of hiv status as a predictor of contraceptive use . in uganda disclosure of hiv status was a predictor of modern contraceptive use in women living with hiv . disclosure of one s hiv status to partner would ensure that both parties would understand the importance of using the condom and other modern contraceptives to prevent unintended pregnancies . women living with hiv could therefore be counselled and encouraged to disclose their hiv status to their partners since it may improve their contraceptive use to prevent unintended pregnancies and the transmission of hiv / sti . disclosure of hiv status to partner is a dilemma because of the stigma and discrimination[9 , 10 ] associated with the infection and more so if the woman is the first to know of her status it sometimes presupposes that she is the one who got the infection first . interventions that promote hiv status disclosure and issues relating to non - disclosure could be assessed[11 , 12 ] since it may improve uptake of contraceptives . contraceptive use among women living with hiv is generally higher than the general population[8 , 13 ] . this is mainly because of the high use of the condom which has a dual purpose of preventing the transmission of hiv and preventing unintended pregnancies . this compares with a study done in soweto , south africa were 84% women living with hiv were currently using at least one contraceptive method . a cross - sectional study in kenya reported 44.2% of respondents currently using a contraceptive method . a study in uganda , even reported as low as 25.2% of women currently using a contraceptive method and this could be due to the fact that the study was conducted in a post conflict era . in lusaka , zambia , 59.2% of women living with hiv reported current use of a modern contraceptive and this is due to the fact that more than half of the respondents had not disclosed their hiv status to their partners . the differences in the level of contraceptive use can be explained by the differences in the characteristics of the populations sampled . in the current study , more than three - out - of - four women were using the male condom . this is mainly because of its dual purpose in preventing the transmission of sti / hiv and pregnancy , and this is the main contraceptive promoted at hiv / aids clinics in kumasi . similarly in other african countries the commonest contraceptive used by women living with hiv is the male condom[7 , 8 , 13 ] . the female condom which has the advantage of being female controlled and can be used if the man is reluctant to use the male condom was however very low ( 0.5% ) . it can be argued that the female condom has not been well promoted ; it was recently re - launched by the ghana health service in october 2012 with the aim that it will help with its integration into sector wide policies as well as within hiv / aids programming . similarly in uganda , female condom use was low ( 0.8% ) . female condom use in kenya was higher ( 10.5% ) unlike that in kumasi , ghana but this is still low when compared with male condom use . further research into the low use of female condom among women living with hiv will help clarify the barriers to its use and probably improve its uptake . dual contraceptive method use which is the male condom and any other method was low ( 4.4% ) in the sexually active women living with hiv . this study recorded lower dual contraceptive method use and it may be due to the fact that much attention has not been given to the other modern contraceptive methods among women living with hiv . dual contraceptive method use has the additional advantage of being an effective method of preventing unintended pregnancies and this is important especially for couples who have completed their families or who do not desire children . the integration of family planning services into hiv / aids clinics creates an ideal opportunity to promote dual method use and this will go a long way in the prevention of mother to child transmission and the transmission of hiv . in the united states however sterilisation was one of the commonest contraceptives ( 44.4% ) used among women living with hiv / aids . in counselling women on contraceptive methods , care must be taken so as not to coerce women living with hiv to accept permanent contraceptive methods . women living with hiv have reproductive rights to decide freely on the number of children and spacing of their children and counselling on contraceptive methods should be done in a supportive environment . in kumasi , about one tenth of sexually active women living with hiv used other family planning methods without condom . since the condom is the only contraceptive that has the additional benefit of preventing hiv / sti transmission , its continuous promotion at hiv / aids clinics the clients attending the clinic may be compliant clients who adhere to treatment and counselling . this will affect assessing the health status and contraceptive use of all women living with hiv in general . these limitations notwithstanding the findings from this study provide a good source of information for improving the services to women living with hiv . women who were very sick or in who clinical stage iv were excluded from the study . this will affect assessing the health status and contraceptive use of all women living with hiv in general . these limitations notwithstanding the findings from this study provide a good source of information for improving the services to women living with hiv . women who were very sick or in who clinical stage iv were excluded from the study . this will affect assessing the health status and contraceptive use of all women living with hiv in general . these limitations notwithstanding the findings from this study provide a good source of information for improving the services to women living with hiv . overall , the use of contraceptives among women living with hiv in the kumasi metropolis , ghana is high . condom is the main contraceptive used because of its dual purpose in preventing the transmission of hiv / sti and preventing unintended pregnancy . factors associated with contraceptive use at univariate analysis : are ever had children , desire for a child , hiv status of partner and hiv disclosure to partner . women who have not disclosed their hiv status to their partners were less likely to use contraceptive . women living with hiv could be encouraged to disclose their hiv status to their partners and encourage their partners to also go for hiv testing . women who had not disclosed their hiv status could be as a result of stigma and discrimination associated with the infection and they could also be lacking condom negotiation skills . there may be the need of improving the condom negotiation skills of women living with hiv . as part of the integration of family planning services into hiv / aids services in ghana , attention could be given to temporary long term and permanent contraceptives whilst emphasising condom use as well .

human immunodeficiency virus prevalence in the world has levelled - off in most regions in the world . women formed about 50% of the adult population ( 15 years or older ) infected with hiv / aids in the world . in sub - saharan africa , women living with hiv make up 59% of the adult population living with the infection . in ghana , the estimated hiv prevalence is 1.5 with about 226,000 people living with hiv / aids . most hiv infections in children are transmitted through mother - to - child during pregnancy , labor and breastfeeding . condom use among women living with hiv is also important to prevent the transmission of hiv / sti to the partner . even for those who desire to have a child , , the current contraceptive use of any method among married women ( 15- 49 years ) in the general population is 23.5% and modern contraceptive use is 23.0% . condom use among married women aged 15 49 years is 2.4% in the general population in ghana . the sexual behaviour and contraceptive use among women living with hiv are important in reducing the transmission of hiv . it was projected that about 10,300 mothers need prevention of mother - to - child transmission services and there would be 1,100 new hiv infections in children in 2012 in ghana . condoms have been the main contraceptive that has been promoted for people living with hiv / aids because of its dual purpose of preventing the transmission of hiv / sti and preventing pregnancies . it is therefore important to study the contraceptive use and the factors associated with contraceptive uptake in women living with hiv in order to inform programs designed to reduce the transmission of hiv and prevent unintended pregnancies among this sub - population . this was hospital - based cross - sectional descriptive study conducted from the 10th of july 2012 to the 15th of august 2012 . the study was conducted in the kumasi metropolitan area which has a total population of 2,035,064 ; females constitute 52.2% of the population . for health purposes , the metropolis is divided into five sub - metropolitan areas : bantama , asokwa , manhyia north , manhyia south and subin . two public health institutions in the metropolis with hiv / aids clinics , namely the kumasi south hospital in the asokwa sub - metropolitan health area and the suntreso government hospital in the bantama sub - metropolitan health area were used for this study . the hiv / aids clinics at the kumasi south hospital and the suntreso government hospital are the largest hiv / aids clinics of the ghana health service facilities in the metropolis and they have about 4000 and 2000 clients respectively . the hiv / aids clinics offer counselling and testing , care and treatment , and provision of antiretroviral drugs . women living with hiv / aids within the reproductive age 18 to 49 years attending clinics at the above mentioned facilities were recruited for the study . to be eligible for the study , newly diagnosed hiv / aids clients were excluded in the study since they are usually emotionally unstable and not sexually active or had lost interest in sex . individuals in the who clinical stage iv and the very sick were also excluded three hundred women living with hiv / aids were approached to be interviewed . we aimed to recruit all eligible hiv / aids clients presenting at the clinic within the study period . if the client agreed to participate in the study , a consent form was signed and interviewed conducted . in all two hundred and ninety five women living with hiv agreed and questionnaires were administered to them . the questionnaire covered socio - demographic characteristics , contraceptive use , haart use , reproductive history , partner s characteristics and desire for children , knowledge of pmtct and contraceptive methods discussed at the hiv / aids clinic . hospital records were reviewed to collect information on who clinical stage and date of diagnosis of hiv of the study participant . questions on contraceptive knowledge and use were adopted from the 2008 ghana demographic health survey . interviews were conducted in english or translated into twi ( local dialect ) depending on preference of the participant , and it lasted between 10 to 15 minutes . analysis was performed on 183 study participants who were sexually active ; frequencies , percentages and odds ratio were calculated . multivariate logistic regression model was fitted to simultaneously adjust for the effect of other covariates . ethical clearance was obtained from the committee on human research , publications and ethics , school of medical sciences , kwame nkrumah university of science and technology , kumasi , ghana . this was hospital - based cross - sectional descriptive study conducted from the 10th of july 2012 to the 15th of august 2012 . the study was conducted in the kumasi metropolitan area which has a total population of 2,035,064 ; females constitute 52.2% of the population . for health purposes , the metropolis is divided into five sub - metropolitan areas : bantama , asokwa , manhyia north , manhyia south and subin . two public health institutions in the metropolis with hiv / aids clinics , namely the kumasi south hospital in the asokwa sub - metropolitan health area and the suntreso government hospital in the bantama sub - metropolitan health area were used for this study . the hiv / aids clinics at the kumasi south hospital and the suntreso government hospital are the largest hiv / aids clinics of the ghana health service facilities in the metropolis and they have about 4000 and 2000 clients respectively . the hiv / aids clinics offer counselling and testing , care and treatment , and provision of antiretroviral drugs . women living with hiv / aids within the reproductive age 18 to 49 years attending clinics at the above mentioned facilities were recruited for the study . to be eligible for the study , newly diagnosed hiv / aids clients were excluded in the study since they are usually emotionally unstable and not sexually active or had lost interest in sex . individuals in the who clinical stage iv and the very sick were also excluded three hundred women living with hiv / aids were approached to be interviewed . we aimed to recruit all eligible hiv / aids clients presenting at the clinic within the study period . if the client agreed to participate in the study , a consent form was signed and interviewed conducted . in all two hundred and ninety five women living with hiv agreed and questionnaires were administered to them . the questionnaire covered socio - demographic characteristics , contraceptive use , haart use , reproductive history , partner s characteristics and desire for children , knowledge of pmtct and contraceptive methods discussed at the hiv / aids clinic . hospital records were reviewed to collect information on who clinical stage and date of diagnosis of hiv of the study participant . interviews were conducted in english or translated into twi ( local dialect ) depending on preference of the participant , and it lasted between 10 to 15 minutes . multivariate logistic regression model was fitted to simultaneously adjust for the effect of other covariates . ethical clearance was obtained from the committee on human research , publications and ethics , school of medical sciences , kwame nkrumah university of science and technology , kumasi , ghana . this was hospital - based cross - sectional descriptive study conducted from the 10th of july 2012 to the 15th of august 2012 . the study was conducted in the kumasi metropolitan area which has a total population of 2,035,064 ; females constitute 52.2% of the population . two public health institutions in the metropolis with hiv / aids clinics , namely the kumasi south hospital in the asokwa sub - metropolitan health area and the suntreso government hospital in the bantama sub - metropolitan health area were used for this study . the hiv / aids clinics at the kumasi south hospital and the suntreso government hospital are the largest hiv / aids clinics of the ghana health service facilities in the metropolis and they have about 4000 and 2000 clients respectively . the hiv / aids clinics offer counselling and testing , care and treatment , and provision of antiretroviral drugs . women living with hiv / aids within the reproductive age 18 to 49 years attending clinics at the above mentioned facilities were recruited for the study . to be eligible for the study newly diagnosed hiv / aids clients were excluded in the study since they are usually emotionally unstable and not sexually active or had lost interest in sex . individuals in the who clinical stage iv and the very sick were also excluded three hundred women living with hiv / aids were approached to be interviewed . we aimed to recruit all eligible hiv / aids clients presenting at the clinic within the study period . if the client agreed to participate in the study , a consent form was signed and interviewed conducted . the questionnaire covered socio - demographic characteristics , contraceptive use , haart use , reproductive history , partner s characteristics and desire for children , knowledge of pmtct and contraceptive methods discussed at the hiv / aids clinic . hospital records were reviewed to collect information on who clinical stage and date of diagnosis of hiv of the study participant . interviews were conducted in english or translated into twi ( local dialect ) depending on preference of the participant , and it lasted between 10 to 15 minutes . multivariate logistic regression model was fitted to simultaneously adjust for the effect of other covariates . ethical clearance was obtained from the committee on human research , publications and ethics , school of medical sciences , kwame nkrumah university of science and technology , kumasi , ghana . about 79% of the respondents were married and of those who were using a contraceptive 82.2% were married . the average monthly income of most of the respondents who were employed was below us$ 53 ( ghcedi ( ) 1.90 is equivalent to us$ 1.00 ) ( see table 1 ) . socio - demographic and health characteristics of women living with hiv other christian= all christian organisations ( methodist , presbyterian , pentecost , charismatic ) except catholic , basic education = primary high education= middle / junior high school , secondary / senior high school and tertiary , among those who use contraceptive ( n=125 ) and those who do not use contraceptive ( n=21 ) most of the respondents ( 80.3% ) were menstruating regularly and 94% had ever had children . majority of the women and their partners desire to have children and about 85% of the respondents had disclosed their hiv status to their partners . among the women currently using contraceptive 61.2% desire to have children in the future and 59.2% of their partners also desire to have children ( table 2 ) . reproductive characteristics and intentions of women living with hiv by contraceptive use in the univariate analysis , however , women who were cohabitating were less likely to use contraceptives ( unadjusted odds ratio [ or ] = 0.32 ; 95% ci = 0.12 - 0.88 , p = 0.03 ) compared with those who were married . participants who did not have children were significantly less likely to use a contraceptive ( unadjusted 0r = 0.17 ; 95% ci = 0.05 - 0.60 , p = 0.02 ) compared with women with children . the hiv status of the partner was associated with contraceptive use ; partners whose hiv status were unknown compared with those who were hiv positive were less likely to use a contraceptive ( unadjusted or = 0.27 ; 95% ci = 0.08 - 0.86 , p = 0.03 ) . multivariate analysis showed that , disclosure of hiv status to partner was the only variable that significantly predicted contraceptive use , suggesting that women who have not disclosed their status to their partners were less likely to use contraceptive ( aor = 0.25 ; 95% ci = 0.07 - 0.87 , p = 0.03 ) ( table 3 ) . univariate and adjusted analysis of factors associated with contraceptive use among women living with hiv eighty - five percent of the respondents were using a modern contraceptive method . the distribution of the various contraceptive methods are male condom(77.0% ) , injectables(5.5% ) , female sterilisation(1.1% ) , female condom(0.5% ) , implants(0.5 ) and other non modern methods which is the rhythm(1.1% ) . majority of the women and their partners desire to have children and about 85% of the respondents had disclosed their hiv status to their partners . among the women currently using contraceptive 61.2% desire to have children in the future and 59.2% of their partners also desire to have children ( table 2 ) . reproductive characteristics and intentions of women living with hiv by contraceptive use in the univariate analysis , age group and educational status were not associated with the use of contraceptives . however , women who were cohabitating were less likely to use contraceptives ( unadjusted odds ratio [ or ] = 0.32 ; 95% ci = 0.12 - 0.88 , p = 0.03 ) compared with those who were married . participants who did not have children were significantly less likely to use a contraceptive ( unadjusted 0r = 0.17 ; 95% ci = 0.05 - 0.60 , p = 0.02 ) compared with women with children . the hiv status of the partner was associated with contraceptive use ; partners whose hiv status were unknown compared with those who were hiv positive were less likely to use a contraceptive ( unadjusted or = 0.27 ; 95% ci = 0.08 - 0.86 , p = 0.03 ) . multivariate analysis showed that , disclosure of hiv status to partner was the only variable that significantly predicted contraceptive use , suggesting that women who have not disclosed their status to their partners were less likely to use contraceptive ( aor = 0.25 ; 95% ci = 0.07 - 0.87 , p = 0.03 ) ( table 3 ) . the distribution of the various contraceptive methods are male condom(77.0% ) , injectables(5.5% ) , female sterilisation(1.1% ) , female condom(0.5% ) , implants(0.5 ) and other non modern methods which is the rhythm(1.1% ) . this cross - sectional study serves as a pilot study on the contraceptive characteristics of women living with hiv in the kumasi metropolis , ghana . the results showed that disclosure of hiv sero - status to one s partner was a significant predictor of contraceptive use ; women who had not disclosed their status to their partners were significantly less likely to use contraceptive . in uganda disclosure of hiv status was a predictor of modern contraceptive use in women living with hiv . women living with hiv could therefore be counselled and encouraged to disclose their hiv status to their partners since it may improve their contraceptive use to prevent unintended pregnancies and the transmission of hiv / sti . disclosure of hiv status to partner is a dilemma because of the stigma and discrimination[9 , 10 ] associated with the infection and more so if the woman is the first to know of her status it sometimes presupposes that she is the one who got the infection first . women in many places in africa are expected to be modest and chaste , and be with only one partner . interventions that promote hiv status disclosure and issues relating to non - disclosure could be assessed[11 , 12 ] since it may improve uptake of contraceptives . this is mainly because of the high use of the condom which has a dual purpose of preventing the transmission of hiv and preventing unintended pregnancies . this compares with a study done in soweto , south africa were 84% women living with hiv were currently using at least one contraceptive method . a study in uganda , even reported as low as 25.2% of women currently using a contraceptive method and this could be due to the fact that the study was conducted in a post conflict era . in lusaka , zambia , 59.2% of women living with hiv reported current use of a modern contraceptive and this is due to the fact that more than half of the respondents had not disclosed their hiv status to their partners . the differences in the level of contraceptive use can be explained by the differences in the characteristics of the populations sampled . in the current study , more than three - out - of - four women were using the male condom . this is mainly because of its dual purpose in preventing the transmission of sti / hiv and pregnancy , and this is the main contraceptive promoted at hiv / aids clinics in kumasi . similarly in other african countries the commonest contraceptive used by women living with hiv is the male condom[7 , 8 , 13 ] . the female condom which has the advantage of being female controlled and can be used if the man is reluctant to use the male condom was however very low ( 0.5% ) . it can be argued that the female condom has not been well promoted ; it was recently re - launched by the ghana health service in october 2012 with the aim that it will help with its integration into sector wide policies as well as within hiv / aids programming . female condom use in kenya was higher ( 10.5% ) unlike that in kumasi , ghana but this is still low when compared with male condom use . further research into the low use of female condom among women living with hiv will help clarify the barriers to its use and probably improve its uptake . dual contraceptive method use which is the male condom and any other method was low ( 4.4% ) in the sexually active women living with hiv . this study recorded lower dual contraceptive method use and it may be due to the fact that much attention has not been given to the other modern contraceptive methods among women living with hiv . the integration of family planning services into hiv / aids clinics creates an ideal opportunity to promote dual method use and this will go a long way in the prevention of mother to child transmission and the transmission of hiv . in the united states however sterilisation was one of the commonest contraceptives ( 44.4% ) used among women living with hiv / aids . women living with hiv have reproductive rights to decide freely on the number of children and spacing of their children and counselling on contraceptive methods should be done in a supportive environment . in kumasi , about one tenth of sexually active women living with hiv used other family planning methods without condom . since the condom is the only contraceptive that has the additional benefit of preventing hiv / sti transmission , its continuous promotion at hiv / aids clinics the clients attending the clinic may be compliant clients who adhere to treatment and counselling . this will affect assessing the health status and contraceptive use of all women living with hiv in general . these limitations notwithstanding the findings from this study provide a good source of information for improving the services to women living with hiv . this will affect assessing the health status and contraceptive use of all women living with hiv in general . these limitations notwithstanding the findings from this study provide a good source of information for improving the services to women living with hiv . this will affect assessing the health status and contraceptive use of all women living with hiv in general . these limitations notwithstanding the findings from this study provide a good source of information for improving the services to women living with hiv . overall , the use of contraceptives among women living with hiv in the kumasi metropolis , ghana is high . factors associated with contraceptive use at univariate analysis : are ever had children , desire for a child , hiv status of partner and hiv disclosure to partner . as part of the integration of family planning services into hiv / aids services in ghana , attention could be given to temporary long term and permanent contraceptives whilst emphasising condom use as well .

the development of regenerative medicine has enthralled researchers to study and exploit its usage and therapeutic effects . different types of stem cells exhibit a potential that has helped improving symptoms of various intractable neuronal diseases , such as stroke . bone marrow - derived mononuclear stem cells ( bm - mnc ) have been used in preclinical studies suggesting increased angiogenesis in penumbral tissue following cd34 + cell transplantation , whether given systemically ( intra - arterial , intravenous or intrathecal ) or by the intracerebral route . stem cells actively contribute to their environment by secreting cytokines , growth factors and extracellular matrix molecules that act either by themselves ( autocrine actions ) or on human body and other tissues ( paracrine ) for regeneration . in addition , these cells secrete angiogenic factors , antifibrotic factors , extracellular matrix homeostasis proteins such as collagens , matrix metalloproteinases and other inhibitors . brain - derived neurotrophic growth factor ( bdnf ) crucially promotes the synaptic and axonal plasticity associated with learning , memory and sensorimotor recovery . it has also been used to induce neurogenesis after focal ischemia , thereby increasing the number of newborn neurons in several regions of the brain enhancing neural structural plasticity . vascular endothelial growth factor ( vegf ) it has been shown to increase vascular permeability ; it can induce chemotaxis in monocytes in pathological conditions as well as inhibit endothelial cell apoptosis . recently , it was shown that both vegf and its receptor flt-1 are upregulated in both neurons and blood vessels in the penumbra after transient or permanent occlusion of the middle cerebral artery in the rat . cell treatment or treatment with a stem cell - containing population is nascent in the current stage and has met enormous skepticism in the field of cell therapeutics . since the realization that the beneficial effects of stem cells may be due to localized or generalized release of trophic factors , and not attributed ( in part or entirely ) to stem cell transdifferentiation or homing in to the lesioned cortex , many scientists have focused on harnessing the paracrine actions of stem cells to enhance therapeutic efficacy . repair mechanisms in stroke are related to acute injury ( first epoch ) and they are said to occur in the initial few hours after the acute event when changes in blood flow , metabolism and ischemic cascade are most active . a second epoch is related to the upregulation of growth factors which continues for days to weeks and is referred to as endogenous repair - related events . a third epoch occurs weeks to months after stroke when spontaneous recovery mechanisms plateau representing a stable but modifiable early and late chronic phase . the objectives were ( a ) to study safety , feasibility and efficacy ( if any ) of bm - mnc infusion in chronic ischemic stroke ; ( b ) to study early / late upregulation of serum growth factors ( vegf and bdnf ) after bm - mnc and neuromotor rehabilitation after stroke , and ( c ) to study the correlation of serum vegf and bdnf with functional recovery after stroke . thirty patients were screened who fulfilled the inclusion criteria ; among them , 5 refused to participate in the trial and 5 had deranged baseline laboratory values and were therefore excluded ; hence , 20 patients were recruited in the study . patients diagnosed with ischemic stroke 3 months to 1.5 years after the index event with medical research council ( mrc ) grade of power for wrist and hand muscles of at least 2 , brunnstrom stage of recovery of 2 - 5 , national institutes of health stroke scale ( nihss ) score of between 4 and 15 , being conscious / comprehendible as well as 20 age - matched healthy controls were recruited . the exclusion criteria were hematological disorders , autoimmune disorders , immunocompromised subjects , chronic liver and renal failure , progressive neurological worsening , unilateral neglect , neoplasia , contraindication to mri and pregnancy . one group was infused with bm - mnc and the other group with placebo ( i.e. intravenous saline infusion ) . all patients were administered a neuromotor rehabilitation regime for 8 weeks . prior to stem cell therapy , patients were screened and educated about the stem cells and bone marrow aspiration technique . written informed consent was obtained , complete medical history , examination and baseline laboratory tests were performed . the patients were examined by a neurologist and a neurophysiotherapist for muscle strength / power ( mrc grade ) and tone ( modified ashworth tone scale ) , fugl meyer ( fm ) scale for upper limb , the edinburgh handedness inventory and the modified barthel index ( mbi ) . the study is a randomized placebo - controlled clinical trial which was approved by the ic - scrt ( institute committee for stem cell research and therapy ) and is registered with ctri ( ctri/2014/09/005028 ) . safety and efficacy end points were assessed at 7 days , 8 weeks , 6 months and 1 year after stem cell transplantation . serum vegf and bdnf levels were measured at baseline , 8 weeks , 6 months and 1 year . this paper presents an interim analysis of safety , feasibility and efficacy of bm - mnc in chronic ischemic stroke along with the upregulation of the serum growth factors vegf and bdnf after 2 months of cell infusion . bone marrow ( approximately 40 - 50 ml ) was aspirated under aseptic conditions from the posterior superior iliac crest in 10 chronic stroke patients . bone marrow aspirate was diluted with phosphate - buffered saline , layered over ficoll density medium and centrifuged at 1,800 rpm for 25 min . bm - mnc layers were collected , and the number of cd34 + cells ( flow cytometry method ) was counted for each patient ( table 1 ) . the mean cell viability of bm - mnc at transplantation was 98% , which was performed with trypan blue stain , and the cells were sterile and endotoxin - free during culture and at transplantation . an aseptic technique of infusion was performed with collected bm - mnc in a 50-ml sterile syringe , which was directly dissolved in 250 ml of saline and infused intravenously over 3 h. samples were immediately centrifuged ( 1,500 g/15 min ) and serum was stored at 70c until assayed . serum vegf and bdnf levels were measured by standard quantitative sandwich elisa ( quantikine ) kits , obtained from r&d systems . samples from each individual were analyzed in triplicate and subsequently used in all further statistical analysis . the lower limits of detection were 5.0 pg / ml for vegf and 3 ng / ml for bdnf ( information provided by r&d systems ) . motor imagery training using xbox and mirror therapy were administered to the affected upper limb with bilateral hands focusing on principles of learning . small hand muscle exercises like ball squeezing , lifting an object , rolling a cylindrical shape , and thrust release ( mass finger extension ) were included in the protocol . general aerobic exercise training was administered with 10 min of static cycling and brisk walk ( the extent the patient can ) , partial squats ( hold for 7 s ) , and step up and down . the comparative data between the two groups were expressed as the mean / median with sd and quartiles . parametric paired t test was used for intragroup and independent - samples t test for intergroup comparisons with statistically significant p = 0.05 . two - way repeated - measures anova was used to compare motor function scores at different time points . analysis was done to identify differences in growth factor expression in chronic stroke , in relation to stroke subtypes and etiology , with clinical disability , including short - term follow - up . bone marrow ( approximately 40 - 50 ml ) was aspirated under aseptic conditions from the posterior superior iliac crest in 10 chronic stroke patients . bone marrow aspirate was diluted with phosphate - buffered saline , layered over ficoll density medium and centrifuged at 1,800 rpm for 25 min . bm - mnc layers were collected , and the number of cd34 + cells ( flow cytometry method ) was counted for each patient ( table 1 ) . the mean cell viability of bm - mnc at transplantation was 98% , which was performed with trypan blue stain , and the cells were sterile and endotoxin - free during culture and at transplantation . an aseptic technique of infusion was performed with collected bm - mnc in a 50-ml sterile syringe , which was directly dissolved in 250 ml of saline and infused intravenously over 3 h. samples were immediately centrifuged ( 1,500 g/15 min ) and serum was stored at 70c until assayed . serum vegf and bdnf levels were measured by standard quantitative sandwich elisa ( quantikine ) kits , obtained from r&d systems . samples from each individual were analyzed in triplicate and subsequently used in all further statistical analysis . the lower limits of detection were 5.0 pg / ml for vegf and 3 ng / ml for bdnf ( information provided by r&d systems ) . motor imagery training using xbox and mirror therapy were administered to the affected upper limb with bilateral hands focusing on principles of learning . small hand muscle exercises like ball squeezing , lifting an object , rolling a cylindrical shape , and thrust release ( mass finger extension ) were included in the protocol . general aerobic exercise training was administered with 10 min of static cycling and brisk walk ( the extent the patient can ) , partial squats ( hold for 7 s ) , and step up and down . the comparative data between the two groups were expressed as the mean / median with sd and quartiles . parametric paired t test was used for intragroup and independent - samples t test for intergroup comparisons with statistically significant p = 0.05 . two - way repeated - measures anova was used to compare motor function scores at different time points . the analysis was two - tailed unless otherwise specified . for correlative analysis , the spearman rank correlation coefficient ( r ) analysis was done to identify differences in growth factor expression in chronic stroke , in relation to stroke subtypes and etiology , with clinical disability , including short - term follow - up . different organ systems were tested through various laboratory tests like percent hemoglobin , total leukocyte count , differential leukocyte count , platelets , prothrombin time , liver and kidney function assessed at the 1st , 3rd and 7th day after infusion . the safety outcomes included death , adverse events ( fatal or nonfatal myocardial infarction ) , epileptiform discharges and evidence of any new growth in radiological scans . cell viability , mononuclear stem cell surface markers , bacteria , syphilis , fungi , viral , mycoplasma , and endotoxin levels were tested in expansion process and transplantation . there were no early or late adverse reactions during and after transplantation observed until 8 weeks . as this was an amendment of our last trial , dosage was kept the same , i.e. , 1 million / kg body weight . cd34 + cells were characterized , with a mean count of 0.31% with 62.8 10 cells infused . ischemic stroke classification yielded 8 large - artery ( la ) , 7 small - artery ( sa ) , 1 cardioembolic ( ce ) and 4 other ( determined and undetermined ) strokes . the risk factors were as follows : 55% hypertensive , 25% diabetics , 40% hypercholesterolemia and 4% alcoholism ( table 1 ) . in group 1 ( male - to - female ratio 8:2 , mean age 48.6 7.1 years ) , the mean fm score at baseline was 23.9 7.5 and at 8 weeks it was 39.7 10.3 ( 95% ci 24.341 to 7.259 , p = 0.002 ) , whereas the mean mbi was 46.7 4.3 at baseline and 65.9 10.5 at 2 months ( 95% ci 27 to 11.3 , p = 0.009 ) . in group 2 , the male - to - female ratio was 9:1 , the mean age was 48.1 9.1 years , and the mean fm score was 21.4 6.1 and 34.1 10.3 at baseline and 8 weeks , respectively ( 95% ci 9.7 to 4.3 , p = 0.04 ) ( table 2 ) . the mean mbi at baseline was 46.2 7.8 and 61 9.8 at 2 months of follow - up ( 95% ci 11.4 to 7.8 , p = 0.01 ) . the mrc and ashworth tone scale did not show any significant improvement between baseline and 2 months in all patients . comparing the two groups , no statistically significant difference was observed in the activities of daily living scale mbi ( 95% ci 14.3 - 4.5 , p = 0.31 ) and fm score at 2 months ( 95% ci 15.2 - 5.35 , p = 0.25 ) ( fig . the ashworth tone scale and the mrc grade for hand muscles remained statistically nonsignificant at 2 months between groups 1 and 2 ( p > 0.05 ) . twenty healthy age - matched controls had a mean vegf level of 257 102.3 pg / ml and a bdnf level of 16.6 3.4 ng / ml . the baseline vegf and bdnf levels were similar ( p = 0.42 and p = 0.56 , respectively ) and hence were compared at 8 weeks . mean vegf at baseline was 336 78.6 pg / ml in group 1 and 370 91.3 pg / ml in group 2 . all patients showed improvement within the groups at 8 weeks ( p < 0.05 ) , but no statistically significant difference was observed between the two groups at 8 weeks ( mean 453.5 89.1 vs. 408.4 93.3 pg / ml , 95% ci 13.3 - 6.7 , p = 0.96 ) . the median values for vegf between the groups 1 and 2 were also nonsignificant at 8 weeks ( fig . 2 ) ( 442.1 vs. 400.3 pg / ml , p = 0.67 ) . in group 1 , mean bdnf at baseline was 18.7 6.6 and 16.1 7.6 ng / ml in group 2 . there was no difference observed within the groups at 8 weeks ( p = 0.45 and p = 0.68 for group 1 and group 2 , respectively ) . no statistically significant improvement was observed between the groups ( mean 32.8 9.2 vs. 27.3 9.1 ng / ml ; 95% ci 5.7 - 1.2 , p = 0.78 ) after 8 weeks of cell transplantation . out of 20 patients , all patients with la disease were found to have a mean baseline vegf of 500.9 pg / ml followed by small - vessel ( sv ) ( p = 0.01 , mann - whitney u test , one - tailed ) and other stroke types , whereas 1 patient with ce stroke had a vegf level of 316.4 pg / ml . bdnf showed the opposite , i.e , a high bdnf level was observed in stroke patients with other undetermined etiology followed by la and sa strokes , with 28.2 ng / ml in 1 ce stroke ( fig . 3 ) . there was no causal relationship observed with risk factors , infarct size , or other laboratory parameters ( platelets ) for both bdnf and vegf ( spearman coefficient r = 0.34 , p = 0.56 ) . we divided patients according to severity : severely affected ( mrc grade = 1 with an fm score 25 ) and moderately affected ( mrc grade > 2 3 with an fm score of 25 - 45 ) and correlated them with the serum factors . eleven of 20 patients were severely affected , with baseline median values of vegf and bdnf concentrations of 316.4 ( interquartile range 471.8 - 166.1 ) and 12.5 ng / ml ( interquartile range 23.7 - 4.05 ) , respectively . on the other hand , mean concentrations of vegf in moderately affected patients at 2 months were 314.8 versus 289.8 pg / ml ( 95% ci 116.4 - 66.5 , p = 0.45 ) and in the severely affected group 592.1 versus 487.6 pg / ml ( p = 0.76 ) without any statistically significant improvement . bdnf also showed statistically insignificant results , with the severely affected group having a mean bdnf level of 28.1 ng / ml in group 1 and 23.5 ng / ml in group 2 , and the same was observed in the moderately affected group ( 38.1 vs. 30.4 ng / ml , p = 0.32 ) ( fig . different organ systems were tested through various laboratory tests like percent hemoglobin , total leukocyte count , differential leukocyte count , platelets , prothrombin time , liver and kidney function assessed at the 1st , 3rd and 7th day after infusion . the safety outcomes included death , adverse events ( fatal or nonfatal myocardial infarction ) , epileptiform discharges and evidence of any new growth in radiological scans . cell viability , mononuclear stem cell surface markers , bacteria , syphilis , fungi , viral , mycoplasma , and endotoxin levels were tested in expansion process and transplantation . there were no early or late adverse reactions during and after transplantation observed until 8 weeks . as this was an amendment of our last trial , dosage was kept the same , i.e. , 1 million / kg body weight . cd34 + cells were characterized , with a mean count of 0.31% with 62.8 10 cells infused . ischemic stroke classification yielded 8 large - artery ( la ) , 7 small - artery ( sa ) , 1 cardioembolic ( ce ) and 4 other ( determined and undetermined ) strokes . the risk factors were as follows : 55% hypertensive , 25% diabetics , 40% hypercholesterolemia and 4% alcoholism ( table 1 ) . in group 1 ( male - to - female ratio 8:2 , mean age 48.6 7.1 years ) , the mean fm score at baseline was 23.9 7.5 and at 8 weeks it was 39.7 10.3 ( 95% ci 24.341 to 7.259 , p = 0.002 ) , whereas the mean mbi was 46.7 4.3 at baseline and 65.9 10.5 at 2 months ( 95% ci 27 to 11.3 , p = 0.009 ) . in group 2 , the male - to - female ratio was 9:1 , the mean age was 48.1 9.1 years , and the mean fm score was 21.4 6.1 and 34.1 10.3 at baseline and 8 weeks , respectively ( 95% ci 9.7 to 4.3 , p = 0.04 ) ( table 2 ) . the mean mbi at baseline was 46.2 7.8 and 61 9.8 at 2 months of follow - up ( 95% ci 11.4 to 7.8 , p = 0.01 ) . the mrc and ashworth tone scale did not show any significant improvement between baseline and 2 months in all patients . comparing the two groups , no statistically significant difference was observed in the activities of daily living scale mbi ( 95% ci 14.3 - 4.5 , p = 0.31 ) and fm score at 2 months ( 95% ci 15.2 - 5.35 , p = 0.25 ) ( fig . the ashworth tone scale and the mrc grade for hand muscles remained statistically nonsignificant at 2 months between groups 1 and 2 ( p > 0.05 ) . twenty healthy age - matched controls had a mean vegf level of 257 102.3 pg / ml and a bdnf level of 16.6 3.4 ng / ml . the baseline vegf and bdnf levels were similar ( p = 0.42 and p = 0.56 , respectively ) and hence were compared at 8 weeks . mean vegf at baseline was 336 78.6 pg / ml in group 1 and 370 91.3 pg / ml in group 2 . all patients showed improvement within the groups at 8 weeks ( p < 0.05 ) , but no statistically significant difference was observed between the two groups at 8 weeks ( mean 453.5 89.1 vs. 408.4 93.3 pg / ml , 95% ci 13.3 - 6.7 , p = 0.96 ) . the median values for vegf between the groups 1 and 2 were also nonsignificant at 8 weeks ( fig . 2 ) ( 442.1 vs. 400.3 pg / ml , p = 0.67 ) . in group 1 , mean bdnf at baseline was 18.7 6.6 and 16.1 7.6 ng / ml in group 2 . there was no difference observed within the groups at 8 weeks ( p = 0.45 and p = 0.68 for group 1 and group 2 , respectively ) . no statistically significant improvement was observed between the groups ( mean 32.8 9.2 vs. 27.3 9.1 ng / ml ; 95% ci 5.7 - 1.2 , p = 0.78 ) after 8 weeks of cell transplantation . out of 20 patients , all patients with la disease were found to have a mean baseline vegf of 500.9 pg / ml followed by small - vessel ( sv ) ( p = 0.01 , mann - whitney u test , one - tailed ) and other stroke types , whereas 1 patient with ce stroke had a vegf level of 316.4 pg / ml . bdnf showed the opposite , i.e , a high bdnf level was observed in stroke patients with other undetermined etiology followed by la and sa strokes , with 28.2 ng / ml in 1 ce stroke ( fig . 3 ) . there was no causal relationship observed with risk factors , infarct size , or other laboratory parameters ( platelets ) for both bdnf and vegf ( spearman coefficient r = 0.34 , p = 0.56 ) . we divided patients according to severity : severely affected ( mrc grade = 1 with an fm score 25 ) and moderately affected ( mrc grade > 2 3 with an fm score of 25 - 45 ) and correlated them with the serum factors . eleven of 20 patients were severely affected , with baseline median values of vegf and bdnf concentrations of 316.4 ( interquartile range 471.8 - 166.1 ) and 12.5 ng / ml ( interquartile range 23.7 - 4.05 ) , respectively . on the other hand , mean concentrations of vegf in moderately affected patients at 2 months were 314.8 versus 289.8 pg / ml ( 95% ci 116.4 - 66.5 , p = 0.45 ) and in the severely affected group 592.1 versus 487.6 pg / ml ( p = 0.76 ) without any statistically significant improvement . bdnf also showed statistically insignificant results , with the severely affected group having a mean bdnf level of 28.1 ng / ml in group 1 and 23.5 ng / ml in group 2 , and the same was observed in the moderately affected group ( 38.1 vs. 30.4 ng / ml , p = 0.32 ) ( fig . it is defined as a communication between two different cells where one releases chemical mediators to its immediate environment , which leads to a change in behavior of a cell in its adjacent territory . autologous intravenous stem cell therapy is safe and feasible in chronic stroke as evidenced by this report and has been established by us in our previous trials as well in which we used both naive , i.e. , mononuclear , and culture - expanded , i.e. , mesenchymal , stem cells . the endogeneous or intrinsic recovery mechanisms start just after an acute insult and are completed by 3 months . the exogenous recovery continues over a longer period , through cell - based therapies , gene therapy , neuromotor rehabilitation and others . we recruited patients between 3 months and 1.5 years after the index event , so that the spontaneous recovery is over . as gliosis and scarring of the brain tissue appears beyond 2 years , we had screened patients within this range . a mean of 59.9 10 mononuclear stem cells ( each ) were easily procured from 10 bone marrow samples in 1 - 2 h of culture procedure . earlier studies received 50 million cells twice , 200 - 400 million cells , 34.6 million cells and 5 - 10 million cells . compared to our last trial , savitz et al . , in a recent study , conducted autologous mononuclear stem cell transplantation in 10 subjects . eight received 10 million cells / kg body weight , 1 received 7 million cells / kg body weight , and 1 received 8 million / kg body weight . starting-2 collaborators have suggested autologous culture - expanded mesenchymal stem cells to a dose of 1 10 cells / kg , the equivalent human dose found to be effective in a rat stroke model ( 1 10 to 3 10 cells / rat ) based on mean body weight . cell - enhanced recovery has been reported with chronic delivery of cells even at 1 month after ischemia . the best route of transplantation still needs to be established although many clinical trials are opting intravenous as the mode of transplantation being safe and easily tolerable . a very recent study published by prasad et al . investigated the safety and efficacy of intravenous mononuclear stem cell transplantation in subacute ischemic stroke in which 58 patients received a mean of 280.75 million bone marrow stem cells at a median of 18.5 days after stroke onset . there was no significant difference between the bone marrow stem cell arm and the control arm in the barthel index score ( 63.1 vs. 63.6 , p = 0.92 ) , modified rankin scale shift analysis ( p = 0.53 ) or score > 3 ( 47.5 vs. 49.2% , p = 0.85 ) , and nihss score ( 6.3 vs. 7.0 , p = 0.53 ) , which is similar to our study , as we did not observe any statistically significant improvement between both groups on ashworth tone scale , mrc grade , fm scale and mbi ( 95% ci 15.2 - 5.35 , p = 0.25 ; 95% ci 14.3 - 4.5 , p = 0.31 , respectively ) with a short follow - up until 2 months . this ongoing study was planned to examine the efficacy not only on clinical , functional and radiological outcome measures but on the release of growth factors ( vegf and bdnf ) after 2 months . the growth factors such as bfgf , vegf , and angiopoietin-1 , and cardiac levels of angiogenic ligands are released as a consequence of neural repair after brain insult . bdnf is involved in many facets of brain function , including neuroplastic changes that underlie motor learning . it exerts its effects on neuroplasticity by facilitating long - term potentiation , by promoting dendritic growth and remodeling . this was observed in all the patients who had been administered a neuromotor regime for 2 months . patients in group 2 showed a statistically significant increase ( baseline to 8 weeks ) in bdnf as compared to vegf ( p = 0.01 vs. p = 0.53 ) indicating that bdnf is more likely to be involved in neuroplastic changes , which is supported by the literature . several animal studies have demonstrated significantly increased bdnf mrna levels in the rat hippocampus after a single bout of 6 h voluntary wheel running . the percent gain in bdnf was high compared to vegf ( 68 vs. 20% ) , suggesting that unlike other growth factors , bdnf is secreted in the central nervous system and blood stream through both repair initiated and an activity - dependent pathway . the activity - dependent secretion is crucial to the role of bdnf in promoting neuroplasticity in circuits activated in response to enrichened experience , i.e. , stem cells along with exercise . in a related work animals exceeding this threshold exhibited forelimb recovery and showed significant increases in motor cortex bdnf levels , while rats receiving less rehabilitation did not recover and bdnf levels remained at control levels . all groups received motor imagery and aerobic form of training for 8 weeks , which was 1 h in the hospital settings and rest exercises were advised at home . these findings indicate that bdnf also plays a key role in poststroke rehabilitation and consequently bdnf may be an important target to enhance recovery in stroke patients . serum bdnf levels rise during aerobic exercise , and quickly return to baseline levels upon exercise cessation , approximately 10 - 15 min after exercise offset . the mean concentration of both vegf and bdnf in the serum of patients with stroke was significantly higher than that of the healthy controls . vegf is a key mediator of angiogenesis , which is an important process leading to reperfusion of ischemic brain tissue after acute stroke . an increase in vegf was observed from baseline to 2 months but did not reach statistically significant levels in both groups . mean vegf expression was lowest in the serum of patients with sv , increasing in stroke with other origin and being the greatest in la stroke in both groups , suggesting that vegf could be a marker indicating the size of the infarct , which is supported by previous literature . it has been observed that transplantation of bone marrow mesenchymal stem cells into ischemic brain resulted in gain of coordinated function in rats due to bone marrow cell soluble factor release , resulting in inhibited scar formation , increased angiogenesis and neuronal commitment . posited that increased plasma vegf values last for at least 90 days in all stroke subtypes , and the clinical significance varies for different stroke types . it was found that the severely affected group had a high mean vegf of 592.1 versus 487.6 pg / ml ( p = 0.76 ) , without any statistically significant improvement between groups 1 and 2 ( fig . serum vegf at baseline was higher in severely affected patients than in moderately affected patients ( 316.1 vs. 257.4 pg / ml ) , which remained high at 2 months predicting a good functional recovery , suggesting that in chronic strokes ( without classification into stroke subtype and volume ) , vegf might have been increased already at acute onset in severely affected patients and function as an angiogenic and neuroprotective molecule . thus , higher vegf values would attenuate neuronal death and reduce infarct volume , although infarct volumes are not depicted in this analysis . senescence in stem cells is one phenomenon that is currently being studied and recently published data suggest that their efficacy is limited by natural aging . the impact of aging on stem cell populations differs between tissues and depends on a number of intrinsic / extrinsic factors , including systemic changes associated with immune system alterations . the mean age of patients in our study was a relatively younger population ( 48.6 9.6 years ) , so we assume that efficacy was not greatly compromised . comorbid or risk factors like age , hypertension , or diabetes were observed in our group , but we did not find any correlation with the cell effects nor did we find a low yield in the correlation with these risk factors . the bone marrow harvest from each patient was not compromised in spite of all the risk factors . owing to ethical concerns and rampant use of stem cells without regulation , the institute ethics committee gave approval for 10 patients initially . these patients will be followed up for up to 6 months to 2 years to gauge the efficacy in terms of serum growth factor , spectroscopic analysis and functional imaging . both groups showed improvement with increased clinical and activity of daily living scores , which would urge the researchers to ponder over psychoimmunological / placebo effects of mononuclear stem cells . doll et al . , in a review article , reported that an increase in the production of pro - inflammatory cytokines and a decrease in the production of anti - inflammatory cytokines are correlated with a larger infarct size and worse clinical outcome . tnf- , il-1 , il-6 and il-10 are some cytokines that are known to get upregulated after acute stroke . il-6 is elevated in plasma during the first week after stroke , while il-1 is elevated in the cerebrospinal fluid of severe stroke patients with peak levels at 2 - 3 days . tnf--positive neurons peak between 2 and 3 days after stroke and tnf--positive astrocytes peak between 15 h and 14 days . pro - inflammatory cytokines after mononuclear stem cell infusion has also been reported . since this trial studies mononuclear stem cell infusion in the chronic stage of stroke , where acute inflammatory pathways might have stabilized , cytokines released from mononuclear stem cells can be measured and may account as another outcome measure to study efficacy in future trials . there is evidence suggesting that human neural stem cells , human umbilical cord blood and bm - mncs secrete glial cell - derived neurotrophic factor and bdnf , igf-1 and vegf , which may protect dysfunctional motor neurons , thereby prolonging the lifespan of the stroke - induced animals . they are known to secrete nt-3 , which supports the survival , and differentiation of existing stroke - induced damage to the brain and central nervous system caused by focal or global ischemia . the pathophysiology of stroke within the first few days includes infarct progression , active inflammation and edema formation and loss of autoregulatory control from the brain . stem cell research is in the nascent stage and is being studied in all stages of stroke . in chronic stroke , the rationale of cell infusion is upregulation of growth factors , prevention of ongoing cell death , enhancement of synaptic connectivity between the host and graft , cell differentiation and integration acting as scaffolds and chaperons . a very small sample size , dose of cells , site / mode of transplantation , no fmri data analyzed and lack of in vivo monitoring of the intravenously transplanted cells are a few limitations which do not infer efficacy of bm - mnc in chronic stroke patients . the heterogeneity of stroke , infarct etiologies , topography , injury location and size are hurdles to prove the same . the interim analysis shows that the research may be continued as no risk was observed in both groups , which indicates that cell transplantation was safe in 10 of these patients . novel research directions aspire to use hematopoietic stem cells as biologically active drug delivery vehicles to facilitate tissue regeneration . it is likely that the paracrine mediators are expressed / released in a temporal and spatial manner after stem cell infusion and exercise training exerting different effects depending on the microenvironment of the host after injury .

backgroundthe emerging role of stem cell technology and transplantation has helped scientists to study their potential role in neural repair and regeneration . the fate of stem cells is determined by their niche , consisting of surrounding cells and the secreted trophic growth factors . this interim report evaluates the safety , feasibility and efficacy ( if any ) of bone marrow - derived mononuclear stem cells ( bm - mnc ) in chronic ischemic stroke by studying the release of serum vascular endothelial growth factor ( vegf ) and brain - derived neurotrophic growth factor ( bdnf).methodstwenty stroke patients and 20 age - matched healthy controls were recruited with the following inclusion criteria : 3 months to 1.5 years from the index event , medical research council ( mrc ) grade of hand muscles of at least 2 , brunnstrom stage 2 - 5 , conscious , and comprehendible . they were randomized to one group receiving autologous bm - mnc ( mean 60 - 70 million ) and to another group receiving saline infusion ( placebo ) . all patients were administered a neuromotor rehabilitation regime for 8 weeks . clinical assessments [ fugl meyer scale ( fm ) , modified barthel index ( mbi ) , mrc grade , ashworth tone scale ] were carried out and serum vegf and bdnf levels were assessed at baseline and at 8 weeks.resultsno serious adverse events were observed during the study . there was no statistically significant clinical improvement between the groups ( fm : 95% ci 15.2 - 5.35 , p = 0.25 ; mbi : 95% ci 14.3 - 4.5 , p = 0.31 ) . vegf and bdnf expression was found to be greater in group 1 compared to group 2 ( vegf : 442.1 vs. 400.3 pg / ml , p = 0.67 ; bdnf : 21.3 vs. 19.5 ng / ml ) without any statistically significant difference.conclusionautologous mononuclear stem cell infusion is safe and tolerable by chronic ischemic stroke patients . the released growth factors ( vegf and bdnf ) in the microenvironment could be due to the paracrine hypothesis of stem cell niche and neurorehabilitation regime .

Introduction Methods Bone Marrow Aspiration, Separation and Transplantation Serum Sampling for VEGF and BDNF Neurorehabilitation Regime Statistics Results Safety and Feasibility: Monitoring of Infusion-Related Toxicity Cell Dosing and Characterization Clinical Scores Serum VEGF and BDNF Expression Levels Correlation of Growth Factors with Stroke Subtype and Motor Recovery Discussion Conclusion Disclosure Statement

bone marrow - derived mononuclear stem cells ( bm - mnc ) have been used in preclinical studies suggesting increased angiogenesis in penumbral tissue following cd34 + cell transplantation , whether given systemically ( intra - arterial , intravenous or intrathecal ) or by the intracerebral route . brain - derived neurotrophic growth factor ( bdnf ) crucially promotes the synaptic and axonal plasticity associated with learning , memory and sensorimotor recovery . vascular endothelial growth factor ( vegf ) it has been shown to increase vascular permeability ; it can induce chemotaxis in monocytes in pathological conditions as well as inhibit endothelial cell apoptosis . since the realization that the beneficial effects of stem cells may be due to localized or generalized release of trophic factors , and not attributed ( in part or entirely ) to stem cell transdifferentiation or homing in to the lesioned cortex , many scientists have focused on harnessing the paracrine actions of stem cells to enhance therapeutic efficacy . the objectives were ( a ) to study safety , feasibility and efficacy ( if any ) of bm - mnc infusion in chronic ischemic stroke ; ( b ) to study early / late upregulation of serum growth factors ( vegf and bdnf ) after bm - mnc and neuromotor rehabilitation after stroke , and ( c ) to study the correlation of serum vegf and bdnf with functional recovery after stroke . thirty patients were screened who fulfilled the inclusion criteria ; among them , 5 refused to participate in the trial and 5 had deranged baseline laboratory values and were therefore excluded ; hence , 20 patients were recruited in the study . patients diagnosed with ischemic stroke 3 months to 1.5 years after the index event with medical research council ( mrc ) grade of power for wrist and hand muscles of at least 2 , brunnstrom stage of recovery of 2 - 5 , national institutes of health stroke scale ( nihss ) score of between 4 and 15 , being conscious / comprehendible as well as 20 age - matched healthy controls were recruited . one group was infused with bm - mnc and the other group with placebo ( i.e. all patients were administered a neuromotor rehabilitation regime for 8 weeks . prior to stem cell therapy , patients were screened and educated about the stem cells and bone marrow aspiration technique . the patients were examined by a neurologist and a neurophysiotherapist for muscle strength / power ( mrc grade ) and tone ( modified ashworth tone scale ) , fugl meyer ( fm ) scale for upper limb , the edinburgh handedness inventory and the modified barthel index ( mbi ) . safety and efficacy end points were assessed at 7 days , 8 weeks , 6 months and 1 year after stem cell transplantation . serum vegf and bdnf levels were measured at baseline , 8 weeks , 6 months and 1 year . this paper presents an interim analysis of safety , feasibility and efficacy of bm - mnc in chronic ischemic stroke along with the upregulation of the serum growth factors vegf and bdnf after 2 months of cell infusion . bm - mnc layers were collected , and the number of cd34 + cells ( flow cytometry method ) was counted for each patient ( table 1 ) . the mean cell viability of bm - mnc at transplantation was 98% , which was performed with trypan blue stain , and the cells were sterile and endotoxin - free during culture and at transplantation . an aseptic technique of infusion was performed with collected bm - mnc in a 50-ml sterile syringe , which was directly dissolved in 250 ml of saline and infused intravenously over 3 h. samples were immediately centrifuged ( 1,500 g/15 min ) and serum was stored at 70c until assayed . serum vegf and bdnf levels were measured by standard quantitative sandwich elisa ( quantikine ) kits , obtained from r&d systems . the lower limits of detection were 5.0 pg / ml for vegf and 3 ng / ml for bdnf ( information provided by r&d systems ) . bone marrow ( approximately 40 - 50 ml ) was aspirated under aseptic conditions from the posterior superior iliac crest in 10 chronic stroke patients . bm - mnc layers were collected , and the number of cd34 + cells ( flow cytometry method ) was counted for each patient ( table 1 ) . the mean cell viability of bm - mnc at transplantation was 98% , which was performed with trypan blue stain , and the cells were sterile and endotoxin - free during culture and at transplantation . an aseptic technique of infusion was performed with collected bm - mnc in a 50-ml sterile syringe , which was directly dissolved in 250 ml of saline and infused intravenously over 3 h. samples were immediately centrifuged ( 1,500 g/15 min ) and serum was stored at 70c until assayed . serum vegf and bdnf levels were measured by standard quantitative sandwich elisa ( quantikine ) kits , obtained from r&d systems . cell viability , mononuclear stem cell surface markers , bacteria , syphilis , fungi , viral , mycoplasma , and endotoxin levels were tested in expansion process and transplantation . in group 1 ( male - to - female ratio 8:2 , mean age 48.6 7.1 years ) , the mean fm score at baseline was 23.9 7.5 and at 8 weeks it was 39.7 10.3 ( 95% ci 24.341 to 7.259 , p = 0.002 ) , whereas the mean mbi was 46.7 4.3 at baseline and 65.9 10.5 at 2 months ( 95% ci 27 to 11.3 , p = 0.009 ) . in group 2 , the male - to - female ratio was 9:1 , the mean age was 48.1 9.1 years , and the mean fm score was 21.4 6.1 and 34.1 10.3 at baseline and 8 weeks , respectively ( 95% ci 9.7 to 4.3 , p = 0.04 ) ( table 2 ) . the mean mbi at baseline was 46.2 7.8 and 61 9.8 at 2 months of follow - up ( 95% ci 11.4 to 7.8 , p = 0.01 ) . the mrc and ashworth tone scale did not show any significant improvement between baseline and 2 months in all patients . comparing the two groups , no statistically significant difference was observed in the activities of daily living scale mbi ( 95% ci 14.3 - 4.5 , p = 0.31 ) and fm score at 2 months ( 95% ci 15.2 - 5.35 , p = 0.25 ) ( fig . the ashworth tone scale and the mrc grade for hand muscles remained statistically nonsignificant at 2 months between groups 1 and 2 ( p > 0.05 ) . twenty healthy age - matched controls had a mean vegf level of 257 102.3 pg / ml and a bdnf level of 16.6 3.4 ng / ml . the baseline vegf and bdnf levels were similar ( p = 0.42 and p = 0.56 , respectively ) and hence were compared at 8 weeks . mean vegf at baseline was 336 78.6 pg / ml in group 1 and 370 91.3 pg / ml in group 2 . all patients showed improvement within the groups at 8 weeks ( p < 0.05 ) , but no statistically significant difference was observed between the two groups at 8 weeks ( mean 453.5 89.1 vs. 408.4 93.3 pg / ml , 95% ci 13.3 - 6.7 , p = 0.96 ) . the median values for vegf between the groups 1 and 2 were also nonsignificant at 8 weeks ( fig . 2 ) ( 442.1 vs. 400.3 pg / ml , p = 0.67 ) . in group 1 , mean bdnf at baseline was 18.7 6.6 and 16.1 7.6 ng / ml in group 2 . there was no difference observed within the groups at 8 weeks ( p = 0.45 and p = 0.68 for group 1 and group 2 , respectively ) . no statistically significant improvement was observed between the groups ( mean 32.8 9.2 vs. 27.3 9.1 ng / ml ; 95% ci 5.7 - 1.2 , p = 0.78 ) after 8 weeks of cell transplantation . out of 20 patients , all patients with la disease were found to have a mean baseline vegf of 500.9 pg / ml followed by small - vessel ( sv ) ( p = 0.01 , mann - whitney u test , one - tailed ) and other stroke types , whereas 1 patient with ce stroke had a vegf level of 316.4 pg / ml . there was no causal relationship observed with risk factors , infarct size , or other laboratory parameters ( platelets ) for both bdnf and vegf ( spearman coefficient r = 0.34 , p = 0.56 ) . eleven of 20 patients were severely affected , with baseline median values of vegf and bdnf concentrations of 316.4 ( interquartile range 471.8 - 166.1 ) and 12.5 ng / ml ( interquartile range 23.7 - 4.05 ) , respectively . on the other hand , mean concentrations of vegf in moderately affected patients at 2 months were 314.8 versus 289.8 pg / ml ( 95% ci 116.4 - 66.5 , p = 0.45 ) and in the severely affected group 592.1 versus 487.6 pg / ml ( p = 0.76 ) without any statistically significant improvement . bdnf also showed statistically insignificant results , with the severely affected group having a mean bdnf level of 28.1 ng / ml in group 1 and 23.5 ng / ml in group 2 , and the same was observed in the moderately affected group ( 38.1 vs. 30.4 ng / ml , p = 0.32 ) ( fig . cell viability , mononuclear stem cell surface markers , bacteria , syphilis , fungi , viral , mycoplasma , and endotoxin levels were tested in expansion process and transplantation . in group 1 ( male - to - female ratio 8:2 , mean age 48.6 7.1 years ) , the mean fm score at baseline was 23.9 7.5 and at 8 weeks it was 39.7 10.3 ( 95% ci 24.341 to 7.259 , p = 0.002 ) , whereas the mean mbi was 46.7 4.3 at baseline and 65.9 10.5 at 2 months ( 95% ci 27 to 11.3 , p = 0.009 ) . in group 2 , the male - to - female ratio was 9:1 , the mean age was 48.1 9.1 years , and the mean fm score was 21.4 6.1 and 34.1 10.3 at baseline and 8 weeks , respectively ( 95% ci 9.7 to 4.3 , p = 0.04 ) ( table 2 ) . the mean mbi at baseline was 46.2 7.8 and 61 9.8 at 2 months of follow - up ( 95% ci 11.4 to 7.8 , p = 0.01 ) . the mrc and ashworth tone scale did not show any significant improvement between baseline and 2 months in all patients . comparing the two groups , no statistically significant difference was observed in the activities of daily living scale mbi ( 95% ci 14.3 - 4.5 , p = 0.31 ) and fm score at 2 months ( 95% ci 15.2 - 5.35 , p = 0.25 ) ( fig . the ashworth tone scale and the mrc grade for hand muscles remained statistically nonsignificant at 2 months between groups 1 and 2 ( p > 0.05 ) . twenty healthy age - matched controls had a mean vegf level of 257 102.3 pg / ml and a bdnf level of 16.6 3.4 ng / ml . the baseline vegf and bdnf levels were similar ( p = 0.42 and p = 0.56 , respectively ) and hence were compared at 8 weeks . mean vegf at baseline was 336 78.6 pg / ml in group 1 and 370 91.3 pg / ml in group 2 . all patients showed improvement within the groups at 8 weeks ( p < 0.05 ) , but no statistically significant difference was observed between the two groups at 8 weeks ( mean 453.5 89.1 vs. 408.4 93.3 pg / ml , 95% ci 13.3 - 6.7 , p = 0.96 ) . 2 ) ( 442.1 vs. 400.3 pg / ml , p = 0.67 ) . in group 1 , mean bdnf at baseline was 18.7 6.6 and 16.1 7.6 ng / ml in group 2 . there was no difference observed within the groups at 8 weeks ( p = 0.45 and p = 0.68 for group 1 and group 2 , respectively ) . no statistically significant improvement was observed between the groups ( mean 32.8 9.2 vs. 27.3 9.1 ng / ml ; 95% ci 5.7 - 1.2 , p = 0.78 ) after 8 weeks of cell transplantation . out of 20 patients , all patients with la disease were found to have a mean baseline vegf of 500.9 pg / ml followed by small - vessel ( sv ) ( p = 0.01 , mann - whitney u test , one - tailed ) and other stroke types , whereas 1 patient with ce stroke had a vegf level of 316.4 pg / ml . there was no causal relationship observed with risk factors , infarct size , or other laboratory parameters ( platelets ) for both bdnf and vegf ( spearman coefficient r = 0.34 , p = 0.56 ) . we divided patients according to severity : severely affected ( mrc grade = 1 with an fm score 25 ) and moderately affected ( mrc grade > 2 3 with an fm score of 25 - 45 ) and correlated them with the serum factors . eleven of 20 patients were severely affected , with baseline median values of vegf and bdnf concentrations of 316.4 ( interquartile range 471.8 - 166.1 ) and 12.5 ng / ml ( interquartile range 23.7 - 4.05 ) , respectively . on the other hand , mean concentrations of vegf in moderately affected patients at 2 months were 314.8 versus 289.8 pg / ml ( 95% ci 116.4 - 66.5 , p = 0.45 ) and in the severely affected group 592.1 versus 487.6 pg / ml ( p = 0.76 ) without any statistically significant improvement . bdnf also showed statistically insignificant results , with the severely affected group having a mean bdnf level of 28.1 ng / ml in group 1 and 23.5 ng / ml in group 2 , and the same was observed in the moderately affected group ( 38.1 vs. 30.4 ng / ml , p = 0.32 ) ( fig . we recruited patients between 3 months and 1.5 years after the index event , so that the spontaneous recovery is over . a mean of 59.9 10 mononuclear stem cells ( each ) were easily procured from 10 bone marrow samples in 1 - 2 h of culture procedure . investigated the safety and efficacy of intravenous mononuclear stem cell transplantation in subacute ischemic stroke in which 58 patients received a mean of 280.75 million bone marrow stem cells at a median of 18.5 days after stroke onset . there was no significant difference between the bone marrow stem cell arm and the control arm in the barthel index score ( 63.1 vs. 63.6 , p = 0.92 ) , modified rankin scale shift analysis ( p = 0.53 ) or score > 3 ( 47.5 vs. 49.2% , p = 0.85 ) , and nihss score ( 6.3 vs. 7.0 , p = 0.53 ) , which is similar to our study , as we did not observe any statistically significant improvement between both groups on ashworth tone scale , mrc grade , fm scale and mbi ( 95% ci 15.2 - 5.35 , p = 0.25 ; 95% ci 14.3 - 4.5 , p = 0.31 , respectively ) with a short follow - up until 2 months . this ongoing study was planned to examine the efficacy not only on clinical , functional and radiological outcome measures but on the release of growth factors ( vegf and bdnf ) after 2 months . patients in group 2 showed a statistically significant increase ( baseline to 8 weeks ) in bdnf as compared to vegf ( p = 0.01 vs. p = 0.53 ) indicating that bdnf is more likely to be involved in neuroplastic changes , which is supported by the literature . the percent gain in bdnf was high compared to vegf ( 68 vs. 20% ) , suggesting that unlike other growth factors , bdnf is secreted in the central nervous system and blood stream through both repair initiated and an activity - dependent pathway . the mean concentration of both vegf and bdnf in the serum of patients with stroke was significantly higher than that of the healthy controls . it was found that the severely affected group had a high mean vegf of 592.1 versus 487.6 pg / ml ( p = 0.76 ) , without any statistically significant improvement between groups 1 and 2 ( fig . serum vegf at baseline was higher in severely affected patients than in moderately affected patients ( 316.1 vs. 257.4 pg / ml ) , which remained high at 2 months predicting a good functional recovery , suggesting that in chronic strokes ( without classification into stroke subtype and volume ) , vegf might have been increased already at acute onset in severely affected patients and function as an angiogenic and neuroprotective molecule . since this trial studies mononuclear stem cell infusion in the chronic stage of stroke , where acute inflammatory pathways might have stabilized , cytokines released from mononuclear stem cells can be measured and may account as another outcome measure to study efficacy in future trials . there is evidence suggesting that human neural stem cells , human umbilical cord blood and bm - mncs secrete glial cell - derived neurotrophic factor and bdnf , igf-1 and vegf , which may protect dysfunctional motor neurons , thereby prolonging the lifespan of the stroke - induced animals . in chronic stroke , the rationale of cell infusion is upregulation of growth factors , prevention of ongoing cell death , enhancement of synaptic connectivity between the host and graft , cell differentiation and integration acting as scaffolds and chaperons .

bone marrow - derived mononuclear stem cells ( bm - mnc ) have been used in preclinical studies suggesting increased angiogenesis in penumbral tissue following cd34 + cell transplantation , whether given systemically ( intra - arterial , intravenous or intrathecal ) or by the intracerebral route . brain - derived neurotrophic growth factor ( bdnf ) crucially promotes the synaptic and axonal plasticity associated with learning , memory and sensorimotor recovery . it has also been used to induce neurogenesis after focal ischemia , thereby increasing the number of newborn neurons in several regions of the brain enhancing neural structural plasticity . recently , it was shown that both vegf and its receptor flt-1 are upregulated in both neurons and blood vessels in the penumbra after transient or permanent occlusion of the middle cerebral artery in the rat . cell treatment or treatment with a stem cell - containing population is nascent in the current stage and has met enormous skepticism in the field of cell therapeutics . since the realization that the beneficial effects of stem cells may be due to localized or generalized release of trophic factors , and not attributed ( in part or entirely ) to stem cell transdifferentiation or homing in to the lesioned cortex , many scientists have focused on harnessing the paracrine actions of stem cells to enhance therapeutic efficacy . repair mechanisms in stroke are related to acute injury ( first epoch ) and they are said to occur in the initial few hours after the acute event when changes in blood flow , metabolism and ischemic cascade are most active . a second epoch is related to the upregulation of growth factors which continues for days to weeks and is referred to as endogenous repair - related events . the objectives were ( a ) to study safety , feasibility and efficacy ( if any ) of bm - mnc infusion in chronic ischemic stroke ; ( b ) to study early / late upregulation of serum growth factors ( vegf and bdnf ) after bm - mnc and neuromotor rehabilitation after stroke , and ( c ) to study the correlation of serum vegf and bdnf with functional recovery after stroke . patients diagnosed with ischemic stroke 3 months to 1.5 years after the index event with medical research council ( mrc ) grade of power for wrist and hand muscles of at least 2 , brunnstrom stage of recovery of 2 - 5 , national institutes of health stroke scale ( nihss ) score of between 4 and 15 , being conscious / comprehendible as well as 20 age - matched healthy controls were recruited . prior to stem cell therapy , patients were screened and educated about the stem cells and bone marrow aspiration technique . the patients were examined by a neurologist and a neurophysiotherapist for muscle strength / power ( mrc grade ) and tone ( modified ashworth tone scale ) , fugl meyer ( fm ) scale for upper limb , the edinburgh handedness inventory and the modified barthel index ( mbi ) . the study is a randomized placebo - controlled clinical trial which was approved by the ic - scrt ( institute committee for stem cell research and therapy ) and is registered with ctri ( ctri/2014/09/005028 ) . this paper presents an interim analysis of safety , feasibility and efficacy of bm - mnc in chronic ischemic stroke along with the upregulation of the serum growth factors vegf and bdnf after 2 months of cell infusion . the mean cell viability of bm - mnc at transplantation was 98% , which was performed with trypan blue stain , and the cells were sterile and endotoxin - free during culture and at transplantation . general aerobic exercise training was administered with 10 min of static cycling and brisk walk ( the extent the patient can ) , partial squats ( hold for 7 s ) , and step up and down . analysis was done to identify differences in growth factor expression in chronic stroke , in relation to stroke subtypes and etiology , with clinical disability , including short - term follow - up . the mean cell viability of bm - mnc at transplantation was 98% , which was performed with trypan blue stain , and the cells were sterile and endotoxin - free during culture and at transplantation . general aerobic exercise training was administered with 10 min of static cycling and brisk walk ( the extent the patient can ) , partial squats ( hold for 7 s ) , and step up and down . for correlative analysis , the spearman rank correlation coefficient ( r ) analysis was done to identify differences in growth factor expression in chronic stroke , in relation to stroke subtypes and etiology , with clinical disability , including short - term follow - up . cell viability , mononuclear stem cell surface markers , bacteria , syphilis , fungi , viral , mycoplasma , and endotoxin levels were tested in expansion process and transplantation . in group 1 ( male - to - female ratio 8:2 , mean age 48.6 7.1 years ) , the mean fm score at baseline was 23.9 7.5 and at 8 weeks it was 39.7 10.3 ( 95% ci 24.341 to 7.259 , p = 0.002 ) , whereas the mean mbi was 46.7 4.3 at baseline and 65.9 10.5 at 2 months ( 95% ci 27 to 11.3 , p = 0.009 ) . in group 2 , the male - to - female ratio was 9:1 , the mean age was 48.1 9.1 years , and the mean fm score was 21.4 6.1 and 34.1 10.3 at baseline and 8 weeks , respectively ( 95% ci 9.7 to 4.3 , p = 0.04 ) ( table 2 ) . the mean mbi at baseline was 46.2 7.8 and 61 9.8 at 2 months of follow - up ( 95% ci 11.4 to 7.8 , p = 0.01 ) . comparing the two groups , no statistically significant difference was observed in the activities of daily living scale mbi ( 95% ci 14.3 - 4.5 , p = 0.31 ) and fm score at 2 months ( 95% ci 15.2 - 5.35 , p = 0.25 ) ( fig . twenty healthy age - matched controls had a mean vegf level of 257 102.3 pg / ml and a bdnf level of 16.6 3.4 ng / ml . all patients showed improvement within the groups at 8 weeks ( p < 0.05 ) , but no statistically significant difference was observed between the two groups at 8 weeks ( mean 453.5 89.1 vs. 408.4 93.3 pg / ml , 95% ci 13.3 - 6.7 , p = 0.96 ) . no statistically significant improvement was observed between the groups ( mean 32.8 9.2 vs. 27.3 9.1 ng / ml ; 95% ci 5.7 - 1.2 , p = 0.78 ) after 8 weeks of cell transplantation . out of 20 patients , all patients with la disease were found to have a mean baseline vegf of 500.9 pg / ml followed by small - vessel ( sv ) ( p = 0.01 , mann - whitney u test , one - tailed ) and other stroke types , whereas 1 patient with ce stroke had a vegf level of 316.4 pg / ml . bdnf showed the opposite , i.e , a high bdnf level was observed in stroke patients with other undetermined etiology followed by la and sa strokes , with 28.2 ng / ml in 1 ce stroke ( fig . we divided patients according to severity : severely affected ( mrc grade = 1 with an fm score 25 ) and moderately affected ( mrc grade > 2 3 with an fm score of 25 - 45 ) and correlated them with the serum factors . eleven of 20 patients were severely affected , with baseline median values of vegf and bdnf concentrations of 316.4 ( interquartile range 471.8 - 166.1 ) and 12.5 ng / ml ( interquartile range 23.7 - 4.05 ) , respectively . on the other hand , mean concentrations of vegf in moderately affected patients at 2 months were 314.8 versus 289.8 pg / ml ( 95% ci 116.4 - 66.5 , p = 0.45 ) and in the severely affected group 592.1 versus 487.6 pg / ml ( p = 0.76 ) without any statistically significant improvement . bdnf also showed statistically insignificant results , with the severely affected group having a mean bdnf level of 28.1 ng / ml in group 1 and 23.5 ng / ml in group 2 , and the same was observed in the moderately affected group ( 38.1 vs. 30.4 ng / ml , p = 0.32 ) ( fig . cell viability , mononuclear stem cell surface markers , bacteria , syphilis , fungi , viral , mycoplasma , and endotoxin levels were tested in expansion process and transplantation . in group 1 ( male - to - female ratio 8:2 , mean age 48.6 7.1 years ) , the mean fm score at baseline was 23.9 7.5 and at 8 weeks it was 39.7 10.3 ( 95% ci 24.341 to 7.259 , p = 0.002 ) , whereas the mean mbi was 46.7 4.3 at baseline and 65.9 10.5 at 2 months ( 95% ci 27 to 11.3 , p = 0.009 ) . in group 2 , the male - to - female ratio was 9:1 , the mean age was 48.1 9.1 years , and the mean fm score was 21.4 6.1 and 34.1 10.3 at baseline and 8 weeks , respectively ( 95% ci 9.7 to 4.3 , p = 0.04 ) ( table 2 ) . the mean mbi at baseline was 46.2 7.8 and 61 9.8 at 2 months of follow - up ( 95% ci 11.4 to 7.8 , p = 0.01 ) . comparing the two groups , no statistically significant difference was observed in the activities of daily living scale mbi ( 95% ci 14.3 - 4.5 , p = 0.31 ) and fm score at 2 months ( 95% ci 15.2 - 5.35 , p = 0.25 ) ( fig . twenty healthy age - matched controls had a mean vegf level of 257 102.3 pg / ml and a bdnf level of 16.6 3.4 ng / ml . all patients showed improvement within the groups at 8 weeks ( p < 0.05 ) , but no statistically significant difference was observed between the two groups at 8 weeks ( mean 453.5 89.1 vs. 408.4 93.3 pg / ml , 95% ci 13.3 - 6.7 , p = 0.96 ) . no statistically significant improvement was observed between the groups ( mean 32.8 9.2 vs. 27.3 9.1 ng / ml ; 95% ci 5.7 - 1.2 , p = 0.78 ) after 8 weeks of cell transplantation . out of 20 patients , all patients with la disease were found to have a mean baseline vegf of 500.9 pg / ml followed by small - vessel ( sv ) ( p = 0.01 , mann - whitney u test , one - tailed ) and other stroke types , whereas 1 patient with ce stroke had a vegf level of 316.4 pg / ml . bdnf showed the opposite , i.e , a high bdnf level was observed in stroke patients with other undetermined etiology followed by la and sa strokes , with 28.2 ng / ml in 1 ce stroke ( fig . we divided patients according to severity : severely affected ( mrc grade = 1 with an fm score 25 ) and moderately affected ( mrc grade > 2 3 with an fm score of 25 - 45 ) and correlated them with the serum factors . eleven of 20 patients were severely affected , with baseline median values of vegf and bdnf concentrations of 316.4 ( interquartile range 471.8 - 166.1 ) and 12.5 ng / ml ( interquartile range 23.7 - 4.05 ) , respectively . on the other hand , mean concentrations of vegf in moderately affected patients at 2 months were 314.8 versus 289.8 pg / ml ( 95% ci 116.4 - 66.5 , p = 0.45 ) and in the severely affected group 592.1 versus 487.6 pg / ml ( p = 0.76 ) without any statistically significant improvement . bdnf also showed statistically insignificant results , with the severely affected group having a mean bdnf level of 28.1 ng / ml in group 1 and 23.5 ng / ml in group 2 , and the same was observed in the moderately affected group ( 38.1 vs. 30.4 ng / ml , p = 0.32 ) ( fig . autologous intravenous stem cell therapy is safe and feasible in chronic stroke as evidenced by this report and has been established by us in our previous trials as well in which we used both naive , i.e. starting-2 collaborators have suggested autologous culture - expanded mesenchymal stem cells to a dose of 1 10 cells / kg , the equivalent human dose found to be effective in a rat stroke model ( 1 10 to 3 10 cells / rat ) based on mean body weight . investigated the safety and efficacy of intravenous mononuclear stem cell transplantation in subacute ischemic stroke in which 58 patients received a mean of 280.75 million bone marrow stem cells at a median of 18.5 days after stroke onset . there was no significant difference between the bone marrow stem cell arm and the control arm in the barthel index score ( 63.1 vs. 63.6 , p = 0.92 ) , modified rankin scale shift analysis ( p = 0.53 ) or score > 3 ( 47.5 vs. 49.2% , p = 0.85 ) , and nihss score ( 6.3 vs. 7.0 , p = 0.53 ) , which is similar to our study , as we did not observe any statistically significant improvement between both groups on ashworth tone scale , mrc grade , fm scale and mbi ( 95% ci 15.2 - 5.35 , p = 0.25 ; 95% ci 14.3 - 4.5 , p = 0.31 , respectively ) with a short follow - up until 2 months . this ongoing study was planned to examine the efficacy not only on clinical , functional and radiological outcome measures but on the release of growth factors ( vegf and bdnf ) after 2 months . patients in group 2 showed a statistically significant increase ( baseline to 8 weeks ) in bdnf as compared to vegf ( p = 0.01 vs. p = 0.53 ) indicating that bdnf is more likely to be involved in neuroplastic changes , which is supported by the literature . the percent gain in bdnf was high compared to vegf ( 68 vs. 20% ) , suggesting that unlike other growth factors , bdnf is secreted in the central nervous system and blood stream through both repair initiated and an activity - dependent pathway . the activity - dependent secretion is crucial to the role of bdnf in promoting neuroplasticity in circuits activated in response to enrichened experience , i.e. the mean concentration of both vegf and bdnf in the serum of patients with stroke was significantly higher than that of the healthy controls . mean vegf expression was lowest in the serum of patients with sv , increasing in stroke with other origin and being the greatest in la stroke in both groups , suggesting that vegf could be a marker indicating the size of the infarct , which is supported by previous literature . it has been observed that transplantation of bone marrow mesenchymal stem cells into ischemic brain resulted in gain of coordinated function in rats due to bone marrow cell soluble factor release , resulting in inhibited scar formation , increased angiogenesis and neuronal commitment . it was found that the severely affected group had a high mean vegf of 592.1 versus 487.6 pg / ml ( p = 0.76 ) , without any statistically significant improvement between groups 1 and 2 ( fig . serum vegf at baseline was higher in severely affected patients than in moderately affected patients ( 316.1 vs. 257.4 pg / ml ) , which remained high at 2 months predicting a good functional recovery , suggesting that in chronic strokes ( without classification into stroke subtype and volume ) , vegf might have been increased already at acute onset in severely affected patients and function as an angiogenic and neuroprotective molecule . the impact of aging on stem cell populations differs between tissues and depends on a number of intrinsic / extrinsic factors , including systemic changes associated with immune system alterations . comorbid or risk factors like age , hypertension , or diabetes were observed in our group , but we did not find any correlation with the cell effects nor did we find a low yield in the correlation with these risk factors . these patients will be followed up for up to 6 months to 2 years to gauge the efficacy in terms of serum growth factor , spectroscopic analysis and functional imaging . , in a review article , reported that an increase in the production of pro - inflammatory cytokines and a decrease in the production of anti - inflammatory cytokines are correlated with a larger infarct size and worse clinical outcome . il-6 is elevated in plasma during the first week after stroke , while il-1 is elevated in the cerebrospinal fluid of severe stroke patients with peak levels at 2 - 3 days . since this trial studies mononuclear stem cell infusion in the chronic stage of stroke , where acute inflammatory pathways might have stabilized , cytokines released from mononuclear stem cells can be measured and may account as another outcome measure to study efficacy in future trials . there is evidence suggesting that human neural stem cells , human umbilical cord blood and bm - mncs secrete glial cell - derived neurotrophic factor and bdnf , igf-1 and vegf , which may protect dysfunctional motor neurons , thereby prolonging the lifespan of the stroke - induced animals . they are known to secrete nt-3 , which supports the survival , and differentiation of existing stroke - induced damage to the brain and central nervous system caused by focal or global ischemia . in chronic stroke , the rationale of cell infusion is upregulation of growth factors , prevention of ongoing cell death , enhancement of synaptic connectivity between the host and graft , cell differentiation and integration acting as scaffolds and chaperons . a very small sample size , dose of cells , site / mode of transplantation , no fmri data analyzed and lack of in vivo monitoring of the intravenously transplanted cells are a few limitations which do not infer efficacy of bm - mnc in chronic stroke patients . it is likely that the paracrine mediators are expressed / released in a temporal and spatial manner after stem cell infusion and exercise training exerting different effects depending on the microenvironment of the host after injury .

vulvar and vaginal atrophy ( vva ) is a chronic and progressive medical condition that develops as a result of the decline of estrogen levels . the process of atrophy is universal following the decline in estrogen with physiological changes taking place in the vulval , vaginal and urogenital epithelia . these changes underlie a broad range of genital and urinary symptoms , including dryness , burning , dyspareunia , loss of vaginal secretions , leukorrhea , vulvar pruritus , feeling of pressure , itching , urethral discomfort , frequency and urgency of urination , hematuria , urinary tract infection and dysuria . up to 50% of postmenopausal women suffer from the symptoms of vva , with the onset of these symptoms varying from one woman to another . the presence and severity of these symptoms are variable , ranging from mild discomfort to great impairment and can be influenced by age , time and type of menopause as well as sexual activity and other personal history elements . although vva has a negative impact on sexual health and quality of life , only a minority of postmenopausal women will ever seek medical treatment for their condition . the principles of treatment in women with established vaginal atrophy are to restore urogenital physiology and to alleviate symptoms . treatment recommendations for vva include first - line therapy with non - hormonal lubricants with intercourse and , if indicated , regular use of long - acting vaginal moisturizers . however , these approaches seldom restore premenopausal anatomy or physiology and do not provide a long - term solution . local estrogen therapy may be used in postmenopausal women with symptomatic vva who do not respond to lubricants and moisturizers . however , long - term safety , hormone exposure and side - effects in general have been cited as concerns that women have associated with use of prescription vaginal products and a potential systemic effect of local estrogens can not totally be excluded . furthermore , studies have suggested that satisfaction with the available treatment options is low , with common reasons for dissatisfaction including messiness , ritual and inconvenience of administration and interference with the spontaneity of sex . therefore , there is a need for novel pharmacological treatment options that are easy to administer by women with symptoms of vva . recent clinical trial programs for drugs to treat vva are based on draft guidance from the us food and drug administration ( fda ) , which specifies that a new product must demonstrate efficacy at three co - primary endpoints , namely a change in : ( 1 ) maturation index ( decrease in the percentage of parabasal vaginal cells and increase in the percentage of superficial vaginal cells ) , ( 2 ) vaginal ph , and ( 3 ) severity of the patient - reported most bothersome symptom ( mbs ) . the severity of mbs is based on self - assessment of the symptoms of vva by the patient and includes vaginal dryness , vaginal pain associated with sexual activity ( dyspareunia ) , vaginal and/or vulvar irritation / itching , dysuria and vaginal bleeding associated with sexual activity . patients are required to rate the severity of the first four symptoms as none , mild , moderate or severe and the presence of vaginal bleeding after intercourse as either present or absent . the severity score is usually given a numerical value ( none = 0 , mild = 1 , moderate = 2 and severe = 3 ) enabling a mean change from baseline to be calculated . the mbs must be chosen at baseline from the symptoms rated as moderate or severe and the mean change in mbs score from baseline is used to evaluate symptomatic improvement . although this approach is an important primary measure of efficacy and is a major step forward in standardizing measurement of self - assessed changes in the severity of vva to validate potential new treatments , it is not easy to interpret what the resulting ospemifene is a novel selective estrogen receptor modulator ( serm ) , licensed in the usa for oral treatment of moderate to severe dyspareunia , a symptom of vva , due to menopause , and current recommendations from the north american menopause society include the use of ospemifene for treatment of these symptoms . pivotal studies with ospemifene have demonstrated the tolerability and efficacy of 12 weeks of ospemifene treatment in improving the mbs of dyspareunia and vaginal dryness . these studies of ospemifene have reported the mean change from baseline ( range : ospemifene , 1.19 to 1.5 ; placebo , 0.84 to 1.2 ) . while these data are important to show the statistical significance of ospemifene treatment , it would be beneficial to have data that are applicable to everyday clinical practice to assist health - care professionals in understanding what can be expected from prescribed treatments . this will be useful for defining the patient 's expectation of potential benefits and limitations of treatment . in the ospemifene clinical trial program , the majority of the symptoms reported as mbs were either vaginal dryness or dyspareunia . the objective of the current analysis was to explore clinically relevant differences in the severity of vaginal dryness or dyspareunia in postmenopausal women treated with ospemifene 60 mg / day compared with placebo , using data of the primary endpoint from the two previously published studies . the safety and tolerability profile of 12 weeks of ospemifene in postmenopausal women , including a risk benefit discussion , has been reported previously and is therefore not included in this current analysis . furthermore , safety data on ospemifene for the treatment of vva in postmenopausal women for 52 weeks and up to 64 weeks for women without a uterus have also been reported elsewhere . the current study analyzes the results of two previously conducted trials evaluating the safety and efficacy of oral ospemifene 60 mg / day for the treatment of the symptoms of vva ( nct00276094/sponsor protocol no . 1550310 , referred to herein as study 310 ; nct00729469/sponsor protocol no . 15 - 50821 , referred to herein as study 821 ) . both studies were multicenter , randomized , double - blind , 12-week phase - iii studies in postmenopausal women , aged 4080 years , with the following criteria of vva : ( 1 ) 5% or less superficial cells on the vaginal smear ( maturation index ) ; ( 2 ) vaginal ph > 5.0 ; ( 3 ) at least one moderate or severe symptom of vva ( study 310 ) , or moderate to severe vaginal dryness or vaginal pain associated with sexual activity ( study 821 ) . a total of 826 postmenopausal women were randomized ( 1 : 1 : 1 ) and treated with either ospemifene 30 mg / day ( n = 282 ) , ospemifene 60 mg / day ( n = 276 ) or placebo ( n = 268 ) for up to 12 weeks in study 310 . a total of 919 postmenopausal women with either vaginal dryness or dyspareunia as their mbs were randomized ( 1 : 1 ) and treated with either ospemifene 60 mg / day ( n = 463 ) or placebo ( n = 456 ) for up to 12 weeks in study 821 . a four - point scoring system ( none = 0 ; mild = 1 ; moderate = 2 ; severe = 3 ) was used to evaluate the severity of the co - primary endpoints of dryness and dyspareunia in these studies ; the mean change from baseline , based on this scoring system for the co - primary endpoint in each study has been reported elsewhere and is shown in table 1 . in both studies , the women in all treatment groups were supplied with a non - hormonal lubricant ( k - y jelly , mcneil - ppc , inc . , nj , usa ) and were instructed to use as needed . primary efficacy analysis : change from baseline to week 12 in most bothersome symptom ( itt , locf ) a last - observation - carried - forward ( locf ) approach was used for those discontinuing prematurely for the analyses of efficacy variables . both studies were approved by the institutional review board or ethics committee for each site , the declaration of helsinki was followed , and informed consent obtained from all patients . this current analysis focuses on the women who received either ospemifene 60 mg / day or placebo in both the 310 and 821 studies . using the data from the co - primary endpoints , analysis of clinically meaningful differences in the patient 's condition was assessed by the concepts of improvement , substantial improvement and relief ( figure 1 ) of symptoms in the 60 mg ospemifene group compared to placebo : improvement was defined as a reduction in one or more units on the four - point severity scoring system ( this includes patients whose baseline score changed from severe to none , mild or moderate , from moderate to mild or none , and from mild to none ) . substantial improvement was defined as a reduction in two or three units on the four - point severity scoring system ( this includes patients whose baseline score changed from severe or moderate to none , or from severe to mild ) . relief was defined as having a severity score at week 12 of mild or none ( i.e. does not signify a change , but records the final score ) . the concepts of improvement , relief and substantial improvement in symptoms of vulvar and vaginal atrophy to assess clinically meaningful differences in the patient 's condition . severity was determined using a four - point scoring system ( none = 0 ; mild = 1 ; moderate = 2 ; severe = 3 ) efficacy analysis included the change from baseline to week 4 and week 12 in the severity of symptoms . within this model , the response variables were the change from baseline to week 4/week 12 ; for missing values , the last observation was used ( the locf ) . the baseline value was the covariate , and treatment and study center were the fixed effects . the relative difference of the effect of ospemifene versus placebo in improvement , substantial improvement or relief in dryness or dyspareunia was determined using the following calculation per effect : ( proportion of patients ( % ) on ospemifene 60 mg proportion of patients ( % ) on placebo)/proportion of patients ( % ) on placebo . change from baseline to week 4 and week 12 in the severity of symptoms was analyzed using a cochran mantel haenszel row mean scores test , controlling for uterine status ( intact uterus or post - hysterectomy ) and study center . improvement , relief and substantial improvement were analyzed using the fisher 's exact two - sided test . the current study analyzes the results of two previously conducted trials evaluating the safety and efficacy of oral ospemifene 60 mg / day for the treatment of the symptoms of vva ( nct00276094/sponsor protocol no . 1550310 , referred to herein as study 310 ; nct00729469/sponsor protocol no . 15 - 50821 , referred to herein as study 821 ) . both studies were multicenter , randomized , double - blind , 12-week phase - iii studies in postmenopausal women , aged 4080 years , with the following criteria of vva : ( 1 ) 5% or less superficial cells on the vaginal smear ( maturation index ) ; ( 2 ) vaginal ph > 5.0 ; ( 3 ) at least one moderate or severe symptom of vva ( study 310 ) , or moderate to severe vaginal dryness or vaginal pain associated with sexual activity ( study 821 ) . a total of 826 postmenopausal women were randomized ( 1 : 1 : 1 ) and treated with either ospemifene 30 mg / day ( n = 282 ) , ospemifene 60 mg / day ( n = 276 ) or placebo ( n = 268 ) for up to 12 weeks in study 310 . a total of 919 postmenopausal women with either vaginal dryness or dyspareunia as their mbs were randomized ( 1 : 1 ) and treated with either ospemifene 60 mg / day ( n = 463 ) or placebo ( n = 456 ) for up to 12 weeks in study 821 . a four - point scoring system ( none = 0 ; mild = 1 ; moderate = 2 ; severe = 3 ) was used to evaluate the severity of the co - primary endpoints of dryness and dyspareunia in these studies ; the mean change from baseline , based on this scoring system for the co - primary endpoint in each study has been reported elsewhere and is shown in table 1 . in both studies , the women in all treatment groups were supplied with a non - hormonal lubricant ( k - y jelly , mcneil - ppc , inc . , nj , usa ) and were instructed to use as needed . primary efficacy analysis : change from baseline to week 12 in most bothersome symptom ( itt , locf ) a last - observation - carried - forward ( locf ) approach was used for those discontinuing prematurely for the analyses of efficacy variables . both studies were approved by the institutional review board or ethics committee for each site , the declaration of helsinki was followed , and informed consent obtained from all patients . this current analysis focuses on the women who received either ospemifene 60 mg / day or placebo in both the 310 and 821 studies . using the data from the co - primary endpoints , analysis of clinically meaningful differences in the patient 's condition was assessed by the concepts of improvement , substantial improvement and relief ( figure 1 ) of symptoms in the 60 mg ospemifene group compared to placebo : improvement was defined as a reduction in one or more units on the four - point severity scoring system ( this includes patients whose baseline score changed from severe to none , mild or moderate , from moderate to mild or none , and from mild to none ) . substantial improvement was defined as a reduction in two or three units on the four - point severity scoring system ( this includes patients whose baseline score changed from severe or moderate to none , or from severe to mild ) . relief was defined as having a severity score at week 12 of mild or none ( i.e. does not signify a change , but records the final score ) . the concepts of improvement , relief and substantial improvement in symptoms of vulvar and vaginal atrophy to assess clinically meaningful differences in the patient 's condition . severity was determined using a four - point scoring system ( none = 0 ; mild = 1 ; moderate = 2 ; severe = 3 ) efficacy analysis included the change from baseline to week 4 and week 12 in the severity of symptoms . within this model , the response variables were the change from baseline to week 4/week 12 ; for missing values , the last observation was used ( the locf ) . the baseline value was the covariate , and treatment and study center were the fixed effects . the relative difference of the effect of ospemifene versus placebo in improvement , substantial improvement or relief in dryness or dyspareunia was determined using the following calculation per effect : ( proportion of patients ( % ) on ospemifene 60 mg proportion of patients ( % ) on placebo)/proportion of patients ( % ) on placebo . change from baseline to week 4 and week 12 in the severity of symptoms was analyzed using a cochran mantel haenszel row mean scores test , controlling for uterine status ( intact uterus or post - hysterectomy ) and study center . improvement , relief and substantial improvement were analyzed using the fisher 's exact two - sided test . the rate of discontinuation was low in both studies : 84.8% of women taking ospemifene 60 mg / day and 85.8% of women taking placebo completed the 12 weeks of study 310 ; 89.8% of women taking ospemifene 60 mg / day and 88.4% of women taking placebo completed the 12 weeks of study 821 . after 4 weeks of treatment , the frequency of lubricant application decreased slightly in the ospemifene group with no change in the placebo group ( both studies ) , while the frequency of sexual activity remained consistent in both treatment groups ( only recorded in study 821 ) . the clinical relevance of ospemifene treatment assessed using the definitions of improvement , substantial improvement and relief of vaginal dryness associated with vva is shown in figure 2 . clinically relevant differences based on the most bothersome symptom of vaginal dryness in study 310 ( n = 222 ) and study 821 ( n = 214 ) ( itt , locf ) . p values for treatment comparisons ( ospemifene 60 mg / day vs. placebo ) from fisher 's exact two - sided test for women with a mbs of vaginal dryness in study 310 , significantly more women who received ospemifene 60 mg / day for 12 weeks reported improvement ( p = 0.0101 ) , substantial improvement ( p = 0.0172 ) and relief ( p = 0.0140 ) of vaginal dryness from baseline levels compared with placebo . these proportions were similar in study 821 although the only statistical difference was with women ( p = 0.0385 ) experiencing a substantial improvement in vaginal dryness with ospemifene 60 mg / day compared with placebo ( figure 2 ) . these differences in the proportion of women responding to treatment were noticeable after 4 weeks of treatment with ospemifene 60 mg / day . the relative differences in the proportion of women with improvement , substantial improvement or relief of their mbs of vaginal dryness following treatment with ospemifene 60 mg compared with placebo are shown in table 2 . the relative differences in the proportion of women responding to treatment with ospemifene 60 mg over placebo are also apparent after 4 weeks of treatment , with a further increase in the percentage of women showing improvement , substantial improvement or relief in dryness in study 310 and substantial improvement in study 821 at week 12 . relative differences in the proportion of women with improvement , substantial improvement or relief of vaginal dryness or dyspareunia with ospemifene 60 mg compared with placebo , calculated as ( % on 60 mg ospemifene - % on placebo)/% on placebo the differences in the severity of dyspareunia associated with vva , as assessed using the definitions of improvement , substantial improvement and relief are shown in figure 3 . clinically relevant differences based on the most bothersome symptom of dyspareunia in study 310 ( n = 242 ) and study 821 ( n = 605 ) ( itt , locf ) . p values for treatment comparisons ( ospemifene 60 mg / day vs. placebo ) from fisher 's exact two - sided test among the women reporting dyspareunia as their mbs in study 310 , after 12 weeks of treatment with ospemifene 60 mg / day , there were significantly more women with improvement ( p = 0.0255 ) and relief ( p = 0.0205 ) in the severity of dyspareunia from baseline compared with placebo . there was also a trend towards significance in the proportion of women with substantial improvement ( p = 0.0799 ) in the severity of dyspareunia from baseline . these proportions were similar in study 821 , with significantly greater improvement ( p < 0.0001 ) , substantial improvement ( p = 0.0006 ) and relief ( p = 0.0001 ) in the severity of symptoms after 12 weeks of treatment with ospemifene 60 mg / day compared with placebo ( figure 3 ) . the relative differences in the proportion of women with improvement , substantial improvement or relief of their mbs of dyspareunia following treatment with ospemifene 60 mg compared with placebo are shown in table 2 . the relative increases in improvement , substantial improvement or relief of treatment with ospemifene 60 mg compared with placebo are apparent after 4 weeks of treatment and continue to increase up to 12 weeks . the clinical relevance of ospemifene treatment assessed using the definitions of improvement , substantial improvement and relief of vaginal dryness associated with vva is shown in figure 2 . clinically relevant differences based on the most bothersome symptom of vaginal dryness in study 310 ( n = 222 ) and study 821 ( n = 214 ) ( itt , locf ) . p values for treatment comparisons ( ospemifene 60 mg / day vs. placebo ) from fisher 's exact two - sided test for women with a mbs of vaginal dryness in study 310 , significantly more women who received ospemifene 60 mg / day for 12 weeks reported improvement ( p = 0.0101 ) , substantial improvement ( p = 0.0172 ) and relief ( p = 0.0140 ) of vaginal dryness from baseline levels compared with placebo . these proportions were similar in study 821 although the only statistical difference was with women ( p = 0.0385 ) experiencing a substantial improvement in vaginal dryness with ospemifene 60 mg / day compared with placebo ( figure 2 ) . these differences in the proportion of women responding to treatment were noticeable after 4 weeks of treatment with ospemifene 60 mg / day . the relative differences in the proportion of women with improvement , substantial improvement or relief of their mbs of vaginal dryness following treatment with ospemifene 60 mg compared with placebo are shown in table 2 . the relative differences in the proportion of women responding to treatment with ospemifene 60 mg over placebo are also apparent after 4 weeks of treatment , with a further increase in the percentage of women showing improvement , substantial improvement or relief in dryness in study 310 and substantial improvement in study 821 at week 12 . relative differences in the proportion of women with improvement , substantial improvement or relief of vaginal dryness or dyspareunia with ospemifene 60 mg compared with placebo , calculated as ( % on 60 mg ospemifene - % on placebo)/% on placebo the differences in the severity of dyspareunia associated with vva , as assessed using the definitions of improvement , substantial improvement and relief are shown in figure 3 . clinically relevant differences based on the most bothersome symptom of dyspareunia in study 310 ( n = 242 ) and study 821 ( n = 605 ) ( itt , locf ) . p values for treatment comparisons ( ospemifene 60 mg / day vs. placebo ) from fisher 's exact two - sided test among the women reporting dyspareunia as their mbs in study 310 , after 12 weeks of treatment with ospemifene 60 mg / day , there were significantly more women with improvement ( p = 0.0255 ) and relief ( p = 0.0205 ) in the severity of dyspareunia from baseline compared with placebo . there was also a trend towards significance in the proportion of women with substantial improvement ( p = 0.0799 ) in the severity of dyspareunia from baseline . these proportions were similar in study 821 , with significantly greater improvement ( p < 0.0001 ) , substantial improvement ( p = 0.0006 ) and relief ( p = 0.0001 ) in the severity of symptoms after 12 weeks of treatment with ospemifene 60 mg / day compared with placebo ( figure 3 ) . the relative differences in the proportion of women with improvement , substantial improvement or relief of their mbs of dyspareunia following treatment with ospemifene 60 mg compared with placebo are shown in table 2 . the relative increases in improvement , substantial improvement or relief of treatment with ospemifene 60 mg compared with placebo are apparent after 4 weeks of treatment and continue to increase up to 12 weeks . this current analysis demonstrates that approximately three - quarters of women treated for 12 weeks with ospemifene 60 mg / day experienced improvement of vaginal dryness and dyspareunia associated with vva , compared with 5060% of those who received placebo . although previous studies have demonstrated the efficacy of ospemifene on the mean change in vaginal dryness from baseline ( range : ospemifene , 1.26 to 1.3 ; placebo , 0.84 to 1.1 ) and dyspareunia ( range : ospemifene , 1.19 to 1.5 ; placebo , 0.89 to 1.2 ) , this is the first analysis to show the clinical relevance of such changes . this is particularly important as numerical measures of severity are not routinely used in clinical practice and gynecologists and their patients may therefore find it difficult to apply such knowledge to understand how treatments for vva may benefit women with symptoms attributable to the condition . the data presented here therefore provide clinically meaningful information as to the proportion of women who might expect to experience improvement or relief of their symptoms with ospemifene treatment . a number of other studies investigating treatment of vva in postmenopausal women have been published in recent years using the mbs as a co - primary endpoint . however , although the concept of the clinical relevance of changes in the severity score for the mbs with treatment has been suggested through the analysis of improvement and relief of such symptoms , this is the first manuscript to report such changes in this way . first , a one - unit improvement in severity score is often spontaneous and can occur without intervention . for example , in the ospemifene pivotal efficacy trials , patients selected their mbs at screening and again at randomization ( baseline ) . the period between screening and randomization was no more than 6 weeks , with no therapeutic intervention during this time . however , in study 821 , 14.9% of patients who described their mbs as severe at screening showed improvement by randomization , but only 0.2% showed substantial improvement at this point in time . if a substantial improvement is observed , different from placebo , it is more likely to reflect a true effect of a treatment in the patient population since a change of that magnitude in severity is rare without intervention . furthermore , in the absence of a strict definition of the degree of severity ( none , mild , moderate or severe ) , it is possible to question the significance of a change in severity score of one unit . for example , what may be perceived as moderate one day can be perceived as severe another day . however , there is little uncertainty about a change from moderate to none or from severe to mild or none . the efficacy of ospemifene in improving objective measures of vva , i.e. increasing the maturation index and decreasing the vaginal ph , has been shown in studies 310 and 821 . in both pivotal studies , 12 weeks of treatment with ospemifene 60 mg / day increased the percentage of superficial cells by approximately 10% compared with placebo ( 2% ) and decreased the percentage of parabasal cells by 3040% compared with placebo ( 04% ) . notably , in both studies , improvements in maturation index were observed after 4 weeks of treatment . the clinically relevant changes in vaginal dryness and dyspareunia with ospemifene 60 mg / day reported in this current analysis , also observed after 4 weeks of treatment , suggest that symptomatic relief follows the objective signs of improvement reported previously . taken together , these data indicate that ospemifene is treating the cause of vaginal atrophy , not just ameliorating the associated symptoms . furthermore , the onset of these benefits may be experienced within 4 weeks of treatment . in the current analysis , there is a substantial placebo effect , with improvement of dyspareunia and vaginal dryness associated with vva in approximately 50% of women who received placebo . however , it is important to note that the design of the two studies reported here allowed all participants to use a non - hormonal vaginal lubricant as needed and therefore the placebo effect also includes the effect of the lubricant . indeed , in the current analysis , the placebo effect was apparent after 4 weeks of the study this early onset of the placebo effect has also been reported previously . this early placebo effect usually wears off , as demonstrated by the continuing improvement in the ospemifene 60 mg / day group compared with placebo . not only did ospemifene 60 mg / day show efficacy over and above lubricant , but lubricant use was reduced in the ospemifene treatment group towards the end of the study . there is a need for more stringent definitions for the degree of severity for the symptoms of vva , particularly when conducting clinical trials and for comparisons of the efficacy of different products . furthermore , while such subjective measurements are essential to ensure the clinical effectiveness of new treatments that address both the underlying physiology and symptoms experienced by postmenopausal women to improve and maintain their quality of life in older age , it would also be beneficial to include a formal assessment of quality of life in the assessment of new treatments to understand fully the impact of the symptoms of vva in women . in the absence of specific vva quality - of - life rating scales , sexual quality - of - life scales are currently used as surrogates to assess the effect of vva symptoms . establishing a practical , validated questionnaire , which takes into account the impact of vva symptoms on personal , social and professional aspects of quality of life , should now be prioritized to assist in assessing the effectiveness of new treatment options for vva . this analysis provides a clinically meaningful analysis of the efficacy of ospemifene 60 mg / day on vaginal dryness and dyspareunia as a result of vva . approximately three - quarters of women treated for 12 weeks with ospemifene 60 mg / day experienced improvement of dyspareunia and vaginal dryness associated with vva , compared with 5060% who received placebo . this compares favorably with previous analyses of ospemifene 60 mg / day which demonstrate significant improvements relative to placebo in the objective measurements of vva as well as the severity of vaginal dryness and dyspareunia reported as the most bothersome symptom . in conclusion , this analysis provides informative and clinically relevant data that will enable gynecologists and their patients to assess the effectiveness that patients can expect to achieve when prescribed ospemifene for the improvement of their symptoms . conflict of interest during the past 2 years r.e.n . has had financial relationships ( lecturer , member of advisory boards and/or consultant ) with bayer - schering pharma , ely lilly , gedeon - richter , hra pharma , merck sharpe & dohme , novo nordisk , pfizer inc . has received honoraria for consultancy work and lectures from a number of pharmaceutical companies including bayer , besins , novo nordisk , pfizer , se cur and shionogi ltd . n.b . is a consultant working for shionogi ltd . during the past 2 years c.c - b . has had financial relationships ( lecturer , member of advisory boards and/or consultant ) with amgen , pierre - fabre , servier , merck sharpe & dohme , isdin , pfizer inc . v. has had financial relationships ( lecturer , member of advisory boards and/or consultant ) with bayer - schering pharma , merck sharpe & dohme , pfizer inc . , j.a.s . has served ( within the last year ) or is currently serving as a consultant to or on the advisory boards of abbvie , inc . ( thousand oaks , ca ) , amneal pharmaceuticals ( bridgewater , nj ) , apotex , inc . ( toronto , canada ) , ascend therapeutics ( herndon , va ) , depomed , inc . ( west orange , nj ) , lupin pharmaceuticals ( baltimore , md ) , meda pharmaceuticals inc . ( whitehouse station , nj ) , novartis pharmaceuticals corporation ( east hanover , nj ) , noven pharmaceuticals , inc . ( new york , ny ) , novo nordisk ( bagsvrerd , denmark ) , pfizer inc . ( florham park , nj ) , shippan point advisors llc ( upper saddle river , nj ) , sprout pharmaceuticals ( raleigh , nc ) , therapeuticsmd ( boca raton , fl ) , teva pharmaceutical industries ltd ( jerusalem , israel ) . in the last year he has received or is currently receiving grant / research support from abbvie , inc . ( quebec , quebec ) , novo nordisk ( bagsvrerd , denmark ) , novogyne ( east hanover , nj ) , palatin technologies ( cranbury , nj ) , and teva pharmaceutical industries ltd ( jerusalem , israel ) . he has also served or is currently serving on the speakers bureaux of amgen inc . ( woodcliff lake , nj ) , merck ( whitehouse station , nj ) , noven pharmaceuticals , inc . ( florham park , nj ) , and teva pharmaceutical industries ltd ( jerusalem , israel ) . j.a.s . was the chief medical officer for sprout pharmaceuticals ( raleigh , nc ) until 4 january 2013 . editorial assistance was provided by dr marion james of apothecom scopemedical ( supported by shionogi ) .

objectivesto explore clinically relevant differences in severity of vulvar and vaginal atrophy ( vva ) in postmenopausal women treated with ospemifene compared with placebo.methodsanalysis of two multicenter , randomized , double - blind , 12-week phase - iii studies in postmenopausal women ( 4080 years , with vva , treated with ospemifene 60 mg / day or placebo ( study 310 and study 821 ) ) . severity of vaginal dryness and dyspareunia were evaluated using a four - point scoring system and clinically relevant differences between ospemifene and placebo were analyzed and are presented as improvement ( reduction in 1 unit on four - point scoring system ) , substantial improvement ( reduction in 23 units on four - point scoring system ) and relief ( severity score of mild / none after 12 weeks).resultsin study 310 , significantly more women with a most bothersome symptom of dyspareunia had improvement ( 68.3% vs. 54.1% ; p = 0.0255 ) or relief ( 57.5% vs. 41.8% ; p = 0.0205 ) in the severity of dyspareunia from baseline to week 12 with ospemifene compared with placebo . for those with a most bothersome symptom of vaginal dryness , significantly more experienced improvement ( 74.6% vs. 57.7% ; p = 0.0101 ) , substantial improvement ( 42.4% vs. 26.9% ; p = 0.0172 ) and relief ( 66.1% vs. 49.0% ; p = 0.0140 ) of vaginal dryness from baseline to week 12 with ospemifene compared with placebo . proportions of women with improvement / substantial improvement / relief of symptoms of vaginal dryness or dyspareunia were similar in study 821 . clinically relevant differences were noticeable by week 4.conclusionstreatment with ospemifene was consistently associated with greater improvement , substantial improvement or relief in the severity of the most bothersome symptoms of vaginal dryness or dyspareunia compared with placebo .

INTRODUCTION METHODS Patients and study design Definition of improvement, substantial improvement and relief RESULTS Vaginal dryness Dyspareunia DISCUSSION CONCLUSION None

vulvar and vaginal atrophy ( vva ) is a chronic and progressive medical condition that develops as a result of the decline of estrogen levels . recent clinical trial programs for drugs to treat vva are based on draft guidance from the us food and drug administration ( fda ) , which specifies that a new product must demonstrate efficacy at three co - primary endpoints , namely a change in : ( 1 ) maturation index ( decrease in the percentage of parabasal vaginal cells and increase in the percentage of superficial vaginal cells ) , ( 2 ) vaginal ph , and ( 3 ) severity of the patient - reported most bothersome symptom ( mbs ) . the severity of mbs is based on self - assessment of the symptoms of vva by the patient and includes vaginal dryness , vaginal pain associated with sexual activity ( dyspareunia ) , vaginal and/or vulvar irritation / itching , dysuria and vaginal bleeding associated with sexual activity . although this approach is an important primary measure of efficacy and is a major step forward in standardizing measurement of self - assessed changes in the severity of vva to validate potential new treatments , it is not easy to interpret what the resulting ospemifene is a novel selective estrogen receptor modulator ( serm ) , licensed in the usa for oral treatment of moderate to severe dyspareunia , a symptom of vva , due to menopause , and current recommendations from the north american menopause society include the use of ospemifene for treatment of these symptoms . the objective of the current analysis was to explore clinically relevant differences in the severity of vaginal dryness or dyspareunia in postmenopausal women treated with ospemifene 60 mg / day compared with placebo , using data of the primary endpoint from the two previously published studies . the current study analyzes the results of two previously conducted trials evaluating the safety and efficacy of oral ospemifene 60 mg / day for the treatment of the symptoms of vva ( nct00276094/sponsor protocol no . both studies were multicenter , randomized , double - blind , 12-week phase - iii studies in postmenopausal women , aged 4080 years , with the following criteria of vva : ( 1 ) 5% or less superficial cells on the vaginal smear ( maturation index ) ; ( 2 ) vaginal ph > 5.0 ; ( 3 ) at least one moderate or severe symptom of vva ( study 310 ) , or moderate to severe vaginal dryness or vaginal pain associated with sexual activity ( study 821 ) . a total of 826 postmenopausal women were randomized ( 1 : 1 : 1 ) and treated with either ospemifene 30 mg / day ( n = 282 ) , ospemifene 60 mg / day ( n = 276 ) or placebo ( n = 268 ) for up to 12 weeks in study 310 . a total of 919 postmenopausal women with either vaginal dryness or dyspareunia as their mbs were randomized ( 1 : 1 ) and treated with either ospemifene 60 mg / day ( n = 463 ) or placebo ( n = 456 ) for up to 12 weeks in study 821 . a four - point scoring system ( none = 0 ; mild = 1 ; moderate = 2 ; severe = 3 ) was used to evaluate the severity of the co - primary endpoints of dryness and dyspareunia in these studies ; the mean change from baseline , based on this scoring system for the co - primary endpoint in each study has been reported elsewhere and is shown in table 1 . primary efficacy analysis : change from baseline to week 12 in most bothersome symptom ( itt , locf ) a last - observation - carried - forward ( locf ) approach was used for those discontinuing prematurely for the analyses of efficacy variables . this current analysis focuses on the women who received either ospemifene 60 mg / day or placebo in both the 310 and 821 studies . using the data from the co - primary endpoints , analysis of clinically meaningful differences in the patient 's condition was assessed by the concepts of improvement , substantial improvement and relief ( figure 1 ) of symptoms in the 60 mg ospemifene group compared to placebo : improvement was defined as a reduction in one or more units on the four - point severity scoring system ( this includes patients whose baseline score changed from severe to none , mild or moderate , from moderate to mild or none , and from mild to none ) . substantial improvement was defined as a reduction in two or three units on the four - point severity scoring system ( this includes patients whose baseline score changed from severe or moderate to none , or from severe to mild ) . the concepts of improvement , relief and substantial improvement in symptoms of vulvar and vaginal atrophy to assess clinically meaningful differences in the patient 's condition . severity was determined using a four - point scoring system ( none = 0 ; mild = 1 ; moderate = 2 ; severe = 3 ) efficacy analysis included the change from baseline to week 4 and week 12 in the severity of symptoms . the relative difference of the effect of ospemifene versus placebo in improvement , substantial improvement or relief in dryness or dyspareunia was determined using the following calculation per effect : ( proportion of patients ( % ) on ospemifene 60 mg proportion of patients ( % ) on placebo)/proportion of patients ( % ) on placebo . change from baseline to week 4 and week 12 in the severity of symptoms was analyzed using a cochran mantel haenszel row mean scores test , controlling for uterine status ( intact uterus or post - hysterectomy ) and study center . the current study analyzes the results of two previously conducted trials evaluating the safety and efficacy of oral ospemifene 60 mg / day for the treatment of the symptoms of vva ( nct00276094/sponsor protocol no . both studies were multicenter , randomized , double - blind , 12-week phase - iii studies in postmenopausal women , aged 4080 years , with the following criteria of vva : ( 1 ) 5% or less superficial cells on the vaginal smear ( maturation index ) ; ( 2 ) vaginal ph > 5.0 ; ( 3 ) at least one moderate or severe symptom of vva ( study 310 ) , or moderate to severe vaginal dryness or vaginal pain associated with sexual activity ( study 821 ) . a total of 826 postmenopausal women were randomized ( 1 : 1 : 1 ) and treated with either ospemifene 30 mg / day ( n = 282 ) , ospemifene 60 mg / day ( n = 276 ) or placebo ( n = 268 ) for up to 12 weeks in study 310 . a total of 919 postmenopausal women with either vaginal dryness or dyspareunia as their mbs were randomized ( 1 : 1 ) and treated with either ospemifene 60 mg / day ( n = 463 ) or placebo ( n = 456 ) for up to 12 weeks in study 821 . a four - point scoring system ( none = 0 ; mild = 1 ; moderate = 2 ; severe = 3 ) was used to evaluate the severity of the co - primary endpoints of dryness and dyspareunia in these studies ; the mean change from baseline , based on this scoring system for the co - primary endpoint in each study has been reported elsewhere and is shown in table 1 . primary efficacy analysis : change from baseline to week 12 in most bothersome symptom ( itt , locf ) a last - observation - carried - forward ( locf ) approach was used for those discontinuing prematurely for the analyses of efficacy variables . this current analysis focuses on the women who received either ospemifene 60 mg / day or placebo in both the 310 and 821 studies . using the data from the co - primary endpoints , analysis of clinically meaningful differences in the patient 's condition was assessed by the concepts of improvement , substantial improvement and relief ( figure 1 ) of symptoms in the 60 mg ospemifene group compared to placebo : improvement was defined as a reduction in one or more units on the four - point severity scoring system ( this includes patients whose baseline score changed from severe to none , mild or moderate , from moderate to mild or none , and from mild to none ) . the concepts of improvement , relief and substantial improvement in symptoms of vulvar and vaginal atrophy to assess clinically meaningful differences in the patient 's condition . severity was determined using a four - point scoring system ( none = 0 ; mild = 1 ; moderate = 2 ; severe = 3 ) efficacy analysis included the change from baseline to week 4 and week 12 in the severity of symptoms . the relative difference of the effect of ospemifene versus placebo in improvement , substantial improvement or relief in dryness or dyspareunia was determined using the following calculation per effect : ( proportion of patients ( % ) on ospemifene 60 mg proportion of patients ( % ) on placebo)/proportion of patients ( % ) on placebo . change from baseline to week 4 and week 12 in the severity of symptoms was analyzed using a cochran mantel haenszel row mean scores test , controlling for uterine status ( intact uterus or post - hysterectomy ) and study center . the rate of discontinuation was low in both studies : 84.8% of women taking ospemifene 60 mg / day and 85.8% of women taking placebo completed the 12 weeks of study 310 ; 89.8% of women taking ospemifene 60 mg / day and 88.4% of women taking placebo completed the 12 weeks of study 821 . the clinical relevance of ospemifene treatment assessed using the definitions of improvement , substantial improvement and relief of vaginal dryness associated with vva is shown in figure 2 . clinically relevant differences based on the most bothersome symptom of vaginal dryness in study 310 ( n = 222 ) and study 821 ( n = 214 ) ( itt , locf ) . p values for treatment comparisons ( ospemifene 60 mg / day vs. placebo ) from fisher 's exact two - sided test for women with a mbs of vaginal dryness in study 310 , significantly more women who received ospemifene 60 mg / day for 12 weeks reported improvement ( p = 0.0101 ) , substantial improvement ( p = 0.0172 ) and relief ( p = 0.0140 ) of vaginal dryness from baseline levels compared with placebo . these proportions were similar in study 821 although the only statistical difference was with women ( p = 0.0385 ) experiencing a substantial improvement in vaginal dryness with ospemifene 60 mg / day compared with placebo ( figure 2 ) . these differences in the proportion of women responding to treatment were noticeable after 4 weeks of treatment with ospemifene 60 mg / day . the relative differences in the proportion of women with improvement , substantial improvement or relief of their mbs of vaginal dryness following treatment with ospemifene 60 mg compared with placebo are shown in table 2 . the relative differences in the proportion of women responding to treatment with ospemifene 60 mg over placebo are also apparent after 4 weeks of treatment , with a further increase in the percentage of women showing improvement , substantial improvement or relief in dryness in study 310 and substantial improvement in study 821 at week 12 . relative differences in the proportion of women with improvement , substantial improvement or relief of vaginal dryness or dyspareunia with ospemifene 60 mg compared with placebo , calculated as ( % on 60 mg ospemifene - % on placebo)/% on placebo the differences in the severity of dyspareunia associated with vva , as assessed using the definitions of improvement , substantial improvement and relief are shown in figure 3 . clinically relevant differences based on the most bothersome symptom of dyspareunia in study 310 ( n = 242 ) and study 821 ( n = 605 ) ( itt , locf ) . p values for treatment comparisons ( ospemifene 60 mg / day vs. placebo ) from fisher 's exact two - sided test among the women reporting dyspareunia as their mbs in study 310 , after 12 weeks of treatment with ospemifene 60 mg / day , there were significantly more women with improvement ( p = 0.0255 ) and relief ( p = 0.0205 ) in the severity of dyspareunia from baseline compared with placebo . there was also a trend towards significance in the proportion of women with substantial improvement ( p = 0.0799 ) in the severity of dyspareunia from baseline . these proportions were similar in study 821 , with significantly greater improvement ( p < 0.0001 ) , substantial improvement ( p = 0.0006 ) and relief ( p = 0.0001 ) in the severity of symptoms after 12 weeks of treatment with ospemifene 60 mg / day compared with placebo ( figure 3 ) . the relative differences in the proportion of women with improvement , substantial improvement or relief of their mbs of dyspareunia following treatment with ospemifene 60 mg compared with placebo are shown in table 2 . the relative increases in improvement , substantial improvement or relief of treatment with ospemifene 60 mg compared with placebo are apparent after 4 weeks of treatment and continue to increase up to 12 weeks . the clinical relevance of ospemifene treatment assessed using the definitions of improvement , substantial improvement and relief of vaginal dryness associated with vva is shown in figure 2 . clinically relevant differences based on the most bothersome symptom of vaginal dryness in study 310 ( n = 222 ) and study 821 ( n = 214 ) ( itt , locf ) . p values for treatment comparisons ( ospemifene 60 mg / day vs. placebo ) from fisher 's exact two - sided test for women with a mbs of vaginal dryness in study 310 , significantly more women who received ospemifene 60 mg / day for 12 weeks reported improvement ( p = 0.0101 ) , substantial improvement ( p = 0.0172 ) and relief ( p = 0.0140 ) of vaginal dryness from baseline levels compared with placebo . these proportions were similar in study 821 although the only statistical difference was with women ( p = 0.0385 ) experiencing a substantial improvement in vaginal dryness with ospemifene 60 mg / day compared with placebo ( figure 2 ) . these differences in the proportion of women responding to treatment were noticeable after 4 weeks of treatment with ospemifene 60 mg / day . the relative differences in the proportion of women with improvement , substantial improvement or relief of their mbs of vaginal dryness following treatment with ospemifene 60 mg compared with placebo are shown in table 2 . the relative differences in the proportion of women responding to treatment with ospemifene 60 mg over placebo are also apparent after 4 weeks of treatment , with a further increase in the percentage of women showing improvement , substantial improvement or relief in dryness in study 310 and substantial improvement in study 821 at week 12 . relative differences in the proportion of women with improvement , substantial improvement or relief of vaginal dryness or dyspareunia with ospemifene 60 mg compared with placebo , calculated as ( % on 60 mg ospemifene - % on placebo)/% on placebo the differences in the severity of dyspareunia associated with vva , as assessed using the definitions of improvement , substantial improvement and relief are shown in figure 3 . clinically relevant differences based on the most bothersome symptom of dyspareunia in study 310 ( n = 242 ) and study 821 ( n = 605 ) ( itt , locf ) . p values for treatment comparisons ( ospemifene 60 mg / day vs. placebo ) from fisher 's exact two - sided test among the women reporting dyspareunia as their mbs in study 310 , after 12 weeks of treatment with ospemifene 60 mg / day , there were significantly more women with improvement ( p = 0.0255 ) and relief ( p = 0.0205 ) in the severity of dyspareunia from baseline compared with placebo . there was also a trend towards significance in the proportion of women with substantial improvement ( p = 0.0799 ) in the severity of dyspareunia from baseline . these proportions were similar in study 821 , with significantly greater improvement ( p < 0.0001 ) , substantial improvement ( p = 0.0006 ) and relief ( p = 0.0001 ) in the severity of symptoms after 12 weeks of treatment with ospemifene 60 mg / day compared with placebo ( figure 3 ) . the relative differences in the proportion of women with improvement , substantial improvement or relief of their mbs of dyspareunia following treatment with ospemifene 60 mg compared with placebo are shown in table 2 . the relative increases in improvement , substantial improvement or relief of treatment with ospemifene 60 mg compared with placebo are apparent after 4 weeks of treatment and continue to increase up to 12 weeks . this current analysis demonstrates that approximately three - quarters of women treated for 12 weeks with ospemifene 60 mg / day experienced improvement of vaginal dryness and dyspareunia associated with vva , compared with 5060% of those who received placebo . although previous studies have demonstrated the efficacy of ospemifene on the mean change in vaginal dryness from baseline ( range : ospemifene , 1.26 to 1.3 ; placebo , 0.84 to 1.1 ) and dyspareunia ( range : ospemifene , 1.19 to 1.5 ; placebo , 0.89 to 1.2 ) , this is the first analysis to show the clinical relevance of such changes . however , although the concept of the clinical relevance of changes in the severity score for the mbs with treatment has been suggested through the analysis of improvement and relief of such symptoms , this is the first manuscript to report such changes in this way . in both pivotal studies , 12 weeks of treatment with ospemifene 60 mg / day increased the percentage of superficial cells by approximately 10% compared with placebo ( 2% ) and decreased the percentage of parabasal cells by 3040% compared with placebo ( 04% ) . the clinically relevant changes in vaginal dryness and dyspareunia with ospemifene 60 mg / day reported in this current analysis , also observed after 4 weeks of treatment , suggest that symptomatic relief follows the objective signs of improvement reported previously . in the current analysis , there is a substantial placebo effect , with improvement of dyspareunia and vaginal dryness associated with vva in approximately 50% of women who received placebo . this early placebo effect usually wears off , as demonstrated by the continuing improvement in the ospemifene 60 mg / day group compared with placebo . not only did ospemifene 60 mg / day show efficacy over and above lubricant , but lubricant use was reduced in the ospemifene treatment group towards the end of the study . this analysis provides a clinically meaningful analysis of the efficacy of ospemifene 60 mg / day on vaginal dryness and dyspareunia as a result of vva . approximately three - quarters of women treated for 12 weeks with ospemifene 60 mg / day experienced improvement of dyspareunia and vaginal dryness associated with vva , compared with 5060% who received placebo . this compares favorably with previous analyses of ospemifene 60 mg / day which demonstrate significant improvements relative to placebo in the objective measurements of vva as well as the severity of vaginal dryness and dyspareunia reported as the most bothersome symptom .

these changes underlie a broad range of genital and urinary symptoms , including dryness , burning , dyspareunia , loss of vaginal secretions , leukorrhea , vulvar pruritus , feeling of pressure , itching , urethral discomfort , frequency and urgency of urination , hematuria , urinary tract infection and dysuria . however , long - term safety , hormone exposure and side - effects in general have been cited as concerns that women have associated with use of prescription vaginal products and a potential systemic effect of local estrogens can not totally be excluded . recent clinical trial programs for drugs to treat vva are based on draft guidance from the us food and drug administration ( fda ) , which specifies that a new product must demonstrate efficacy at three co - primary endpoints , namely a change in : ( 1 ) maturation index ( decrease in the percentage of parabasal vaginal cells and increase in the percentage of superficial vaginal cells ) , ( 2 ) vaginal ph , and ( 3 ) severity of the patient - reported most bothersome symptom ( mbs ) . the severity of mbs is based on self - assessment of the symptoms of vva by the patient and includes vaginal dryness , vaginal pain associated with sexual activity ( dyspareunia ) , vaginal and/or vulvar irritation / itching , dysuria and vaginal bleeding associated with sexual activity . patients are required to rate the severity of the first four symptoms as none , mild , moderate or severe and the presence of vaginal bleeding after intercourse as either present or absent . although this approach is an important primary measure of efficacy and is a major step forward in standardizing measurement of self - assessed changes in the severity of vva to validate potential new treatments , it is not easy to interpret what the resulting ospemifene is a novel selective estrogen receptor modulator ( serm ) , licensed in the usa for oral treatment of moderate to severe dyspareunia , a symptom of vva , due to menopause , and current recommendations from the north american menopause society include the use of ospemifene for treatment of these symptoms . pivotal studies with ospemifene have demonstrated the tolerability and efficacy of 12 weeks of ospemifene treatment in improving the mbs of dyspareunia and vaginal dryness . the objective of the current analysis was to explore clinically relevant differences in the severity of vaginal dryness or dyspareunia in postmenopausal women treated with ospemifene 60 mg / day compared with placebo , using data of the primary endpoint from the two previously published studies . the safety and tolerability profile of 12 weeks of ospemifene in postmenopausal women , including a risk benefit discussion , has been reported previously and is therefore not included in this current analysis . the current study analyzes the results of two previously conducted trials evaluating the safety and efficacy of oral ospemifene 60 mg / day for the treatment of the symptoms of vva ( nct00276094/sponsor protocol no . both studies were multicenter , randomized , double - blind , 12-week phase - iii studies in postmenopausal women , aged 4080 years , with the following criteria of vva : ( 1 ) 5% or less superficial cells on the vaginal smear ( maturation index ) ; ( 2 ) vaginal ph > 5.0 ; ( 3 ) at least one moderate or severe symptom of vva ( study 310 ) , or moderate to severe vaginal dryness or vaginal pain associated with sexual activity ( study 821 ) . a total of 826 postmenopausal women were randomized ( 1 : 1 : 1 ) and treated with either ospemifene 30 mg / day ( n = 282 ) , ospemifene 60 mg / day ( n = 276 ) or placebo ( n = 268 ) for up to 12 weeks in study 310 . a total of 919 postmenopausal women with either vaginal dryness or dyspareunia as their mbs were randomized ( 1 : 1 ) and treated with either ospemifene 60 mg / day ( n = 463 ) or placebo ( n = 456 ) for up to 12 weeks in study 821 . a four - point scoring system ( none = 0 ; mild = 1 ; moderate = 2 ; severe = 3 ) was used to evaluate the severity of the co - primary endpoints of dryness and dyspareunia in these studies ; the mean change from baseline , based on this scoring system for the co - primary endpoint in each study has been reported elsewhere and is shown in table 1 . in both studies , the women in all treatment groups were supplied with a non - hormonal lubricant ( k - y jelly , mcneil - ppc , inc . primary efficacy analysis : change from baseline to week 12 in most bothersome symptom ( itt , locf ) a last - observation - carried - forward ( locf ) approach was used for those discontinuing prematurely for the analyses of efficacy variables . using the data from the co - primary endpoints , analysis of clinically meaningful differences in the patient 's condition was assessed by the concepts of improvement , substantial improvement and relief ( figure 1 ) of symptoms in the 60 mg ospemifene group compared to placebo : improvement was defined as a reduction in one or more units on the four - point severity scoring system ( this includes patients whose baseline score changed from severe to none , mild or moderate , from moderate to mild or none , and from mild to none ) . the relative difference of the effect of ospemifene versus placebo in improvement , substantial improvement or relief in dryness or dyspareunia was determined using the following calculation per effect : ( proportion of patients ( % ) on ospemifene 60 mg proportion of patients ( % ) on placebo)/proportion of patients ( % ) on placebo . change from baseline to week 4 and week 12 in the severity of symptoms was analyzed using a cochran mantel haenszel row mean scores test , controlling for uterine status ( intact uterus or post - hysterectomy ) and study center . the current study analyzes the results of two previously conducted trials evaluating the safety and efficacy of oral ospemifene 60 mg / day for the treatment of the symptoms of vva ( nct00276094/sponsor protocol no . both studies were multicenter , randomized , double - blind , 12-week phase - iii studies in postmenopausal women , aged 4080 years , with the following criteria of vva : ( 1 ) 5% or less superficial cells on the vaginal smear ( maturation index ) ; ( 2 ) vaginal ph > 5.0 ; ( 3 ) at least one moderate or severe symptom of vva ( study 310 ) , or moderate to severe vaginal dryness or vaginal pain associated with sexual activity ( study 821 ) . a total of 826 postmenopausal women were randomized ( 1 : 1 : 1 ) and treated with either ospemifene 30 mg / day ( n = 282 ) , ospemifene 60 mg / day ( n = 276 ) or placebo ( n = 268 ) for up to 12 weeks in study 310 . a total of 919 postmenopausal women with either vaginal dryness or dyspareunia as their mbs were randomized ( 1 : 1 ) and treated with either ospemifene 60 mg / day ( n = 463 ) or placebo ( n = 456 ) for up to 12 weeks in study 821 . a four - point scoring system ( none = 0 ; mild = 1 ; moderate = 2 ; severe = 3 ) was used to evaluate the severity of the co - primary endpoints of dryness and dyspareunia in these studies ; the mean change from baseline , based on this scoring system for the co - primary endpoint in each study has been reported elsewhere and is shown in table 1 . in both studies , the women in all treatment groups were supplied with a non - hormonal lubricant ( k - y jelly , mcneil - ppc , inc . primary efficacy analysis : change from baseline to week 12 in most bothersome symptom ( itt , locf ) a last - observation - carried - forward ( locf ) approach was used for those discontinuing prematurely for the analyses of efficacy variables . using the data from the co - primary endpoints , analysis of clinically meaningful differences in the patient 's condition was assessed by the concepts of improvement , substantial improvement and relief ( figure 1 ) of symptoms in the 60 mg ospemifene group compared to placebo : improvement was defined as a reduction in one or more units on the four - point severity scoring system ( this includes patients whose baseline score changed from severe to none , mild or moderate , from moderate to mild or none , and from mild to none ) . the relative difference of the effect of ospemifene versus placebo in improvement , substantial improvement or relief in dryness or dyspareunia was determined using the following calculation per effect : ( proportion of patients ( % ) on ospemifene 60 mg proportion of patients ( % ) on placebo)/proportion of patients ( % ) on placebo . change from baseline to week 4 and week 12 in the severity of symptoms was analyzed using a cochran mantel haenszel row mean scores test , controlling for uterine status ( intact uterus or post - hysterectomy ) and study center . the rate of discontinuation was low in both studies : 84.8% of women taking ospemifene 60 mg / day and 85.8% of women taking placebo completed the 12 weeks of study 310 ; 89.8% of women taking ospemifene 60 mg / day and 88.4% of women taking placebo completed the 12 weeks of study 821 . after 4 weeks of treatment , the frequency of lubricant application decreased slightly in the ospemifene group with no change in the placebo group ( both studies ) , while the frequency of sexual activity remained consistent in both treatment groups ( only recorded in study 821 ) . the clinical relevance of ospemifene treatment assessed using the definitions of improvement , substantial improvement and relief of vaginal dryness associated with vva is shown in figure 2 . clinically relevant differences based on the most bothersome symptom of vaginal dryness in study 310 ( n = 222 ) and study 821 ( n = 214 ) ( itt , locf ) . p values for treatment comparisons ( ospemifene 60 mg / day vs. placebo ) from fisher 's exact two - sided test for women with a mbs of vaginal dryness in study 310 , significantly more women who received ospemifene 60 mg / day for 12 weeks reported improvement ( p = 0.0101 ) , substantial improvement ( p = 0.0172 ) and relief ( p = 0.0140 ) of vaginal dryness from baseline levels compared with placebo . the relative differences in the proportion of women with improvement , substantial improvement or relief of their mbs of vaginal dryness following treatment with ospemifene 60 mg compared with placebo are shown in table 2 . the relative differences in the proportion of women responding to treatment with ospemifene 60 mg over placebo are also apparent after 4 weeks of treatment , with a further increase in the percentage of women showing improvement , substantial improvement or relief in dryness in study 310 and substantial improvement in study 821 at week 12 . relative differences in the proportion of women with improvement , substantial improvement or relief of vaginal dryness or dyspareunia with ospemifene 60 mg compared with placebo , calculated as ( % on 60 mg ospemifene - % on placebo)/% on placebo the differences in the severity of dyspareunia associated with vva , as assessed using the definitions of improvement , substantial improvement and relief are shown in figure 3 . clinically relevant differences based on the most bothersome symptom of dyspareunia in study 310 ( n = 242 ) and study 821 ( n = 605 ) ( itt , locf ) . p values for treatment comparisons ( ospemifene 60 mg / day vs. placebo ) from fisher 's exact two - sided test among the women reporting dyspareunia as their mbs in study 310 , after 12 weeks of treatment with ospemifene 60 mg / day , there were significantly more women with improvement ( p = 0.0255 ) and relief ( p = 0.0205 ) in the severity of dyspareunia from baseline compared with placebo . there was also a trend towards significance in the proportion of women with substantial improvement ( p = 0.0799 ) in the severity of dyspareunia from baseline . these proportions were similar in study 821 , with significantly greater improvement ( p < 0.0001 ) , substantial improvement ( p = 0.0006 ) and relief ( p = 0.0001 ) in the severity of symptoms after 12 weeks of treatment with ospemifene 60 mg / day compared with placebo ( figure 3 ) . the relative differences in the proportion of women with improvement , substantial improvement or relief of their mbs of dyspareunia following treatment with ospemifene 60 mg compared with placebo are shown in table 2 . clinically relevant differences based on the most bothersome symptom of vaginal dryness in study 310 ( n = 222 ) and study 821 ( n = 214 ) ( itt , locf ) . p values for treatment comparisons ( ospemifene 60 mg / day vs. placebo ) from fisher 's exact two - sided test for women with a mbs of vaginal dryness in study 310 , significantly more women who received ospemifene 60 mg / day for 12 weeks reported improvement ( p = 0.0101 ) , substantial improvement ( p = 0.0172 ) and relief ( p = 0.0140 ) of vaginal dryness from baseline levels compared with placebo . the relative differences in the proportion of women with improvement , substantial improvement or relief of their mbs of vaginal dryness following treatment with ospemifene 60 mg compared with placebo are shown in table 2 . the relative differences in the proportion of women responding to treatment with ospemifene 60 mg over placebo are also apparent after 4 weeks of treatment , with a further increase in the percentage of women showing improvement , substantial improvement or relief in dryness in study 310 and substantial improvement in study 821 at week 12 . relative differences in the proportion of women with improvement , substantial improvement or relief of vaginal dryness or dyspareunia with ospemifene 60 mg compared with placebo , calculated as ( % on 60 mg ospemifene - % on placebo)/% on placebo the differences in the severity of dyspareunia associated with vva , as assessed using the definitions of improvement , substantial improvement and relief are shown in figure 3 . clinically relevant differences based on the most bothersome symptom of dyspareunia in study 310 ( n = 242 ) and study 821 ( n = 605 ) ( itt , locf ) . p values for treatment comparisons ( ospemifene 60 mg / day vs. placebo ) from fisher 's exact two - sided test among the women reporting dyspareunia as their mbs in study 310 , after 12 weeks of treatment with ospemifene 60 mg / day , there were significantly more women with improvement ( p = 0.0255 ) and relief ( p = 0.0205 ) in the severity of dyspareunia from baseline compared with placebo . there was also a trend towards significance in the proportion of women with substantial improvement ( p = 0.0799 ) in the severity of dyspareunia from baseline . these proportions were similar in study 821 , with significantly greater improvement ( p < 0.0001 ) , substantial improvement ( p = 0.0006 ) and relief ( p = 0.0001 ) in the severity of symptoms after 12 weeks of treatment with ospemifene 60 mg / day compared with placebo ( figure 3 ) . the relative differences in the proportion of women with improvement , substantial improvement or relief of their mbs of dyspareunia following treatment with ospemifene 60 mg compared with placebo are shown in table 2 . this current analysis demonstrates that approximately three - quarters of women treated for 12 weeks with ospemifene 60 mg / day experienced improvement of vaginal dryness and dyspareunia associated with vva , compared with 5060% of those who received placebo . although previous studies have demonstrated the efficacy of ospemifene on the mean change in vaginal dryness from baseline ( range : ospemifene , 1.26 to 1.3 ; placebo , 0.84 to 1.1 ) and dyspareunia ( range : ospemifene , 1.19 to 1.5 ; placebo , 0.89 to 1.2 ) , this is the first analysis to show the clinical relevance of such changes . however , although the concept of the clinical relevance of changes in the severity score for the mbs with treatment has been suggested through the analysis of improvement and relief of such symptoms , this is the first manuscript to report such changes in this way . furthermore , in the absence of a strict definition of the degree of severity ( none , mild , moderate or severe ) , it is possible to question the significance of a change in severity score of one unit . in both pivotal studies , 12 weeks of treatment with ospemifene 60 mg / day increased the percentage of superficial cells by approximately 10% compared with placebo ( 2% ) and decreased the percentage of parabasal cells by 3040% compared with placebo ( 04% ) . the clinically relevant changes in vaginal dryness and dyspareunia with ospemifene 60 mg / day reported in this current analysis , also observed after 4 weeks of treatment , suggest that symptomatic relief follows the objective signs of improvement reported previously . however , it is important to note that the design of the two studies reported here allowed all participants to use a non - hormonal vaginal lubricant as needed and therefore the placebo effect also includes the effect of the lubricant . indeed , in the current analysis , the placebo effect was apparent after 4 weeks of the study this early onset of the placebo effect has also been reported previously . furthermore , while such subjective measurements are essential to ensure the clinical effectiveness of new treatments that address both the underlying physiology and symptoms experienced by postmenopausal women to improve and maintain their quality of life in older age , it would also be beneficial to include a formal assessment of quality of life in the assessment of new treatments to understand fully the impact of the symptoms of vva in women . in the absence of specific vva quality - of - life rating scales , sexual quality - of - life scales are currently used as surrogates to assess the effect of vva symptoms . establishing a practical , validated questionnaire , which takes into account the impact of vva symptoms on personal , social and professional aspects of quality of life , should now be prioritized to assist in assessing the effectiveness of new treatment options for vva . approximately three - quarters of women treated for 12 weeks with ospemifene 60 mg / day experienced improvement of dyspareunia and vaginal dryness associated with vva , compared with 5060% who received placebo . this compares favorably with previous analyses of ospemifene 60 mg / day which demonstrate significant improvements relative to placebo in the objective measurements of vva as well as the severity of vaginal dryness and dyspareunia reported as the most bothersome symptom . in conclusion , this analysis provides informative and clinically relevant data that will enable gynecologists and their patients to assess the effectiveness that patients can expect to achieve when prescribed ospemifene for the improvement of their symptoms .

obstructive sleep apnea syndrome ( osas ) is a complex clinical syndrome characterized by apnea episodes , sleep fragmentation , oxygen desaturation , and increased daytime somnolence as a result of recurrent obstructive episodes in upper airways during sleep . previous studies have revealed that osas is related with many cardiovascular diseases including hypertension , coronary artery disease , arrhythmias , and congestive heart failure . basic hemodynamic mechanisms responsible from cardiovascular diseases are increased negative intrathoracic pressure , hypoxemia , and increased catecholamine release . in osas both ventricles are subjected to hemodynamic stress that induces various structural changes . the electrocardiogram ( ecg ) represents the sum of electrical activity of all myocytes within the heart and hemodynamic changes produce various ecg changes . qrs complexes mainly represent ventricular depolarization and morphological assessment of qrs complex may give important prognostic information . it has been shown in previous trials that qrs fragmentation is a predictor of sudden cardiac death and total mortality . in addition , a prolonged qrs duration has been implicated in cardiovascular events , sudden death , and total mortality . in this study , zonguldak uzun mehmet gs hastalklar hospital and admitted to sleep laboratory in order to undergo polysomnography test . each of these patients were diagnosed with osas ( apnea - hypopnea index [ ahi ] 5 events / h ) and they had no previous treatment . thirty - four consecutive participants who were shown to have no osas ( ahi < 5 events / h ) following polysomnopgraphy test were included as our control group . patients with following conditions were excluded from the study : chronic obstructive pulmonary disease on pulmonary function testing , previous history of continuous positive airway pressure treatment , stage 23 hypertension , a positive stress test , coronary artery disease and/or previous myocardial infarction , left ventricular ( lv ) dysfunction , echocardiographically proven lv hypertrophy , moderate - to - severe valve disease , renal or hepatic dysfunction , impairment of thyroid function tests , atrial fibrillation , history of dysrhythmia , patients with permanent pacemakers , bundle branch block or fascicular block , and electrolyte abnormalities , presence of infections or inflammations , hematologic disease or any known malignancy , individuals who have systemic and metabolic diseases that could adversely affect the cardiac or pulmonary structure and functions . none of the participants were taking antiarrhythmic drugs , digitalis , beta - blockers , nondihydropyridine calcium - channel blockers or any other qt prolonging medications . in this study , all of the procedures that took place were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the declaration of helsinki ( 1975 ) , as revised in 2000 . overnight polysomnography was performed in all patients by a computerized system ( alice 5 diagnostic sleep system , 55 channels ; respironics , usa ) . the test included the following variables : electrooculogram ( two channels ) , electroencephalogram ( four channels ) , electromyogram of submental muscles ( two channels ) , electromyogram of the anterior tibialis muscle of both legs ( two channels ) , thoracic and abdominal movements , pulse oximeter oxygen saturation , ecg , airflow ( with a nasal cannula ) , body position detector , and tracheal sound . the recordings were conducted at a speed of 10 mm / s , and sleep stage was scored according to the standard criteria of rechtschaffen and kales . the ahi was obtained by dividing the total number of apneas and hypopneas by the total sleep time . apneas were defined as complete cessation of airflow 10 s. hypopneas were defined as a reduction of > 50% in one of the three respiratory signals , airflow signal or either respiratory or abdominal signals of respiratory inductance plethysmography , with an associated decrease of 3% in oxygen saturation or an arousal . according to the recently updated international classification of sleep disorders published by the american academy of sleep medicine , a diagnosis of osas is to be made if the ahi is 15 events / h , independent of occurrence of symptoms , or whenever an ahi > 5 events / h is associated with any of the following : ( 1 ) sleep attacks or excessive daytime sleepiness , unrefreshing sleep , fatigue or ( 2 ) insomnia , or ( 3 ) witnessed heavy snoring and/or breathing pauses referred by the partner . osas was excluded by a negative history of sleep - related symptoms ( snoring , witnessed apneas , and excessive daytime sleepiness ) and with an ahi < 5 events / h at overnight polysomnopgraphy . patients with sleep disorders except osas such as upper airway resistance syndrome , periodic leg movement syndrome , or narcolepsy were excluded . all standard 12-lead ecgs were obtained simultaneously using a recorder set at a 2550 mm / s paper speed and a voltage calibration of 1 mv / cm . all examinations were carried out in a quiet room during spontaneous breathing , following 10 min of rest in the supine position . the ecgs were each numbered and presented to the analyzing investigators who were blind both to patient name and group information . ecg measurements were taken by two medically qualified observers blind to the name and group of patients . ecg parameters were measured using a digital caliper ( sensitivity : 1/100 mm ) by magnifying lens . the highest value for each parameter was used . fragmented qrs was diagnosed with the presence of an additional r wave ( r ' ) or notching in the nadir of the r wave or the s wave , or the presence of more than one r ( fragmentation ) in two consecutive leads , corresponding to a certain myocardial territory . echocardiographic examination was carried out with the patient resting in a supine left lateral decubitus position . lv dimensions and wall thickness were obtained from the parasternal long axis view with the m - mode cursor positioned just below the mitral leaflet tips , perpendicular to the long axis of the lv . lv end - diastolic diameter and end - systolic diameter , thickness of the interventricular septum , and posterior wall of the lv were all measured using a single operator by the standards of the american society of echocardiography . categorical data were presented as number and frequency ( % ) . in order for the suitable analysis technique to be chosen , kolmogorov smirnov and homogeneity of variance tests were undertaken . independent samples t - test was used for the two - group comparison of the normally distributed variables and mann whitney u - test for the two - group comparison of the variables without normal distribution . the coefficient of variation is calculated as the sd of the differences between the repeated measurements divided by the averages of the repeated measurements , and is expressed as a percentage . the intraobserver variabilities were 4.08% for pq , 5.60% for qrs , and 4.26% for qtc . the interobserver variabilities were 4.74% for pq , 6.65% for qrs , and 4.21% for qtc . zonguldak uzun mehmet gs hastalklar hospital and admitted to sleep laboratory in order to undergo polysomnography test . each of these patients were diagnosed with osas ( apnea - hypopnea index [ ahi ] 5 events / h ) and they had no previous treatment . thirty - four consecutive participants who were shown to have no osas ( ahi < 5 events / h ) following polysomnopgraphy test were included as our control group . patients with following conditions were excluded from the study : chronic obstructive pulmonary disease on pulmonary function testing , previous history of continuous positive airway pressure treatment , stage 23 hypertension , a positive stress test , coronary artery disease and/or previous myocardial infarction , left ventricular ( lv ) dysfunction , echocardiographically proven lv hypertrophy , moderate - to - severe valve disease , renal or hepatic dysfunction , impairment of thyroid function tests , atrial fibrillation , history of dysrhythmia , patients with permanent pacemakers , bundle branch block or fascicular block , and electrolyte abnormalities , presence of infections or inflammations , hematologic disease or any known malignancy , individuals who have systemic and metabolic diseases that could adversely affect the cardiac or pulmonary structure and functions . none of the participants were taking antiarrhythmic drugs , digitalis , beta - blockers , nondihydropyridine calcium - channel blockers or any other qt prolonging medications . in this study , all of the procedures that took place were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the declaration of helsinki ( 1975 ) , as revised in 2000 . overnight polysomnography was performed in all patients by a computerized system ( alice 5 diagnostic sleep system , 55 channels ; respironics , usa ) . the test included the following variables : electrooculogram ( two channels ) , electroencephalogram ( four channels ) , electromyogram of submental muscles ( two channels ) , electromyogram of the anterior tibialis muscle of both legs ( two channels ) , thoracic and abdominal movements , pulse oximeter oxygen saturation , ecg , airflow ( with a nasal cannula ) , body position detector , and tracheal sound . the recordings were conducted at a speed of 10 mm / s , and sleep stage was scored according to the standard criteria of rechtschaffen and kales . the ahi was obtained by dividing the total number of apneas and hypopneas by the total sleep time . apneas were defined as complete cessation of airflow 10 s. hypopneas were defined as a reduction of > 50% in one of the three respiratory signals , airflow signal or either respiratory or abdominal signals of respiratory inductance plethysmography , with an associated decrease of 3% in oxygen saturation or an arousal . according to the recently updated international classification of sleep disorders published by the american academy of sleep medicine , a diagnosis of osas is to be made if the ahi is 15 events / h , independent of occurrence of symptoms , or whenever an ahi > 5 events / h is associated with any of the following : ( 1 ) sleep attacks or excessive daytime sleepiness , unrefreshing sleep , fatigue or ( 2 ) insomnia , or ( 3 ) witnessed heavy snoring and/or breathing pauses referred by the partner . osas was excluded by a negative history of sleep - related symptoms ( snoring , witnessed apneas , and excessive daytime sleepiness ) and with an ahi < 5 events / h at overnight polysomnopgraphy . patients with sleep disorders except osas such as upper airway resistance syndrome , periodic leg movement syndrome , or narcolepsy were excluded . all standard 12-lead ecgs were obtained simultaneously using a recorder set at a 2550 mm / s paper speed and a voltage calibration of 1 mv / cm . all examinations were carried out in a quiet room during spontaneous breathing , following 10 min of rest in the supine position . the ecgs were each numbered and presented to the analyzing investigators who were blind both to patient name and group information . ecg measurements were taken by two medically qualified observers blind to the name and group of patients . ecg parameters were measured using a digital caliper ( sensitivity : 1/100 mm ) by magnifying lens . fragmented qrs was diagnosed with the presence of an additional r wave ( r ' ) or notching in the nadir of the r wave or the s wave , or the presence of more than one r ( fragmentation ) in two consecutive leads , corresponding to a certain myocardial territory . echocardiographic examination was carried out with the patient resting in a supine left lateral decubitus position . lv dimensions and wall thickness were obtained from the parasternal long axis view with the m - mode cursor positioned just below the mitral leaflet tips , perpendicular to the long axis of the lv . lv end - diastolic diameter and end - systolic diameter , thickness of the interventricular septum , and posterior wall of the lv were all measured using a single operator by the standards of the american society of echocardiography . categorical data were presented as number and frequency ( % ) . in order for the suitable analysis technique to be chosen , kolmogorov smirnov and homogeneity of variance tests were undertaken . independent samples t - test was used for the two - group comparison of the normally distributed variables and mann whitney u - test for the two - group comparison of the variables without normal distribution . the coefficient of variation is calculated as the sd of the differences between the repeated measurements divided by the averages of the repeated measurements , and is expressed as a percentage . the intraobserver variabilities were 4.08% for pq , 5.60% for qrs , and 4.26% for qtc . the interobserver variabilities were 4.74% for pq , 6.65% for qrs , and 4.21% for qtc . clinical and demographic features of both groups * independent t - test ; mann whitney u - test ; chi - square test . ahi : apnea - hypopnea index ; bmi : body mass index ; crp : c - reactive protein ; dm : diabetes mellitus ; fbg : fasting blood glucose ; hdl - c : high - density lipoprotein cholesterol ; hl : hyperlipidemia ; ht : hypertension ; ldl - c : low - density lipoprotein cholesterol ; nc : neck circumference ; tc : total cholesterol ; tg : triglycerides ; tsh : thyroid stimulating hormone ; wc : waist circumference ; osas : obstructive sleep apnea syndrome . fragmented qrs frequency was significantly higher in patients with osas ( n = 31 [ 61% ] vs. n = 12 [ 35% ] , fqrs frequency was 2.8 times higher in patients compared to controls after adjustment for gender [ table 3 ] . when osas patients were grouped according to presence of fragmented qrs , it was found that the fragmented qrs group had a longer qrs duration ( 95.0 11.6 ms vs. 102.9 14.5 ms , p = 0.045 ) [ table 4 ] . furthermore , crp level was also higher in the fragmented qrs group ( 0.26 0.32 mg / dl vs. 0.39 0.36 mg / dl , p = 0.027 ) . comparison of the electrocardiographic and echocardiographic parameters * independent t - test ; mann whitney u - test . ci : cornell index ; ef : ejection fraction ; fqrs : fragmented qrs ; ivs : interventricular septum ; la : left atrium ; lv - edd : left ventricular end - diastolic diameter ; lv - esd : left ventricular end - systolic diameter ; pq : pq interval duration ; pw : posterior wall ; qrs : qrs duration ; qtc : corrected qt interval duration ; sli : skolow - lyon index ; osas : obstructive sleep apnea syndrome . a logistic regression model for fqrs fqrs : fragmented qrs ; se : standard error ; df : degree of freedom ; ci : confidence interval ; osas : obstructive sleep apnea syndrome . clinical , demographic , electrocardiographic , and echocardiographic parameters for fragmented and non - fqrs * independent t - test ; mann whitney u - test ; chi - square test . ahi : apnea - hypopnea index ; bmi : body mass index ; ci : cornell index ; dm : diabetes mellitus ; ef : ejection fraction ; fqrs : fragmented qrs ; fbg : fasting blood glucose ; hdl - c : high - density lipoprotein cholesterol ; hl : hyperlipidemia ; ht : hypertension ; ivs : interventricular septum ; la : left atrium ; ldl - c : low - density lipoprotein cholesterol ; lv - edd : left ventricular end - diastolic diameter ; lv - esd : left ventricular end - systolic diameter ; nc : neck circumference ; pq : pq interval duration ; pw : posterior wall ; qrs : qrs duration ; qtc : corrected qt interval duration ; sli : skolow - lyon index ; tc : total cholesterol ; tg : triglycerides ; tsh : thyroid stimulating hormone ; wc : waist circumference . qrs and qtc durations were significantly longer in osas group compared to the control group ( 99.8 13.9 ms vs. 84.7 14.3 ms , p < 0.0001 ; 411.4 26.9 ms vs. 390.1 32.2 ms , p = 0.001 , respectively ) [ figures 1 and 2 ] . qrs and qtc durations were also longer in patients compared to controls after adjustment for gender ( 99.7 14.4 ms vs. 84.8 14.6 ms , p < 0.001 ; 413.3 29.1 ms vs. 387.3 29.4 ms , p < 0.001 , respectively ) . echocardiographic parameters were within normal limits in both groups , although osas group had increased lv thickness and diameters , increased left atrial diameter , and a lower ejection fraction . analysis of the patient and control groups combined revealed a weak - moderate correlation between ahi and qrs duration ( r = 0.292 , p = 0.070 ) and between ahi and qtc duration ( r = 0.338 , p = 0.020 ) . however , there was no correlation between ahi and qrs duration ( r = 0.231 , p = 0.203 ) and between ahi and qtc duration ( r = 0.004 , p = 0.980 ) in osas group . we found an increased fragmented qrs frequency , qrs duration and qtc duration in patients with osas compared to the control group . obstructive sleep apnea syndrome is an important public health problem since it is both prevalent and associated with disastrous outcomes including , but not limited to , cardiovascular morbidity and mortality . so far , certain causative factors have been implicated in osas 's deleterious effects on myocardial structure and function . hypoxia and hypercapnia that are common during sleep ; increased negative intrathoracic pressure during the arousal phase and excessive hemodynamic changes as a result of sympathetic stimulation ; oxidative stress , systemic inflammation , endothelial dysfunction , increased myocardial oxygen supply - demand mismatch all lead to myocardial injury . a study performed in rats demonstrated that chronic intermittent hypoxia as in osas was associated with lv damage in large scale and myocyte hypertrophy and cellular apoptosis at the cellular level . another study demonstrated an increased cardiomyocyte diameter and amount of interstitial fibrosis in lv myocardium partly as a result of oxidative stress in mice subjected to intermittent hypoxic stress . another study reported an increased amount of type iii aminoterminal peptide , a peptide used as an indirect marker of myocardial fibrosis , and suggested that its amount is related with disease severity . myocardial scar or fibrosis does not necessarily lead to qrs prolongation , but rather they alter qrs shape without prolonging it by producing a previously absent r or a notch in either or both of r or s waves . it has been shown in previous trials that qrs fragmentation is a predictor of sudden cardiac death and total mortality . an increased fragmented qrs rate in osas patients in our study may have been related to cellular apoptosis and interstitial fibrosis in cardiac structure , which may be secondary to chronic hypoxia and other hemodynamic changes . cetin et al . , in a study in patients with stable angina pectoris , showed that fragmented qrs and crp levels were inter - related . we also found that osas patients with fragmented qrs had higher crp levels than those without . a higher crp level in osas patients with fragmented qrs may suggest that inflammation could also play a role in the alterations of the qrs morphology in osas patients . irrespective of its cause , lv hypertrophy is associated with increased cardiovascular morbidity and mortality . the causative factor of lv hypertrophy in osas is the increased afterload which may be due to increased negative intrathoracic pressure secondary to forceful breathing attempts against a closed upper airway or to heightened blood pressure because of sympathetic activation , hypoxemia , and arousal from sleep . in our study lv thickness was found to be increased in osas patients compared to controls despite the fact that there are a very low number of hypertensive patients and besides they had only mild hypertension . it is well - known that increased lv mural thickness may lead to prolonged qrs complexes . in the life trial qrs duration was independently predictive of cardiovascular and all - cause mortality . desai et al . reported that qrs duration was a significant and independent predictor of cardiovascular mortality in general medical population . similar to the previous one , our study also found that osas patients had prolonged qrs duration . another point to be considered was the higher numbers of male patients in the osa group . showed that , in the adults , the principal differences were an increased qrs duration in men compared with women both in the standard and signal - averaged ecg . but qrs duration was also longer in patients compared to controls after adjustment for gender in our study . however , the osas group 's qrs duration , which was within normal limits although it was prolonged as compared to the control group , may have altered secondary to above mentioned factors . our study also showed that , although in normal limits , qtc duration was longer in patients with osas . while osa patients revealed longer qt durations similar to our study in literature , in the study of gupta et al . secondly , in our patient group , we had more male patients than female patients . because osas is diagnosed more frequently in male patients , and we have consecutively selected patients , we believe that our group study group supports the actual profile . thirdly , we calculated measurements with electronic caliper by magnifying lens instead of computer - assisted calculations . although we carefully assessed the patients for the presence of the coronary artery disease , we do not used invasive methods such as coronary angiography . thus , it is possible that our cohort included patients with undetected coronary artery disease at entry . furthermore , the sleep clinic population used here may not reflect the findings in the general community , and the results should be further confirmed with several longitudinal studies . in conclusion , in our study fragmented qrs frequency and qrs duration were found to increase in osas patients . both parameters are related with increased cardiovascular mortality . considering the prognostic importance of ecg parameters , it may be reasonable to recommend more detailed evaluation of osas patients with fragmented or prolonged qrs complexes with respect to presence of cardiovascular diseases . secondly , in our patient group , we had more male patients than female patients . because osas is diagnosed more frequently in male patients , and we have consecutively selected patients , we believe that our group study group supports the actual profile . thirdly , we calculated measurements with electronic caliper by magnifying lens instead of computer - assisted calculations . although we carefully assessed the patients for the presence of the coronary artery disease , we do not used invasive methods such as coronary angiography . thus , it is possible that our cohort included patients with undetected coronary artery disease at entry . furthermore , the sleep clinic population used here may not reflect the findings in the general community , and the results should be further confirmed with several longitudinal studies . in conclusion , in our study fragmented qrs frequency and qrs duration were found to increase in osas patients . both parameters are related with increased cardiovascular mortality . considering the prognostic importance of ecg parameters , it may be reasonable to recommend more detailed evaluation of osas patients with fragmented or prolonged qrs complexes with respect to presence of cardiovascular diseases .

background : obstructive sleep apnea syndrome ( osas ) is a disease with increasing prevalence , which is mainly characterized by increased cardiopulmonary mortality and morbidity . it is well - known that osas patients have increased prevalence of cardiovascular diseases including coronary heart disease , heart failure , and arrhythmias . the aim of this study was to evaluate the presence of prolonged and fragmented qrs complexes , which have previously been associated with cardiovascular mortality , in osas patients.methods:our study included 51 patients ( mean age 41.6 10.1 years ) who were recently diagnosed with osas ( apnea - hypopnea index [ ahi ] 5 events / h ) and never received therapy . the control group consisted of 34 volunteers ( mean age 43.1 11.6 years ) in whom osas was excluded ( ahi < 5 events / h ) . the longest qrs complexes was measured in the 12-lead electrocardiogram ( ecg ) and the presence of fragmentation in qrs complexes was investigated.results:fragmented qrs frequency was significantly higher in patients with osas ( n = 31 [ 61% ] vs. n = 12 [ 35% ] , p = 0.021 ) . qrs and qtc durations were also significantly longer in osas patients than controls ( 99.8 13.9 ms vs. 84.7 14.3 ms , p < 0.001 ; 411.4 26.9 ms vs. 390.1 32.2 ms , p = 0.001 , respectively ) . analysis of the patient and controls groups combined revealed a weak - moderate correlation between ahi and qrs duration ( r = 0.292 , p = 0.070 ) . osas group had no correlation between ahi and qrs duration ( r = 0.231 , p = 0.203).conclusions : in our study fragmented qrs frequency and qrs duration were found to increase in osas patients . both parameters are related with increased cardiovascular mortality . considering the prognostic importance of ecg parameters , it may be reasonable to recommend more detailed evaluation of osas patients with fragmented or prolonged qrs complexes with respect to presence of cardiovascular diseases .

I M Study design Study population Polysomnography Electrocardiographic assessment Echocardiographic assessment Statistical analysis Reproducibility R D Study limitations

obstructive sleep apnea syndrome ( osas ) is a complex clinical syndrome characterized by apnea episodes , sleep fragmentation , oxygen desaturation , and increased daytime somnolence as a result of recurrent obstructive episodes in upper airways during sleep . previous studies have revealed that osas is related with many cardiovascular diseases including hypertension , coronary artery disease , arrhythmias , and congestive heart failure . basic hemodynamic mechanisms responsible from cardiovascular diseases are increased negative intrathoracic pressure , hypoxemia , and increased catecholamine release . in osas both ventricles are subjected to hemodynamic stress that induces various structural changes . the electrocardiogram ( ecg ) represents the sum of electrical activity of all myocytes within the heart and hemodynamic changes produce various ecg changes . qrs complexes mainly represent ventricular depolarization and morphological assessment of qrs complex may give important prognostic information . it has been shown in previous trials that qrs fragmentation is a predictor of sudden cardiac death and total mortality . in addition , a prolonged qrs duration has been implicated in cardiovascular events , sudden death , and total mortality . each of these patients were diagnosed with osas ( apnea - hypopnea index [ ahi ] 5 events / h ) and they had no previous treatment . thirty - four consecutive participants who were shown to have no osas ( ahi < 5 events / h ) following polysomnopgraphy test were included as our control group . patients with following conditions were excluded from the study : chronic obstructive pulmonary disease on pulmonary function testing , previous history of continuous positive airway pressure treatment , stage 23 hypertension , a positive stress test , coronary artery disease and/or previous myocardial infarction , left ventricular ( lv ) dysfunction , echocardiographically proven lv hypertrophy , moderate - to - severe valve disease , renal or hepatic dysfunction , impairment of thyroid function tests , atrial fibrillation , history of dysrhythmia , patients with permanent pacemakers , bundle branch block or fascicular block , and electrolyte abnormalities , presence of infections or inflammations , hematologic disease or any known malignancy , individuals who have systemic and metabolic diseases that could adversely affect the cardiac or pulmonary structure and functions . in this study , all of the procedures that took place were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the declaration of helsinki ( 1975 ) , as revised in 2000 . the test included the following variables : electrooculogram ( two channels ) , electroencephalogram ( four channels ) , electromyogram of submental muscles ( two channels ) , electromyogram of the anterior tibialis muscle of both legs ( two channels ) , thoracic and abdominal movements , pulse oximeter oxygen saturation , ecg , airflow ( with a nasal cannula ) , body position detector , and tracheal sound . the recordings were conducted at a speed of 10 mm / s , and sleep stage was scored according to the standard criteria of rechtschaffen and kales . according to the recently updated international classification of sleep disorders published by the american academy of sleep medicine , a diagnosis of osas is to be made if the ahi is 15 events / h , independent of occurrence of symptoms , or whenever an ahi > 5 events / h is associated with any of the following : ( 1 ) sleep attacks or excessive daytime sleepiness , unrefreshing sleep , fatigue or ( 2 ) insomnia , or ( 3 ) witnessed heavy snoring and/or breathing pauses referred by the partner . osas was excluded by a negative history of sleep - related symptoms ( snoring , witnessed apneas , and excessive daytime sleepiness ) and with an ahi < 5 events / h at overnight polysomnopgraphy . patients with sleep disorders except osas such as upper airway resistance syndrome , periodic leg movement syndrome , or narcolepsy were excluded . all examinations were carried out in a quiet room during spontaneous breathing , following 10 min of rest in the supine position . the ecgs were each numbered and presented to the analyzing investigators who were blind both to patient name and group information . fragmented qrs was diagnosed with the presence of an additional r wave ( r ' ) or notching in the nadir of the r wave or the s wave , or the presence of more than one r ( fragmentation ) in two consecutive leads , corresponding to a certain myocardial territory . echocardiographic examination was carried out with the patient resting in a supine left lateral decubitus position . lv dimensions and wall thickness were obtained from the parasternal long axis view with the m - mode cursor positioned just below the mitral leaflet tips , perpendicular to the long axis of the lv . lv end - diastolic diameter and end - systolic diameter , thickness of the interventricular septum , and posterior wall of the lv were all measured using a single operator by the standards of the american society of echocardiography . independent samples t - test was used for the two - group comparison of the normally distributed variables and mann whitney u - test for the two - group comparison of the variables without normal distribution . the coefficient of variation is calculated as the sd of the differences between the repeated measurements divided by the averages of the repeated measurements , and is expressed as a percentage . the intraobserver variabilities were 4.08% for pq , 5.60% for qrs , and 4.26% for qtc . each of these patients were diagnosed with osas ( apnea - hypopnea index [ ahi ] 5 events / h ) and they had no previous treatment . thirty - four consecutive participants who were shown to have no osas ( ahi < 5 events / h ) following polysomnopgraphy test were included as our control group . patients with following conditions were excluded from the study : chronic obstructive pulmonary disease on pulmonary function testing , previous history of continuous positive airway pressure treatment , stage 23 hypertension , a positive stress test , coronary artery disease and/or previous myocardial infarction , left ventricular ( lv ) dysfunction , echocardiographically proven lv hypertrophy , moderate - to - severe valve disease , renal or hepatic dysfunction , impairment of thyroid function tests , atrial fibrillation , history of dysrhythmia , patients with permanent pacemakers , bundle branch block or fascicular block , and electrolyte abnormalities , presence of infections or inflammations , hematologic disease or any known malignancy , individuals who have systemic and metabolic diseases that could adversely affect the cardiac or pulmonary structure and functions . in this study , all of the procedures that took place were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the declaration of helsinki ( 1975 ) , as revised in 2000 . the test included the following variables : electrooculogram ( two channels ) , electroencephalogram ( four channels ) , electromyogram of submental muscles ( two channels ) , electromyogram of the anterior tibialis muscle of both legs ( two channels ) , thoracic and abdominal movements , pulse oximeter oxygen saturation , ecg , airflow ( with a nasal cannula ) , body position detector , and tracheal sound . according to the recently updated international classification of sleep disorders published by the american academy of sleep medicine , a diagnosis of osas is to be made if the ahi is 15 events / h , independent of occurrence of symptoms , or whenever an ahi > 5 events / h is associated with any of the following : ( 1 ) sleep attacks or excessive daytime sleepiness , unrefreshing sleep , fatigue or ( 2 ) insomnia , or ( 3 ) witnessed heavy snoring and/or breathing pauses referred by the partner . osas was excluded by a negative history of sleep - related symptoms ( snoring , witnessed apneas , and excessive daytime sleepiness ) and with an ahi < 5 events / h at overnight polysomnopgraphy . all examinations were carried out in a quiet room during spontaneous breathing , following 10 min of rest in the supine position . the ecgs were each numbered and presented to the analyzing investigators who were blind both to patient name and group information . ecg parameters were measured using a digital caliper ( sensitivity : 1/100 mm ) by magnifying lens . fragmented qrs was diagnosed with the presence of an additional r wave ( r ' ) or notching in the nadir of the r wave or the s wave , or the presence of more than one r ( fragmentation ) in two consecutive leads , corresponding to a certain myocardial territory . echocardiographic examination was carried out with the patient resting in a supine left lateral decubitus position . lv dimensions and wall thickness were obtained from the parasternal long axis view with the m - mode cursor positioned just below the mitral leaflet tips , perpendicular to the long axis of the lv . lv end - diastolic diameter and end - systolic diameter , thickness of the interventricular septum , and posterior wall of the lv were all measured using a single operator by the standards of the american society of echocardiography . independent samples t - test was used for the two - group comparison of the normally distributed variables and mann whitney u - test for the two - group comparison of the variables without normal distribution . the coefficient of variation is calculated as the sd of the differences between the repeated measurements divided by the averages of the repeated measurements , and is expressed as a percentage . the intraobserver variabilities were 4.08% for pq , 5.60% for qrs , and 4.26% for qtc . the interobserver variabilities were 4.74% for pq , 6.65% for qrs , and 4.21% for qtc . ahi : apnea - hypopnea index ; bmi : body mass index ; crp : c - reactive protein ; dm : diabetes mellitus ; fbg : fasting blood glucose ; hdl - c : high - density lipoprotein cholesterol ; hl : hyperlipidemia ; ht : hypertension ; ldl - c : low - density lipoprotein cholesterol ; nc : neck circumference ; tc : total cholesterol ; tg : triglycerides ; tsh : thyroid stimulating hormone ; wc : waist circumference ; osas : obstructive sleep apnea syndrome . fragmented qrs frequency was significantly higher in patients with osas ( n = 31 [ 61% ] vs. n = 12 [ 35% ] , fqrs frequency was 2.8 times higher in patients compared to controls after adjustment for gender [ table 3 ] . when osas patients were grouped according to presence of fragmented qrs , it was found that the fragmented qrs group had a longer qrs duration ( 95.0 11.6 ms vs. 102.9 14.5 ms , p = 0.045 ) [ table 4 ] . furthermore , crp level was also higher in the fragmented qrs group ( 0.26 0.32 mg / dl vs. 0.39 0.36 mg / dl , p = 0.027 ) . comparison of the electrocardiographic and echocardiographic parameters * independent t - test ; mann whitney u - test . ci : cornell index ; ef : ejection fraction ; fqrs : fragmented qrs ; ivs : interventricular septum ; la : left atrium ; lv - edd : left ventricular end - diastolic diameter ; lv - esd : left ventricular end - systolic diameter ; pq : pq interval duration ; pw : posterior wall ; qrs : qrs duration ; qtc : corrected qt interval duration ; sli : skolow - lyon index ; osas : obstructive sleep apnea syndrome . a logistic regression model for fqrs fqrs : fragmented qrs ; se : standard error ; df : degree of freedom ; ci : confidence interval ; osas : obstructive sleep apnea syndrome . ahi : apnea - hypopnea index ; bmi : body mass index ; ci : cornell index ; dm : diabetes mellitus ; ef : ejection fraction ; fqrs : fragmented qrs ; fbg : fasting blood glucose ; hdl - c : high - density lipoprotein cholesterol ; hl : hyperlipidemia ; ht : hypertension ; ivs : interventricular septum ; la : left atrium ; ldl - c : low - density lipoprotein cholesterol ; lv - edd : left ventricular end - diastolic diameter ; lv - esd : left ventricular end - systolic diameter ; nc : neck circumference ; pq : pq interval duration ; pw : posterior wall ; qrs : qrs duration ; qtc : corrected qt interval duration ; sli : skolow - lyon index ; tc : total cholesterol ; tg : triglycerides ; tsh : thyroid stimulating hormone ; wc : waist circumference . qrs and qtc durations were significantly longer in osas group compared to the control group ( 99.8 13.9 ms vs. 84.7 14.3 ms , p < 0.0001 ; 411.4 26.9 ms vs. 390.1 32.2 ms , p = 0.001 , respectively ) [ figures 1 and 2 ] . qrs and qtc durations were also longer in patients compared to controls after adjustment for gender ( 99.7 14.4 ms vs. 84.8 14.6 ms , p < 0.001 ; 413.3 29.1 ms vs. 387.3 29.4 ms , p < 0.001 , respectively ) . echocardiographic parameters were within normal limits in both groups , although osas group had increased lv thickness and diameters , increased left atrial diameter , and a lower ejection fraction . analysis of the patient and control groups combined revealed a weak - moderate correlation between ahi and qrs duration ( r = 0.292 , p = 0.070 ) and between ahi and qtc duration ( r = 0.338 , p = 0.020 ) . however , there was no correlation between ahi and qrs duration ( r = 0.231 , p = 0.203 ) and between ahi and qtc duration ( r = 0.004 , p = 0.980 ) in osas group . we found an increased fragmented qrs frequency , qrs duration and qtc duration in patients with osas compared to the control group . obstructive sleep apnea syndrome is an important public health problem since it is both prevalent and associated with disastrous outcomes including , but not limited to , cardiovascular morbidity and mortality . a study performed in rats demonstrated that chronic intermittent hypoxia as in osas was associated with lv damage in large scale and myocyte hypertrophy and cellular apoptosis at the cellular level . another study reported an increased amount of type iii aminoterminal peptide , a peptide used as an indirect marker of myocardial fibrosis , and suggested that its amount is related with disease severity . it has been shown in previous trials that qrs fragmentation is a predictor of sudden cardiac death and total mortality . an increased fragmented qrs rate in osas patients in our study may have been related to cellular apoptosis and interstitial fibrosis in cardiac structure , which may be secondary to chronic hypoxia and other hemodynamic changes . , in a study in patients with stable angina pectoris , showed that fragmented qrs and crp levels were inter - related . we also found that osas patients with fragmented qrs had higher crp levels than those without . a higher crp level in osas patients with fragmented qrs may suggest that inflammation could also play a role in the alterations of the qrs morphology in osas patients . irrespective of its cause , lv hypertrophy is associated with increased cardiovascular morbidity and mortality . the causative factor of lv hypertrophy in osas is the increased afterload which may be due to increased negative intrathoracic pressure secondary to forceful breathing attempts against a closed upper airway or to heightened blood pressure because of sympathetic activation , hypoxemia , and arousal from sleep . in our study lv thickness was found to be increased in osas patients compared to controls despite the fact that there are a very low number of hypertensive patients and besides they had only mild hypertension . it is well - known that increased lv mural thickness may lead to prolonged qrs complexes . in the life trial qrs duration was independently predictive of cardiovascular and all - cause mortality . reported that qrs duration was a significant and independent predictor of cardiovascular mortality in general medical population . similar to the previous one , our study also found that osas patients had prolonged qrs duration . another point to be considered was the higher numbers of male patients in the osa group . showed that , in the adults , the principal differences were an increased qrs duration in men compared with women both in the standard and signal - averaged ecg . but qrs duration was also longer in patients compared to controls after adjustment for gender in our study . however , the osas group 's qrs duration , which was within normal limits although it was prolonged as compared to the control group , may have altered secondary to above mentioned factors . our study also showed that , although in normal limits , qtc duration was longer in patients with osas . while osa patients revealed longer qt durations similar to our study in literature , in the study of gupta et al . secondly , in our patient group , we had more male patients than female patients . because osas is diagnosed more frequently in male patients , and we have consecutively selected patients , we believe that our group study group supports the actual profile . although we carefully assessed the patients for the presence of the coronary artery disease , we do not used invasive methods such as coronary angiography . thus , it is possible that our cohort included patients with undetected coronary artery disease at entry . furthermore , the sleep clinic population used here may not reflect the findings in the general community , and the results should be further confirmed with several longitudinal studies . in conclusion , in our study fragmented qrs frequency and qrs duration were found to increase in osas patients . both parameters are related with increased cardiovascular mortality . considering the prognostic importance of ecg parameters , it may be reasonable to recommend more detailed evaluation of osas patients with fragmented or prolonged qrs complexes with respect to presence of cardiovascular diseases . secondly , in our patient group , we had more male patients than female patients . because osas is diagnosed more frequently in male patients , and we have consecutively selected patients , we believe that our group study group supports the actual profile . although we carefully assessed the patients for the presence of the coronary artery disease , we do not used invasive methods such as coronary angiography . thus , it is possible that our cohort included patients with undetected coronary artery disease at entry . furthermore , the sleep clinic population used here may not reflect the findings in the general community , and the results should be further confirmed with several longitudinal studies . in conclusion , in our study fragmented qrs frequency and qrs duration were found to increase in osas patients . both parameters are related with increased cardiovascular mortality . considering the prognostic importance of ecg parameters , it may be reasonable to recommend more detailed evaluation of osas patients with fragmented or prolonged qrs complexes with respect to presence of cardiovascular diseases .

obstructive sleep apnea syndrome ( osas ) is a complex clinical syndrome characterized by apnea episodes , sleep fragmentation , oxygen desaturation , and increased daytime somnolence as a result of recurrent obstructive episodes in upper airways during sleep . previous studies have revealed that osas is related with many cardiovascular diseases including hypertension , coronary artery disease , arrhythmias , and congestive heart failure . basic hemodynamic mechanisms responsible from cardiovascular diseases are increased negative intrathoracic pressure , hypoxemia , and increased catecholamine release . the electrocardiogram ( ecg ) represents the sum of electrical activity of all myocytes within the heart and hemodynamic changes produce various ecg changes . qrs complexes mainly represent ventricular depolarization and morphological assessment of qrs complex may give important prognostic information . it has been shown in previous trials that qrs fragmentation is a predictor of sudden cardiac death and total mortality . in addition , a prolonged qrs duration has been implicated in cardiovascular events , sudden death , and total mortality . each of these patients were diagnosed with osas ( apnea - hypopnea index [ ahi ] 5 events / h ) and they had no previous treatment . thirty - four consecutive participants who were shown to have no osas ( ahi < 5 events / h ) following polysomnopgraphy test were included as our control group . patients with following conditions were excluded from the study : chronic obstructive pulmonary disease on pulmonary function testing , previous history of continuous positive airway pressure treatment , stage 23 hypertension , a positive stress test , coronary artery disease and/or previous myocardial infarction , left ventricular ( lv ) dysfunction , echocardiographically proven lv hypertrophy , moderate - to - severe valve disease , renal or hepatic dysfunction , impairment of thyroid function tests , atrial fibrillation , history of dysrhythmia , patients with permanent pacemakers , bundle branch block or fascicular block , and electrolyte abnormalities , presence of infections or inflammations , hematologic disease or any known malignancy , individuals who have systemic and metabolic diseases that could adversely affect the cardiac or pulmonary structure and functions . in this study , all of the procedures that took place were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the declaration of helsinki ( 1975 ) , as revised in 2000 . the test included the following variables : electrooculogram ( two channels ) , electroencephalogram ( four channels ) , electromyogram of submental muscles ( two channels ) , electromyogram of the anterior tibialis muscle of both legs ( two channels ) , thoracic and abdominal movements , pulse oximeter oxygen saturation , ecg , airflow ( with a nasal cannula ) , body position detector , and tracheal sound . the recordings were conducted at a speed of 10 mm / s , and sleep stage was scored according to the standard criteria of rechtschaffen and kales . the ahi was obtained by dividing the total number of apneas and hypopneas by the total sleep time . apneas were defined as complete cessation of airflow 10 s. hypopneas were defined as a reduction of > 50% in one of the three respiratory signals , airflow signal or either respiratory or abdominal signals of respiratory inductance plethysmography , with an associated decrease of 3% in oxygen saturation or an arousal . according to the recently updated international classification of sleep disorders published by the american academy of sleep medicine , a diagnosis of osas is to be made if the ahi is 15 events / h , independent of occurrence of symptoms , or whenever an ahi > 5 events / h is associated with any of the following : ( 1 ) sleep attacks or excessive daytime sleepiness , unrefreshing sleep , fatigue or ( 2 ) insomnia , or ( 3 ) witnessed heavy snoring and/or breathing pauses referred by the partner . osas was excluded by a negative history of sleep - related symptoms ( snoring , witnessed apneas , and excessive daytime sleepiness ) and with an ahi < 5 events / h at overnight polysomnopgraphy . all standard 12-lead ecgs were obtained simultaneously using a recorder set at a 2550 mm / s paper speed and a voltage calibration of 1 mv / cm . fragmented qrs was diagnosed with the presence of an additional r wave ( r ' ) or notching in the nadir of the r wave or the s wave , or the presence of more than one r ( fragmentation ) in two consecutive leads , corresponding to a certain myocardial territory . lv dimensions and wall thickness were obtained from the parasternal long axis view with the m - mode cursor positioned just below the mitral leaflet tips , perpendicular to the long axis of the lv . lv end - diastolic diameter and end - systolic diameter , thickness of the interventricular septum , and posterior wall of the lv were all measured using a single operator by the standards of the american society of echocardiography . in order for the suitable analysis technique to be chosen , kolmogorov smirnov and homogeneity of variance tests were undertaken . independent samples t - test was used for the two - group comparison of the normally distributed variables and mann whitney u - test for the two - group comparison of the variables without normal distribution . the coefficient of variation is calculated as the sd of the differences between the repeated measurements divided by the averages of the repeated measurements , and is expressed as a percentage . the intraobserver variabilities were 4.08% for pq , 5.60% for qrs , and 4.26% for qtc . the interobserver variabilities were 4.74% for pq , 6.65% for qrs , and 4.21% for qtc . each of these patients were diagnosed with osas ( apnea - hypopnea index [ ahi ] 5 events / h ) and they had no previous treatment . thirty - four consecutive participants who were shown to have no osas ( ahi < 5 events / h ) following polysomnopgraphy test were included as our control group . patients with following conditions were excluded from the study : chronic obstructive pulmonary disease on pulmonary function testing , previous history of continuous positive airway pressure treatment , stage 23 hypertension , a positive stress test , coronary artery disease and/or previous myocardial infarction , left ventricular ( lv ) dysfunction , echocardiographically proven lv hypertrophy , moderate - to - severe valve disease , renal or hepatic dysfunction , impairment of thyroid function tests , atrial fibrillation , history of dysrhythmia , patients with permanent pacemakers , bundle branch block or fascicular block , and electrolyte abnormalities , presence of infections or inflammations , hematologic disease or any known malignancy , individuals who have systemic and metabolic diseases that could adversely affect the cardiac or pulmonary structure and functions . in this study , all of the procedures that took place were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the declaration of helsinki ( 1975 ) , as revised in 2000 . the test included the following variables : electrooculogram ( two channels ) , electroencephalogram ( four channels ) , electromyogram of submental muscles ( two channels ) , electromyogram of the anterior tibialis muscle of both legs ( two channels ) , thoracic and abdominal movements , pulse oximeter oxygen saturation , ecg , airflow ( with a nasal cannula ) , body position detector , and tracheal sound . the recordings were conducted at a speed of 10 mm / s , and sleep stage was scored according to the standard criteria of rechtschaffen and kales . the ahi was obtained by dividing the total number of apneas and hypopneas by the total sleep time . apneas were defined as complete cessation of airflow 10 s. hypopneas were defined as a reduction of > 50% in one of the three respiratory signals , airflow signal or either respiratory or abdominal signals of respiratory inductance plethysmography , with an associated decrease of 3% in oxygen saturation or an arousal . according to the recently updated international classification of sleep disorders published by the american academy of sleep medicine , a diagnosis of osas is to be made if the ahi is 15 events / h , independent of occurrence of symptoms , or whenever an ahi > 5 events / h is associated with any of the following : ( 1 ) sleep attacks or excessive daytime sleepiness , unrefreshing sleep , fatigue or ( 2 ) insomnia , or ( 3 ) witnessed heavy snoring and/or breathing pauses referred by the partner . osas was excluded by a negative history of sleep - related symptoms ( snoring , witnessed apneas , and excessive daytime sleepiness ) and with an ahi < 5 events / h at overnight polysomnopgraphy . all standard 12-lead ecgs were obtained simultaneously using a recorder set at a 2550 mm / s paper speed and a voltage calibration of 1 mv / cm . fragmented qrs was diagnosed with the presence of an additional r wave ( r ' ) or notching in the nadir of the r wave or the s wave , or the presence of more than one r ( fragmentation ) in two consecutive leads , corresponding to a certain myocardial territory . lv dimensions and wall thickness were obtained from the parasternal long axis view with the m - mode cursor positioned just below the mitral leaflet tips , perpendicular to the long axis of the lv . lv end - diastolic diameter and end - systolic diameter , thickness of the interventricular septum , and posterior wall of the lv were all measured using a single operator by the standards of the american society of echocardiography . in order for the suitable analysis technique to be chosen , kolmogorov smirnov and homogeneity of variance tests were undertaken . independent samples t - test was used for the two - group comparison of the normally distributed variables and mann whitney u - test for the two - group comparison of the variables without normal distribution . the coefficient of variation is calculated as the sd of the differences between the repeated measurements divided by the averages of the repeated measurements , and is expressed as a percentage . the intraobserver variabilities were 4.08% for pq , 5.60% for qrs , and 4.26% for qtc . ahi : apnea - hypopnea index ; bmi : body mass index ; crp : c - reactive protein ; dm : diabetes mellitus ; fbg : fasting blood glucose ; hdl - c : high - density lipoprotein cholesterol ; hl : hyperlipidemia ; ht : hypertension ; ldl - c : low - density lipoprotein cholesterol ; nc : neck circumference ; tc : total cholesterol ; tg : triglycerides ; tsh : thyroid stimulating hormone ; wc : waist circumference ; osas : obstructive sleep apnea syndrome . fragmented qrs frequency was significantly higher in patients with osas ( n = 31 [ 61% ] vs. n = 12 [ 35% ] , fqrs frequency was 2.8 times higher in patients compared to controls after adjustment for gender [ table 3 ] . when osas patients were grouped according to presence of fragmented qrs , it was found that the fragmented qrs group had a longer qrs duration ( 95.0 11.6 ms vs. 102.9 14.5 ms , p = 0.045 ) [ table 4 ] . furthermore , crp level was also higher in the fragmented qrs group ( 0.26 0.32 mg / dl vs. 0.39 0.36 mg / dl , p = 0.027 ) . ci : cornell index ; ef : ejection fraction ; fqrs : fragmented qrs ; ivs : interventricular septum ; la : left atrium ; lv - edd : left ventricular end - diastolic diameter ; lv - esd : left ventricular end - systolic diameter ; pq : pq interval duration ; pw : posterior wall ; qrs : qrs duration ; qtc : corrected qt interval duration ; sli : skolow - lyon index ; osas : obstructive sleep apnea syndrome . a logistic regression model for fqrs fqrs : fragmented qrs ; se : standard error ; df : degree of freedom ; ci : confidence interval ; osas : obstructive sleep apnea syndrome . ahi : apnea - hypopnea index ; bmi : body mass index ; ci : cornell index ; dm : diabetes mellitus ; ef : ejection fraction ; fqrs : fragmented qrs ; fbg : fasting blood glucose ; hdl - c : high - density lipoprotein cholesterol ; hl : hyperlipidemia ; ht : hypertension ; ivs : interventricular septum ; la : left atrium ; ldl - c : low - density lipoprotein cholesterol ; lv - edd : left ventricular end - diastolic diameter ; lv - esd : left ventricular end - systolic diameter ; nc : neck circumference ; pq : pq interval duration ; pw : posterior wall ; qrs : qrs duration ; qtc : corrected qt interval duration ; sli : skolow - lyon index ; tc : total cholesterol ; tg : triglycerides ; tsh : thyroid stimulating hormone ; wc : waist circumference . qrs and qtc durations were significantly longer in osas group compared to the control group ( 99.8 13.9 ms vs. 84.7 14.3 ms , p < 0.0001 ; 411.4 26.9 ms vs. 390.1 32.2 ms , p = 0.001 , respectively ) [ figures 1 and 2 ] . qrs and qtc durations were also longer in patients compared to controls after adjustment for gender ( 99.7 14.4 ms vs. 84.8 14.6 ms , p < 0.001 ; 413.3 29.1 ms vs. 387.3 29.4 ms , p < 0.001 , respectively ) . echocardiographic parameters were within normal limits in both groups , although osas group had increased lv thickness and diameters , increased left atrial diameter , and a lower ejection fraction . analysis of the patient and control groups combined revealed a weak - moderate correlation between ahi and qrs duration ( r = 0.292 , p = 0.070 ) and between ahi and qtc duration ( r = 0.338 , p = 0.020 ) . however , there was no correlation between ahi and qrs duration ( r = 0.231 , p = 0.203 ) and between ahi and qtc duration ( r = 0.004 , p = 0.980 ) in osas group . we found an increased fragmented qrs frequency , qrs duration and qtc duration in patients with osas compared to the control group . obstructive sleep apnea syndrome is an important public health problem since it is both prevalent and associated with disastrous outcomes including , but not limited to , cardiovascular morbidity and mortality . hypoxia and hypercapnia that are common during sleep ; increased negative intrathoracic pressure during the arousal phase and excessive hemodynamic changes as a result of sympathetic stimulation ; oxidative stress , systemic inflammation , endothelial dysfunction , increased myocardial oxygen supply - demand mismatch all lead to myocardial injury . a study performed in rats demonstrated that chronic intermittent hypoxia as in osas was associated with lv damage in large scale and myocyte hypertrophy and cellular apoptosis at the cellular level . another study demonstrated an increased cardiomyocyte diameter and amount of interstitial fibrosis in lv myocardium partly as a result of oxidative stress in mice subjected to intermittent hypoxic stress . another study reported an increased amount of type iii aminoterminal peptide , a peptide used as an indirect marker of myocardial fibrosis , and suggested that its amount is related with disease severity . an increased fragmented qrs rate in osas patients in our study may have been related to cellular apoptosis and interstitial fibrosis in cardiac structure , which may be secondary to chronic hypoxia and other hemodynamic changes . , in a study in patients with stable angina pectoris , showed that fragmented qrs and crp levels were inter - related . we also found that osas patients with fragmented qrs had higher crp levels than those without . a higher crp level in osas patients with fragmented qrs may suggest that inflammation could also play a role in the alterations of the qrs morphology in osas patients . the causative factor of lv hypertrophy in osas is the increased afterload which may be due to increased negative intrathoracic pressure secondary to forceful breathing attempts against a closed upper airway or to heightened blood pressure because of sympathetic activation , hypoxemia , and arousal from sleep . in our study lv thickness was found to be increased in osas patients compared to controls despite the fact that there are a very low number of hypertensive patients and besides they had only mild hypertension . in the life trial qrs duration was independently predictive of cardiovascular and all - cause mortality . reported that qrs duration was a significant and independent predictor of cardiovascular mortality in general medical population . showed that , in the adults , the principal differences were an increased qrs duration in men compared with women both in the standard and signal - averaged ecg . but qrs duration was also longer in patients compared to controls after adjustment for gender in our study . however , the osas group 's qrs duration , which was within normal limits although it was prolonged as compared to the control group , may have altered secondary to above mentioned factors . while osa patients revealed longer qt durations similar to our study in literature , in the study of gupta et al . because osas is diagnosed more frequently in male patients , and we have consecutively selected patients , we believe that our group study group supports the actual profile . although we carefully assessed the patients for the presence of the coronary artery disease , we do not used invasive methods such as coronary angiography . furthermore , the sleep clinic population used here may not reflect the findings in the general community , and the results should be further confirmed with several longitudinal studies . in conclusion , in our study fragmented qrs frequency and qrs duration were found to increase in osas patients . considering the prognostic importance of ecg parameters , it may be reasonable to recommend more detailed evaluation of osas patients with fragmented or prolonged qrs complexes with respect to presence of cardiovascular diseases . because osas is diagnosed more frequently in male patients , and we have consecutively selected patients , we believe that our group study group supports the actual profile . although we carefully assessed the patients for the presence of the coronary artery disease , we do not used invasive methods such as coronary angiography . furthermore , the sleep clinic population used here may not reflect the findings in the general community , and the results should be further confirmed with several longitudinal studies . in conclusion , in our study fragmented qrs frequency and qrs duration were found to increase in osas patients . considering the prognostic importance of ecg parameters , it may be reasonable to recommend more detailed evaluation of osas patients with fragmented or prolonged qrs complexes with respect to presence of cardiovascular diseases .

solution ph is one of the most important environmental variables that affects the structural and dynamic properties of biomolecules . various biological events such as protein folding / unfolding , ligand binding , and enzyme activity heavily depend on solution ph . solution ph affects protein denaturation , aggregation , and regulates many ph - dependent membrane proteins and channels . in cells , the ph in different compartments varies significantly ; for example , the ph in the cytoplasm and nucleus is neutral ( around 7.2 ) , in vacuoles and golgi , it is acidic ( between 4.8 and 6.5 ) , and in mitochondria , it is basic ( around 8.0 ) . at the same time , the ph of each compartment is tightly regulated , and a small ph change can lead to serious diseases . solution ph affects proteins by changing the protonation states of ionizable / titratable residues . the protonation state of an ionizable residue is a function of its pka value . however , the pka values of ionizable residues in protein environments can be shifted from the standard pka values experienced in water . the shift is especially large for groups found in protein interiors , and as a result , such buried groups can sometimes change protonation state even at physiological ph . changes in the protonation state can be exploited for function , for example , when they are coupled to conformational reorganization , such as in the case of atp synthase , bacteriorhodopsin , cytochrome c oxidase , or the photoactive yellow protein . to understand the mechanisms of such proteins , it is essential to know the pka values of functionally important residues . experimental determination of pka values of such residues can be a challenge , and carefully calibrated computational methods offer a possibility to obtain them . however , current computational methods have limitations when large scale structural reorganization is coupled to a change in protonation state . a widely used methodology for pka calculation is based on the solutions of the poisson the limitation of pb - based methods is that they may not properly represent the reorganization / response of protein induced by the titration of ionizable groups . most pb - based methods use only a single conformation or allow perturbation of side - chain or hydrogen atoms . also , the conformational response of a protein is modeled by a single value of a dielectric constant , which is dubious considering the inhomogeneous environment of the interior and surface of a protein . this approximation can also be problematic when water molecules are tightly coupled with ionizable groups of interest , which is commonly observed in many transmembrane proteins . a more accurate description of protein environment and polarizability can be achieved through quantum mechanical ( qm ) or mixed quantum mechanics / molecular mechanics ( qm / mm ) approaches . however , such calculations are computationally very expensive , and proper description of conformational changes triggered by protonation / deprotonation may not be achieved on a relevant time scale . protein conformational changes triggered by ionization can be considered explicitly using constant ph molecular dynamics simulations . constant ph simulation methods can be categorized into two groups : ( 1 ) discrete protonation state models , and ( 2 ) continuous protonation state models . constant ph methods based on discrete protonation state models generally use a hybrid approach combining molecular dynamics ( md ) and monte carlo ( mc ) . in the hybrid mc / md methods , a mc procedure is performed to determine the protonation states of ionizable groups at a regular interval over the course of a md simulation with either implicit or explicit water . during the mc procedure , a random walk between different protonation states is performed , and a state is determined based on the estimated free energy difference and metropolis criteria . with a continuum electrostatic model , dlugosz and antosiewicz developed a mc / md method based on the analytic continuum electrostatic method . used a generalized born ( gb ) solvation model to calculate ( de)protonation free energies . in contrast to the implicit solvent methods above , the md / mc method with explicit water requires a more sophisticated mc move due to solvent reorganization . without solvent reorganization , a sudden change of charge distribution of a side - chain is likely to result in a large electrostatic penalty , which leads to an extremely low mc acceptance ratio . to increase the acceptance ratio of mc moves , for example , baptista et al . evaluated ( de)protonation free energy by pb and performed a short md run to relax the solvent . brgi et al . performed short thermodynamic integration ( ti ) calculations for mc moves , which is extremely expensive . stern proposed a similar approach where mc moves consist of short md simulations ( not free energy calculations ) using a time - dependent hamiltonian that interpolates two protonation states . most of these types of approaches require approximations that result in a loss of rigor that distorts the final ensemble . in the continuous protonation state models , a protonation state is represented as a titration variable considered as an independent dynamic variable . baptista et al . performed md simulations with the average charges of ionizable groups obtained by a mean field approximation . brjesson and hnenberger devised a model in which the extent of ( de)protonation is equilibrated by weak coupling to a proton bath . . applied , and khandoghin et al . improved the dynamics approach to constant - ph simulation with the gb solvation model the extent of protonation is parametrized by a fictitious particle in the hamiltonian , whose value fluctuates between 0 and 1 . during the postprocessing of trajectories , conformations whose value is higher or lower than a threshold value are assigned to a protonation state , and other unphysical conformations are discarded . to avoid sampling of unphysical states , this potential has to be carefully adjusted to maximize the number of transitions and minimize sampling of unphysical states , however , even with this approach , most of the sampled conformations are different from physical states . recently this approach has been extended to perform constant - ph md simulations in explicit water . to enhance the accuracy of simulations that depend heavily on the accuracy of conformational sampling , constant ph methods with both discrete and continuous protonation states the gb - based constant ph methods have been coupled with the temperature replica exchange and accelerated md methods . a ph based replica exchange scheme has also been implemented where ph values are exchanged between replicas . to further increase sampling efficiency , the ph - exchange approach has been combined with reservoirs of conformations and protonation states within the framework of the double reservoir ph replica exchange method ( work submitted for publication ) . in this study , we develop a new constant - ph method that yields the correct description of protonation states by combining the enveloping distribution sampling ( eds ) and hamiltonian replica exchange ( hrex ) methods . the eds method was devised to allow sampling of multiple end states from a single md simulation of a hybrid hamiltonian . similar mixing schemes have been proposed by others . in the context of a constant ph simulation , the hybrid hamiltonian is the sum of boltzmann factors of multiple hamiltonians , each corresponding to a different protonation state . the effect of solution ph is considered by the relative free energy differences between protonation states . to ensure proper sampling of all protonation states , a smoothness parameter , s , that controls the height of energy barriers when s 0 , the hybrid hamiltonian becomes highly smoothed , which makes energy barriers disappear . on the other hand , as s , no smoothing is applied to the hybrid hamiltonian , and it follows the minimum energy surface among the multiple end states , which corresponds to the physical hamiltonian . to obtain the correct ensemble of multiple protonation states while enhancing sampling efficiency , we coupled the eds simulations with different smoothness parameter through the hrex method . the hrex method , often called the bias - exchange method , facilitates diverse conformational sampling by modifying a physical hamiltonian or introducing various biasing potentials . to enhance conformational sampling efficiency , various hamiltonian modification schemes have been suggested : scaling hydrophobic , long - range , solvent - related interactions , or biasing backbone dihedral angles . in the context of the eds - hrex method , we use multiple eds potentials with different smoothness parameter , including an eds potential without smoothing that follows the minimum energy of multiple end states , and perform exchanges between them periodically . we assessed the performance of our method with titratable amino acid monomers : aspartic acid , glutamic acid , lysine , and histidine . we also tested out method with a small four - residue peptide ( kaae ) and snake cardiotoxin . the results show that our method can successfully and efficiently reproduce the correct distribution of different protonation states at a given ph . we briefly review the enveloping distribution sampling ( eds ) and hamiltonian replica exchange ( hrex ) methods and subsequently describe how these two methods are combined to sample the correct ensemble of different protonation states at a given ph . the free energy difference between two states a and b is given by1where z is the partition function of state and is the inverse of the thermodynamic temperature . in the eds approach , a hybrid hamiltonian enveloping both states is defined as follows,2where ea and eb are the hamiltonians of states a and b. in principle , a simulation performed on eh allows sampling of the important phase space of both state a and b , and their free energy difference can be estimated by3where however , if the energy difference between the minima of ea and eb is too large , the simulation will be trapped in the lowest energy basin of a single hamiltonian . additionally , if the energy barrier between minima is too high , transitions between the two states will be observed rarely , which can lead to large errors in the free energy result . to alleviate these problems , a modified eds scheme with smoothing was suggested as follows,4where n is the number of end states ( e.g. , in the case of two states , n = 2 ) , s is a smoothness parameter , and eioffset are energy offset parameters . the schematic representations of mixing two protonation states , ha and a , with different s values are illustrated in figure 1 . the relative energy difference between state ha and a depends on the solution ph , which can be adjusted by the energy offset parameters . the ph dependence of energy offset values will be discussed in more detail in the following section . a lower s value leads to a lower energy barrier in the hybrid hamiltonian , which can facilitate spontaneous state transitions . however , if s becomes too small , eh adopts a single energy minimum , which deviates from the original energy minima of ea and eb . a simulation on eh with such a small s value results in an ensemble comprising of only unphysical intermediate conformations . an iterative parameter optimization procedure was suggested to determine appropriate parameters for an efficient eds simulation . in this paper , we address this problem by performing simulations at multiple s values and performing hamiltonian replica between these simulations to enhance sampling of conformational transitions in the physically realistic hamiltonian . schematic representation of eds hamiltonians ( solid lines ) mixing protonated ( ha , blue dashed line ) and deprotonated ( a , green dashed line ) states for constant ph simulations under various ph conditions : ( a ) ph = pka , ( b ) ph > pka , and ( c ) ph < pka . the difference between energy minima of each protonation state is determined by eq 8 . five eds hamiltonians constructed with different smoothness parameters are illustrated : s = ( red ) , s = 0.7 ( cyan ) , s = 0.22 ( purple ) , s = 0.15 ( yellow ) , s = 0.08 ( black ) . note that a smaller s value leads to a smoother eds hamiltonian with a lower energy barrier . if s is small enough , an eds hamiltonian has a single energy minimum , which is different from the energy minima of either original end state . one goal of constant - ph simulation is to sample the equilibrium distribution of the protonated ( ha ) and deprotonated ( a ) states of a titratable group of a biomolecule at a given ph . the equilibrium distribution of the two protonation states is determined by their free energy difference . however , this free energy difference can not be calculated by a conventional molecular mechanics ( mm ) approach because it can not account for two factors : ( 1 ) the quantum mechanical energy of bond breaking and formation and ( 2 ) the contribution of proton solvation , which is affected by external ph . following mongan et al . , we assume that the total protonation free energy of a titratable group in a protein ( gprotein ) consists of the molecular mechanics ( gprotienmm ) and nonmolecular mechanics ( gprotiennon - mm ) contributions:5 the non - mm component can be estimated by introducing the model compound , which has the same titratable group as the protein but with a known experimental pka value . in this study , a model compound is defined as a solvated amino acid monomer with capped termini . based on the known pka of the model compound ( pka , model ) , its protonation free energy istherefore , the non - mm component of the protonation free energy of the model compound is8with the assumption that the non - mm component of the model compound is identical with that of the protein , gprotiennon - mm = gmodelnon - mm , eq 5 can be expressed as9where gmodelmm can be readily obtained by conventional free energy calculation methods . for a single ionizable group , when no other ionizable groups are titrated , gproteinmm can be calculated by performing an eds simulation with hamiltonians of two protonation states , eproteinmm ( ha ) and eproteinmm ( a ) . the gproteinmm gmodelmm part in eq 9 can be viewed as a shift in free energy of the model compound that is due to the change in the environment of nonbonded interactions of the ionizable group when it is transferred from the solvent to the protein environment . by using eq 9 , the sampling of different protonation states of a titratable group in a protein environment is performed in a ph dependent manner . equation 9 is practically implemented such that the protonated state is considered the reference state and is not experiencing any energy offset , while the deprotonated state is experiencing the ph dependent energy offset gmodelmm + kbt ln 10 ( ph pka , model ) , as also depicted in figure 1 . when multiple ionizable groups in a protein are titrated , the number of states considered has to be increased , e.g. for two ionizable groups , four different states need to be considered , and for three ionizable groups , eight different states need to be considered . the advantage of this method , as opposed to free energy methods such as thermodynamic integration in which only two states are considered , is that it can estimate the ph dependent populations of multiple states in a single eds simulation . for example , in the case of two titratable groups , four states need to be considered . while one of the states ( say the state in which both titratable groups are protonated ) can be considered to be the model state , the ph dependent offset will be applied to the three other states . for a state in which titratable group 1 is deprotonated and group 2 is protonated , the ph dependent energy offset is gmodel1 mm + kbt ln 10 ( ph pka , model1 ) ; for a state in which titratable group 1 is protonated and group 2 is deprotonated , the ph dependent energy offset is gmodel2 mm + kbt ln 10 ( ph pka , model2 ) ; for a state in which both groups are deprotonated , the offset is a sum of the two offsets . if the two titratable groups are of different nature , say lys and glu , the two model compound free energies gmodelmm and pka , model values will be different , but if two groups are the same , these energies and pka , model values will be the same . in hamiltonian replica exchange ( hrex ) each hamiltonian corresponds to a different environmental condition or representation of a system , such as external fields in ising spin system or the strength of hydrophobic interaction in protein folding simulation . generally , a proper exchange between different hamiltonians can enhance the sampling efficiency while preserving the boltzmann distribution . if the mth replica follows the hamiltonian em ( x ) , its boltzmann distribution is10where zm is the partition function of em . because replicas are noninteracting , the joint probability of having configuration x in in the mth replica and configuration x in the nth replica is defined as11we define the probability of exchanging x in mth replica with x in nth replica as w(x , em;x,en ) , and the probability of the reverse process is w(x,em;x , en ) . to satisfy the detailed balance condition , the exchange probability between the two replica must follow the relation:12combining eq 10 with eq 12 leads to13where14this condition can be satisfied by using the metropolis - type criteria for exchanges , the original eds method with smoothing ( eq 4 ) allows sampling of parts of the important phase space of multiple states in a single simulation , and it may lead to poor sampling of physical states . the conformations sampled in the middle of potential energy minima correspond to virtual intermediates between physical states , which may be similar to conformations with fractional charges ( e.g. , 0.5 ) in -dynamics . however , these mixed states are never included in analysis of the ensemble . to increase the efficiency of simulation , residence time at intermediate states as shown in figure 1 , when s is small , the corresponding eds simulation will mainly reside in an intermediate region in the phase space , which deviates substantially from the physical energy minima . to address this problem while preserving the sampling efficiency of eds , we combined the eds method with the hamiltonian exchange method ( figure 2 ) . in this study , we introduce the baseline eds hamiltonian without smoothing for conformational sampling , e(x;s = ) , which follows the minimum potential surface among the original states . assume that state i with hamiltonian ei has the lowest energy at x0 among { e1, ... ,en},all exponential terms in eq 18 vanish as s because s(ej therefore , all conformations sampled with e exactly correspond to one of the original end states , and we denote the corresponding ensemble 0 . in other words , e connects multiple hamiltonians in a way that maximizes the correspondence to the original end states . this effect is more prominent near transition state regions , where the original potential energy surfaces are distorted most by a positive s ( figure 1 ) . in addition , e enables a more accurate sampling of equilibrium ensembles of given hamiltonians than the original eds method , which uses an effective s = 1.0 and slightly deviates from the original hamiltonians near the transition state region . the contribution of a given conformation x in to each partition function can be obtained by simply calculating a corresponding boltzmann factor . these properties make this minimum energy surface the logical choice for collecting the accurate constant ph ensemble . generally , e has a high energy barrier in explicit solvent simulations due to solvent reorganization , which makes it impossible to observe spontaneous protonation state transitions within a computationally accessible time scale . thus , to accelerate transitions , we introduce additional hybrid hamiltonians with smaller s , which lowers the energy barriers in transition regions ( figure 2 ) and perform exchanges between the hamiltonians at a regular interval . at the potential with the smallest s value these nonphysical conformations will be filtered through successive exchanges with more physical hamiltonians , and eventually , only physically accessible conformations will be collected in the e trajectory . schematic representation of the combination of eds and hamiltonian exchange methods for constant ph simulation . an eds hamiltonian with s = , e(x;s = ) , follows the minimum of either hamiltonian of protonated or deprotonated state , min(eprot(x),edeprot(x ) ) . the other hybrid hamiltonians with positive s values have lower energy barriers , which enhances protonation state transitions . in this study , we used five test systems : an aspartic acid , glutamic acid , lysine monomers , a kaae peptide , and snake cardiotoxin v from naja naja atra ( ctx a5 , pdb i d : 1cvo ) . the input structures of all test systems were generated from the charmm22 topology files using the charmming server . the n - termini and c - termini of all test systems are capped with the neutral acetyl and n - methyl groups , respectively . the amino acid monomers and the kaae peptide are solvated in a 30 cubic box with explicit tip3p water molecules , and the snake cardiotoxin is solvated in a 60 cubic box . the protonated and deprotonated states only differ in the partial charges of their side chains . the deprotonated state has a dummy nonzero mass hydrogen atom without charge while keeping the bond , angle , and van der waals interactions . as discussed in the previous section , the contributions of these terms cancel out because the non - mm free energy components of the model compound and protein environment are almost identical . the eds calculation is performed with the eds command of the mscale facility , which can run simulations of multiple independent but connected systems . their potential energy values are calculated in subprocesses and are used to calculate the eds energy and associated gradients in the main processes . the hrex calculation is performed by the repd facility . for all md simulations in this study , exchanges between replicas are attempted every 1000 md steps , and the shake algorithm is used to constrain the bond length of hydrogen atoms . a time step of 1 fs is used , and the nos hoover thermostat is employed to maintain a temperature of 300 k. a nonbonded cutoff of 15 is used , electrostatic interactions are truncated by the force shift method , and van der waals interactions are truncated with a switching function between 10 and 12 . all initial solvated systems are minimized by 200 steps of steepest descent followed by 200 steps with the adopted basis newton raphson method . after minimization , the systems are equilibrated for 1 ns with constant pressure simulations at 1 atm . the last snapshot of the equilibration run is used as the initial structure for the constant ph simulations , and the size of water box is kept constant . to carry out a constant ph simulation , the mm contribution to the protonation free energy of the model compound is required , which effectively arises from the change of electrostatic interactions . in this study , a model compound is defined as a solvated amino acid monomer with capped termini . the gmodelmm values of model compounds are obtained by ti,20where represents a coupling parameter between the protonated and deprotonated states . eele ( ) is defined as21where eele , prot and eele , deprot are the electrostatic potentials of protonated and deprotonated states , respectively . the value changes from 0 to 1 in increments of 0.05 at every 120 ps , using 20 ps of equilibration and 100 ps for gathering statistics . the calculated gmodelmm values are summarized in table 1 . with a given pka value , the fraction of deprotonated samples depends on the ph value and can be obtained with the hill equation:22where fd ( ph ) is the fraction of deprotonated states at a given ph , and n is the hill coefficient . in our method , the fd value is estimated by comparing the boltzmann factors of the protonated and deprotonated states of the conformations in 0 sampled with the baseline hamiltonian e ( eq 19 ) . the fd value can be obtained with23where n is the number of configurations in , xi , and e = e e. to assess the accuracy of eds - hrex constant - ph simulation , we estimated the pka values of several two - state systems , including aspartic acid , glutamic acid , and lysine monomers in explicit water . for each system , we performed three independent constant - ph simulations for 1 ns at different ph values . aspartic acid was run for 5 ns to investigate the convergence of a small system with our method . each eds - hrex simulation consists of 4 eds replicas , e to e , using s values of , 0.027 , 0.020 , and 0.01 . the pka values were estimated by fitting the baseline ensembles to the hill equation . the average pka values and standard deviations of all benchmark systems are summarized in table 2 . for aspartic acid , we carried out 3 independent sets of 6 eds - hrex simulations with ph values ranging from 2 to 7 with an interval of 1 . figure 3a illustrates the average deprotonated fraction of the aspartic acid at each ph condition obtained from 3 independent eds - hrex simulations , along with the corresponding hill equation . the average pka value of the aspartic acids is estimated to be 3.92 , which agrees well with the experimental pka value of 4.0 . deprotonated fractions of ( a ) aspartic acid , ( b ) glutamic acid , and ( c ) lysine by eds - hrex constant ph simulation with explicit water molecules . the average deprotonated fractions of three independent 1 ns simulations are shown as red dots . the fitted titration curves are shown as solid lines . to test the convergence and accuracy of the eds - hrex method , we performed constant ph simulations of aspartic acid for 5 ns at 6 different ph conditions : 18 constant ph simulations in total . the average estimated pka values and standard deviations are calculated for 1 ns time windows ( table 3 ) . the results show that a deviation from experiment of only 0.08 pka units , corresponding to 0.11 kcal / mol , can be achieved even with a 1 ns simulation . starting from a standard deviation of 0.094 pka unit , the value decreases to 0.032 after 2 ns . little change in the standard deviation after 2 ns indicates that the simulations are converged within 2 ns . these results demonstrate that the eds - hrex method can give a reliable pka estimate . to verify that the exchange with smoothed eds potentials enhances the state transitions , we traced the protonation state transition of replica 0 of the aspartic acid from one simulation over time ( figure 4 ) . to determine the state of a conformation x in the smoothed eds hamiltonians , we define the likelihood of state i , i ( x ) , that is , being protonated or deprotonated , as follows:24where e = e e , which is adopted from the zwanzig equation ( eq 3 ) . in figure 4a , if i ( x ) is larger than 0.9999 , x is considered to be the state i. if both i ( x ) and j ( x ) are less than 0.9999 , x is considered as an intermediate state , which could be unphysical . it can be observed that , after multiple exchanges between the eds potentials , replica 0 of aspartic acid returns to e and its protonation state is changed from the protonated to the deprotonated state at 350 ps . after 80 ps , replica 0 reaches the eds hamiltonian with the smallest s , e3 . at e3 , intermediate states are dominantly sampled due to a lowered energy barrier . in addition , fast spontaneous protonation state transitions without hamiltonian exchange are readily observed . these results demonstrate that an eds potential with a small s facilitates state transitions through nonphysical intermediate states , as expected . ( top ) protonation states and the visited eds potentials of replica 0 during 1 ns eds - hrex simulation at ph = 4 . based on the state likelihood i , the protonated ( red circle ) , deprotonated ( blue marks ) , and intermediate ( green triangle ) states are assigned . ( bottom ) difference between the adjusted potential energies of two protonation states , e = ( eprot eprotoffset ) because e follows the lower energy between ( eprot eprotoffset ) and ( edeprot edeprotoffset ) , if e is negative , a configuration corresponds to the protonated state ( red ) . the sampling efficiency of eds - hrex can be estimated from the number of protonation state transitions observed in the production ensemble 0 ( figure 4b ) . during the 1 ns simulation of aspartic acid at ph 4 , we observe an average of 83 protonation state transitions , which is comparable to previous -dynamics based approaches . donnini et al . observed 100 transitions during 20 ns of the titration of imidazole , corresponding to 5 transitions per ns , and goh et al . achieved 50 transitions per ns for the titrations of adenine and cytosine . to verify that the ensemble consists only of physical states , we compare the radial distribution functions ( rdfs ) of water molecules around the od atoms of aspartic acid in each protonation state obtained by our constant - ph simulation with those produced by conventional md simulations with fixed charges ( figure 5 ) . the rdfs obtained by our constant - ph simulations are almost identical with those from conventional fixed - charge md simulations , which demonstrates that our method samples physical states rather than approximate states with noninteger charges obtained by -dynamics . the rdfs between the water hydrogen atoms and the od atoms of aspartic acids are significantly different depending on the protonation state . in the deprotonated state , due to the negatively charged od atoms , the water hydrogen atoms form a sharp peak of the first solvation shell at 1.8 , and the second solvation shell is observed at 3.1 . however , in the protonated state , a hydrogen atom with + 0.44e charge is bonded to an od atom , which repels water hydrogen atoms and reduces the peak height of first solvation shell substantially . the first and second solvation shells are clearly observed at 2.7 and 4.8 , while only the first solvation shell is observed in the protonated state . the rdfs obtained from the ensemble sampled with e ( x;s = 0.01 ) show much less difference between the protonation states . the protonation state is determined based on the state likelihood criterion , ( x ) > 0.99 . the peaks of the deprotonated aspartic acid become lower , and those of the protonated state become higher , which converges to the average of the rdfs of the two protonation states . this shows that the eds sampling with a positive s leads to a significant deviation from the original states . thus , one should be cautious when interpreting an ensemble obtained with a modified hamiltonian , such as -dynamics or an eds potential with s > 0 , because the estimated thermodynamic properties can significantly diverge from the true values . radial distribution functions ( rdfs ) between the od atoms of aspartic acid and water molecules obtained by eds - hrex constant ph simulation at ( a ) e(x;s = ) and ( b ) e(x;s = 0.01 ) with a state likelihood threshold of 0.99 . the subplot c is obtained from 2 ns md simulations with the fixed charges for the protonated and deprotonated states . the rdfs of protonated and deprotonated states are shown as solid and dashed lines , respectively . to verify that our method can be extended to a chemically coupled multistate titration straightforwardly , we performed a constant ph simulation of histidine , which has two coupled titratable sites , nd1 and ne2 , leading to four possible tautomeric states . the titration of histidine is one of the most important goals of constant ph simulations because its experimental pka value is in the range of physiological conditions . in this work , we consider three protonation states defined in the charmm22 force field , the doubly protonated state ( residue type hsp ) , the nd1-protonated state ( hsd ) , and the he2-protonated state ( hse ) . from the given microscopic equilibrium constants , k1 and k2 , for the reactions hsp hsd and hsp hse , the macroscopic equilibrium constant , k , for histidine can be derived as25by using the definition of pka , the macroscopic experimental pka value of histidine can be obtained from the microscopic pka values , pka,1 and pka,2 , as follows26from the given experimental pka value of 6.5 for hsd and 7.1 for hse , the macroscopic experimental pka value is determined to be 6.4 . we carried out 1 ns constant ph simulations of histidine at 5 different ph values ranging from 5 to 9 . because histidine has three protonation states , more state transitions are required for convergence than for the two protonation state systems . we used 6 eds potentials with s = , 0.06 , 0.05 , 0.04 , 0.024 , and 0.01 . figure 6 shows the macroscopic and two microscopic titration curves of histidine , and the estimated pka values are listed in table 4 . the estimated macroscopic pka value is 6.24 with a standard deviation of 0.033 , which agrees well with the experimental pka . the estimated microscopic pka values of hsd and hse are 6.33 and 7.20 , respectively , which also agree with the experimental pka values . these results demonstrate that our method can successfully perform constant ph simulations of multiple titratable sites with chemical coupling . titration curves of histidine obtained by eds - hrex constant ph simulation with explicit water molecules . ( a ) the macroscopic , the total deprotonated fraction of n and n , and two microscopic titration curves of ( b ) n and ( c ) n are illustrated . the average deprotonated fractions of three 1 ns simulations are shown as red dots . for this peptide , four different protonation states were considered : state 1 , with both groups protonated , state 2 with lys-1 deprotonated and glu-4 protonated , state 3 with lys-1 protonated and glu-4 deprotonated , and state 4 with both groups deprotonated . because the pka of a lys model compound is 10.4 and the pka value of glu model compound is 4.4 , state 2 with deprotonated lys and protonated glu is improbable and could have been omitted . however , for consistency , we kept it in the calculations . simulations of the kaae peptide were performed at ph values 2.4 to 13.4 in steps of 1 ph unit . three different sets of simulations were performed , each with different initial velocities and using slightly different eds potentials . the first set of simulations was performed at s = , 0.027 , 0.021 , 0.016 , and 0.012 . the acceptance ratios in replica exchange simulations between replicas 3 and 4 were 45% , that is , larger than the target acceptance ratio of 20% . thus , the second set of simulations was performed with the same s value of the highest replica decreased from 0.012 to 0.0086 . the acceptance ratios for replica exchange between all replicas were still higher than 20% ( the target acceptance ratio ) , except at a few ph values between replicas 0 and 1 , and at one ph value between replicas 1 and 2 . thus , we performed the third set of simulations at s = , 0.03 , 0.022 , 0.016 , and 0.0086 . the titration curves for the peptide were determined by averaging the three simulations ( figure 7 ) . based on the standard deviations , we conclude that the change in distribution of eds potentials had virtually no effect on the population of the four states and the calculated pka values and hill coefficients . ( a ) the macroscopic , the sum of deprotonated fractions of glutamic acid and lysine , and two microscopic titration curves of ( b ) glutamic acid and ( c ) lysine are illustrated . the average deprotonated fractions of three 1 ns simulations are shown as red dots . the pka value of lys-1 is 11.38 ; that is , it is shifted by 1 ph unit from the model compound pka value of 10.4 , while the pka of glu-4 is 4.23 , only 0.2 ph units lower than that of the model compound . hydrogen bond analysis was performed with vmd for simulations at ph values 2.4 , 7.4 , and 13.4 , which corresponded to states 1 , 3 , and 4 , respectively , being predominately populated . as a measure of ion - pair interactions for ph values 2.4 and 13.4 , these two atoms were never closer than 4 to each other , but at ph 7 , they were within 4 6% of the time . in terms of hydrogen bonding interactions with the rest of the peptide , glu-4 did not engage in any , while lys-1 was hydrogen bonded 8% of the time at ph 2.4 , 20% of the time at ph 7.4 , and 2% of the time at ph 13.4 . these hydrogen bonding interactions may explain why the calculated pka value of lys was shifted more than that of glu . finally , we performed a constant ph simulation of snake cardiotoxin v from naja naja atra ( ctx a5 , pdb i d : 1cvo ) . ctx a5 has three titratable residues , glu-17 , asp-42 , and asp-59 that affect the stability of the protein between ph values 2 and 5 . we considered all possible protonation state combinations of these residues ( 8 total ) . three sets of 1 ns eds - hrex simulations were performed at ph values ranging from 1 to 6 in steps of 1 ph unit . each eds - hrex simulation consists of 6 replicas with s values of , 0.033 , 0.027 , 0.022 , 0.018 , and 0.01 corresponding to a total simulation time of 108 ns . the deprotonated fractions of titratable residues were obtained from the average of three sets of simulations , and the variables for the hill equation were obtained by fitting the average data points to the hill equation ( figure 8) . the calculated pka values and hill coefficients of three titratable residues titration curves of three titratable residues of snake cardiotoxin ( ctx a5 ) are shown . the deprotonated fractions of ( a ) glu-17 , ( b ) asp-42 , and ( c ) asp-59 are illustrated . the average deprotonated fractions of three 1 ns simulations are shown as red dots . the calculated pka values , directions of the pka shifts , and hill coefficients are in accordance with the experiment . it is known that asp-59 strongly interacts with the adjacent lys-2 , which results in a large shift of pka value of asp-59 , from 4.0 to less than 2.3 . in our result , the pka is calculated to be 1.4 , which is consistent with this . the pka of asp-42 is calculated to be 3.0 , which is close to the experimental value of 3.2 . the largest error is observed in glu-17 , whose calculated pka value is lower than the experiment by 1.6 pka units . one possible source of this error may be limited conformational sampling . to obtain accurate pka estimates , multiple transitions between different protonation states should be sampled . generally , the titrations of residues are strongly coupled with protein conformational changes . therefore , sufficient conformational sampling is important to reproduce experimental results . to check the convergence of our simulations , we counted the average number of protonation transitions of replicas sampled with the s = hamiltonian at ph 2 and ph 3 ( figure 9 ) . the protonation states of three residues , glu-17 , asp-42 , and asp-59 , are denoted by three letters ( e.g. , pdd ) . p and d represent the protonated and deprotonated state , respectively , and thus pdd would correspond to glu-17 protonated , asp-42 deprotonated , and asp-59 deprotonated . the majority of state transitions are observed between a subset of states , while the rest of the states are rarely visited . this indicates that the simulations are not fully converged , possibly due to limited conformational sampling . at ph 2 , most protonation transitions occur between states ppp , ppd , dpp , and dpd , while the transition to state ddp is sampled only once during all 3 simulations ( figure 9a and b ) . when the external ph is 3 , transitions between two pairs of states the current eds scheme lowers energy barriers caused by different energy terms between end states . in constant ph simulations , in other words , the energy barriers originating from other energy terms ( i.e. , van der waals or dihedral terms ) are conserved after mixing by eds , which can limit the conformational sampling of titratable groups . this sampling issue may be solved by combining the current eds scheme with other accelerated sampling methods that preserve the canonical ensemble , such as self - guided langevin dynamics with reweighting or orthogonal space random walk . in addition , introducing additional dimensions of hamiltonian exchange to allow exchanges between ph values can also improve the convergence rate of simulations . another source of error may originate from an imperfect representation of electrostatistics : using the classical mm model with fixed partial charges . for glu-17 , three positively charged lysines , lys-2 , lys-13 , and lys-19 , are located in the vicinity of glu-17 and can affect the titration behavior . lys-2 is considered to be especially important in controlling the stability of ctx a5 through interaction with glu-17 . if electrostatic interactions between glu-17 and these three neighboring lysines are overestimated with the current mm force field , it may result in overpopulation of deprotonated glu-17 leading to a lower calculated pka value . this issue can be addressed by using polarizable force fields or qm / mm approaches , which can treat electrostatic interactions more accurately . recently , wallace and shen have shown that a charge - leveling by simultaneous ionization or neutralization of a ion in solution can help to reproduce an experimental pka value more accurately . summary of protonation state transitions at ph ( a ) 2 and ( b ) 3 are shown . each node represents a protonation state and the width of the edge is proportional to the average number of transitions observed . the thinnest edge corresponds to only one transition from three sets of eds - hrex simulations . the eds - hrex method can be readily extended to titratable groups with chemically coupled moieties , such as histidine . this approach solves a problem inherent in -dynamic based approaches , where a new model or coordinate must be implemented to control the interconversion between such states . for example , histidine has two titratable sites , n and n , and their atomic charges depend on each other . this dependence can not be represented properly by a single titration coordinate . to address this issue , khandogin and brooks introduced a tautomeric state variable in addition to , and donnini et al . performed linear - interpolations between all possible combinations of protonation states explicitly . in the eds - hrex method , transitions between any pair of protonation states are automatically considered by performing md simulation with the hybrid hamiltonian . the eds - hrex method is compatible with any existing force field because the energy and forces of the hybrid hamiltonian can be readily obtained from those of end states , which are calculated independently . in most current force fields , an atom type and its associated force field parameters our method can consider the change of atomic parameters other than charge , such as the van der waals or generalized born solvation radius parameters . to apply the -dynamics approach to the change of general force field parameters rigorously , the parameters have to be interpolated linearly with respect to . otherwise , this can be an inherent source of error as discussed in previous constant ph simulation with the gbsw implicit solvent model . the linear interpolation of parameters also requires the analytic derivatives of energy functions associated with , which can be highly complicated to compute . the eds - hrex method can be used for any free - energy calculation , not only those involving ph . it yields accurate free energy estimates because the e replica does not smooth the eds hamiltonian at all ( s = ) . in the original eds method , however , this equation only converges if energy differences between the original hybrid states are small . therefore , a trade - off must be made between efficiency of sampling ( small s ) and convergence of the result ( achievable with large s ) . to address this , an iterative parameter optimization scheme has been proposed to find the ideal s value , which optimizes accuracy . however , this method still samples some number of unphysical system states . in the eds - hrex method , the e replica always has a s of , while other replicas are used to explore different conformations . therefore , the free energy differences between states can be directly calculated by comparing their boltzmann factors from replica e because this replica only samples physical states . another potentially significant advantage of eds - hrex is that the resultant ensemble has only discrete protonation states , and these can be coupled to a high quality quantum mechanics ( qm ) or qm / mm surface using a non - boltzmann bennett approach . determining the optimal replica distribution and smoothness parameters efficiently requires further investigation . in this study , the parameters were determined by trial - and - error , using a series of short simulations . we plan to devise an automatic procedure to determine the optimal parameter set for a given problem , which is similar to an iterative procedure that optimizes parameters for a single eds simulation . as general guidelines to determine parameters , two conditions should be satisfied . first , spontaneous state transitions should be observed with highly smoothed eds potentials , as shown in figure 4a . this ensures that an eds - hrex simulation actually samples important protonation states , which is essential in constant ph simulations . second , an average exchange rate between replicas should be in the range 2030% for an efficient sampling of various protonation state . however , this increased cost is offset by the advantage that every time step may be used to collect the final ensemble . currently , the eds method is implemented via the mscale facility in charmm , which requires the independent energy evaluation of each end state . therefore , the apparent cost of our method is simply proportional to the number of possible protonation states times the number of replicas used . for example , if there are x titratable groups with two protonation states , we need at most 2nr times more computational resources than a single hamiltonian simulation , where nr is the number of replicas . however , in some cases , at any given ph , the number of states considered can be reduced . for example , a histidine residue has four possible protonation states but the only three of those states need to be considered under physiological conditions ; the fully deprotonated state will not contribute . in the case of the kaae peptide , the state in which glu is protonated and lys is deprotonated could have also been omitted , reducing the total number of states from four to three . finally , ctx a5 has multiple lysines on its surface , which were assumed to be positive in this study because they are almost fully exposed and expected to experience little pka shifts . therefore , the cost may be reduced by eliminating rarely populated charge states . on a similar note , we point out that the scalability of -dynamics may not be completely linear with the number of titratable groups . when there are n independent titratable groups , and if we assume that a probability to obtain a physical charge state of a single titratable site , that is , > 0.8 or < 0.2 , is p , the fraction of snapshots that all titratable groups are in physical charge states becomes p. our method focuses on obtaining accurate configurations of different protonation states . as shown in figure 5 , even with a rather strict state likelihood value > 0.99 , the radial distribution functions between titratable sites and water molecules obtained from a smoothed eds potential are significantly distorted . thus , the major contribution of our method is in the improved quality of ensembles , because our method samples original end states with the s = hamiltonian . the computational cost can also be reduced with an improved eds scheme when only a few atom charges change in a large system . the eds equation only requires energy differences . instead of calculating the full energy of each charge state , computing just the energy differences in other words , instead of calculating e1 , e2 , e3 , and e4 , we can perform an eds simulation by calculating e1 , e2 the energies of different states differ only in electrostatic interactions between titratable groups and their neighboring atoms within a cutoff radius . therefore , the energies and gradients of a subset of nonbonded pairs should be recalculated with different charge sets . we devised a new computational approach for constant - ph simulations in explicit solvent by combining the eds and hamiltonian replica exchange algorithms . we showed that this method can reproduce the correct description of multiple protonation states with frequent state transitions . a comparison of radial distribution functions between aspartic acid and water molecules demonstrates that the ensemble obtained with the baseline eds hamiltonian agrees well with those of md simulations with fixed charges . in terms of sampling efficiency , we observed over 80 protonation state transitions of blocked aspartic acid during 1 ns of simulation , which is comparable to the -dynamics based approaches . we also showed that the eds - hrex method can be easily extended to multiple protonation state cases with the titration of histidine , kaae peptide , and snake cardiotoxin . due to the generality of the eds - hrex method , it can be applied to free energy calculations in various problems that require frequent transitions between multiple states separated by large energy barriers .

we present a new computational approach for constant ph simulations in explicit solvent based on the combination of the enveloping distribution sampling ( eds ) and hamiltonian replica exchange ( hrex ) methods . unlike constant ph methods based on variable and continuous charge models , our method is based on discrete protonation states . eds generates a hybrid hamiltonian of different protonation states . a smoothness parameter s is used to control the heights of energy barriers of the hybrid - state energy landscape . a small s value facilitates state transitions by lowering energy barriers . replica exchange between eds potentials with different s values allows us to readily obtain a thermodynamically accurate ensemble of multiple protonation states with frequent state transitions . the analysis is performed with an ensemble obtained from an eds hamiltonian without smoothing , s = , which strictly follows the minimum energy surface of the end states . the accuracy and efficiency of this method is tested on aspartic acid , lysine , and glutamic acid , which have two protonation states , a histidine with three states , a four - residue peptide with four states , and snake cardiotoxin with eight states . the pka values estimated with the eds - hrex method agree well with the experimental pka values . the mean absolute errors of small benchmark systems range from 0.03 to 0.17 pka units , and those of three titratable groups of snake cardiotoxin range from 0.2 to 1.6 pka units . this study demonstrates that eds - hrex is a potent theoretical framework , which gives the correct description of multiple protonation states and good calculated pka values .

Introduction Theory Methods Results and Discussion Conclusion

a widely used methodology for pka calculation is based on the solutions of the poisson the limitation of pb - based methods is that they may not properly represent the reorganization / response of protein induced by the titration of ionizable groups . also , the conformational response of a protein is modeled by a single value of a dielectric constant , which is dubious considering the inhomogeneous environment of the interior and surface of a protein . constant ph simulation methods can be categorized into two groups : ( 1 ) discrete protonation state models , and ( 2 ) continuous protonation state models . constant ph methods based on discrete protonation state models generally use a hybrid approach combining molecular dynamics ( md ) and monte carlo ( mc ) . in the hybrid mc / md methods , a mc procedure is performed to determine the protonation states of ionizable groups at a regular interval over the course of a md simulation with either implicit or explicit water . during the mc procedure , a random walk between different protonation states is performed , and a state is determined based on the estimated free energy difference and metropolis criteria . to enhance the accuracy of simulations that depend heavily on the accuracy of conformational sampling , constant ph methods with both discrete and continuous protonation states the gb - based constant ph methods have been coupled with the temperature replica exchange and accelerated md methods . in this study , we develop a new constant - ph method that yields the correct description of protonation states by combining the enveloping distribution sampling ( eds ) and hamiltonian replica exchange ( hrex ) methods . the eds method was devised to allow sampling of multiple end states from a single md simulation of a hybrid hamiltonian . in the context of a constant ph simulation , the hybrid hamiltonian is the sum of boltzmann factors of multiple hamiltonians , each corresponding to a different protonation state . to ensure proper sampling of all protonation states , a smoothness parameter , s , that controls the height of energy barriers when s 0 , the hybrid hamiltonian becomes highly smoothed , which makes energy barriers disappear . on the other hand , as s , no smoothing is applied to the hybrid hamiltonian , and it follows the minimum energy surface among the multiple end states , which corresponds to the physical hamiltonian . to obtain the correct ensemble of multiple protonation states while enhancing sampling efficiency , we coupled the eds simulations with different smoothness parameter through the hrex method . in the context of the eds - hrex method , we use multiple eds potentials with different smoothness parameter , including an eds potential without smoothing that follows the minimum energy of multiple end states , and perform exchanges between them periodically . we assessed the performance of our method with titratable amino acid monomers : aspartic acid , glutamic acid , lysine , and histidine . we also tested out method with a small four - residue peptide ( kaae ) and snake cardiotoxin . the results show that our method can successfully and efficiently reproduce the correct distribution of different protonation states at a given ph . we briefly review the enveloping distribution sampling ( eds ) and hamiltonian replica exchange ( hrex ) methods and subsequently describe how these two methods are combined to sample the correct ensemble of different protonation states at a given ph . in the eds approach , a hybrid hamiltonian enveloping both states is defined as follows,2where ea and eb are the hamiltonians of states a and b. in principle , a simulation performed on eh allows sampling of the important phase space of both state a and b , and their free energy difference can be estimated by3where however , if the energy difference between the minima of ea and eb is too large , the simulation will be trapped in the lowest energy basin of a single hamiltonian . , in the case of two states , n = 2 ) , s is a smoothness parameter , and eioffset are energy offset parameters . the schematic representations of mixing two protonation states , ha and a , with different s values are illustrated in figure 1 . a lower s value leads to a lower energy barrier in the hybrid hamiltonian , which can facilitate spontaneous state transitions . a simulation on eh with such a small s value results in an ensemble comprising of only unphysical intermediate conformations . schematic representation of eds hamiltonians ( solid lines ) mixing protonated ( ha , blue dashed line ) and deprotonated ( a , green dashed line ) states for constant ph simulations under various ph conditions : ( a ) ph = pka , ( b ) ph > pka , and ( c ) ph < pka . if s is small enough , an eds hamiltonian has a single energy minimum , which is different from the energy minima of either original end state . , we assume that the total protonation free energy of a titratable group in a protein ( gprotein ) consists of the molecular mechanics ( gprotienmm ) and nonmolecular mechanics ( gprotiennon - mm ) contributions:5 the non - mm component can be estimated by introducing the model compound , which has the same titratable group as the protein but with a known experimental pka value . for a single ionizable group , when no other ionizable groups are titrated , gproteinmm can be calculated by performing an eds simulation with hamiltonians of two protonation states , eproteinmm ( ha ) and eproteinmm ( a ) . by using eq 9 , the sampling of different protonation states of a titratable group in a protein environment is performed in a ph dependent manner . the advantage of this method , as opposed to free energy methods such as thermodynamic integration in which only two states are considered , is that it can estimate the ph dependent populations of multiple states in a single eds simulation . in hamiltonian replica exchange ( hrex ) each hamiltonian corresponds to a different environmental condition or representation of a system , such as external fields in ising spin system or the strength of hydrophobic interaction in protein folding simulation . in this study , we introduce the baseline eds hamiltonian without smoothing for conformational sampling , e(x;s = ) , which follows the minimum potential surface among the original states . assume that state i with hamiltonian ei has the lowest energy at x0 among { e1, ... ,en},all exponential terms in eq 18 vanish as s because s(ej therefore , all conformations sampled with e exactly correspond to one of the original end states , and we denote the corresponding ensemble 0 . generally , e has a high energy barrier in explicit solvent simulations due to solvent reorganization , which makes it impossible to observe spontaneous protonation state transitions within a computationally accessible time scale . thus , to accelerate transitions , we introduce additional hybrid hamiltonians with smaller s , which lowers the energy barriers in transition regions ( figure 2 ) and perform exchanges between the hamiltonians at a regular interval . schematic representation of the combination of eds and hamiltonian exchange methods for constant ph simulation . an eds hamiltonian with s = , e(x;s = ) , follows the minimum of either hamiltonian of protonated or deprotonated state , min(eprot(x),edeprot(x ) ) . the other hybrid hamiltonians with positive s values have lower energy barriers , which enhances protonation state transitions . in this study , we used five test systems : an aspartic acid , glutamic acid , lysine monomers , a kaae peptide , and snake cardiotoxin v from naja naja atra ( ctx a5 , pdb i d : 1cvo ) . the eds calculation is performed with the eds command of the mscale facility , which can run simulations of multiple independent but connected systems . for all md simulations in this study , exchanges between replicas are attempted every 1000 md steps , and the shake algorithm is used to constrain the bond length of hydrogen atoms . the last snapshot of the equilibration run is used as the initial structure for the constant ph simulations , and the size of water box is kept constant . with a given pka value , the fraction of deprotonated samples depends on the ph value and can be obtained with the hill equation:22where fd ( ph ) is the fraction of deprotonated states at a given ph , and n is the hill coefficient . the fd value can be obtained with23where n is the number of configurations in , xi , and e = e e. to assess the accuracy of eds - hrex constant - ph simulation , we estimated the pka values of several two - state systems , including aspartic acid , glutamic acid , and lysine monomers in explicit water . each eds - hrex simulation consists of 4 eds replicas , e to e , using s values of , 0.027 , 0.020 , and 0.01 . for aspartic acid , we carried out 3 independent sets of 6 eds - hrex simulations with ph values ranging from 2 to 7 with an interval of 1 . figure 3a illustrates the average deprotonated fraction of the aspartic acid at each ph condition obtained from 3 independent eds - hrex simulations , along with the corresponding hill equation . the average pka value of the aspartic acids is estimated to be 3.92 , which agrees well with the experimental pka value of 4.0 . deprotonated fractions of ( a ) aspartic acid , ( b ) glutamic acid , and ( c ) lysine by eds - hrex constant ph simulation with explicit water molecules . to test the convergence and accuracy of the eds - hrex method , we performed constant ph simulations of aspartic acid for 5 ns at 6 different ph conditions : 18 constant ph simulations in total . these results demonstrate that the eds - hrex method can give a reliable pka estimate . to verify that the exchange with smoothed eds potentials enhances the state transitions , we traced the protonation state transition of replica 0 of the aspartic acid from one simulation over time ( figure 4 ) . these results demonstrate that an eds potential with a small s facilitates state transitions through nonphysical intermediate states , as expected . ( top ) protonation states and the visited eds potentials of replica 0 during 1 ns eds - hrex simulation at ph = 4 . ( bottom ) difference between the adjusted potential energies of two protonation states , e = ( eprot eprotoffset ) because e follows the lower energy between ( eprot eprotoffset ) and ( edeprot edeprotoffset ) , if e is negative , a configuration corresponds to the protonated state ( red ) . the sampling efficiency of eds - hrex can be estimated from the number of protonation state transitions observed in the production ensemble 0 ( figure 4b ) . during the 1 ns simulation of aspartic acid at ph 4 , we observe an average of 83 protonation state transitions , which is comparable to previous -dynamics based approaches . the rdfs obtained by our constant - ph simulations are almost identical with those from conventional fixed - charge md simulations , which demonstrates that our method samples physical states rather than approximate states with noninteger charges obtained by -dynamics . the peaks of the deprotonated aspartic acid become lower , and those of the protonated state become higher , which converges to the average of the rdfs of the two protonation states . thus , one should be cautious when interpreting an ensemble obtained with a modified hamiltonian , such as -dynamics or an eds potential with s > 0 , because the estimated thermodynamic properties can significantly diverge from the true values . radial distribution functions ( rdfs ) between the od atoms of aspartic acid and water molecules obtained by eds - hrex constant ph simulation at ( a ) e(x;s = ) and ( b ) e(x;s = 0.01 ) with a state likelihood threshold of 0.99 . the titration of histidine is one of the most important goals of constant ph simulations because its experimental pka value is in the range of physiological conditions . because histidine has three protonation states , more state transitions are required for convergence than for the two protonation state systems . we used 6 eds potentials with s = , 0.06 , 0.05 , 0.04 , 0.024 , and 0.01 . the estimated macroscopic pka value is 6.24 with a standard deviation of 0.033 , which agrees well with the experimental pka . the estimated microscopic pka values of hsd and hse are 6.33 and 7.20 , respectively , which also agree with the experimental pka values . these results demonstrate that our method can successfully perform constant ph simulations of multiple titratable sites with chemical coupling . thus , the second set of simulations was performed with the same s value of the highest replica decreased from 0.012 to 0.0086 . thus , we performed the third set of simulations at s = , 0.03 , 0.022 , 0.016 , and 0.0086 . based on the standard deviations , we conclude that the change in distribution of eds potentials had virtually no effect on the population of the four states and the calculated pka values and hill coefficients . each eds - hrex simulation consists of 6 replicas with s values of , 0.033 , 0.027 , 0.022 , 0.018 , and 0.01 corresponding to a total simulation time of 108 ns . the calculated pka values and hill coefficients of three titratable residues titration curves of three titratable residues of snake cardiotoxin ( ctx a5 ) are shown . the calculated pka values , directions of the pka shifts , and hill coefficients are in accordance with the experiment . the largest error is observed in glu-17 , whose calculated pka value is lower than the experiment by 1.6 pka units . in constant ph simulations , in other words , the energy barriers originating from other energy terms ( i.e. the eds - hrex method can be readily extended to titratable groups with chemically coupled moieties , such as histidine . in the eds - hrex method , transitions between any pair of protonation states are automatically considered by performing md simulation with the hybrid hamiltonian . the eds - hrex method is compatible with any existing force field because the energy and forces of the hybrid hamiltonian can be readily obtained from those of end states , which are calculated independently . the eds - hrex method can be used for any free - energy calculation , not only those involving ph . therefore , a trade - off must be made between efficiency of sampling ( small s ) and convergence of the result ( achievable with large s ) . in the eds - hrex method , the e replica always has a s of , while other replicas are used to explore different conformations . another potentially significant advantage of eds - hrex is that the resultant ensemble has only discrete protonation states , and these can be coupled to a high quality quantum mechanics ( qm ) or qm / mm surface using a non - boltzmann bennett approach . this ensures that an eds - hrex simulation actually samples important protonation states , which is essential in constant ph simulations . for example , if there are x titratable groups with two protonation states , we need at most 2nr times more computational resources than a single hamiltonian simulation , where nr is the number of replicas . when there are n independent titratable groups , and if we assume that a probability to obtain a physical charge state of a single titratable site , that is , > 0.8 or < 0.2 , is p , the fraction of snapshots that all titratable groups are in physical charge states becomes p. our method focuses on obtaining accurate configurations of different protonation states . thus , the major contribution of our method is in the improved quality of ensembles , because our method samples original end states with the s = hamiltonian . instead of calculating the full energy of each charge state , computing just the energy differences in other words , instead of calculating e1 , e2 , e3 , and e4 , we can perform an eds simulation by calculating e1 , e2 the energies of different states differ only in electrostatic interactions between titratable groups and their neighboring atoms within a cutoff radius . we devised a new computational approach for constant - ph simulations in explicit solvent by combining the eds and hamiltonian replica exchange algorithms . we showed that this method can reproduce the correct description of multiple protonation states with frequent state transitions . a comparison of radial distribution functions between aspartic acid and water molecules demonstrates that the ensemble obtained with the baseline eds hamiltonian agrees well with those of md simulations with fixed charges . we also showed that the eds - hrex method can be easily extended to multiple protonation state cases with the titration of histidine , kaae peptide , and snake cardiotoxin . due to the generality of the eds - hrex method , it can be applied to free energy calculations in various problems that require frequent transitions between multiple states separated by large energy barriers .

in cells , the ph in different compartments varies significantly ; for example , the ph in the cytoplasm and nucleus is neutral ( around 7.2 ) , in vacuoles and golgi , it is acidic ( between 4.8 and 6.5 ) , and in mitochondria , it is basic ( around 8.0 ) . changes in the protonation state can be exploited for function , for example , when they are coupled to conformational reorganization , such as in the case of atp synthase , bacteriorhodopsin , cytochrome c oxidase , or the photoactive yellow protein . a widely used methodology for pka calculation is based on the solutions of the poisson the limitation of pb - based methods is that they may not properly represent the reorganization / response of protein induced by the titration of ionizable groups . also , the conformational response of a protein is modeled by a single value of a dielectric constant , which is dubious considering the inhomogeneous environment of the interior and surface of a protein . in the hybrid mc / md methods , a mc procedure is performed to determine the protonation states of ionizable groups at a regular interval over the course of a md simulation with either implicit or explicit water . in contrast to the implicit solvent methods above , the md / mc method with explicit water requires a more sophisticated mc move due to solvent reorganization . improved the dynamics approach to constant - ph simulation with the gb solvation model the extent of protonation is parametrized by a fictitious particle in the hamiltonian , whose value fluctuates between 0 and 1 . to avoid sampling of unphysical states , this potential has to be carefully adjusted to maximize the number of transitions and minimize sampling of unphysical states , however , even with this approach , most of the sampled conformations are different from physical states . to enhance the accuracy of simulations that depend heavily on the accuracy of conformational sampling , constant ph methods with both discrete and continuous protonation states the gb - based constant ph methods have been coupled with the temperature replica exchange and accelerated md methods . to further increase sampling efficiency , the ph - exchange approach has been combined with reservoirs of conformations and protonation states within the framework of the double reservoir ph replica exchange method ( work submitted for publication ) . in this study , we develop a new constant - ph method that yields the correct description of protonation states by combining the enveloping distribution sampling ( eds ) and hamiltonian replica exchange ( hrex ) methods . in the context of a constant ph simulation , the hybrid hamiltonian is the sum of boltzmann factors of multiple hamiltonians , each corresponding to a different protonation state . in the context of the eds - hrex method , we use multiple eds potentials with different smoothness parameter , including an eds potential without smoothing that follows the minimum energy of multiple end states , and perform exchanges between them periodically . we briefly review the enveloping distribution sampling ( eds ) and hamiltonian replica exchange ( hrex ) methods and subsequently describe how these two methods are combined to sample the correct ensemble of different protonation states at a given ph . in the eds approach , a hybrid hamiltonian enveloping both states is defined as follows,2where ea and eb are the hamiltonians of states a and b. in principle , a simulation performed on eh allows sampling of the important phase space of both state a and b , and their free energy difference can be estimated by3where however , if the energy difference between the minima of ea and eb is too large , the simulation will be trapped in the lowest energy basin of a single hamiltonian . one goal of constant - ph simulation is to sample the equilibrium distribution of the protonated ( ha ) and deprotonated ( a ) states of a titratable group of a biomolecule at a given ph . however , this free energy difference can not be calculated by a conventional molecular mechanics ( mm ) approach because it can not account for two factors : ( 1 ) the quantum mechanical energy of bond breaking and formation and ( 2 ) the contribution of proton solvation , which is affected by external ph . , we assume that the total protonation free energy of a titratable group in a protein ( gprotein ) consists of the molecular mechanics ( gprotienmm ) and nonmolecular mechanics ( gprotiennon - mm ) contributions:5 the non - mm component can be estimated by introducing the model compound , which has the same titratable group as the protein but with a known experimental pka value . based on the known pka of the model compound ( pka , model ) , its protonation free energy istherefore , the non - mm component of the protonation free energy of the model compound is8with the assumption that the non - mm component of the model compound is identical with that of the protein , gprotiennon - mm = gmodelnon - mm , eq 5 can be expressed as9where gmodelmm can be readily obtained by conventional free energy calculation methods . the gproteinmm gmodelmm part in eq 9 can be viewed as a shift in free energy of the model compound that is due to the change in the environment of nonbonded interactions of the ionizable group when it is transferred from the solvent to the protein environment . equation 9 is practically implemented such that the protonated state is considered the reference state and is not experiencing any energy offset , while the deprotonated state is experiencing the ph dependent energy offset gmodelmm + kbt ln 10 ( ph pka , model ) , as also depicted in figure 1 . the advantage of this method , as opposed to free energy methods such as thermodynamic integration in which only two states are considered , is that it can estimate the ph dependent populations of multiple states in a single eds simulation . while one of the states ( say the state in which both titratable groups are protonated ) can be considered to be the model state , the ph dependent offset will be applied to the three other states . for a state in which titratable group 1 is deprotonated and group 2 is protonated , the ph dependent energy offset is gmodel1 mm + kbt ln 10 ( ph pka , model1 ) ; for a state in which titratable group 1 is protonated and group 2 is deprotonated , the ph dependent energy offset is gmodel2 mm + kbt ln 10 ( ph pka , model2 ) ; for a state in which both groups are deprotonated , the offset is a sum of the two offsets . if the two titratable groups are of different nature , say lys and glu , the two model compound free energies gmodelmm and pka , model values will be different , but if two groups are the same , these energies and pka , model values will be the same . because replicas are noninteracting , the joint probability of having configuration x in in the mth replica and configuration x in the nth replica is defined as11we define the probability of exchanging x in mth replica with x in nth replica as w(x , em;x,en ) , and the probability of the reverse process is w(x,em;x , en ) . to satisfy the detailed balance condition , the exchange probability between the two replica must follow the relation:12combining eq 10 with eq 12 leads to13where14this condition can be satisfied by using the metropolis - type criteria for exchanges , the original eds method with smoothing ( eq 4 ) allows sampling of parts of the important phase space of multiple states in a single simulation , and it may lead to poor sampling of physical states . the conformations sampled in the middle of potential energy minima correspond to virtual intermediates between physical states , which may be similar to conformations with fractional charges ( e.g. to increase the efficiency of simulation , residence time at intermediate states as shown in figure 1 , when s is small , the corresponding eds simulation will mainly reside in an intermediate region in the phase space , which deviates substantially from the physical energy minima . in this study , we introduce the baseline eds hamiltonian without smoothing for conformational sampling , e(x;s = ) , which follows the minimum potential surface among the original states . assume that state i with hamiltonian ei has the lowest energy at x0 among { e1, ... ,en},all exponential terms in eq 18 vanish as s because s(ej therefore , all conformations sampled with e exactly correspond to one of the original end states , and we denote the corresponding ensemble 0 . thus , to accelerate transitions , we introduce additional hybrid hamiltonians with smaller s , which lowers the energy barriers in transition regions ( figure 2 ) and perform exchanges between the hamiltonians at a regular interval . in this study , we used five test systems : an aspartic acid , glutamic acid , lysine monomers , a kaae peptide , and snake cardiotoxin v from naja naja atra ( ctx a5 , pdb i d : 1cvo ) . as discussed in the previous section , the contributions of these terms cancel out because the non - mm free energy components of the model compound and protein environment are almost identical . the fd value can be obtained with23where n is the number of configurations in , xi , and e = e e. to assess the accuracy of eds - hrex constant - ph simulation , we estimated the pka values of several two - state systems , including aspartic acid , glutamic acid , and lysine monomers in explicit water . to test the convergence and accuracy of the eds - hrex method , we performed constant ph simulations of aspartic acid for 5 ns at 6 different ph conditions : 18 constant ph simulations in total . to verify that the exchange with smoothed eds potentials enhances the state transitions , we traced the protonation state transition of replica 0 of the aspartic acid from one simulation over time ( figure 4 ) . to determine the state of a conformation x in the smoothed eds hamiltonians , we define the likelihood of state i , i ( x ) , that is , being protonated or deprotonated , as follows:24where e = e e , which is adopted from the zwanzig equation ( eq 3 ) . it can be observed that , after multiple exchanges between the eds potentials , replica 0 of aspartic acid returns to e and its protonation state is changed from the protonated to the deprotonated state at 350 ps . ( bottom ) difference between the adjusted potential energies of two protonation states , e = ( eprot eprotoffset ) because e follows the lower energy between ( eprot eprotoffset ) and ( edeprot edeprotoffset ) , if e is negative , a configuration corresponds to the protonated state ( red ) . during the 1 ns simulation of aspartic acid at ph 4 , we observe an average of 83 protonation state transitions , which is comparable to previous -dynamics based approaches . to verify that the ensemble consists only of physical states , we compare the radial distribution functions ( rdfs ) of water molecules around the od atoms of aspartic acid in each protonation state obtained by our constant - ph simulation with those produced by conventional md simulations with fixed charges ( figure 5 ) . in the deprotonated state , due to the negatively charged od atoms , the water hydrogen atoms form a sharp peak of the first solvation shell at 1.8 , and the second solvation shell is observed at 3.1 . the peaks of the deprotonated aspartic acid become lower , and those of the protonated state become higher , which converges to the average of the rdfs of the two protonation states . radial distribution functions ( rdfs ) between the od atoms of aspartic acid and water molecules obtained by eds - hrex constant ph simulation at ( a ) e(x;s = ) and ( b ) e(x;s = 0.01 ) with a state likelihood threshold of 0.99 . to verify that our method can be extended to a chemically coupled multistate titration straightforwardly , we performed a constant ph simulation of histidine , which has two coupled titratable sites , nd1 and ne2 , leading to four possible tautomeric states . in this work , we consider three protonation states defined in the charmm22 force field , the doubly protonated state ( residue type hsp ) , the nd1-protonated state ( hsd ) , and the he2-protonated state ( hse ) . from the given microscopic equilibrium constants , k1 and k2 , for the reactions hsp hsd and hsp hse , the macroscopic equilibrium constant , k , for histidine can be derived as25by using the definition of pka , the macroscopic experimental pka value of histidine can be obtained from the microscopic pka values , pka,1 and pka,2 , as follows26from the given experimental pka value of 6.5 for hsd and 7.1 for hse , the macroscopic experimental pka value is determined to be 6.4 . ( a ) the macroscopic , the total deprotonated fraction of n and n , and two microscopic titration curves of ( b ) n and ( c ) n are illustrated . for this peptide , four different protonation states were considered : state 1 , with both groups protonated , state 2 with lys-1 deprotonated and glu-4 protonated , state 3 with lys-1 protonated and glu-4 deprotonated , and state 4 with both groups deprotonated . because the pka of a lys model compound is 10.4 and the pka value of glu model compound is 4.4 , state 2 with deprotonated lys and protonated glu is improbable and could have been omitted . based on the standard deviations , we conclude that the change in distribution of eds potentials had virtually no effect on the population of the four states and the calculated pka values and hill coefficients . ( a ) the macroscopic , the sum of deprotonated fractions of glutamic acid and lysine , and two microscopic titration curves of ( b ) glutamic acid and ( c ) lysine are illustrated . in terms of hydrogen bonding interactions with the rest of the peptide , glu-4 did not engage in any , while lys-1 was hydrogen bonded 8% of the time at ph 2.4 , 20% of the time at ph 7.4 , and 2% of the time at ph 13.4 . at ph 2 , most protonation transitions occur between states ppp , ppd , dpp , and dpd , while the transition to state ddp is sampled only once during all 3 simulations ( figure 9a and b ) . in most current force fields , an atom type and its associated force field parameters our method can consider the change of atomic parameters other than charge , such as the van der waals or generalized born solvation radius parameters . another potentially significant advantage of eds - hrex is that the resultant ensemble has only discrete protonation states , and these can be coupled to a high quality quantum mechanics ( qm ) or qm / mm surface using a non - boltzmann bennett approach . in the case of the kaae peptide , the state in which glu is protonated and lys is deprotonated could have also been omitted , reducing the total number of states from four to three . when there are n independent titratable groups , and if we assume that a probability to obtain a physical charge state of a single titratable site , that is , > 0.8 or < 0.2 , is p , the fraction of snapshots that all titratable groups are in physical charge states becomes p. our method focuses on obtaining accurate configurations of different protonation states . thus , the major contribution of our method is in the improved quality of ensembles , because our method samples original end states with the s = hamiltonian . instead of calculating the full energy of each charge state , computing just the energy differences in other words , instead of calculating e1 , e2 , e3 , and e4 , we can perform an eds simulation by calculating e1 , e2 the energies of different states differ only in electrostatic interactions between titratable groups and their neighboring atoms within a cutoff radius . a comparison of radial distribution functions between aspartic acid and water molecules demonstrates that the ensemble obtained with the baseline eds hamiltonian agrees well with those of md simulations with fixed charges . we also showed that the eds - hrex method can be easily extended to multiple protonation state cases with the titration of histidine , kaae peptide , and snake cardiotoxin . due to the generality of the eds - hrex method , it can be applied to free energy calculations in various problems that require frequent transitions between multiple states separated by large energy barriers .

our modelling suggests that the use of measles , mumps , rubella , and varicella combination vaccines ( mmrv ) instead of measles , mumps , and rubella vaccine with varicella vaccine ( mmr + v ) can substantially reduce the number of hospitalisation days via higher vaccination coverage against varicella , despite the observed increased risk of febrile convulsions when mmrv is used as a first dose of measles - containing vaccine.the net result of these two opposing effects is one of the trade - offs between the two vaccination schemes that needs to be considered when making decisions on their use in immunisation programmes.this proof - of - concept analysis has demonstrated the feasibility and usefulness of quantitative modelling approaches , based on the combination of heterogeneous data sources , to provide objective , rational , and transparent information . combination vaccines are used to simplify the recommended immunisation schedules , decrease the number of healthcare visits and injections in children , and improve vaccination coverage and compliance [ 1 , 2 ] . therefore , measles , mumps , rubella , and varicella combination vaccines ( mmrv ) have been developed to replace the separate administration of the trivalent measles , mumps , and rubella vaccine ( mmr ) with the monovalent varicella vaccine ( v ) . as seen with other combination vaccines , mmrv has been a key driver in improving the vaccination coverage against varicella in various countries , such as germany and the usa [ 38 ] . german data suggest that v uptake was suboptimal when separate administration of mmr + v was recommended . indeed , some parents are willing to vaccinate their child against mmr , but do not accept separate v and mmr vaccination . the main factors associated with the low acceptance rate of v vaccination by parents are the recommendations of paediatricians , who often have doubts about the benefits of varicella vaccination as they rarely observe severe complications in their practices ; a negative attitude towards vaccination in general ; parents doubts about vaccine safety and effectiveness ; and the perception that varicella is a mild disease [ 5 , 7 , 8 ] . data from germany , where mmrv was introduced in 2006 , also showed that , even when mmrv was widely used , varicella vaccination coverage and timeliness of vaccination could still be improved . data from a national sentinel network led by the robert koch institute suggested that varicella vaccination coverage in germany was 78 % in 2008 ; however , this could be an overestimation of the real coverage since the network may not be representative of the entire country . in children , the risk of febrile convulsions ( fc ) is increased during the first 2 weeks following mmr vaccination [ 11 , 12 ] . in addition , more recent studies have shown that this risk was about two times higher in children vaccinated with the first mmrv vaccine licensed worldwide ( proquad , merck & co. , inc . , whitehouse station , new jersey , usa ) compared with the separate administration of mmr + v [ 13 , 14 ] . later , a study conducted by the bremen institute for epidemiology and prevention research showed that the risk of developing fc within 512 days post - vaccination was also higher in children younger than 5 years who received the other licensed mmrv vaccine ( priorix - tetra , gsk , belgium ) compared with separate injections of mmr + v or mmr alone . these observations suggest that both mmrv vaccines , when used as a first dose of measles - containing vaccine , are associated with an increased risk of fc . while combined mmrv vaccines can potentially increase the number of fc cases , they can decrease the number of varicella cases through increased coverage for the v component . here , we present an attempt to assess the net result of these two opposing effects ( fig . 1 ) . this comparison would ideally be made by counting the hospitalisations for fc and varicella , and their duration , in a number of large populations randomly allocated to mmrv and mmr + v schemes , over several years , and with everything else being equal . as this is hardly feasible , we selected a modelling approach and the recent experience in germany as a starting point . in germany , a general varicella immunisation for infants from the age of 11 months was introduced in 2004 , followed by the subsequent recommendation of a second vaccine dose at 1523 months of age in 2009 . priorix - tetra was licensed in july 2006 , and both vaccination schemes ( mmr + v and mmrv ) have been widely used ; this has favoured comparison between the two vaccination regimens in a previous study conducted by the bremen institute for epidemiology and prevention research . in our analysis , we first used german data to put the increased risk of fc associated with mmrv ( priorix - tetra ) into perspective by comparing it with the higher risk of varicella infection associated with the mmr + v regimen . 1schematic representation of the two competing vaccination schemes and their associated risks and benefits as selected for the current analysis . * the ( small ) fraction of the total population who is not properly vaccinated against mmr . fc febrile convulsions , mmr measles , mumps , and rubella vaccine , mmrv measles , mumps , rubella , and varicella vaccine , v varicella vaccine schematic representation of the two competing vaccination schemes and their associated risks and benefits as selected for the current analysis . * the ( small ) fraction of the total population who is not properly vaccinated against mmr . fc febrile convulsions , mmr measles , mumps , and rubella vaccine , mmrv measles , mumps , rubella , and varicella vaccine , v varicella vaccine in this quantitative analysis , the increased risk of fc associated with mmrv ( priorix - tetra ) was contrasted with the higher risk of varicella infections associated with the separate mmr + v vaccination approach ( fig . 1 ) . we used a model based on several assumptions to integrate parameters from heterogeneous sources of data from germany ( in the period 20062008 ) . statistical uncertainty was evaluated through a probabilistic uncertainty analysis by monte carlo simulations . in addition , to address the potential variability between countries or over time , structural uncertainty was assessed through a sensitivity analysis . the assumptions , as well as the point estimates for the input parameters , are described in table 1 , and were selected to mimic the situation in germany in the period 20062008 . the risk estimates were derived from the post - marketing study conducted by the bremen institute for epidemiology and prevention research among children who received a first vaccination with mmrv ( priorix - tetra ) , mmr , or mmr + v between january 2006 and december 2008 . whereas the german vaccination programme changed to a two - dose schedule for mmrv or mmr + v in 2009 , only the first doses were taken into account in this population - based analysis.table 1definitions and point estimates of the input parameters , and uncertainty of these parameters observed in the monte carlo simulationsparameterpoint estimatesourcesdistributionsimulation , median ( 95 % ci)p1incidence of fc in days 512 after mmrv ( number of fc per child)51/82,436poisson61.9 ( 46.178.9 ) p2incidence of fc in days 512 after mmr ( number of fc per child)21/82,469poisson25.5 ( 15.836.4 ) p3relative probability of hospitalisation for fc as compared with the german data from 200620081.0constant1 ( )p4number of hospitalisations for varicella per year ( before the introduction of routine vaccination against varicella , across all age groups)1996normal1998 ( 13652633)p5median los for fc ( number of days)1constant1 ( )p6median los for varicella ( number of days)5poisson5 ( 110)p7mmr coverage for the first dose0.9constant0.9 ( )p8probability of v vaccination along with mmr vaccination0.78constant0.78 ( )p9german birth cohort size ( year 2005)685,795national statisticsconstant685,795 ( ) ci confidence interval , fc febrile convulsions , los length of stay in hospital , mmr measles , mumps , and rubella vaccine , mmrv measles , mumps , rubella , and varicella vaccine , p parameter , v varicella vaccine cumulative incidence per 100,000 children definitions and point estimates of the input parameters , and uncertainty of these parameters observed in the monte carlo simulations ci confidence interval , fc febrile convulsions , los length of stay in hospital , mmr measles , mumps , and rubella vaccine , mmrv measles , mumps , rubella , and varicella vaccine , p parameter , v varicella vaccine cumulative incidence per 100,000 children the primary health outcome in this analysis was the duration of hospitalisation ( median length of stay ) , which was chosen as a measure of the burden of both fc and varicella infection . the model was based on the following assumptions : vaccination with v occurred only in co - administration with mmr.mmr vaccination coverage and effectiveness were considered as identical between the mmr + v and the mmrv regimens.vaccination coverage of v varied between the two vaccination regimens.vaccine effectiveness of v was considered identical under both vaccination regimens [ 1820].one dose of v conferred identical and total protection against varicella - related hospitalisation under both vaccination regimens .the two vaccination regimens were compared as if they had been implemented for a sufficient amount of time as to reach a steady state in the dynamics of the disease , and all effects were assumed to be linear .the incidence of adverse events leading to hospitalisation , other than fc in the period of 512 days after the first dose , were considered identical under both vaccination regimens [ 18 , 23].there were no health consequences later in life from hospitalisation for fc or varicella [ 24 , 25].the number of hospitalisations for varicella after introduction of routine varicella vaccination was linearly proportional to the proportion of unvaccinated children . mmr vaccination coverage and effectiveness were considered as identical between the mmr + v and the mmrv regimens . one dose of v conferred identical and total protection against varicella - related hospitalisation under both vaccination regimens . the two vaccination regimens were compared as if they had been implemented for a sufficient amount of time as to reach a steady state in the dynamics of the disease , and all effects were assumed to be linear . the incidence of adverse events leading to hospitalisation , other than fc in the period of 512 days after the first dose , were considered identical under both vaccination regimens [ 18 , 23 ] . there were no health consequences later in life from hospitalisation for fc or varicella [ 24 , 25 ] . the number of hospitalisations for varicella after introduction of routine varicella vaccination was linearly proportional to the proportion of unvaccinated children . to quantify the benefit , we calculated the number of hospitalisation days and the number of hospitalisations for varicella that could be averted when using mmrv instead of mmr + v. the hospitalisation rate for varicella in unvaccinated children was based on the yearly number of varicella - related hospitalisations observed in germany before the introduction of routine varicella vaccination . the yearly number of varicella - related hospitalisation days and hospitalisations attributable to mmr + v compared with mmrv were estimated as follows ( table 1 ) : ( p6 ) ( p4 ) ( p7 ) [ 1 ( p8 ) ] and ( p4 ) ( p7 ) [ 1 ( p8 ) ] , respectively . to quantify the risk , we calculated the yearly number of hospitalisation days and the number of hospitalisations for fc that were attributable to mmrv compared with mmr or mmr + v. the yearly population at risk was the fraction of the birth cohort vaccinated with mmrv . the yearly number of hospitalisation days and hospitalisations for fc attributable to mmrv compared with mmr + v were estimated from the differences between the incidence of fc under both vaccination regimens as follows ( table 1 ) : ( p5 ) ( p9 ) ( p7 ) [ ( p1 ) ( p2 ) ] ( p3 ) and ( p9 ) ( p7 ) [ ( p1 ) ( p2 ) ] ( p3 ) , respectively . the hospitalisation ratio ( hr ) was the ratio between the excess number of hospitalisation days ( primary outcome ) or hospitalisations ( secondary outcome ) attributable to varicella when vaccinating with mmr + v compared with mmrv , and the excess number of hospitalisation days ( primary outcome ) or hospitalisations ( secondary outcome ) attributable to vaccine - related fc when vaccinating with mmrv compared with mmr + v. the statistical variability of each non - constant input parameter of the model was used to evaluate the statistical uncertainty of the hr through a probabilistic analysis based on monte carlo simulations . the most relevant statistical distribution was selected for each input parameter , and 100,000 sets with randomly generated values for all input parameters were produced . the point estimate from the monte carlo simulation was defined as the median of the 100,000 values , and the 95 % confidence interval ( ci ) was derived from the observed percentiles . rather surprisingly , data from the bremen institute for epidemiology and prevention research indicated a median duration of hospitalisation for fc of 23 days during the first month following mmr or mmrv vaccination in 20062008 , with several cases having a relatively long duration of up to 17 days . however , since the most recent german guidelines include no indication on whether children with fc should be hospitalised or not , the duration of hospitalisation for fc was based on the most recent international recommendations and set to a value of 1 day in this analysis ( table 1 ) . a study conducted in 1985 in the uk already suggested that children who had been observed for 24 h after fc could be discharged from hospital if the diagnosis of the cause of fever had been established and the children were medically fit , even if they were still febrile . with current vaccination schedules , the risk of meningitis has become extremely low , and it is not recommended anymore to carry out unnecessary investigations in most children presenting with fc . current international recommendations for the management of fc advocate minimal intervention [ 2629 ] . in germany , the bremen institute for epidemiology and prevention research reported that hospitalisation rates for fc in 20062008 were around 60 % in 9- to 17-month - old children ; this estimation was based on different case definitions for hospitalised and non - hospitalised children , and did not take into account the timing of mmr or mmrv vaccination . in a study conducted in the usa , hospitalisation rates for fc were 6 and 17 % during 710 days following mmrv and mmr + v vaccination , respectively . in another study , hospitalisation rates dropped significantly after implementation of updated guidelines for the management of children with fc ( from 57.3 to 20.5 % and from 16.9 to 3.2 % in two large hospitals in france and italy , respectively ) . we identified the two parameters that are most likely to vary largely between countries or over time and have a large effect on the hr : rates of hospitalisation for fc and vaccination coverage for v under mmr + v. a sensitivity analysis was conducted to explore the structural uncertainty of hr in situations where the values of these two parameters were changed over their potential range . monte carlo simulations were used to derive the different values of both the relative probability of hospitalisation for fc as compared with the german data from 20062008 ( parameter p3 ) and the vaccination coverage for v under mmr + v ( parameter p8 ) , for which the point estimates for the hr were 1 . statistical uncertainty was estimated as explained above . in the study of the bremen institute for epidemiology and prevention research , the probability of hospitalisation for fc was 100 % by definition since only hospitalised cases were included in the analysis . however , in the german national context , the probability of hospitalisation for fc is not 100 % and is considered to be close to 80 % . to allow for generalisation , the model was slightly modified in order to use the national probability of hospitalisation for fc instead of the one related to the specific study of the bremen institute for epidemiology and prevention research . in order to differentiate the parameter space where the hr could be considered to be significantly higher than 1 , we also derived the parameter values for which the lower limit of the 95 % ci around the hr was 1 . the model was based on the following assumptions : vaccination with v occurred only in co - administration with mmr.mmr vaccination coverage and effectiveness were considered as identical between the mmr + v and the mmrv regimens.vaccination coverage of v varied between the two vaccination regimens.vaccine effectiveness of v was considered identical under both vaccination regimens [ 1820].one dose of v conferred identical and total protection against varicella - related hospitalisation under both vaccination regimens .the two vaccination regimens were compared as if they had been implemented for a sufficient amount of time as to reach a steady state in the dynamics of the disease , and all effects were assumed to be linear .the incidence of adverse events leading to hospitalisation , other than fc in the period of 512 days after the first dose , were considered identical under both vaccination regimens [ 18 , 23].there were no health consequences later in life from hospitalisation for fc or varicella [ 24 , 25].the number of hospitalisations for varicella after introduction of routine varicella vaccination was linearly proportional to the proportion of unvaccinated children . mmr vaccination coverage and effectiveness were considered as identical between the mmr + v and the mmrv regimens . one dose of v conferred identical and total protection against varicella - related hospitalisation under both vaccination regimens . the two vaccination regimens were compared as if they had been implemented for a sufficient amount of time as to reach a steady state in the dynamics of the disease , and all effects were assumed to be linear . the incidence of adverse events leading to hospitalisation , other than fc in the period of 512 days after the first dose , were considered identical under both vaccination regimens [ 18 , 23 ] . there were no health consequences later in life from hospitalisation for fc or varicella [ 24 , 25 ] . the number of hospitalisations for varicella after introduction of routine varicella vaccination was linearly proportional to the proportion of unvaccinated children . to quantify the benefit , we calculated the number of hospitalisation days and the number of hospitalisations for varicella that could be averted when using mmrv instead of mmr + v. the hospitalisation rate for varicella in unvaccinated children was based on the yearly number of varicella - related hospitalisations observed in germany before the introduction of routine varicella vaccination . the yearly number of varicella - related hospitalisation days and hospitalisations attributable to mmr + v compared with mmrv were estimated as follows ( table 1 ) : ( p6 ) ( p4 ) ( p7 ) [ 1 ( p8 ) ] and ( p4 ) ( p7 ) [ 1 ( p8 ) ] , respectively . to quantify the risk , we calculated the yearly number of hospitalisation days and the number of hospitalisations for fc that were attributable to mmrv compared with mmr or mmr + v. the yearly population at risk was the fraction of the birth cohort vaccinated with mmrv . the yearly number of hospitalisation days and hospitalisations for fc attributable to mmrv compared with mmr + v were estimated from the differences between the incidence of fc under both vaccination regimens as follows ( table 1 ) : ( p5 ) ( p9 ) ( p7 ) [ ( p1 ) ( p2 ) ] ( p3 ) and ( p9 ) ( p7 ) [ ( p1 ) ( p2 ) ] ( p3 ) , respectively . the hospitalisation ratio ( hr ) was the ratio between the excess number of hospitalisation days ( primary outcome ) or hospitalisations ( secondary outcome ) attributable to varicella when vaccinating with mmr + v compared with mmrv , and the excess number of hospitalisation days ( primary outcome ) or hospitalisations ( secondary outcome ) attributable to vaccine - related fc when vaccinating with mmrv compared with mmr + v. the statistical variability of each non - constant input parameter of the model was used to evaluate the statistical uncertainty of the hr through a probabilistic analysis based on monte carlo simulations . the most relevant statistical distribution was selected for each input parameter , and 100,000 sets with randomly generated values for all input parameters were produced . the point estimate from the monte carlo simulation was defined as the median of the 100,000 values , and the 95 % confidence interval ( ci ) was derived from the observed percentiles . rather surprisingly , data from the bremen institute for epidemiology and prevention research indicated a median duration of hospitalisation for fc of 23 days during the first month following mmr or mmrv vaccination in 20062008 , with several cases having a relatively long duration of up to 17 days . however , since the most recent german guidelines include no indication on whether children with fc should be hospitalised or not , the duration of hospitalisation for fc was based on the most recent international recommendations and set to a value of 1 day in this analysis ( table 1 ) . a study conducted in 1985 in the uk already suggested that children who had been observed for 24 h after fc could be discharged from hospital if the diagnosis of the cause of fever had been established and the children were medically fit , even if they were still febrile . with current vaccination schedules , the risk of meningitis has become extremely low , and it is not recommended anymore to carry out unnecessary investigations in most children presenting with fc . current international recommendations for the management of fc advocate minimal intervention [ 2629 ] . in germany , the bremen institute for epidemiology and prevention research reported that hospitalisation rates for fc in 20062008 were around 60 % in 9- to 17-month - old children ; this estimation was based on different case definitions for hospitalised and non - hospitalised children , and did not take into account the timing of mmr or mmrv vaccination . in a study conducted in the usa , hospitalisation rates for fc were 6 and 17 % during 710 days following mmrv and mmr + v vaccination , respectively . in another study , hospitalisation rates dropped significantly after implementation of updated guidelines for the management of children with fc ( from 57.3 to 20.5 % and from 16.9 to 3.2 % in two large hospitals in france and italy , respectively ) . we identified the two parameters that are most likely to vary largely between countries or over time and have a large effect on the hr : rates of hospitalisation for fc and vaccination coverage for v under mmr + v. a sensitivity analysis was conducted to explore the structural uncertainty of hr in situations where the values of these two parameters were changed over their potential range . monte carlo simulations were used to derive the different values of both the relative probability of hospitalisation for fc as compared with the german data from 20062008 ( parameter p3 ) and the vaccination coverage for v under mmr + v ( parameter p8 ) , for which the point estimates for the hr were 1 . statistical uncertainty was estimated as explained above . in the study of the bremen institute for epidemiology and prevention research , the probability of hospitalisation for fc was 100 % by definition since only hospitalised cases were included in the analysis . however , in the german national context , the probability of hospitalisation for fc is not 100 % and is considered to be close to 80 % . to allow for generalisation , the model was slightly modified in order to use the national probability of hospitalisation for fc instead of the one related to the specific study of the bremen institute for epidemiology and prevention research . in order to differentiate the parameter space where the hr could be considered to be significantly higher than 1 , we also derived the parameter values for which the lower limit of the 95 % ci around the hr was 1 . the estimates for the different input parameters used in the model were selected from heterogeneous sources of data as described in table 1 [ 8 , 10 , 15 , 24 ] . when using these point estimates for all parameters , the model predicted 1976 days of hospitalisation ( 395.4 hospitalisations ) for varicella averted when the mmrv vaccination regimen was used , compared with 225 days of hospitalisation ( 225.0 hospitalisations ) for fc attributable to mmrv . the point estimate of the hr was close to 11 in the primary analysis , which was based on the number of hospitalisation days . when the random variability of each non - constant input parameter of the model was estimated by a monte carlo probabilistic uncertainty analysis , the median hr was 8.48 ( 95 % ci : 1.9925.22 ) in terms of hospitalisation days and 1.76 ( 0.984.02 ) in terms of number of hospitalisations ( table 2 ) . the difference with the point estimate presented above may be explained by the non - symmetrical distribution of the hr . in the primary analysis , the lower limit of the 95 % ci around the hr was above 1 , indicating that the number of hospitalisation days attributable to non - prevented varicella when using mmr + v as compared with mmrv was significantly higher than the number of hospitalisation days attributable to fc after mmrv as compared with mmr + v at a 95 % ci ( table 2).table 2benefit and risk estimates in terms of number of hospitalisation days in germanynamemeanmedian95 % ciyearly number of hospitalisation days for fc attributable to mmrv ( risk)225.0224.7104.9351.9yearly number of hospitalisation days for varicella prevented under mmrv ( benefit)19891879433.44125hospitalisation ratio8.51.99525.22 ci confidence interval , fc febrile convulsions , mmrv measles , mumps , rubella , and varicella vaccine benefit and risk estimates in terms of number of hospitalisation days in germany ci confidence interval , fc febrile convulsions , mmrv measles , mumps , rubella , and varicella vaccine expected outcomes in other countries or under different scenarios were derived from a sensitivity analysis , which was based on two key parameters : the vaccination coverage of v when co - administered with mmr and the probability of being hospitalised for fc . the point estimate of the hr in terms of hospitalisation days was higher than 1 for scenarios where the probability of being hospitalised for fc was less than 40 times the probability of not receiving v along with mmr ( fig . ci confidence interval , fc febrile convulsions , hr hospitalisation ratio , mmr measles , mumps , and rubella vaccine , v varicella vaccine estimates of the hr in terms of number of hospitalisation days under different scenarios . ci confidence interval , fc febrile convulsions , hr hospitalisation ratio , mmr measles , mumps , and rubella vaccine , v varicella vaccine in countries where the mean duration of hospitalisation for varicella was different than in germany ( e.g. 3 days instead of 5 days ) , the slope of the discriminating line , which is shown in fig . 2 , would be corrected by the same ratio ( e.g. 40 3/5 = 24 ) . when considering the line for the 95 % ci limit in fig . 2 , the estimates for the different input parameters used in the model were selected from heterogeneous sources of data as described in table 1 [ 8 , 10 , 15 , 24 ] . when using these point estimates for all parameters , the model predicted 1976 days of hospitalisation ( 395.4 hospitalisations ) for varicella averted when the mmrv vaccination regimen was used , compared with 225 days of hospitalisation ( 225.0 hospitalisations ) for fc attributable to mmrv . the point estimate of the hr was close to 11 in the primary analysis , which was based on the number of hospitalisation days . when the random variability of each non - constant input parameter of the model was estimated by a monte carlo probabilistic uncertainty analysis , the median hr was 8.48 ( 95 % ci : 1.9925.22 ) in terms of hospitalisation days and 1.76 ( 0.984.02 ) in terms of number of hospitalisations ( table 2 ) . the difference with the point estimate presented above may be explained by the non - symmetrical distribution of the hr . in the primary analysis , the lower limit of the 95 % ci around the hr was above 1 , indicating that the number of hospitalisation days attributable to non - prevented varicella when using mmr + v as compared with mmrv was significantly higher than the number of hospitalisation days attributable to fc after mmrv as compared with mmr + v at a 95 % ci ( table 2).table 2benefit and risk estimates in terms of number of hospitalisation days in germanynamemeanmedian95 % ciyearly number of hospitalisation days for fc attributable to mmrv ( risk)225.0224.7104.9351.9yearly number of hospitalisation days for varicella prevented under mmrv ( benefit)19891879433.44125hospitalisation ratio8.51.99525.22 ci confidence interval , fc febrile convulsions , mmrv measles , mumps , rubella , and varicella vaccine benefit and risk estimates in terms of number of hospitalisation days in germany ci confidence interval , fc febrile convulsions , mmrv measles , mumps , rubella , and varicella vaccine expected outcomes in other countries or under different scenarios were derived from a sensitivity analysis , which was based on two key parameters : the vaccination coverage of v when co - administered with mmr and the probability of being hospitalised for fc . the point estimate of the hr in terms of hospitalisation days was higher than 1 for scenarios where the probability of being hospitalised for fc was less than 40 times the probability of not receiving v along with mmr ( fig . ci confidence interval , fc febrile convulsions , hr hospitalisation ratio , mmr measles , mumps , and rubella vaccine , v varicella vaccine estimates of the hr in terms of number of hospitalisation days under different scenarios . ci confidence interval , fc febrile convulsions , hr hospitalisation ratio , mmr measles , mumps , and rubella vaccine , v varicella vaccine in countries where the mean duration of hospitalisation for varicella was different than in germany ( e.g. 3 days instead of 5 days ) , the slope of the discriminating line , which is shown in fig . 2 , would be corrected by the same ratio ( e.g. 40 3/5 = 24 ) . when considering the line for the 95 % ci limit in fig . 2 , the results of our analysis using german data suggested that the predicted number of hospitalisation days induced by the increase risk of fc associated with mmrv is lower than that induced by higher risk of varicella infection associated with the mmr + v regimen . the expansion of this analysis to other scenarios beyond the german experience suggested that transitioning from mmr + v to mmrv would be favourable in situations where mmrv use would significantly impact varicella vaccination uptake . for parameter values compatible with the recent experience in germany , the model suggested that transitioning from mmr + v to mmrv would reduce the yearly number of hospitalisation days ; although there may be 225 hospitalisation days attributable to vaccine - related fc , at the same time , 1976 hospitalisation days due to severe varicella could be averted by using mmrv instead of mmr + v. when monte carlo simulations were used to evaluate the statistical uncertainty , the median hr was estimated to be 8.5 ( 95 % ci 1.9925.22 ) . since these estimations were derived from a model based on several assumptions and using heterogeneous sources of data , this quantitative analysis should be used along with other relevant considerations for any overall benefit / risk assessment of mmrv compared with mmr + v. a sensitivity analysis was performed to estimate hr under different scenarios , which were based on existing or hypothetical situations , and could represent other countries or other times when vaccine recommendations or use were different . this modelling approach , with all its limitations , suggested that , in situations where mmrv use would significantly impact varicella vaccination uptake , the use of mmrv instead of mmr + v would be favourable . for any further use of these results , it is critical to consider the model assumptions and assess their relevance before making any decision on the use of mmrv in immunisation programmes . the results are based on a modelling approach with several limitations , including : the model did not consider situations where v was co - administered with the second mmr dose ( either as mmr + v or mmrv ) if it was not co - administered with the first mmr dose.the assumption of total protection against hospitalisation could have overestimated the benefit measure of the mmrv compared with the mmr + v vaccination regimen.the measures of the burden of both fc and varicella infection that were selected in this analysis ( days of hospitalisation and number of hospitalisations ) may not have completely captured the severity of the two medical conditions ; hospitalisation for fc is generally more a measure of precaution , while hospitalisation related to varicella infection generally indicates a serious medical concern.the model did not take into account the potential impact of the recommendation to administer the first doses of mmr and v separately in germany since 2011 .the model did not take into account the effect of herd immunity , which is relevant in the context of protection against varicella . this is a conservative approach since such an effect would increase the benefit of mmrv compared with mmr + v. the model did not consider situations where v was co - administered with the second mmr dose ( either as mmr + v or mmrv ) if it was not co - administered with the first mmr dose . the assumption of total protection against hospitalisation could have overestimated the benefit measure of the mmrv compared with the mmr + v vaccination regimen . the measures of the burden of both fc and varicella infection that were selected in this analysis ( days of hospitalisation and number of hospitalisations ) may not have completely captured the severity of the two medical conditions ; hospitalisation for fc is generally more a measure of precaution , while hospitalisation related to varicella infection generally indicates a serious medical concern . the model did not take into account the potential impact of the recommendation to administer the first doses of mmr and v separately in germany since 2011 . the model did not take into account the effect of herd immunity , which is relevant in the context of protection against varicella . this is a conservative approach since such an effect would increase the benefit of mmrv compared with mmr + v. a modelling approach can be used to estimate the overall impact of different vaccine regimens on some specific outcomes . the model used in this analysis suggested that transitioning from mmr + v to mmrv may substantially reduce the number of hospitalisation days , despite the observed increased risk of fc when mmrv was used as a first dose of measles - containing vaccine . this proof - of - concept analysis has demonstrated the feasibility and usefulness of quantitative modelling approaches , based on the combination of heterogeneous data sources , to provide objective , rational , and transparent information . in addition , our model only assessed one of the multiple trade - offs between the two vaccination regimens that need to be considered when making decisions on their use in immunisation programmes . in the future , more extensive research could be performed to address unanswered questions , such as analyses using quality - adjusted life - years as an outcome instead of the length and number of hospitalisations , and analyses taking into account indirect effects , such as herd immunity or increased hesitancy to accept mmrv due to knowledge of the increased risk of fc . vincent bauchau and lionel van holle are employed by the gsk group of companies and own restricted shares in the company . carine cohen was employed by the gsk group of companies at the time of this study and owns restricted shares in the company . glaxosmithkline biologicals sa sponsored this analysis and was involved in all stages of the analysis . glaxosmithkline biologicals sa also took responsibility for all costs associated with the development and publishing of the present manuscript .

introductionmeasles , mumps , rubella , and varicella combination vaccines ( mmrv ) facilitate varicella vaccination uptake compared with separate administration of measles , mumps , and rubella vaccine ( mmr ) with varicella vaccine ( v ) . however , the risk of developing febrile convulsions ( fc ) is higher in children vaccinated with mmrv.objectivesthe aim was to demonstrate how to put the increased fc risk associated with mmrv into perspective by comparing it with the lower v - coverage risk associated with mmr + v.methodsfc and varicella burdens were measured by total numbers or duration of hospitalisations . a model , based on several assumptions and integrating parameters from heterogeneous data sources relevant to germany , was developed to evaluate hospitalisation ratios ( hrs ; ratios between yearly numbers of varicella - related hospitalisation days prevented by mmrv and yearly numbers of fc - related hospitalisation days attributed to mmrv , both compared with mmr + v ) . a sensitivity analysis estimated hr under different scenarios beyond the german experience.resultsfor parameter values compatible with the german experience , where mmrv ( priorix - tetra , gsk , belgium ) was introduced in 2006 , the model predicted that transitioning from mmr + v to mmrv would induce 225 vaccine - related fc hospitalisation days whilst preventing 1976 varicella - related hospitalisation days per year . the hr estimated by monte carlo simulations was 8.5 ( 95 % confidence interval : 1.9925.22 ) . a sensitivity analysis on two key parameters suggested that transitioning from mmr + v to mmrv would be favourable in situations where mmrv use would significantly impact varicella vaccination uptake.conclusionsmmrv use instead of mmr + v can substantially reduce the number of hospitalisation days , despite increased fc risk when mmrv is used as a first dose of measles - containing vaccine .

Key Points Introduction Methods Modelling Assumptions Calculation of the Hospitalisation Ratio Probabilistic Uncertainty Analysis Sensitivity Analysis Results Modelling Based on the German Experience Sensitivity Analysis Discussion Conclusion Trademarks None None

our modelling suggests that the use of measles , mumps , rubella , and varicella combination vaccines ( mmrv ) instead of measles , mumps , and rubella vaccine with varicella vaccine ( mmr + v ) can substantially reduce the number of hospitalisation days via higher vaccination coverage against varicella , despite the observed increased risk of febrile convulsions when mmrv is used as a first dose of measles - containing vaccine.the net result of these two opposing effects is one of the trade - offs between the two vaccination schemes that needs to be considered when making decisions on their use in immunisation programmes.this proof - of - concept analysis has demonstrated the feasibility and usefulness of quantitative modelling approaches , based on the combination of heterogeneous data sources , to provide objective , rational , and transparent information . therefore , measles , mumps , rubella , and varicella combination vaccines ( mmrv ) have been developed to replace the separate administration of the trivalent measles , mumps , and rubella vaccine ( mmr ) with the monovalent varicella vaccine ( v ) . later , a study conducted by the bremen institute for epidemiology and prevention research showed that the risk of developing fc within 512 days post - vaccination was also higher in children younger than 5 years who received the other licensed mmrv vaccine ( priorix - tetra , gsk , belgium ) compared with separate injections of mmr + v or mmr alone . these observations suggest that both mmrv vaccines , when used as a first dose of measles - containing vaccine , are associated with an increased risk of fc . in our analysis , we first used german data to put the increased risk of fc associated with mmrv ( priorix - tetra ) into perspective by comparing it with the higher risk of varicella infection associated with the mmr + v regimen . fc febrile convulsions , mmr measles , mumps , and rubella vaccine , mmrv measles , mumps , rubella , and varicella vaccine , v varicella vaccine schematic representation of the two competing vaccination schemes and their associated risks and benefits as selected for the current analysis . fc febrile convulsions , mmr measles , mumps , and rubella vaccine , mmrv measles , mumps , rubella , and varicella vaccine , v varicella vaccine in this quantitative analysis , the increased risk of fc associated with mmrv ( priorix - tetra ) was contrasted with the higher risk of varicella infections associated with the separate mmr + v vaccination approach ( fig . whereas the german vaccination programme changed to a two - dose schedule for mmrv or mmr + v in 2009 , only the first doses were taken into account in this population - based analysis.table 1definitions and point estimates of the input parameters , and uncertainty of these parameters observed in the monte carlo simulationsparameterpoint estimatesourcesdistributionsimulation , median ( 95 % ci)p1incidence of fc in days 512 after mmrv ( number of fc per child)51/82,436poisson61.9 ( 46.178.9 ) p2incidence of fc in days 512 after mmr ( number of fc per child)21/82,469poisson25.5 ( 15.836.4 ) p3relative probability of hospitalisation for fc as compared with the german data from 200620081.0constant1 ( )p4number of hospitalisations for varicella per year ( before the introduction of routine vaccination against varicella , across all age groups)1996normal1998 ( 13652633)p5median los for fc ( number of days)1constant1 ( )p6median los for varicella ( number of days)5poisson5 ( 110)p7mmr coverage for the first dose0.9constant0.9 ( )p8probability of v vaccination along with mmr vaccination0.78constant0.78 ( )p9german birth cohort size ( year 2005)685,795national statisticsconstant685,795 ( ) ci confidence interval , fc febrile convulsions , los length of stay in hospital , mmr measles , mumps , and rubella vaccine , mmrv measles , mumps , rubella , and varicella vaccine , p parameter , v varicella vaccine cumulative incidence per 100,000 children definitions and point estimates of the input parameters , and uncertainty of these parameters observed in the monte carlo simulations ci confidence interval , fc febrile convulsions , los length of stay in hospital , mmr measles , mumps , and rubella vaccine , mmrv measles , mumps , rubella , and varicella vaccine , p parameter , v varicella vaccine cumulative incidence per 100,000 children the primary health outcome in this analysis was the duration of hospitalisation ( median length of stay ) , which was chosen as a measure of the burden of both fc and varicella infection . the model was based on the following assumptions : vaccination with v occurred only in co - administration with mmr.mmr vaccination coverage and effectiveness were considered as identical between the mmr + v and the mmrv regimens.vaccination coverage of v varied between the two vaccination regimens.vaccine effectiveness of v was considered identical under both vaccination regimens [ 1820].one dose of v conferred identical and total protection against varicella - related hospitalisation under both vaccination regimens .the two vaccination regimens were compared as if they had been implemented for a sufficient amount of time as to reach a steady state in the dynamics of the disease , and all effects were assumed to be linear .the incidence of adverse events leading to hospitalisation , other than fc in the period of 512 days after the first dose , were considered identical under both vaccination regimens [ 18 , 23].there were no health consequences later in life from hospitalisation for fc or varicella [ 24 , 25].the number of hospitalisations for varicella after introduction of routine varicella vaccination was linearly proportional to the proportion of unvaccinated children . to quantify the benefit , we calculated the number of hospitalisation days and the number of hospitalisations for varicella that could be averted when using mmrv instead of mmr + v. the hospitalisation rate for varicella in unvaccinated children was based on the yearly number of varicella - related hospitalisations observed in germany before the introduction of routine varicella vaccination . the yearly number of varicella - related hospitalisation days and hospitalisations attributable to mmr + v compared with mmrv were estimated as follows ( table 1 ) : ( p6 ) ( p4 ) ( p7 ) [ 1 ( p8 ) ] and ( p4 ) ( p7 ) [ 1 ( p8 ) ] , respectively . to quantify the risk , we calculated the yearly number of hospitalisation days and the number of hospitalisations for fc that were attributable to mmrv compared with mmr or mmr + v. the yearly population at risk was the fraction of the birth cohort vaccinated with mmrv . the hospitalisation ratio ( hr ) was the ratio between the excess number of hospitalisation days ( primary outcome ) or hospitalisations ( secondary outcome ) attributable to varicella when vaccinating with mmr + v compared with mmrv , and the excess number of hospitalisation days ( primary outcome ) or hospitalisations ( secondary outcome ) attributable to vaccine - related fc when vaccinating with mmrv compared with mmr + v. the statistical variability of each non - constant input parameter of the model was used to evaluate the statistical uncertainty of the hr through a probabilistic analysis based on monte carlo simulations . monte carlo simulations were used to derive the different values of both the relative probability of hospitalisation for fc as compared with the german data from 20062008 ( parameter p3 ) and the vaccination coverage for v under mmr + v ( parameter p8 ) , for which the point estimates for the hr were 1 . the model was based on the following assumptions : vaccination with v occurred only in co - administration with mmr.mmr vaccination coverage and effectiveness were considered as identical between the mmr + v and the mmrv regimens.vaccination coverage of v varied between the two vaccination regimens.vaccine effectiveness of v was considered identical under both vaccination regimens [ 1820].one dose of v conferred identical and total protection against varicella - related hospitalisation under both vaccination regimens .the two vaccination regimens were compared as if they had been implemented for a sufficient amount of time as to reach a steady state in the dynamics of the disease , and all effects were assumed to be linear .the incidence of adverse events leading to hospitalisation , other than fc in the period of 512 days after the first dose , were considered identical under both vaccination regimens [ 18 , 23].there were no health consequences later in life from hospitalisation for fc or varicella [ 24 , 25].the number of hospitalisations for varicella after introduction of routine varicella vaccination was linearly proportional to the proportion of unvaccinated children . to quantify the benefit , we calculated the number of hospitalisation days and the number of hospitalisations for varicella that could be averted when using mmrv instead of mmr + v. the hospitalisation rate for varicella in unvaccinated children was based on the yearly number of varicella - related hospitalisations observed in germany before the introduction of routine varicella vaccination . the yearly number of varicella - related hospitalisation days and hospitalisations attributable to mmr + v compared with mmrv were estimated as follows ( table 1 ) : ( p6 ) ( p4 ) ( p7 ) [ 1 ( p8 ) ] and ( p4 ) ( p7 ) [ 1 ( p8 ) ] , respectively . to quantify the risk , we calculated the yearly number of hospitalisation days and the number of hospitalisations for fc that were attributable to mmrv compared with mmr or mmr + v. the yearly population at risk was the fraction of the birth cohort vaccinated with mmrv . the hospitalisation ratio ( hr ) was the ratio between the excess number of hospitalisation days ( primary outcome ) or hospitalisations ( secondary outcome ) attributable to varicella when vaccinating with mmr + v compared with mmrv , and the excess number of hospitalisation days ( primary outcome ) or hospitalisations ( secondary outcome ) attributable to vaccine - related fc when vaccinating with mmrv compared with mmr + v. the statistical variability of each non - constant input parameter of the model was used to evaluate the statistical uncertainty of the hr through a probabilistic analysis based on monte carlo simulations . monte carlo simulations were used to derive the different values of both the relative probability of hospitalisation for fc as compared with the german data from 20062008 ( parameter p3 ) and the vaccination coverage for v under mmr + v ( parameter p8 ) , for which the point estimates for the hr were 1 . when the random variability of each non - constant input parameter of the model was estimated by a monte carlo probabilistic uncertainty analysis , the median hr was 8.48 ( 95 % ci : 1.9925.22 ) in terms of hospitalisation days and 1.76 ( 0.984.02 ) in terms of number of hospitalisations ( table 2 ) . in the primary analysis , the lower limit of the 95 % ci around the hr was above 1 , indicating that the number of hospitalisation days attributable to non - prevented varicella when using mmr + v as compared with mmrv was significantly higher than the number of hospitalisation days attributable to fc after mmrv as compared with mmr + v at a 95 % ci ( table 2).table 2benefit and risk estimates in terms of number of hospitalisation days in germanynamemeanmedian95 % ciyearly number of hospitalisation days for fc attributable to mmrv ( risk)225.0224.7104.9351.9yearly number of hospitalisation days for varicella prevented under mmrv ( benefit)19891879433.44125hospitalisation ratio8.51.99525.22 ci confidence interval , fc febrile convulsions , mmrv measles , mumps , rubella , and varicella vaccine benefit and risk estimates in terms of number of hospitalisation days in germany ci confidence interval , fc febrile convulsions , mmrv measles , mumps , rubella , and varicella vaccine expected outcomes in other countries or under different scenarios were derived from a sensitivity analysis , which was based on two key parameters : the vaccination coverage of v when co - administered with mmr and the probability of being hospitalised for fc . ci confidence interval , fc febrile convulsions , hr hospitalisation ratio , mmr measles , mumps , and rubella vaccine , v varicella vaccine estimates of the hr in terms of number of hospitalisation days under different scenarios . ci confidence interval , fc febrile convulsions , hr hospitalisation ratio , mmr measles , mumps , and rubella vaccine , v varicella vaccine in countries where the mean duration of hospitalisation for varicella was different than in germany ( e.g. when the random variability of each non - constant input parameter of the model was estimated by a monte carlo probabilistic uncertainty analysis , the median hr was 8.48 ( 95 % ci : 1.9925.22 ) in terms of hospitalisation days and 1.76 ( 0.984.02 ) in terms of number of hospitalisations ( table 2 ) . in the primary analysis , the lower limit of the 95 % ci around the hr was above 1 , indicating that the number of hospitalisation days attributable to non - prevented varicella when using mmr + v as compared with mmrv was significantly higher than the number of hospitalisation days attributable to fc after mmrv as compared with mmr + v at a 95 % ci ( table 2).table 2benefit and risk estimates in terms of number of hospitalisation days in germanynamemeanmedian95 % ciyearly number of hospitalisation days for fc attributable to mmrv ( risk)225.0224.7104.9351.9yearly number of hospitalisation days for varicella prevented under mmrv ( benefit)19891879433.44125hospitalisation ratio8.51.99525.22 ci confidence interval , fc febrile convulsions , mmrv measles , mumps , rubella , and varicella vaccine benefit and risk estimates in terms of number of hospitalisation days in germany ci confidence interval , fc febrile convulsions , mmrv measles , mumps , rubella , and varicella vaccine expected outcomes in other countries or under different scenarios were derived from a sensitivity analysis , which was based on two key parameters : the vaccination coverage of v when co - administered with mmr and the probability of being hospitalised for fc . ci confidence interval , fc febrile convulsions , hr hospitalisation ratio , mmr measles , mumps , and rubella vaccine , v varicella vaccine estimates of the hr in terms of number of hospitalisation days under different scenarios . ci confidence interval , fc febrile convulsions , hr hospitalisation ratio , mmr measles , mumps , and rubella vaccine , v varicella vaccine in countries where the mean duration of hospitalisation for varicella was different than in germany ( e.g. 2 , the results of our analysis using german data suggested that the predicted number of hospitalisation days induced by the increase risk of fc associated with mmrv is lower than that induced by higher risk of varicella infection associated with the mmr + v regimen . the expansion of this analysis to other scenarios beyond the german experience suggested that transitioning from mmr + v to mmrv would be favourable in situations where mmrv use would significantly impact varicella vaccination uptake . for parameter values compatible with the recent experience in germany , the model suggested that transitioning from mmr + v to mmrv would reduce the yearly number of hospitalisation days ; although there may be 225 hospitalisation days attributable to vaccine - related fc , at the same time , 1976 hospitalisation days due to severe varicella could be averted by using mmrv instead of mmr + v. when monte carlo simulations were used to evaluate the statistical uncertainty , the median hr was estimated to be 8.5 ( 95 % ci 1.9925.22 ) . since these estimations were derived from a model based on several assumptions and using heterogeneous sources of data , this quantitative analysis should be used along with other relevant considerations for any overall benefit / risk assessment of mmrv compared with mmr + v. a sensitivity analysis was performed to estimate hr under different scenarios , which were based on existing or hypothetical situations , and could represent other countries or other times when vaccine recommendations or use were different . this modelling approach , with all its limitations , suggested that , in situations where mmrv use would significantly impact varicella vaccination uptake , the use of mmrv instead of mmr + v would be favourable . the results are based on a modelling approach with several limitations , including : the model did not consider situations where v was co - administered with the second mmr dose ( either as mmr + v or mmrv ) if it was not co - administered with the first mmr dose.the assumption of total protection against hospitalisation could have overestimated the benefit measure of the mmrv compared with the mmr + v vaccination regimen.the measures of the burden of both fc and varicella infection that were selected in this analysis ( days of hospitalisation and number of hospitalisations ) may not have completely captured the severity of the two medical conditions ; hospitalisation for fc is generally more a measure of precaution , while hospitalisation related to varicella infection generally indicates a serious medical concern.the model did not take into account the potential impact of the recommendation to administer the first doses of mmr and v separately in germany since 2011 .the model did not take into account the effect of herd immunity , which is relevant in the context of protection against varicella . the model used in this analysis suggested that transitioning from mmr + v to mmrv may substantially reduce the number of hospitalisation days , despite the observed increased risk of fc when mmrv was used as a first dose of measles - containing vaccine .

our modelling suggests that the use of measles , mumps , rubella , and varicella combination vaccines ( mmrv ) instead of measles , mumps , and rubella vaccine with varicella vaccine ( mmr + v ) can substantially reduce the number of hospitalisation days via higher vaccination coverage against varicella , despite the observed increased risk of febrile convulsions when mmrv is used as a first dose of measles - containing vaccine.the net result of these two opposing effects is one of the trade - offs between the two vaccination schemes that needs to be considered when making decisions on their use in immunisation programmes.this proof - of - concept analysis has demonstrated the feasibility and usefulness of quantitative modelling approaches , based on the combination of heterogeneous data sources , to provide objective , rational , and transparent information . therefore , measles , mumps , rubella , and varicella combination vaccines ( mmrv ) have been developed to replace the separate administration of the trivalent measles , mumps , and rubella vaccine ( mmr ) with the monovalent varicella vaccine ( v ) . the main factors associated with the low acceptance rate of v vaccination by parents are the recommendations of paediatricians , who often have doubts about the benefits of varicella vaccination as they rarely observe severe complications in their practices ; a negative attitude towards vaccination in general ; parents doubts about vaccine safety and effectiveness ; and the perception that varicella is a mild disease [ 5 , 7 , 8 ] . later , a study conducted by the bremen institute for epidemiology and prevention research showed that the risk of developing fc within 512 days post - vaccination was also higher in children younger than 5 years who received the other licensed mmrv vaccine ( priorix - tetra , gsk , belgium ) compared with separate injections of mmr + v or mmr alone . fc febrile convulsions , mmr measles , mumps , and rubella vaccine , mmrv measles , mumps , rubella , and varicella vaccine , v varicella vaccine in this quantitative analysis , the increased risk of fc associated with mmrv ( priorix - tetra ) was contrasted with the higher risk of varicella infections associated with the separate mmr + v vaccination approach ( fig . whereas the german vaccination programme changed to a two - dose schedule for mmrv or mmr + v in 2009 , only the first doses were taken into account in this population - based analysis.table 1definitions and point estimates of the input parameters , and uncertainty of these parameters observed in the monte carlo simulationsparameterpoint estimatesourcesdistributionsimulation , median ( 95 % ci)p1incidence of fc in days 512 after mmrv ( number of fc per child)51/82,436poisson61.9 ( 46.178.9 ) p2incidence of fc in days 512 after mmr ( number of fc per child)21/82,469poisson25.5 ( 15.836.4 ) p3relative probability of hospitalisation for fc as compared with the german data from 200620081.0constant1 ( )p4number of hospitalisations for varicella per year ( before the introduction of routine vaccination against varicella , across all age groups)1996normal1998 ( 13652633)p5median los for fc ( number of days)1constant1 ( )p6median los for varicella ( number of days)5poisson5 ( 110)p7mmr coverage for the first dose0.9constant0.9 ( )p8probability of v vaccination along with mmr vaccination0.78constant0.78 ( )p9german birth cohort size ( year 2005)685,795national statisticsconstant685,795 ( ) ci confidence interval , fc febrile convulsions , los length of stay in hospital , mmr measles , mumps , and rubella vaccine , mmrv measles , mumps , rubella , and varicella vaccine , p parameter , v varicella vaccine cumulative incidence per 100,000 children definitions and point estimates of the input parameters , and uncertainty of these parameters observed in the monte carlo simulations ci confidence interval , fc febrile convulsions , los length of stay in hospital , mmr measles , mumps , and rubella vaccine , mmrv measles , mumps , rubella , and varicella vaccine , p parameter , v varicella vaccine cumulative incidence per 100,000 children the primary health outcome in this analysis was the duration of hospitalisation ( median length of stay ) , which was chosen as a measure of the burden of both fc and varicella infection . the model was based on the following assumptions : vaccination with v occurred only in co - administration with mmr.mmr vaccination coverage and effectiveness were considered as identical between the mmr + v and the mmrv regimens.vaccination coverage of v varied between the two vaccination regimens.vaccine effectiveness of v was considered identical under both vaccination regimens [ 1820].one dose of v conferred identical and total protection against varicella - related hospitalisation under both vaccination regimens .the two vaccination regimens were compared as if they had been implemented for a sufficient amount of time as to reach a steady state in the dynamics of the disease , and all effects were assumed to be linear .the incidence of adverse events leading to hospitalisation , other than fc in the period of 512 days after the first dose , were considered identical under both vaccination regimens [ 18 , 23].there were no health consequences later in life from hospitalisation for fc or varicella [ 24 , 25].the number of hospitalisations for varicella after introduction of routine varicella vaccination was linearly proportional to the proportion of unvaccinated children . to quantify the benefit , we calculated the number of hospitalisation days and the number of hospitalisations for varicella that could be averted when using mmrv instead of mmr + v. the hospitalisation rate for varicella in unvaccinated children was based on the yearly number of varicella - related hospitalisations observed in germany before the introduction of routine varicella vaccination . the yearly number of varicella - related hospitalisation days and hospitalisations attributable to mmr + v compared with mmrv were estimated as follows ( table 1 ) : ( p6 ) ( p4 ) ( p7 ) [ 1 ( p8 ) ] and ( p4 ) ( p7 ) [ 1 ( p8 ) ] , respectively . to quantify the risk , we calculated the yearly number of hospitalisation days and the number of hospitalisations for fc that were attributable to mmrv compared with mmr or mmr + v. the yearly population at risk was the fraction of the birth cohort vaccinated with mmrv . the yearly number of hospitalisation days and hospitalisations for fc attributable to mmrv compared with mmr + v were estimated from the differences between the incidence of fc under both vaccination regimens as follows ( table 1 ) : ( p5 ) ( p9 ) ( p7 ) [ ( p1 ) ( p2 ) ] ( p3 ) and ( p9 ) ( p7 ) [ ( p1 ) ( p2 ) ] ( p3 ) , respectively . the hospitalisation ratio ( hr ) was the ratio between the excess number of hospitalisation days ( primary outcome ) or hospitalisations ( secondary outcome ) attributable to varicella when vaccinating with mmr + v compared with mmrv , and the excess number of hospitalisation days ( primary outcome ) or hospitalisations ( secondary outcome ) attributable to vaccine - related fc when vaccinating with mmrv compared with mmr + v. the statistical variability of each non - constant input parameter of the model was used to evaluate the statistical uncertainty of the hr through a probabilistic analysis based on monte carlo simulations . in another study , hospitalisation rates dropped significantly after implementation of updated guidelines for the management of children with fc ( from 57.3 to 20.5 % and from 16.9 to 3.2 % in two large hospitals in france and italy , respectively ) . we identified the two parameters that are most likely to vary largely between countries or over time and have a large effect on the hr : rates of hospitalisation for fc and vaccination coverage for v under mmr + v. a sensitivity analysis was conducted to explore the structural uncertainty of hr in situations where the values of these two parameters were changed over their potential range . monte carlo simulations were used to derive the different values of both the relative probability of hospitalisation for fc as compared with the german data from 20062008 ( parameter p3 ) and the vaccination coverage for v under mmr + v ( parameter p8 ) , for which the point estimates for the hr were 1 . the model was based on the following assumptions : vaccination with v occurred only in co - administration with mmr.mmr vaccination coverage and effectiveness were considered as identical between the mmr + v and the mmrv regimens.vaccination coverage of v varied between the two vaccination regimens.vaccine effectiveness of v was considered identical under both vaccination regimens [ 1820].one dose of v conferred identical and total protection against varicella - related hospitalisation under both vaccination regimens .the two vaccination regimens were compared as if they had been implemented for a sufficient amount of time as to reach a steady state in the dynamics of the disease , and all effects were assumed to be linear .the incidence of adverse events leading to hospitalisation , other than fc in the period of 512 days after the first dose , were considered identical under both vaccination regimens [ 18 , 23].there were no health consequences later in life from hospitalisation for fc or varicella [ 24 , 25].the number of hospitalisations for varicella after introduction of routine varicella vaccination was linearly proportional to the proportion of unvaccinated children . to quantify the benefit , we calculated the number of hospitalisation days and the number of hospitalisations for varicella that could be averted when using mmrv instead of mmr + v. the hospitalisation rate for varicella in unvaccinated children was based on the yearly number of varicella - related hospitalisations observed in germany before the introduction of routine varicella vaccination . the yearly number of varicella - related hospitalisation days and hospitalisations attributable to mmr + v compared with mmrv were estimated as follows ( table 1 ) : ( p6 ) ( p4 ) ( p7 ) [ 1 ( p8 ) ] and ( p4 ) ( p7 ) [ 1 ( p8 ) ] , respectively . to quantify the risk , we calculated the yearly number of hospitalisation days and the number of hospitalisations for fc that were attributable to mmrv compared with mmr or mmr + v. the yearly population at risk was the fraction of the birth cohort vaccinated with mmrv . the yearly number of hospitalisation days and hospitalisations for fc attributable to mmrv compared with mmr + v were estimated from the differences between the incidence of fc under both vaccination regimens as follows ( table 1 ) : ( p5 ) ( p9 ) ( p7 ) [ ( p1 ) ( p2 ) ] ( p3 ) and ( p9 ) ( p7 ) [ ( p1 ) ( p2 ) ] ( p3 ) , respectively . the hospitalisation ratio ( hr ) was the ratio between the excess number of hospitalisation days ( primary outcome ) or hospitalisations ( secondary outcome ) attributable to varicella when vaccinating with mmr + v compared with mmrv , and the excess number of hospitalisation days ( primary outcome ) or hospitalisations ( secondary outcome ) attributable to vaccine - related fc when vaccinating with mmrv compared with mmr + v. the statistical variability of each non - constant input parameter of the model was used to evaluate the statistical uncertainty of the hr through a probabilistic analysis based on monte carlo simulations . in another study , hospitalisation rates dropped significantly after implementation of updated guidelines for the management of children with fc ( from 57.3 to 20.5 % and from 16.9 to 3.2 % in two large hospitals in france and italy , respectively ) . we identified the two parameters that are most likely to vary largely between countries or over time and have a large effect on the hr : rates of hospitalisation for fc and vaccination coverage for v under mmr + v. a sensitivity analysis was conducted to explore the structural uncertainty of hr in situations where the values of these two parameters were changed over their potential range . monte carlo simulations were used to derive the different values of both the relative probability of hospitalisation for fc as compared with the german data from 20062008 ( parameter p3 ) and the vaccination coverage for v under mmr + v ( parameter p8 ) , for which the point estimates for the hr were 1 . when the random variability of each non - constant input parameter of the model was estimated by a monte carlo probabilistic uncertainty analysis , the median hr was 8.48 ( 95 % ci : 1.9925.22 ) in terms of hospitalisation days and 1.76 ( 0.984.02 ) in terms of number of hospitalisations ( table 2 ) . in the primary analysis , the lower limit of the 95 % ci around the hr was above 1 , indicating that the number of hospitalisation days attributable to non - prevented varicella when using mmr + v as compared with mmrv was significantly higher than the number of hospitalisation days attributable to fc after mmrv as compared with mmr + v at a 95 % ci ( table 2).table 2benefit and risk estimates in terms of number of hospitalisation days in germanynamemeanmedian95 % ciyearly number of hospitalisation days for fc attributable to mmrv ( risk)225.0224.7104.9351.9yearly number of hospitalisation days for varicella prevented under mmrv ( benefit)19891879433.44125hospitalisation ratio8.51.99525.22 ci confidence interval , fc febrile convulsions , mmrv measles , mumps , rubella , and varicella vaccine benefit and risk estimates in terms of number of hospitalisation days in germany ci confidence interval , fc febrile convulsions , mmrv measles , mumps , rubella , and varicella vaccine expected outcomes in other countries or under different scenarios were derived from a sensitivity analysis , which was based on two key parameters : the vaccination coverage of v when co - administered with mmr and the probability of being hospitalised for fc . when the random variability of each non - constant input parameter of the model was estimated by a monte carlo probabilistic uncertainty analysis , the median hr was 8.48 ( 95 % ci : 1.9925.22 ) in terms of hospitalisation days and 1.76 ( 0.984.02 ) in terms of number of hospitalisations ( table 2 ) . in the primary analysis , the lower limit of the 95 % ci around the hr was above 1 , indicating that the number of hospitalisation days attributable to non - prevented varicella when using mmr + v as compared with mmrv was significantly higher than the number of hospitalisation days attributable to fc after mmrv as compared with mmr + v at a 95 % ci ( table 2).table 2benefit and risk estimates in terms of number of hospitalisation days in germanynamemeanmedian95 % ciyearly number of hospitalisation days for fc attributable to mmrv ( risk)225.0224.7104.9351.9yearly number of hospitalisation days for varicella prevented under mmrv ( benefit)19891879433.44125hospitalisation ratio8.51.99525.22 ci confidence interval , fc febrile convulsions , mmrv measles , mumps , rubella , and varicella vaccine benefit and risk estimates in terms of number of hospitalisation days in germany ci confidence interval , fc febrile convulsions , mmrv measles , mumps , rubella , and varicella vaccine expected outcomes in other countries or under different scenarios were derived from a sensitivity analysis , which was based on two key parameters : the vaccination coverage of v when co - administered with mmr and the probability of being hospitalised for fc . for parameter values compatible with the recent experience in germany , the model suggested that transitioning from mmr + v to mmrv would reduce the yearly number of hospitalisation days ; although there may be 225 hospitalisation days attributable to vaccine - related fc , at the same time , 1976 hospitalisation days due to severe varicella could be averted by using mmrv instead of mmr + v. when monte carlo simulations were used to evaluate the statistical uncertainty , the median hr was estimated to be 8.5 ( 95 % ci 1.9925.22 ) . the results are based on a modelling approach with several limitations , including : the model did not consider situations where v was co - administered with the second mmr dose ( either as mmr + v or mmrv ) if it was not co - administered with the first mmr dose.the assumption of total protection against hospitalisation could have overestimated the benefit measure of the mmrv compared with the mmr + v vaccination regimen.the measures of the burden of both fc and varicella infection that were selected in this analysis ( days of hospitalisation and number of hospitalisations ) may not have completely captured the severity of the two medical conditions ; hospitalisation for fc is generally more a measure of precaution , while hospitalisation related to varicella infection generally indicates a serious medical concern.the model did not take into account the potential impact of the recommendation to administer the first doses of mmr and v separately in germany since 2011 .the model did not take into account the effect of herd immunity , which is relevant in the context of protection against varicella . the measures of the burden of both fc and varicella infection that were selected in this analysis ( days of hospitalisation and number of hospitalisations ) may not have completely captured the severity of the two medical conditions ; hospitalisation for fc is generally more a measure of precaution , while hospitalisation related to varicella infection generally indicates a serious medical concern .

asthma is characterized by irreversible obstruction and chronic inflammation of the airways , and is traditionally considered a t helper 2 ( th2-)cell driven inflammatory disorder . however , an important role for the innate immune system in addition to the adaptive immune system is increasingly being recognized in asthma . macrophages are key cells in innate immune responses in the lung : they are among the most abundant cells and one of the first to encounter allergens and other threats to homeostasis . they also have the plasticity to quickly deal with those without endangering normal gas exchange . depending on the signals received , macrophages can be pro- or anti - inflammatory , immunogenic or tolerogenic , and destroying or repairing tissue . each characteristic may belong to a different macrophage phenotype with distinct functions [ 3 , 4 ] . tumor necrosis factor ( tnf ) and interferon ( ifn ) induce , under the influence of the transcription factor interferon - regulatory factor 5 ( irf5 ) , a phenotype of m1 macrophages with increased microbicidal and/or tumoricidal activities [ 4 , 5 ] . exposure to il-4 or il-13 results in a population of m2 macrophages that is involved in anti - parasite and tissue repair responses [ 6 , 7 ] . in mice , these cells are recognized by high production of chitinase and chitinase - like molecules such as ym1 [ 7 , 8 ] . these macrophages can be induced by a variety of stimuli including exposure to a tlr - ligand in the presence of il-10 or many more compounds . the main characteristic of the subtly different m2-like population is the production of il-10 . since il-10 is a potent anti - inflammatory cytokine , these m2-like macrophages are effective inhibitors of inflammation . despite the broad the spectrum of macrophage activation , the role of macrophages in asthma has scarcely been studied . from what is known , all three macrophage phenotypes have been implicated in the development of murine and human asthma [ 1012 ] . in mice , depending on the protocol used , asthma phenotypes can greatly differ [ 13 , 14 ] . we aimed to investigate the distribution of the three main macrophages phenotypes in three different models of hdm - induced asthma and also included the effects of sex on asthma development . first , we show the general differences in airway inflammation in the three hdm models and next we study the distribution of the macrophage phenotypes with regard to severity of allergic airway inflammation . male and female balb / c mice ( aged 68 weeks ) were obtained from harlan ( horst , the netherlands ) . the mice were fed ad libitum with standard food and water and were held under specific pathogen - free conditions in groups of 4 mice per cage . the animal procedures , approved by the institutional animal care and use committee of the university of groningen ( application number 5318 ) , were performed under strict governmental and international guidelines . male ( n = 4 per model ) and female mice ( n = 4 per model ) were anaesthetized with isoflurane and exposed intranasally to whole body hdm extract ( dermatophagoides pteronyssinus , greer laboratories , lenoir , usa ) in 40 l phosphate - buffered saline ( pbs ) according to three different protocols . control animals ( n = 8) were exposed to 40 l pbs according the 21-day protocol described . mice of the first model ( n = 8) received 100 g hdm extract intranasally on day 0 , were subsequently exposed to 10 g hdm on day 711 according to the protocol of hammad et al . and were sacrificed on day 14 ( abbreviated as 14-day hdm ) . in the second model , according to gregory et al . , mice ( n = 8) were exposed to 25 g hdm extract three times a week during three weeks and were sacrificed on day 21 ( abbreviated as 21-day hdm ) . for the last model ( n = 7 , due to illness one female was excluded from the study ) , mice were intranasally exposed to 100 g hdm on days 0 , 7 , 14 and 21 according to the protocol of arora et al . and were sacrificed on day 24 ( abbreviated as 24-day hdm ) . during sacrifice lungs were lavaged three times with 1 ml cold pbs to determine the number of eosinophils and ym1 levels . then , the left lung lobe was collected to isolate lung cells from digested lung for flow cytometry and the right lung was inflated with 0.5 ml 50% tissue - tek o.c.t . zoeterwoude , the netherlands ) in pbs and snap frozen / formalin - fixed for histological analyses . arbitrary elisa units of hdm - specific ige titers were calculated as relative values to the optical density of pooled sera from hdm - exposed mice . balf was collected and total numbers of cells were determined using a casy cell counter ( roche innovatis ag , reutlingen , germany ) . after centrifugation at 300 gfor 10 minutes , balf supernatants were stored at 80c for further analysis ( ym1 elisa ) and the cells were resuspended in rpmi medium ( biowhittaker europe , verviers , belgium ) for preparation of cytospots . approximately 50,000 cells were spotted onto slides using a cytospin 3 ( thermo shandon , waltham , ma , usa ) at 450 rpm for 5 minutes . to determine the percentage of eosinophils in the cytospots , a giemsa staining ( sigma - aldrich , zwijndrecht , the netherlands ) was performed and the number of eosinophils was counted in a total of 300 cells . the level of mecf - l ( ym1 ) in balf supernatants was determined by an elisa kit according to the manufacturer 's instructions ( r&d systems , oxon , uk ) . after bronchoalveolar lavage , the left lung was minced and incubated in rpmi medium supplemented with 10% fetal calf serum ( both lonza , verviers , belgium ) , 10 g / ml dnase i ( grade ii from bovine pancreas , roche applied science , almere , netherlands ) , and 0.7 mg / ml collagenase a ( sigma - aldrich ) for 45 minutes at 37c in a shaking water bath . the digested lung tissue was passed through a 70 m nylon strainer ( bd biosciences , breda , netherlands ) to obtain single cell suspensions . incubation with 10 times diluted pharmlyse ( bd biosciences , breda , the netherlands ) was performed to lyze contaminating erythrocytes . cells were centrifuged through 70 m strainer caps and counted using a casy cell counter ( roche innovatis ag ) . the single lung cell suspensions were stained for t - cell subsets using antibodies for flow cytometry . frequencies of effector t cells ( cd3+cd4+cd25+foxp3 ) and regulatory t cells ( cd3+cd4+cd25+foxp3 + ) were examined using cd3-apc / cy7 ( biolegend , fell , germany ) , cd4-pe / cy7 ( biolegend ) , cd25-pe ( biolegend ) , and foxp3-apc ( ebioscience , vienna , austria ) . an appropriate isotype control was used for the foxp3 staining ( rat igg2ak - apc , ebioscience ) . approximately 10 cells were incubated with the appropriate antibody mix including 1% normal mouse serum for 30 minutes on ice , protected from light . after washing the cells with pbs supplemented with 2% fcs and 5 mm edta , the cells were fixed and permeabilized for 30 minutes using a fixation and permeabilization buffer ( ebioscience ) . then cells were washed with permeabilization buffer and incubated with anti - foxp3 including 1% normal mouse serum for 30 minutes . subsequently , the cells were washed with permeabilization buffer , resuspended in facs lysing solution ( bd biosciences ) and kept in the dark on ice until flow cytometric analysis . the fluorescent staining of the cells was measured on a lsr - ii flow cytometer ( bd biosciences ) and data were analyzed using flowjo software ( tree star , ashland , usa ) . sections of 4 m were cut from the frozen part of the right lung and stained for all macrophages ( rat cd68 , serotec , oxford , uk ) . the numbers of m2 macrophages were determined in frozen sections by staining for ym1 ( goat -mecf - l , r&d systems , oxon , uk ) using standard immunohistochemical procedures . cd68 and ym1 were visualized with 3-amino-9-ethylcarbazole ( aec , sigma aldrich , zwijndrecht , the netherlands ) . the formalin - fixed part of the right lung was embedded in paraffinthen sections of 3 m were cut . to identify the m1 macrophages in tissue sections , antigen retrieval was performed by overnight incubation in tris - hcl buffer ph 9.0 at 80c and then sections were stained for irf5 ( rabbit -irf5 , proteintech europe , manchester uk ) using standard immunohistochemical procedures . to determine the number of il-10 producing cells , antigen retrieval was performed by boiling the sections in citrate buffer ph 6.0 for 10 minutes . the sections were pretreated with 1% bovine serum albumin ( sigma aldrich ) and 5% milk powder in pbs for 30 minutes and incubated with rabbit -il-10 overnight ( hycult biotech , uden , the netherlands ) . irf5 and il-10 were both visualized with immpact novared kit ( vector , burlingame , ca , usa ) . positive cells were quantified by manual counting in parenchymal lung tissue ( thus excluding large airways , vessels , and infiltrates , magnification 200400x ) and the total tissue area was quantified by morphometric analysis using imagescope analysis software ( aperio , vista , ca , usa ) . the numbers of cells were expressed per mm of tissue . to determine if the data were normally distributed a kolmogorov - smirnov test was used . the differences between the models were tested using one - way analysis of variance ( anova ) with tukey 's post - hoc test for multiple comparisons and sex differences were tested with the student 's t test . pearson correlation coefficients were calculated to analyze the correlation between the inflammation parameters and macrophages phenotypes , and correlations within macrophage phenotypes ( graphpad software , la jolla , ca , usa ) . differences were considered significant when p < 0.05 , and p < 0.10 was considered a statistical trend . to test whether exposure to hdm , according to three different protocols , induced allergic airway inflammation differently , we studied a number of general inflammation parameters . higher percentages of eosinophils in balf were found in all three hdm - exposed groups as compared to control mice ( figure 2(a ) ) . no differences in percentage of eosinophils in balf were observed between the three hdm protocols . hdm - specific ige levels in serum were not affected by the different hdm exposures , only a trend of higher levels was found in the group that was exposed to hdm in the 21-day protocol . in all protocols of hdm exposure , hdm - specific ige levels were very low measuring just above the limit of detection in the calibration curve ( figure 2(b ) ) . the higher airway inflammation in the three hdm - exposed groups was accompanied by higher percentages of effector t cells in lung tissue as compared to the control group ( figure 3(a ) ) . the 24-day protocol showed a higher percentage of effector t cells in lungs than the 14- and 21-day protocol . after hdm exposure the percentage of regulatory t cells was also higher in all three protocols as compared to control mice ( figure 3(b ) ) . the 24-day hdm protocol induced higher percentages of regulatory t cells in lungs compared to the 14-day protocol . the ratio of effector t cells to regulatory t cells was higher in the 24-day hdm protocol as compared to the control group and the other two hdm protocols ( figure 3(c ) ) . in females , hdm exposure induced more eosinophilia ( p < 0.01 ) , effector t cells ( p < 0.05 ) , regulatory t cells ( p < 0.05 ) , and higher levels of hdm - specific ige ( p < 0.05 ) than in males . to study the presence of macrophage phenotypes after hdm exposure according to the three different protocols , we stained lung tissue for markers of total macrophages ( cd68 ) , m1 macrophages ( irf5 ) , m2 macrophages ( ym1 ) , and m2-like macrophages ( il-10 ) and counted positive cells in parenchymal tissue . hdm - exposed mice had more cd68-positive cells in lung tissue as compared to control mice ( figure 4(a ) ) . no differences in cd68-positive numbers were observed between the hdm protocols . compared to control mice , irf5-positive cell numbers were higher in 14- and 21-day protocol , but not in mice exposed to hdm according to the 24-day protocol ( figure 4(b ) ) . between the hdm models , lower irf5-positive numbers were found in lungs of mice that were exposed to hdm according to the 24-day protocol as compared to the mice of the 14-day hdm protocol . for ym1 , all hdm protocols induced more ym1-positive cells as compared to control ( figure 4(c ) ) . however , mice that were exposed in the 24-day hdm protocol had higher numbers of ym1-positive cells in lung tissue than the mice of the 14-day hdm protocol . ym1 levels in balf were elevated in all hdm models as compared to control , but no differences were found between the models ( figure 5 ) . interestingly , hdm exposure resulted in significantly lower numbers of il-10-positive cells in all three protocols compared to the control - treated group ( figure 4(d ) ) . there were no differences observed in il-10-positive cell numbers between the three hdm protocols . hdm - exposed females had more cd68-positive cells ( p < 0.05 ) , ym1-positive cells ( p < 0.01 ) , and higher levels of balf ym1 ( p < 0.05 ) than males , whereas no differences were found in irf5- and il-10-positve cells numbers between the two sexes . to assess how severity of airway inflammation is reflected by the presence of the three main macrophage phenotypes , we correlated parameters of allergic airway inflammation with the different macrophage phenotypes in hdm - exposed mice ( table 1 ) . numbers of cd68-positive cells correlated positively with the percentage of eosinophils in balf ( r = 0.58 ) , effector t cells ( trend , r = 0.37 ) and regulatory t cells ( r = 0.42 ) in lungs of hdm - exposed mice , indicating that more severe disease was accompanied by more macrophages . most of these macrophages appear to be ym1-positive as only ym1-positive cell numbers correlated significantly with the percentage of eosinophils in balf ( r = 0.48 , figure 6 ) and the percentage of regulatory t cells ( r = 0.51 ) in lung tissue . no differences were found between males and females . to study the relationship between the different macrophage phenotypes in allergic airway inflammation , correlations were made between ym1-postive , irf5-positive , il-10-positive , and cd68-positive cells in lung tissue of all hdm - exposed mice ( table 2 ) . numbers of irf5-positive cells negatively correlated with cells positive for cd68 ( trend , r = 0.40 ) and ym1 in lung tissue ( r = 0.70 , figure 7(a ) ) . ym1-positive cell numbers correlated negatively with numbers of il-10-positve cells ( r = 0.48 , figure 7(b ) ) and positively with numbers of cd68-positive cells in lung tissue ( r = 0.66 ) . our study has shown that the balance between macrophage phenotype changes as the severity of allergic inflammation increases . higher numbers of m2 macrophages in hdm - exposed mice correlated with higher percentages of eosinophils in balf . at the same time lower numbers of m1 macrophages and m2-like macrophages were found in the mice with more severe inflammation and these therefore correlated negatively with m2 macrophages . in addition , we have confirmed again that females have more pronounced airway inflammation with higher numbers of m2 macrophages as compared to males . the models we used for our study were short - term exposure to hdm and they give us much information about the distribution of the different macrophage phenotypes during induction of asthma . in these models we found that longer exposure to hdm did not induce more severe eosinophilic inflammation but it did lead to higher numbers of m2 macrophages and higher percentages of effector and regulatory t cells in lungs of mice . the fact that we found no differences in eosinophils between the models is probably due to the large variation within the groups . however , when analyzing all hdm - exposed mice separately , eosinophils correlated positively with total macrophages and m2 macrophages , confirming our previous findings in humans that m2 macrophages increase with increasing asthma severity . another important parameter of allergic airway inflammation , serum hdm - specific ige , could barely be detected probably because the duration of the models was too short . it takes around 3 weeks for naive b cells to mature to plasma cells and switch from igm production to ige after first contact with an antigen . our models lasted 24 days at the most and we therefore sacrificed our animals before a full - blown ige response could develop . also , studies from other groups using these models did not show hdm - specific ige in serum [ 1517 ] . to investigate how macrophage phenotypes are distributed during and contribute to more chronic disease , longer models of hdm - exposure need to be used . we sought to phenotype the distinct macrophage subsets in lung tissue using markers that could distinguish each phenotype . we identified m2 macrophages using expression of ym1 , which is unique for this phenotype in the lung . previous studies found that only macrophages express ym1 and the staining is not complicated by other cells staining positive [ 2224 ] . a unique marker for identifying m1 macrophages in lung tissue is , however , more difficult to find . to our knowledge selective surface markers for tissue are not available and therefore the production of il-12 and oxygen radicals have been used in several mouse studies [ 25 , 26 ] . in asthma , oxygen radicals can not be used to stain for m1 macrophages because these are also copiously produced by eosinophils that are present in great numbers . our study was the first to use irf5 as an m1 marker on lung tissue and it is a fairly selective marker . bronchial epithelial cells and incoming leukocytes in infiltrates also stain positive for irf5 but these could be excluded from the quantification of parenchymal tissue based on localization . a double staining with cd68 to make sure only macrophages are included in the quantification is unfortunately at present not possible due to technical incompatibilities . in previous studies , m2-like macrophages are often not distinguished from m2 macrophages because they share many markers , including mannose receptors . the production of the immunosuppressive cytokine il-10 is the most important and reliable characteristic of m2-like macrophages and can be used to identify these cells with . similar to irf5 , bronchial epithelial cells and some cells in infiltrates are also expressing il-10 but these can easily be excluded from the quantification of parenchymal tissue . to exclude other il-10-producing cells from the analysis a double staining with cd68 these limitations in the stainings for irf5 and il-10 may also explain why the total number of ym1- , irf5- and il-10-positive cells is higher than the number of cd68-positive cells . higher numbers of m2 macrophages were found in lungs of hdm - exposed mice , which correlates with previous studies [ 19 , 20 , 28 ] . interestingly , numbers of m2 macrophages and the levels of ym1 in balf correlated strongly with eosinophils in balf . ym1 is also known as eosinophil chemotactic factor ( ecf - l ) , but it was suggested that ym1 only has weak chemotactic properties for eosinophils [ 24 , 30 ] . the chemotactic strength of ym1 is still debated and our findings and those of others do suggest otherwise [ 23 , 31 ] . the number of m2 macrophages also showed a positive correlation with regulatory t cells . in an interesting study by tiemessen et al . , regulatory t cells were shown to promote the induction of m2 macrophages to help with trying to maintain tissue homeostasis and preventing too much tissue damage of the inflammatory response . our data could be explained along these lines with incoming regulatory t cells inducing m2 macrophages in an attempt to restrict inflammation and tissue damage induced by hdm . the m2 macrophages would then be the result of inflammation and be beneficial instead of contributing to allergic airway inflammation , which is still an ongoing debate [ 19 , 33 , 34 ] . asthma is dominated by a th2-driven inflammation , but evidence shows that m1 macrophages are also present in this disease . in two interesting studies , ifn-stimulated macrophages were shown to prevent the onset of allergic airway inflammation [ 35 , 36 ] . our findings with higher numbers of m1 macrophages in the shorter protocols and in less severe diseases suggest that these macrophages are induced as a counterregulatory mechanism to dampen inflammation . however , higher numbers of m1 macrophages in asthma have also been shown in severe asthma and markers of m1 macrophages correlate with asthma severity , suggesting that m1 macrophages play a role in severe asthma as well [ 3739 ] . this appears not to be the case for our results because we find higher numbers of m1 macrophages at the onset of allergic airway inflammation when numbers of effector t cells are lower , and lower numbers of m1 macrophages in the 24-day hdm protocol when effector t - cell numbers are higher . m1 macrophages appear to have a beneficial role in preventing allergic sensitization , but in already established disease they promote the development of a severe phenotype . a similar double role has been reported for the m1 cytokine il-12 in allergic airway inflammation . since our hdm models are short and focus on the onset of the disease , we do not have information on the presence of m1 macrophages in established , more severe disease . since m2-like macrophages have anti - inflammatory functions , we were interested in the presence of this macrophage phenotype in hdm - induced asthma . others have reported that interstitial macrophages are the il-10-producing macrophages and treatment of sensitized mice with these macrophages prevented the development of allergic airway inflammation . our observation of lower numbers of m2-like macrophages in asthma as compared to control is in line with these and previous findings in human asthmatics [ 42 , 43 ] . interestingly , it was shown that il-10 production in severe asthmatics is even lower as compared to moderate asthmatics and that treatment with corticosteroids induces il-10 production by macrophages [ 43 , 44 ] . this suggests that specific stimulation of macrophages to polarize to m2-like macrophages that produce il-10 may reduce asthma symptoms . women suffer from more severe asthma than men and this phenomenon was also found in mouse models of asthma [ 45 , 46 ] . a role for sex hormones has been suggested , but the underlying mechanisms are unknown . in accordance with the previous findings , we show that hdm - exposed female mice had more pronounced airway inflammation than male mice . in macrophage phenotypes , we only found a difference in the m2 macrophages . since we and others found a correlation between m2 macrophages and asthma severity , it may suggest that this phenotype could play a role in the increased airway inflammation in females . taken together , our data suggest that during the development of allergic airway inflammation m1 and m2 macrophages are induced or recruited , whereas m2-like macrophages are prevented from moving in or inhibited . as hdm - induced inflammation progresses , m1 macrophages are diminished in favor of m2 macrophages possibly under the influence of regulatory t cells that try to restrict inflammation . their work may be hampered by the fact that il-10-producing m2-like macrophages do not develop in hdm - induced inflammation and the inflammation can progress . influencing this imbalanced relationship by therapeutic macrophage targeting could be a novel way to treat asthma .

in asthma , an important role for innate immunity is increasingly being recognized . key innate immune cells in the lungs are macrophages . depending on the signals they receive , macrophages can at least have an m1 , m2 , or m2-like phenotype . it is unknown how these macrophage phenotypes behave with regard to ( the severity of ) asthma . we have quantified the phenotypes in three models of house dust mite ( hdm-)induced asthma ( 14 , 21 , and 24 days ) . m1 , m2 , and m2-like phenotypes were identified by interferon regulatory factor 5 ( irf5 ) , ym1 , and il-10 , respectively . we found higher percentages of eosinophils in hdm - exposed mice compared to control but no differences between hdm models . t cell numbers were higher after hdm exposure and were the highest in the 24-day hdm protocol . higher numbers of m2 macrophages after hdm correlated with higher eosinophil numbers . in mice with less severe asthma , m1 macrophage numbers were higher and correlated negatively with m2 macrophages numbers . lower numbers of m2-like macrophages were found after hdm exposure and these correlated negatively with m2 macrophages . the balance between macrophage phenotypes changes as the severity of allergic airway inflammation increases . influencing this imbalanced relationship could be a novel approach to treat asthma .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

asthma is characterized by irreversible obstruction and chronic inflammation of the airways , and is traditionally considered a t helper 2 ( th2-)cell driven inflammatory disorder . however , an important role for the innate immune system in addition to the adaptive immune system is increasingly being recognized in asthma . macrophages are key cells in innate immune responses in the lung : they are among the most abundant cells and one of the first to encounter allergens and other threats to homeostasis . depending on the signals received , macrophages can be pro- or anti - inflammatory , immunogenic or tolerogenic , and destroying or repairing tissue . tumor necrosis factor ( tnf ) and interferon ( ifn ) induce , under the influence of the transcription factor interferon - regulatory factor 5 ( irf5 ) , a phenotype of m1 macrophages with increased microbicidal and/or tumoricidal activities [ 4 , 5 ] . exposure to il-4 or il-13 results in a population of m2 macrophages that is involved in anti - parasite and tissue repair responses [ 6 , 7 ] . in mice , these cells are recognized by high production of chitinase and chitinase - like molecules such as ym1 [ 7 , 8 ] . these macrophages can be induced by a variety of stimuli including exposure to a tlr - ligand in the presence of il-10 or many more compounds . since il-10 is a potent anti - inflammatory cytokine , these m2-like macrophages are effective inhibitors of inflammation . despite the broad the spectrum of macrophage activation , the role of macrophages in asthma has scarcely been studied . from what is known , all three macrophage phenotypes have been implicated in the development of murine and human asthma [ 1012 ] . in mice , depending on the protocol used , asthma phenotypes can greatly differ [ 13 , 14 ] . we aimed to investigate the distribution of the three main macrophages phenotypes in three different models of hdm - induced asthma and also included the effects of sex on asthma development . first , we show the general differences in airway inflammation in the three hdm models and next we study the distribution of the macrophage phenotypes with regard to severity of allergic airway inflammation . the mice were fed ad libitum with standard food and water and were held under specific pathogen - free conditions in groups of 4 mice per cage . the animal procedures , approved by the institutional animal care and use committee of the university of groningen ( application number 5318 ) , were performed under strict governmental and international guidelines . and were sacrificed on day 14 ( abbreviated as 14-day hdm ) . in the second model , according to gregory et al . , mice ( n = 8) were exposed to 25 g hdm extract three times a week during three weeks and were sacrificed on day 21 ( abbreviated as 21-day hdm ) . for the last model ( n = 7 , due to illness one female was excluded from the study ) , mice were intranasally exposed to 100 g hdm on days 0 , 7 , 14 and 21 according to the protocol of arora et al . and were sacrificed on day 24 ( abbreviated as 24-day hdm ) . during sacrifice lungs were lavaged three times with 1 ml cold pbs to determine the number of eosinophils and ym1 levels . arbitrary elisa units of hdm - specific ige titers were calculated as relative values to the optical density of pooled sera from hdm - exposed mice . to determine the percentage of eosinophils in the cytospots , a giemsa staining ( sigma - aldrich , zwijndrecht , the netherlands ) was performed and the number of eosinophils was counted in a total of 300 cells . after bronchoalveolar lavage , the left lung was minced and incubated in rpmi medium supplemented with 10% fetal calf serum ( both lonza , verviers , belgium ) , 10 g / ml dnase i ( grade ii from bovine pancreas , roche applied science , almere , netherlands ) , and 0.7 mg / ml collagenase a ( sigma - aldrich ) for 45 minutes at 37c in a shaking water bath . frequencies of effector t cells ( cd3+cd4+cd25+foxp3 ) and regulatory t cells ( cd3+cd4+cd25+foxp3 + ) were examined using cd3-apc / cy7 ( biolegend , fell , germany ) , cd4-pe / cy7 ( biolegend ) , cd25-pe ( biolegend ) , and foxp3-apc ( ebioscience , vienna , austria ) . subsequently , the cells were washed with permeabilization buffer , resuspended in facs lysing solution ( bd biosciences ) and kept in the dark on ice until flow cytometric analysis . the numbers of m2 macrophages were determined in frozen sections by staining for ym1 ( goat -mecf - l , r&d systems , oxon , uk ) using standard immunohistochemical procedures . positive cells were quantified by manual counting in parenchymal lung tissue ( thus excluding large airways , vessels , and infiltrates , magnification 200400x ) and the total tissue area was quantified by morphometric analysis using imagescope analysis software ( aperio , vista , ca , usa ) . the numbers of cells were expressed per mm of tissue . the differences between the models were tested using one - way analysis of variance ( anova ) with tukey 's post - hoc test for multiple comparisons and sex differences were tested with the student 's t test . pearson correlation coefficients were calculated to analyze the correlation between the inflammation parameters and macrophages phenotypes , and correlations within macrophage phenotypes ( graphpad software , la jolla , ca , usa ) . differences were considered significant when p < 0.05 , and p < 0.10 was considered a statistical trend . to test whether exposure to hdm , according to three different protocols , induced allergic airway inflammation differently , we studied a number of general inflammation parameters . higher percentages of eosinophils in balf were found in all three hdm - exposed groups as compared to control mice ( figure 2(a ) ) . no differences in percentage of eosinophils in balf were observed between the three hdm protocols . hdm - specific ige levels in serum were not affected by the different hdm exposures , only a trend of higher levels was found in the group that was exposed to hdm in the 21-day protocol . in all protocols of hdm exposure , hdm - specific ige levels were very low measuring just above the limit of detection in the calibration curve ( figure 2(b ) ) . the higher airway inflammation in the three hdm - exposed groups was accompanied by higher percentages of effector t cells in lung tissue as compared to the control group ( figure 3(a ) ) . the 24-day protocol showed a higher percentage of effector t cells in lungs than the 14- and 21-day protocol . after hdm exposure the percentage of regulatory t cells was also higher in all three protocols as compared to control mice ( figure 3(b ) ) . the 24-day hdm protocol induced higher percentages of regulatory t cells in lungs compared to the 14-day protocol . the ratio of effector t cells to regulatory t cells was higher in the 24-day hdm protocol as compared to the control group and the other two hdm protocols ( figure 3(c ) ) . in females , hdm exposure induced more eosinophilia ( p < 0.01 ) , effector t cells ( p < 0.05 ) , regulatory t cells ( p < 0.05 ) , and higher levels of hdm - specific ige ( p < 0.05 ) than in males . to study the presence of macrophage phenotypes after hdm exposure according to the three different protocols , we stained lung tissue for markers of total macrophages ( cd68 ) , m1 macrophages ( irf5 ) , m2 macrophages ( ym1 ) , and m2-like macrophages ( il-10 ) and counted positive cells in parenchymal tissue . hdm - exposed mice had more cd68-positive cells in lung tissue as compared to control mice ( figure 4(a ) ) . no differences in cd68-positive numbers were observed between the hdm protocols . compared to control mice , irf5-positive cell numbers were higher in 14- and 21-day protocol , but not in mice exposed to hdm according to the 24-day protocol ( figure 4(b ) ) . between the hdm models , lower irf5-positive numbers were found in lungs of mice that were exposed to hdm according to the 24-day protocol as compared to the mice of the 14-day hdm protocol . for ym1 , all hdm protocols induced more ym1-positive cells as compared to control ( figure 4(c ) ) . however , mice that were exposed in the 24-day hdm protocol had higher numbers of ym1-positive cells in lung tissue than the mice of the 14-day hdm protocol . ym1 levels in balf were elevated in all hdm models as compared to control , but no differences were found between the models ( figure 5 ) . interestingly , hdm exposure resulted in significantly lower numbers of il-10-positive cells in all three protocols compared to the control - treated group ( figure 4(d ) ) . there were no differences observed in il-10-positive cell numbers between the three hdm protocols . hdm - exposed females had more cd68-positive cells ( p < 0.05 ) , ym1-positive cells ( p < 0.01 ) , and higher levels of balf ym1 ( p < 0.05 ) than males , whereas no differences were found in irf5- and il-10-positve cells numbers between the two sexes . to assess how severity of airway inflammation is reflected by the presence of the three main macrophage phenotypes , we correlated parameters of allergic airway inflammation with the different macrophage phenotypes in hdm - exposed mice ( table 1 ) . numbers of cd68-positive cells correlated positively with the percentage of eosinophils in balf ( r = 0.58 ) , effector t cells ( trend , r = 0.37 ) and regulatory t cells ( r = 0.42 ) in lungs of hdm - exposed mice , indicating that more severe disease was accompanied by more macrophages . most of these macrophages appear to be ym1-positive as only ym1-positive cell numbers correlated significantly with the percentage of eosinophils in balf ( r = 0.48 , figure 6 ) and the percentage of regulatory t cells ( r = 0.51 ) in lung tissue . no differences were found between males and females . to study the relationship between the different macrophage phenotypes in allergic airway inflammation , correlations were made between ym1-postive , irf5-positive , il-10-positive , and cd68-positive cells in lung tissue of all hdm - exposed mice ( table 2 ) . numbers of irf5-positive cells negatively correlated with cells positive for cd68 ( trend , r = 0.40 ) and ym1 in lung tissue ( r = 0.70 , figure 7(a ) ) . ym1-positive cell numbers correlated negatively with numbers of il-10-positve cells ( r = 0.48 , figure 7(b ) ) and positively with numbers of cd68-positive cells in lung tissue ( r = 0.66 ) . our study has shown that the balance between macrophage phenotype changes as the severity of allergic inflammation increases . higher numbers of m2 macrophages in hdm - exposed mice correlated with higher percentages of eosinophils in balf . at the same time lower numbers of m1 macrophages and m2-like macrophages were found in the mice with more severe inflammation and these therefore correlated negatively with m2 macrophages . in addition , we have confirmed again that females have more pronounced airway inflammation with higher numbers of m2 macrophages as compared to males . the models we used for our study were short - term exposure to hdm and they give us much information about the distribution of the different macrophage phenotypes during induction of asthma . in these models we found that longer exposure to hdm did not induce more severe eosinophilic inflammation but it did lead to higher numbers of m2 macrophages and higher percentages of effector and regulatory t cells in lungs of mice . the fact that we found no differences in eosinophils between the models is probably due to the large variation within the groups . however , when analyzing all hdm - exposed mice separately , eosinophils correlated positively with total macrophages and m2 macrophages , confirming our previous findings in humans that m2 macrophages increase with increasing asthma severity . another important parameter of allergic airway inflammation , serum hdm - specific ige , could barely be detected probably because the duration of the models was too short . also , studies from other groups using these models did not show hdm - specific ige in serum [ 1517 ] . to investigate how macrophage phenotypes are distributed during and contribute to more chronic disease , longer models of hdm - exposure need to be used . we identified m2 macrophages using expression of ym1 , which is unique for this phenotype in the lung . in asthma , oxygen radicals can not be used to stain for m1 macrophages because these are also copiously produced by eosinophils that are present in great numbers . our study was the first to use irf5 as an m1 marker on lung tissue and it is a fairly selective marker . a double staining with cd68 to make sure only macrophages are included in the quantification is unfortunately at present not possible due to technical incompatibilities . in previous studies , m2-like macrophages are often not distinguished from m2 macrophages because they share many markers , including mannose receptors . the production of the immunosuppressive cytokine il-10 is the most important and reliable characteristic of m2-like macrophages and can be used to identify these cells with . similar to irf5 , bronchial epithelial cells and some cells in infiltrates are also expressing il-10 but these can easily be excluded from the quantification of parenchymal tissue . to exclude other il-10-producing cells from the analysis a double staining with cd68 these limitations in the stainings for irf5 and il-10 may also explain why the total number of ym1- , irf5- and il-10-positive cells is higher than the number of cd68-positive cells . higher numbers of m2 macrophages were found in lungs of hdm - exposed mice , which correlates with previous studies [ 19 , 20 , 28 ] . interestingly , numbers of m2 macrophages and the levels of ym1 in balf correlated strongly with eosinophils in balf . ym1 is also known as eosinophil chemotactic factor ( ecf - l ) , but it was suggested that ym1 only has weak chemotactic properties for eosinophils [ 24 , 30 ] . the number of m2 macrophages also showed a positive correlation with regulatory t cells . , regulatory t cells were shown to promote the induction of m2 macrophages to help with trying to maintain tissue homeostasis and preventing too much tissue damage of the inflammatory response . our data could be explained along these lines with incoming regulatory t cells inducing m2 macrophages in an attempt to restrict inflammation and tissue damage induced by hdm . the m2 macrophages would then be the result of inflammation and be beneficial instead of contributing to allergic airway inflammation , which is still an ongoing debate [ 19 , 33 , 34 ] . in two interesting studies , ifn-stimulated macrophages were shown to prevent the onset of allergic airway inflammation [ 35 , 36 ] . our findings with higher numbers of m1 macrophages in the shorter protocols and in less severe diseases suggest that these macrophages are induced as a counterregulatory mechanism to dampen inflammation . however , higher numbers of m1 macrophages in asthma have also been shown in severe asthma and markers of m1 macrophages correlate with asthma severity , suggesting that m1 macrophages play a role in severe asthma as well [ 3739 ] . this appears not to be the case for our results because we find higher numbers of m1 macrophages at the onset of allergic airway inflammation when numbers of effector t cells are lower , and lower numbers of m1 macrophages in the 24-day hdm protocol when effector t - cell numbers are higher . a similar double role has been reported for the m1 cytokine il-12 in allergic airway inflammation . since our hdm models are short and focus on the onset of the disease , we do not have information on the presence of m1 macrophages in established , more severe disease . since m2-like macrophages have anti - inflammatory functions , we were interested in the presence of this macrophage phenotype in hdm - induced asthma . others have reported that interstitial macrophages are the il-10-producing macrophages and treatment of sensitized mice with these macrophages prevented the development of allergic airway inflammation . our observation of lower numbers of m2-like macrophages in asthma as compared to control is in line with these and previous findings in human asthmatics [ 42 , 43 ] . interestingly , it was shown that il-10 production in severe asthmatics is even lower as compared to moderate asthmatics and that treatment with corticosteroids induces il-10 production by macrophages [ 43 , 44 ] . this suggests that specific stimulation of macrophages to polarize to m2-like macrophages that produce il-10 may reduce asthma symptoms . women suffer from more severe asthma than men and this phenomenon was also found in mouse models of asthma [ 45 , 46 ] . in accordance with the previous findings , we show that hdm - exposed female mice had more pronounced airway inflammation than male mice . in macrophage phenotypes , we only found a difference in the m2 macrophages . since we and others found a correlation between m2 macrophages and asthma severity , it may suggest that this phenotype could play a role in the increased airway inflammation in females . taken together , our data suggest that during the development of allergic airway inflammation m1 and m2 macrophages are induced or recruited , whereas m2-like macrophages are prevented from moving in or inhibited . as hdm - induced inflammation progresses , m1 macrophages are diminished in favor of m2 macrophages possibly under the influence of regulatory t cells that try to restrict inflammation . their work may be hampered by the fact that il-10-producing m2-like macrophages do not develop in hdm - induced inflammation and the inflammation can progress . influencing this imbalanced relationship by therapeutic macrophage targeting could be a novel way to treat asthma .

asthma is characterized by irreversible obstruction and chronic inflammation of the airways , and is traditionally considered a t helper 2 ( th2-)cell driven inflammatory disorder . however , an important role for the innate immune system in addition to the adaptive immune system is increasingly being recognized in asthma . macrophages are key cells in innate immune responses in the lung : they are among the most abundant cells and one of the first to encounter allergens and other threats to homeostasis . depending on the signals received , macrophages can be pro- or anti - inflammatory , immunogenic or tolerogenic , and destroying or repairing tissue . tumor necrosis factor ( tnf ) and interferon ( ifn ) induce , under the influence of the transcription factor interferon - regulatory factor 5 ( irf5 ) , a phenotype of m1 macrophages with increased microbicidal and/or tumoricidal activities [ 4 , 5 ] . exposure to il-4 or il-13 results in a population of m2 macrophages that is involved in anti - parasite and tissue repair responses [ 6 , 7 ] . in mice , these cells are recognized by high production of chitinase and chitinase - like molecules such as ym1 [ 7 , 8 ] . these macrophages can be induced by a variety of stimuli including exposure to a tlr - ligand in the presence of il-10 or many more compounds . the main characteristic of the subtly different m2-like population is the production of il-10 . since il-10 is a potent anti - inflammatory cytokine , these m2-like macrophages are effective inhibitors of inflammation . despite the broad the spectrum of macrophage activation , the role of macrophages in asthma has scarcely been studied . from what is known , all three macrophage phenotypes have been implicated in the development of murine and human asthma [ 1012 ] . in mice , depending on the protocol used , asthma phenotypes can greatly differ [ 13 , 14 ] . we aimed to investigate the distribution of the three main macrophages phenotypes in three different models of hdm - induced asthma and also included the effects of sex on asthma development . first , we show the general differences in airway inflammation in the three hdm models and next we study the distribution of the macrophage phenotypes with regard to severity of allergic airway inflammation . male and female balb / c mice ( aged 68 weeks ) were obtained from harlan ( horst , the netherlands ) . the mice were fed ad libitum with standard food and water and were held under specific pathogen - free conditions in groups of 4 mice per cage . male ( n = 4 per model ) and female mice ( n = 4 per model ) were anaesthetized with isoflurane and exposed intranasally to whole body hdm extract ( dermatophagoides pteronyssinus , greer laboratories , lenoir , usa ) in 40 l phosphate - buffered saline ( pbs ) according to three different protocols . mice of the first model ( n = 8) received 100 g hdm extract intranasally on day 0 , were subsequently exposed to 10 g hdm on day 711 according to the protocol of hammad et al . , mice ( n = 8) were exposed to 25 g hdm extract three times a week during three weeks and were sacrificed on day 21 ( abbreviated as 21-day hdm ) . for the last model ( n = 7 , due to illness one female was excluded from the study ) , mice were intranasally exposed to 100 g hdm on days 0 , 7 , 14 and 21 according to the protocol of arora et al . during sacrifice lungs were lavaged three times with 1 ml cold pbs to determine the number of eosinophils and ym1 levels . then , the left lung lobe was collected to isolate lung cells from digested lung for flow cytometry and the right lung was inflated with 0.5 ml 50% tissue - tek o.c.t . zoeterwoude , the netherlands ) in pbs and snap frozen / formalin - fixed for histological analyses . arbitrary elisa units of hdm - specific ige titers were calculated as relative values to the optical density of pooled sera from hdm - exposed mice . after centrifugation at 300 gfor 10 minutes , balf supernatants were stored at 80c for further analysis ( ym1 elisa ) and the cells were resuspended in rpmi medium ( biowhittaker europe , verviers , belgium ) for preparation of cytospots . to determine the percentage of eosinophils in the cytospots , a giemsa staining ( sigma - aldrich , zwijndrecht , the netherlands ) was performed and the number of eosinophils was counted in a total of 300 cells . the level of mecf - l ( ym1 ) in balf supernatants was determined by an elisa kit according to the manufacturer 's instructions ( r&d systems , oxon , uk ) . after bronchoalveolar lavage , the left lung was minced and incubated in rpmi medium supplemented with 10% fetal calf serum ( both lonza , verviers , belgium ) , 10 g / ml dnase i ( grade ii from bovine pancreas , roche applied science , almere , netherlands ) , and 0.7 mg / ml collagenase a ( sigma - aldrich ) for 45 minutes at 37c in a shaking water bath . frequencies of effector t cells ( cd3+cd4+cd25+foxp3 ) and regulatory t cells ( cd3+cd4+cd25+foxp3 + ) were examined using cd3-apc / cy7 ( biolegend , fell , germany ) , cd4-pe / cy7 ( biolegend ) , cd25-pe ( biolegend ) , and foxp3-apc ( ebioscience , vienna , austria ) . an appropriate isotype control was used for the foxp3 staining ( rat igg2ak - apc , ebioscience ) . the fluorescent staining of the cells was measured on a lsr - ii flow cytometer ( bd biosciences ) and data were analyzed using flowjo software ( tree star , ashland , usa ) . sections of 4 m were cut from the frozen part of the right lung and stained for all macrophages ( rat cd68 , serotec , oxford , uk ) . the numbers of m2 macrophages were determined in frozen sections by staining for ym1 ( goat -mecf - l , r&d systems , oxon , uk ) using standard immunohistochemical procedures . to identify the m1 macrophages in tissue sections , antigen retrieval was performed by overnight incubation in tris - hcl buffer ph 9.0 at 80c and then sections were stained for irf5 ( rabbit -irf5 , proteintech europe , manchester uk ) using standard immunohistochemical procedures . to determine the number of il-10 producing cells , antigen retrieval was performed by boiling the sections in citrate buffer ph 6.0 for 10 minutes . the sections were pretreated with 1% bovine serum albumin ( sigma aldrich ) and 5% milk powder in pbs for 30 minutes and incubated with rabbit -il-10 overnight ( hycult biotech , uden , the netherlands ) . positive cells were quantified by manual counting in parenchymal lung tissue ( thus excluding large airways , vessels , and infiltrates , magnification 200400x ) and the total tissue area was quantified by morphometric analysis using imagescope analysis software ( aperio , vista , ca , usa ) . the differences between the models were tested using one - way analysis of variance ( anova ) with tukey 's post - hoc test for multiple comparisons and sex differences were tested with the student 's t test . pearson correlation coefficients were calculated to analyze the correlation between the inflammation parameters and macrophages phenotypes , and correlations within macrophage phenotypes ( graphpad software , la jolla , ca , usa ) . to test whether exposure to hdm , according to three different protocols , induced allergic airway inflammation differently , we studied a number of general inflammation parameters . higher percentages of eosinophils in balf were found in all three hdm - exposed groups as compared to control mice ( figure 2(a ) ) . no differences in percentage of eosinophils in balf were observed between the three hdm protocols . hdm - specific ige levels in serum were not affected by the different hdm exposures , only a trend of higher levels was found in the group that was exposed to hdm in the 21-day protocol . in all protocols of hdm exposure , hdm - specific ige levels were very low measuring just above the limit of detection in the calibration curve ( figure 2(b ) ) . the higher airway inflammation in the three hdm - exposed groups was accompanied by higher percentages of effector t cells in lung tissue as compared to the control group ( figure 3(a ) ) . the 24-day protocol showed a higher percentage of effector t cells in lungs than the 14- and 21-day protocol . after hdm exposure the percentage of regulatory t cells was also higher in all three protocols as compared to control mice ( figure 3(b ) ) . the 24-day hdm protocol induced higher percentages of regulatory t cells in lungs compared to the 14-day protocol . the ratio of effector t cells to regulatory t cells was higher in the 24-day hdm protocol as compared to the control group and the other two hdm protocols ( figure 3(c ) ) . in females , hdm exposure induced more eosinophilia ( p < 0.01 ) , effector t cells ( p < 0.05 ) , regulatory t cells ( p < 0.05 ) , and higher levels of hdm - specific ige ( p < 0.05 ) than in males . to study the presence of macrophage phenotypes after hdm exposure according to the three different protocols , we stained lung tissue for markers of total macrophages ( cd68 ) , m1 macrophages ( irf5 ) , m2 macrophages ( ym1 ) , and m2-like macrophages ( il-10 ) and counted positive cells in parenchymal tissue . hdm - exposed mice had more cd68-positive cells in lung tissue as compared to control mice ( figure 4(a ) ) . compared to control mice , irf5-positive cell numbers were higher in 14- and 21-day protocol , but not in mice exposed to hdm according to the 24-day protocol ( figure 4(b ) ) . between the hdm models , lower irf5-positive numbers were found in lungs of mice that were exposed to hdm according to the 24-day protocol as compared to the mice of the 14-day hdm protocol . however , mice that were exposed in the 24-day hdm protocol had higher numbers of ym1-positive cells in lung tissue than the mice of the 14-day hdm protocol . ym1 levels in balf were elevated in all hdm models as compared to control , but no differences were found between the models ( figure 5 ) . interestingly , hdm exposure resulted in significantly lower numbers of il-10-positive cells in all three protocols compared to the control - treated group ( figure 4(d ) ) . hdm - exposed females had more cd68-positive cells ( p < 0.05 ) , ym1-positive cells ( p < 0.01 ) , and higher levels of balf ym1 ( p < 0.05 ) than males , whereas no differences were found in irf5- and il-10-positve cells numbers between the two sexes . to assess how severity of airway inflammation is reflected by the presence of the three main macrophage phenotypes , we correlated parameters of allergic airway inflammation with the different macrophage phenotypes in hdm - exposed mice ( table 1 ) . numbers of cd68-positive cells correlated positively with the percentage of eosinophils in balf ( r = 0.58 ) , effector t cells ( trend , r = 0.37 ) and regulatory t cells ( r = 0.42 ) in lungs of hdm - exposed mice , indicating that more severe disease was accompanied by more macrophages . most of these macrophages appear to be ym1-positive as only ym1-positive cell numbers correlated significantly with the percentage of eosinophils in balf ( r = 0.48 , figure 6 ) and the percentage of regulatory t cells ( r = 0.51 ) in lung tissue . to study the relationship between the different macrophage phenotypes in allergic airway inflammation , correlations were made between ym1-postive , irf5-positive , il-10-positive , and cd68-positive cells in lung tissue of all hdm - exposed mice ( table 2 ) . numbers of irf5-positive cells negatively correlated with cells positive for cd68 ( trend , r = 0.40 ) and ym1 in lung tissue ( r = 0.70 , figure 7(a ) ) . ym1-positive cell numbers correlated negatively with numbers of il-10-positve cells ( r = 0.48 , figure 7(b ) ) and positively with numbers of cd68-positive cells in lung tissue ( r = 0.66 ) . our study has shown that the balance between macrophage phenotype changes as the severity of allergic inflammation increases . higher numbers of m2 macrophages in hdm - exposed mice correlated with higher percentages of eosinophils in balf . at the same time lower numbers of m1 macrophages and m2-like macrophages were found in the mice with more severe inflammation and these therefore correlated negatively with m2 macrophages . in addition , we have confirmed again that females have more pronounced airway inflammation with higher numbers of m2 macrophages as compared to males . the models we used for our study were short - term exposure to hdm and they give us much information about the distribution of the different macrophage phenotypes during induction of asthma . in these models we found that longer exposure to hdm did not induce more severe eosinophilic inflammation but it did lead to higher numbers of m2 macrophages and higher percentages of effector and regulatory t cells in lungs of mice . the fact that we found no differences in eosinophils between the models is probably due to the large variation within the groups . however , when analyzing all hdm - exposed mice separately , eosinophils correlated positively with total macrophages and m2 macrophages , confirming our previous findings in humans that m2 macrophages increase with increasing asthma severity . another important parameter of allergic airway inflammation , serum hdm - specific ige , could barely be detected probably because the duration of the models was too short . our models lasted 24 days at the most and we therefore sacrificed our animals before a full - blown ige response could develop . to investigate how macrophage phenotypes are distributed during and contribute to more chronic disease , longer models of hdm - exposure need to be used . we sought to phenotype the distinct macrophage subsets in lung tissue using markers that could distinguish each phenotype . we identified m2 macrophages using expression of ym1 , which is unique for this phenotype in the lung . to our knowledge selective surface markers for tissue are not available and therefore the production of il-12 and oxygen radicals have been used in several mouse studies [ 25 , 26 ] . our study was the first to use irf5 as an m1 marker on lung tissue and it is a fairly selective marker . the production of the immunosuppressive cytokine il-10 is the most important and reliable characteristic of m2-like macrophages and can be used to identify these cells with . to exclude other il-10-producing cells from the analysis a double staining with cd68 these limitations in the stainings for irf5 and il-10 may also explain why the total number of ym1- , irf5- and il-10-positive cells is higher than the number of cd68-positive cells . higher numbers of m2 macrophages were found in lungs of hdm - exposed mice , which correlates with previous studies [ 19 , 20 , 28 ] . interestingly , numbers of m2 macrophages and the levels of ym1 in balf correlated strongly with eosinophils in balf . , regulatory t cells were shown to promote the induction of m2 macrophages to help with trying to maintain tissue homeostasis and preventing too much tissue damage of the inflammatory response . our data could be explained along these lines with incoming regulatory t cells inducing m2 macrophages in an attempt to restrict inflammation and tissue damage induced by hdm . the m2 macrophages would then be the result of inflammation and be beneficial instead of contributing to allergic airway inflammation , which is still an ongoing debate [ 19 , 33 , 34 ] . our findings with higher numbers of m1 macrophages in the shorter protocols and in less severe diseases suggest that these macrophages are induced as a counterregulatory mechanism to dampen inflammation . however , higher numbers of m1 macrophages in asthma have also been shown in severe asthma and markers of m1 macrophages correlate with asthma severity , suggesting that m1 macrophages play a role in severe asthma as well [ 3739 ] . this appears not to be the case for our results because we find higher numbers of m1 macrophages at the onset of allergic airway inflammation when numbers of effector t cells are lower , and lower numbers of m1 macrophages in the 24-day hdm protocol when effector t - cell numbers are higher . m1 macrophages appear to have a beneficial role in preventing allergic sensitization , but in already established disease they promote the development of a severe phenotype . since our hdm models are short and focus on the onset of the disease , we do not have information on the presence of m1 macrophages in established , more severe disease . since m2-like macrophages have anti - inflammatory functions , we were interested in the presence of this macrophage phenotype in hdm - induced asthma . others have reported that interstitial macrophages are the il-10-producing macrophages and treatment of sensitized mice with these macrophages prevented the development of allergic airway inflammation . our observation of lower numbers of m2-like macrophages in asthma as compared to control is in line with these and previous findings in human asthmatics [ 42 , 43 ] . since we and others found a correlation between m2 macrophages and asthma severity , it may suggest that this phenotype could play a role in the increased airway inflammation in females . taken together , our data suggest that during the development of allergic airway inflammation m1 and m2 macrophages are induced or recruited , whereas m2-like macrophages are prevented from moving in or inhibited . as hdm - induced inflammation progresses , m1 macrophages are diminished in favor of m2 macrophages possibly under the influence of regulatory t cells that try to restrict inflammation .

without much risk of controversy , one can safely assume that we all want more people in our society to learn , develop expertise , and innovate ; fewer people to fail to thrive or to become dependent on damaging substances or behaviors ; and for more people to experience a greater sense of well being . but we would also agree that some people in our society learn more , create more , become more emotionally and physically resilient , and have more fun . the question is : why ? for answers to this question we can achieve very little consensus . is it because people are constitutionally endowed with different personal assets native intelligence , motivation , optimism , capacity for pleasure ? . they can be skills ( such as calculation or literacy ) , levels of expertise , knowledge , creativity , emotional or social biases , likes and dislikes ; they are , in short , relatively persistent traits and habits individuality , if you like . questions about the origins of differences among people are of intrinsic interest to many , but from a biomedical perspective , the incentive to study them comes from the well - documented observation that many seriously adverse outcomes evolve disproportionately in the context of specific behavioral phenotypes . risk phenotypes have been linked to many behavioral disorders , including substance use disorders ( volkow , 2005 ; clark et al . , 2013 ) , affective disorders ( klein et al . , 2011 ) , psychosis ( brent et al . , 2014 ) , adhd ( castellanos and tannock , 2002 ) , dyslexia ( peterson and pennington , 2012 ) , and dyscalculia ( butterworth et al . , 2011 ) . one could say that from conception to the onset of these disorders , evolving risk phenotypes are simply forms of individuality , but a deeper understanding of the divergence of high - risk from low - risk phenotypes eludes us . this article will describe some lines of evidence relevant to these issues , including work from behavioral genetics , neurogenetics , and cognitive neuroscience , and will pose important questions that we must answer in future research . finally , impediments to progress in the field , and some recommendations for how to reduce them , are outlined . for example , scores from intelligence tests , performance on cognitive tasks , and self - report scales measuring personality have consistently exhibited moderate heritability in twin studies ( bartels et al . , 2002 ; rettew et al . , 2006 ; kremen et al . , 2007 ) , as have measures of academic skills such as reading and calculation ( luo et al . , 2003 ) . these observations highlight the influence of genetic factors on developing behaviors , but do not identify them . a few behavioral phenotypes have been examined in large , genome wide association studies ( gwas ) , and surprisingly , given reported high estimates of heritability , these studies initially revealed few replicable associations with specific genetic variants , and those variants identified accounted for only very small proportions of the phenotypic variability ( butcher et al . , 2008 ; calboli et al . , 2010 ; rietveld et al . , 2013 ; rietveld et al . , however , more recent studies have applied novel methods that exploit gwas results to estimate heritability attributable to common genetic variants in aggregate ( so called chip heritability ) , and these suggest that substantial variability in behavioral phenotypes may indeed be explained by common variants , but the genetic architecture of these traits is likely to be highly polygenic , and extremely complex . so , in summary , genetic differences probably do play a large role in engendering behavioral individual differences , but it appears that thousands of variants may in some way influence these pheno - types , each with a very small additive effect ( purcell et al . a number of studies have examined the heritability of brain imaging phenotypes , again mostly using twin designs . global measures , such as brain volume ( baar et al . , 2001 ) , gray matter volume ( baar et al . , 2001 ) , and both surface area and thickness of the cortex ( panizzon et al . , 2009 ; eyler et al . , 2011 ) exhibit even higher levels of heritability , sometimes over .9 , with heritability estimates of regional measures significant but more modest . interestingly , cortical surface area and mean thickness of the cortex , both with estimated heritability in the .8.9 range , nevertheless exhibited no evidence for genetic correlation in a study of middle - aged male twins ( panizzon et al . , 2009 ) , suggesting that the genetic factors influencing variability of these brain phenotypes are essentially nonoverlapping . a series of subsequent analyses of imaging data from this study examined more closely the genetic architecture of individual difference variability in cortical regionalization , that is , individual differences in the relative sizes of different cortical regions . work with rodent models has shown that arealization of the developing cortex is linked to gradients of secreted morphogens and expression of transcription factors in the neocortical proliferative zone during the embryonic period ( mallamaci and stoykova , 2006 ; o'leary et al . , 2007 ) . transgenic models have shown that alterations of these gradients are associated with different relative sizes of sensorimotor regions in the later developing cortex . now that surface - based methods are available for examining the relative sizes of human cortical regions more precisely , one might ask whether genetic variation gives rise to individual differences in human cortical regionalization . using data from 400 middle - aged male veterans from the vietnam era twin study cohort , chen et al . ( 2012 ) applied conventional twin methodology to compute the vertex - to - vertex genetic correlations among measures of cortical surface expansion at 2500 vertices ( after smoothing ) . this large matrix of genetic correlations was then further analyzed using a data - driven , fuzzy clustering method to identify 12 sets ( or clusters ) of vertices with relatively higher genetic inter - correlations and relatively lower genetic correlations with other sets ( or clusters ) . these clusters are color - coded in fig . 1 and they represent regions of the cortex where the twin data suggest that individual differences in relative surface area expansion are related to distinct sets of underlying genetic factors . interestingly , this analysis yields a novel method for defining cortical parcels , one that is driven by analysis and simplification of the genetic architecture . after this analysis of cortical surface area variability was published , a similar analysis of genetic correlations for vertex - wise measures of cortical thickness was performed . the results of this analysis ( chen et al . , 2013 ) also revealed regional clustering of genetic inter - correlations , and the 12-cluster solution resembled the regional pattern for surface area expansion . however , the dominant patterns of genetic inter - correlation for thickness measures ( as expressed in the 24 cluster solutions ) differed substantially from those for surface area , and as previously reported for total surface area and mean cortical thickness ( panizzon et al . , 2009 ) , the genetic correlations between areal expansion and thickness within comparable genetic clusters were also very low . these results suggest that genetic factors strongly influence both global area and thickness of the cortex as well as regional differences in these biomarkers ; however , the overlap in genetic factors contributing to the high heritability of area and thickness of the cortex is negligible . attempts to identify specific genetic variants that may contribute to the heritability of imaging phenotypes have been slow to appear because of the paucity of large studies with comparable brain imaging data . in recent years , however , larger studies with imaging and genotyping have been conducted . early attempts to identify variants with effects strong enough to survive gwas significance criteria either failed or led to associations with low replicability , however a few studies have now reported findings in discovery and replication samples . bakken et al . ( 2012 ) performed gwas to identify specific variants associated with the proportional surface area of the visual cortex . this variant was in a regulatory region of a gene that is highly expressed in the occipital cortex ( gpcpd1 ) . ( 2013 ) identified a variant related to lenticular nucleus size that was also replicated in an independent sample . even these important gwas studies , however , examined fewer individuals than have been studied in most successful gwas studies of other complex human phenotypes , including several behavioral phenotypes . recently , the important enhancing neuro imaging genetics through meta - analysis ( enigma ) project has begun to aggregate imaging genomics data across many studies worldwide to facilitate the search for specific variants related to imaging phenotypes . in an early enigma report on gwas of volumes of subcortical structures in over 30 thousand individuals , hibar et al . ( 2015 ) report that several variants contributing to these volumes survived stringent gwas criteria and replicated across samples . so far , however , these early reports suggest that , like heritable behavioral phenotypes , neuroimaging phenotypes may have highly complex genetic architecture , with few variants exerting large effects on the phenotypes , but perhaps many variants contributing strongly in aggregate . together these results suggest that substantial individual differences in the relative sizes of and average thickness in different functional regions of the cortex are genetically mediated to a significant degree , that across regions they are influenced by different sets of genetic factors , and that genetic factors influencing cortical regionalization show little overlap with those influencing thickness variability across the cortex . in other words , the patterns suggest that the genetic architecture of the cortex is exceedingly complex , and each phenotype is likely to be mediated by a different large set of ( probably small additive ) genetic effects . an important question , of course , is whether these differences are associated with individual differences in behavioral phenotypes . whatever the factors that give rise to them , genetic or otherwise , of one thing we can be certain : most behavioral phenotypes do not emerge at a particular point in time , but gradually , through the cumulative effects of factors influencing them over the life of the individual . as an example , the left - hand graph in fig . 2 shows the long time course of developing calculation skills in children between ages 5 and 16 . such a protracted increase in calculation skills is consistent with the expected influence of instruction and practice in formal and informal learning environments . perhaps more surprising is that even the basic capacity to inhibit a primed motor response ( shown in the figure 's right panel ) exhibits a similarly protracted course of development . why , for example , do some children from this study at age 14 exhibit calculation skills more typical of the average 9 year old , and some children at 9 exhibit motor inhibitory functions more typical of 15 year olds ? again , there is evidence for some degree of genetic mediation of variability on both of these kinds of skills , but developing calculation skills are clearly modified by the instruction and learning experiences of children , and by other cultural factors , and even performance on a simple motor inhibitory task like this improves with practice . for simplicity 's sake , we might begin with this basic conceptual model ( fig . 3 ) for an emerging behavioral phenotype in a developing child : that it represents some function of the effects exerted by ( 1 ) the hypothetical domain - relevant neural genotype ; and sometimes by ( 2 ) environmental effects on the neural apparatus relevant to the skill or domain ( e.g. , in cases in which damage or toxic exposure to the brain occurs ) ; but , more generally , also by ( 3 ) cumulative experiences throughout development encountering and manipulating material in the domain ; and finally by ( 4 ) interactions between these factors . much ongoing research in human developmental neuroscience is aimed at improving models like this one . however if we are ever to achieve our translational aims that is , to prevent adverse outcomes and lift the trajectories of well - being in at - risk individuals in our society then we must strive to answer some big questions that strike at the heart of the matter . we might ask : to what degree , and via which biological mechanisms , does common genetic variation constrain or bias functions of maturing circuits in the human brain ? and to what degree , and under which circumstances , are there consequences ( of these gene effects ) for experience - dependent developmental processes , or for responses of the developing brain to neuroactive environmental factors ( such as trauma , toxicity , drug exposure , etc . ) ? only 2 decades ago it seemed unlikely to many that more knowledge about the biological development of the brain across the postnatal period would provide further insight into the nature of individual differences in part because the time course of the evolution of these complex behavioral phenotypes seemed much more protracted than what was thought of as a foreshortened period of the brain 's biological development . it was assumed by many that although early events might fix the range of outcomes to some extent , everything changing in the school age years and beyond was necessarily due to learning in a biologically mature brain . this view began to change with the advent of noninvasive brain imaging . on this occasion , when we honor peter huttenlocher , we revisit one of the first papers published in this area 23 years ago ( jernigan et al . , 1991 ) ; not , certainly , because it is a particularly interesting paper by today 's standards , on the contrary , it will serve to illustrate how far we have come in those 2 decades ; but it is one of the earliest imaging papers to cite peter huttenlocher 's remarkable observations . in those days , jernigan was applying early semi - automated morphometry methods developed in her lab to analyze mr imaging data . the regions of interest ( rois ) for examining the cortex were essentially stereotactically - defined quadrants of the cerebrum , and are a long way back from the sophisticated surface - based methods we apply in our work today . by performing tissue segmentation based on dual echo mr images , jernigan et al . measured cortical gray matter volumes in these large rois ; and in a modest sample of 39 young people ranging in age from 7 to 35 , observed highly significant , and linear , decreases in the cortical gray matter volumes ( adjusted for volumes of the supratentorial cranial vault ) in the two dorsal regions , with no real evidence of change in the ventral areas . obviously , we know a lot more about the age functions and anatomical pattern of these apparent changes in gray matter volume now , but at the time they were quite surprising to many people ; and the biological explanation for them was far from clear . since the one largely postnatal phenomenon of which we were all aware was the protracted course of myelination and oligodendrocyte maturation ( yakovlev and lecours , 1967 ) , we , and others , speculated that signal changes associated with the newly generated myelin sheaths of axons coursing beneath the cortex , or even intracortically , might contribute to these apparent volume reductions in cortical gray . in other words , the apparent cortical gray matter reductions might represent the shifting signal characteristics of immature tissue comprised of more lightly myelinated axons ( i.e. , resembling gray matter ) to the signal characteristics of tissue comprised of more fully myelinated axons . but we also had just become aware of huttenlocher 's observations suggesting synaptic pruning in what seemed to be an anatomically heterochronous pattern across the cortex ( huttenlocher and de courten , 1987 ; huttenlocher , 1990 ; huttenlocher and dabholkar , 1997 ) . we therefore speculated that our observations and those reported by huttenlocher might in both cases reflect some later process of maturation in the dorsal cortical regions during adolescence . before we move into the modern era of neuroimaging , we would emphasize one other result from this early paper , rarely mentioned in the subsequent literature , that seemed to suggest that the mechanisms underlying these apparent cortical volume changes probably involved both loss of tissue volume as well as changing signal contrast on mri . that was the observation that csf volume increased in the adjacent subarachnoid space in a very similar anatomical pattern to the cortical volume reductions , as though nearby tissue loss resulted in expansion of this space , ex vacuo . these early observations demonstrated that the neural architecture itself , like the behavioral phenotypes , continued to show dynamic age - related change over a similarly protracted period of development . furthermore , imaging highlighted the considerable individual difference variability in brain morphology present across the entire age range , for example , the apparent differences in regionalization of the cortex , sometimes reflected in sulcal and gyral patterns , that were later the subject of the bakken et al . and however , little was known about whether the differences in brain morphology might be mirrored in any way in the behavioral variability , particularly in typically developing children . since these early observations , much elegant imaging work has been done revealing robust indices of ongoing biological development of the brain that can be monitored noninvasively in children . many of these neurodevelopmental biomarkers and functional imaging phenotypes show very protracted trajectories of change with age and exhibit regional variation . though a number of studies have now examined age - effects on measures of cortical architecture during the postnatal years , and a few have included longitudinal data , details about the pattern of change have been inconsistent , probably in part because of modest sample sizes , different age - ranges examined , and variable imaging protocols and analysis methods . recently , investigators throughout the country collaborated on the large , multisite pediatric imaging , neurocognition , and genetics ( ping ) project in which well over 1000 children were studied . this imaging genetics study of children between the ages of 3 and 20 enrolled participants at 10 sites throughout the us . the design was cross - sectional and involved only a limited number of developmental and cognitive phenotypes , but the dataset is now shared freely with the research community and has been accessed by people all around the world , through a web - based tool called the ping portal ( pingstudy.ucsd.edu ) . users can apply for access by filing a data use request and a data use agreement on the portal . approved users can download the dataset for offline analysis and/or explore the data using advanced interactive statistical and visualization utilities in the data exploration module ( brown et al . , 2012 ; this dataset provides several advantages for defining postnatal changes on imaging phenotypes , including : the large number of participants studied with harmonized and standardized methods ; the wide age range of the participants ( and therefore the long developmental trajectories that can be estimated ) ; and the availability of genome - wide genotyping , which among other things made it possible to compute sensitive measures of genetic ancestry , in the form of 6 genetic ancestry factors ( alexander et al . , 2009 ; jernigan et al . , thus in this dataset it has been possible to estimate age - differences and extrapolated trajectories while holding constant the scanner used , socioeconomic status of the family , and genetic ancestry , variables that could otherwise introduce cohort effects in a cross - sectional study . application of extended freesurfer methods for computational morphometry produced a set of cortical biomarkers that , for example , isolated variability in surface area from variability in apparent cortical thickness . 4 shows plots produced with the data exploration module of the ping portal of age - differences ( and smooth functions of age ) for two global cortical phenotypes , total surface area and mean thickness ( across the entire cortical surface ) . the effect of age on surface area is nonmonotonic ; surface area expands during early childhood years , and expansion decelerates during middle childhood giving way to gradual contraction during adolescence and thereafter . in contrast , the apparent thickness of the cortex exhibits ( mostly linear ) monotonic decrease across the entire range , from 3 to 21 years . with other features of the portal one can estimate these effects at each vertex on the ( tessellated ) surface of the cortex , and create visualizations that code , for each vertex , an age - specific estimate of annualized rate of change . these estimates of rate of change were calculated from the ping data using a generalized additive model ( gam ) implemented with the r - package mgcv . for each vertex a gam estimated a smooth function of age ( like the one shown above for total surface area ) , controlling for scanner , gender , ses ( family income and highest parental education ) , and a set of six genetic ancestry factors . the predicted values obtained from the age model were sampled at regular intervals ( 100 equally spaced intervals across the age range ) . instantaneous rates of change were calculated from the sampled values using the method of finite differences and normalized to produce estimates for annualized rates of change in surface area and thickness at each vertex . rate of change maps for surface area are shown in fig . 5 and confirm that the global pattern is observed across the entire cortical surface , i.e. , early expansion followed by contraction during adolescence . note that the maps of change at ages 4 and 6 are coded differently than those at ages 8 and above to better visualize regional variability in the generally higher rates of expansion at these earlier ages . to further highlight regional differences we computed the smooth age functions from the gam models for 3 larger rois generated by the 12-cluster genetic parcellation of surface area ( shown in fig . 1 above adapted from chen et al . , 2012 ) . 6 are the trajectories for ( covariate - adjusted ) mean expansion coefficients for parcels in the dorsolateral prefrontal cortex ( blue ) , dorsome - dial frontal cortex ( red ) , and occipital cortex ( green ) ; labeled as parcels 2 , 3 , and 12 , respectively . comparing the models visually suggests that early to middle childhood expansion is more rapid in the dorsolateral prefrontal than in the occipital parcel . thinning of the cortex appears to be present across the entire cortex and across the entire age range from 3 to 21 , contrasting with some previous reports of early childhood cortical thickening . again , the maps suggest some degree of regional heterochronicity in apparent thinning of the cortex , as highlighted by comparing the smooth age functions from the gams for the genetically - derived parcels ( note that mean thickness measures in rois derived from the surface area genetic parcels are used for consistency with fig . 6 ) . 8 are the trajectories for covariate - adjusted mean cortical thickness measures for the parcels in the dorsolateral prefrontal cortex ( blue ) , dorsomedial frontal cortex ( red ) , and occipital cortex ( green ) ; labeled as parcels 2 , 3 , and 12 , respectively , in fig . 1 . the age functions vary slightly , suggesting that unlike in the frontal parcels , where the rate of thinning appears to be fairly constant , thinning may decelerate slightly after age 10 in the occipital parcel . in summary , these results from ping illustrate the advantages of a more integrative , data - sharing approach to research that , in this case , yields more definitive estimates of parameters that relate imaging biomarkers to brain development . they highlight the distinct trajectories that relate age to cortical surface area and apparent cortical thickness , and they suggest that both the non - monotonic surface area function and the monotonic function for apparent thickness may exhibit some regional heterochronicity . however , while apparent differences among the regional trajectories are intriguing , the statistical reliability of these regional patterns is more difficult to assess and may require even larger samples , and certainly longitudinal data , for confirmation . these models describe the modal developmental course of surface area expansion ( and contraction ) and of apparent cortical thinning , but one might ask : what do we know about individual differences in these processes of cortical development and how might they relate to behavioral differences ? in recent years a number of observations in children have associated individual differences in the cortical architecture with individual differences in behavioral phenotypes , and a few suggest that trajectories of biological maturation in specific neural systems may themselves map onto emerging phenotypes . ( 2001 ) using early global regional measures , and more recently by investigators using surface - based methods ( tamnes et al . , 2010 ; porter et al . , 2011 ; squeglia et al . , 2013 ) , regressive changes ( volume loss or thinning ) of the cortex has been associated with better performance on memory , cognitive , and executive functions in developing children and adolescents . these results would seem to suggest that more mature cortical phenotypes are mirrored in more mature performance profiles during development , since thinner cortex is rarely associated with performance improvements in other contexts . in other words , they suggest that phase advance of apparent cortical thinning might be associated with precocious functional development . cortical surface area phenotypes have only rarely been correlated with behavioral measures in developing children , but in two reports from the ping study , such associations have been found . ( 2012 ) observed , in the 512 year old children , an association between greater ( relative ) cortical expansion in the anterior cingulate region and better performance on a flanker task , the latter measured as reduced effects of incongruent cues on reaction time . this association was independent of age . since this region continues to expand in surface area over the 512 year age range ( figs . 5 and 6 ) , one explanation is that earlier anterior cingulate surface area expansion is associated with greater functional maturity of circuitry involved in response conflict resolution on this task . an alternative explanation is that individuals with relatively larger anterior cingulate , e.g. , through regionalization effects , regardless of their developmental status , are more adept at such tasks . that the associations seemed to be absent in older individuals in the study is more consistent with the former than the latter interpretation , but these null effects in older participants could also be due to differences in measurement sensitivity or other factors . a second ping study ( newman et al . , 2015 ) revealed a surface area phenotype related to high self - reported levels of generalized anxiety in 287 ping participants aged 720 years . independent of age , gender , and genetic ancestry factors , anxiety was negatively associated with relative cortical surface area of the ventrome - dial prefrontal cortex , as well as with global cortical thickness , and these associations significantly diminished with age . these findings suggest that higher levels of anxiety , even in typically - developing children , may be characterized by both delayed expansion of the ventromedial prefrontal cortex and an altered trajectory of global cortical thinning . though these early clues about neural correlates of individual differences in children are intriguing , unfortunately , many important questions remain about what they mean and why they occur . for example , for most of the associations reported here there is no direct evidence for or against a role of genetic factors in mediating them , though such effects are plausible given the findings in twin studies . even when genetic factors can be implicated , it is not clear whether this represents variable functional constraint within the circuitry of different individuals , or effects of functional biases rather than constraints per se . in either case , it is unclear when such constraining or biasing effects were exerted during the functional development of the relevant neural systems . thus , we still have little understanding of the meaning of the associations , and therefore they provide little guidance toward promising routes for intervention . an important question , with clear translational implications , is whether the associations are mediated to a greater degree by direct effects of genetic variation on developing neural systems , by direct effects of experience through neuroadaptive , or activity - dependent , processes , or by indirect effects of genetic factors , mediated by experience , through gene - experience correlations ? note that to the degree that the answer is the latter , even phenotypes with high heritability are likely to exhibit robust effects of intelligent behavioral ( or other environmental ) interventions . information about possible gene - experience correlations is lacking , at least in part , because of the absence of large - scale , genetically - informed studies of behavioral phenotypes with sufficiently comprehensive assessments of putative experiential and environmental mediators . almost certainly , the most definitive answers to these questions will require longitudinal observations . to answer these and a significant part of the problem stems from the fact that we do not yet know what the imaging biomarkers represent at a cellular level . focusing on the developmental signals we can monitor in maturing human cortex , we could ask : what differences in the architecture of the cortex attend early cortical surface expansion and later contraction ? could these changes reflect widening or narrowing of cortical columns as might attend expanding and contracting dendritic mass ? although investigators have often hypothesized that synaptic pruning might be related to cortical thinning during development , recent studies of human postmortem material suggest a different pattern of nonmonotonic change in synaptic density and dendritic extent , i.e. , protracted increases into childhood with more modest but similarly protracted decreases through adolescence and adulthood ( glantz et al . , 2007 ; petanjek et al . the time courses reported in these studies are more similar to the age differences in surface area than thickness . other possible contributors to surface area are late maturation of neuronal or glial morphological phenotypes , or even ongoing proliferation and differentiation of glial or microglial populations . similar questions remain about the biological alterations that we measure as apparent cortical thinning . although a role for intracortical and subcortical myelination is often suggested , direct evidence is lacking . in summary , a critical priority for advancing human developmental science should be establishing which cellular changes in the neural architecture attend the changes in imaging biomarkers that allow us to monitor biological development of the brain noninvasively . more information about changing levels and regional distribution of gene expression in the human brain across the developmental age range could provide important clues about the underlying biology . however , longitudinal studies that include careful assessment of plausible experiential moderators of neurodevelopmental processes , and , ideally , prospective behavioral intervention studies , are also needed to examine the role of activity - dependent processes . further study is needed to determine which among the genetically mediated associations are due to effects of early patterning ( e.g. , that may be present throughout the lifespan ) , and which to genetic effects on the time course of brain development ; and of the latter , which exert temporary effects on brain and behavioral development and which exert persisting effects . among the demonstrated experiential effects ( i.e. , of practice , training , or emotion ) , it remains to determine how robust these effects are , how persistent they are , and which specific neural circuits they affect . we would argue that in order to answer these questions we need to take the research to a different level , and that a significant impediment to that aim is an unfortunate degree of fragmentation in developmental science generally , and in the science of the developing human mind and brain in particular . human developmental neuroscience , developmental psychology , child psychiatry , pediatric neurology , education research , and other relevant branches of developmental science are siloed , with few unifying or integrative structures . support for the research is provided through many small programs distributed across multiple institutes , agencies , and foundations . and unfortunately the behavioral disciplines are even further isolated from basic developmental neurobiology , in spite of the fact that both have much to gain from closer integration . above we posed the following as examples of big questions at the heart of our understanding of behavioral phenotypes : to what degree , and via which biological mechanisms , does common genetic variation constrain or bias functions of maturing circuits in the human brain ? and to what degree , and under which circumstances , are there consequences ( of these gene effects ) for experience - dependent developmental processes , or for responses of the developing brain to neuroactive environmental factors ( such as trauma , toxicity , drug exposure , etc . ) ? so how do we achieve the integration needed to answer big questions with this degree of complexity ? first , by beginning to accumulate large , informative , high - dimensional datasets for sharing , populated with harmonized assessments of developing children obtained repeatedly over time as they mature . these studies must attempt to capture genetic and epigenetic variability , and include noninvasive imaging and other neurophysiological measures sensitive to differences in the neural architecture and to the status of the brain 's biological development . they should also include measures of the activity within maturing neural systems as well as behavioral and experiential assessments . these assessments must go beyond conventional forms of formal testing , observation , and parent and self - report methods to include innovative methods for sampling behavior and the environment with mobile devices and quantified life apps . it can not be emphasized enough that we must collect richer data detailing the behaviors and experiences of children , and the physical , social , and cultural contexts in which those experiences occur . data repositories populated with observations like these would yield high scientific dividends for decades , as new methods and conceptual approaches were applied to them over time . whenever opportunities exist we should embed translational arms within these large - scale , longitudinal studies . translation can mean implementation of models derived from observational ( or theoretical ) work in applications that would intervene in the observed process , as well as transfer from basic to clinical , or from clinical to basic , research ; and for human developmental neuro - science all of these forms of translation are needed . first , we should consider implementing the prototyping , testing , and modification of novel interventions for mitigating risks in struggling or other high - risk groups , as well as interventions for enhancing learning and growth more generally , in high - information cohorts of longitudinal studies . thus the additional behavioral , developmental , neurobiological , and genetic information already available for these participants could be leveraged in models of the effects of the interventions . the availability of elective interventions , delivered , for example , in after - school or summer programs held in the same venues as ongoing longitudinal studies , or via web - based interactive applications , could also help to consolidate longer - term relationships with families , and would serve as a catalyst for more interdisciplinary work on translation , e.g. , involving teams of developmental scientists , teachers , and clinicians . at a scientific level , these studies would not only accelerate progress toward more effective prevention and mitigation of adverse developmental outcomes , they would address critical remaining questions about the role of experience - dependent processes , and their interactions with other factors , during development . finally , a more integrative science would involve nested basic science programs explicitly designed to bridge gaps between the results from noninvasive studies in children and those from parallel studies of relevant cellular and molecular phenomena in animals , and in human postmortem and in vitro tissue . interdisciplinary teams should propose studies designed collaboratively by scientists studying human development with others studying animal models . the aims of these studies would be to create new bridging behavioral paradigms for cross - species comparisons and to develop animal models of human developmental biomarkers by applying noninvasive methods parallel to those used in children . developmental neurobiologists , working more directly and closely with human developmental neuroscientists , should be encouraged to generate novel hypotheses about the neurobiological sources of many robust developmental signals that we now monitor with noninvasive assessments . more comprehensive data from human pediatric postmortem material is a critical need , and should be given high priority ; but , again , this work would have larger impact if linked directly to noninvasive biomarkers , for example by retrieving relevant in vivo imaging or neurophysiological data or applying postmortem imaging protocols for bridging the gap . modeling of effects within these new cross - level , multimodality datasets will present significant challenges , and computational scientists with both the biostatistical expertise to implement appropriate mixed - models and sufficient domain knowledge to contribute to the interpretation of the effects must be trained and recruited to a new , more integrative developmental science . in order to achieve these goals , our sponsors also have a role to play , by : creating structures for integrating and leveraging resources within nih and through collaboration with nsf , department of education , and private sponsors.offering new mechanisms for funding of multi - pi , multidisciplinary collaborations ( cross - level , cross species , observations + interventions).providing support for infrastructure and informatics specifically designed for developmental data.revising review criteria to incentivize : method harmonization across studies early data release and sharing integrative elements within programs creating structures for integrating and leveraging resources within nih and through collaboration with nsf , department of education , and private sponsors . offering new mechanisms for funding of multi - pi , multidisciplinary collaborations ( cross - level , cross species , observations + interventions ) . providing support for infrastructure and informatics specifically designed for developmental data . revising review criteria to incentivize : method harmonization across studies early data release and sharing integrative elements within programs method harmonization across studies early data release and sharing integrative elements within programs in conclusion , the remarkable progress made in developmental cognitive neuroscience in the last two decades is undeniable and very exciting but progress toward answering our big questions will languish unless we demand and create a more integrative science of the developing mind and brain . certainly the most daunting challenge we face is the enormous complexity of the subject , but this challenge is all the greater when we are hamstrung by an unnecessary degree of fragmentation in our field . no societal campaign is more universally embraced than the pledge to ensure that each child can reach his or her own potential , but there is no consensus about how to accomplish this , and this situation reflects a sobering fact : insufficient evidence exists to reconcile contradictory assumptions and predictions . the answers to our questions are not beyond our reach , but they may be coming more slowly because we are not deploying our scientific assets in the most powerful way to answer the most important questions .

based on the huttenlocher lecture , this article describes the need for a more integrative scientific paradigm for addressing important questions raised by key observations made over 2 decades ago . among these are the early descriptions by huttenlocher of variability in synaptic density in cortex of postmortem brains of children of different ages and the almost simultaneous reports of cortical volume reductions on mr imaging in children and adolescents . in spite of much progress in developmental neurobiology , developmental cognitive neuroscience , and behavioral and imaging genetics , we still do not know how these early observations relate to each other . it is argued that large scale , collaborative research programs are needed to establish the associations between behavioral differences among children and imaging biomarkers , and to link the latter to cellular changes in the developing brain . examples of progress and challenges remaining are illustrated with data from the pediatric imaging , neurocognition , and genetics project ( ping ) .

1. Introduction 2. Knowing things and becoming an individual take time: the time scale of the research matters 3. Can observing the biological development of the human brain help answer these questions? 4. The Pediatric Imaging, Neurocognition, and Genetics (PING) Project 5. Answering the big questions 6. Fragmentation in the science of the developing mind and brain 7. Conclusion

without much risk of controversy , one can safely assume that we all want more people in our society to learn , develop expertise , and innovate ; fewer people to fail to thrive or to become dependent on damaging substances or behaviors ; and for more people to experience a greater sense of well being . questions about the origins of differences among people are of intrinsic interest to many , but from a biomedical perspective , the incentive to study them comes from the well - documented observation that many seriously adverse outcomes evolve disproportionately in the context of specific behavioral phenotypes . this article will describe some lines of evidence relevant to these issues , including work from behavioral genetics , neurogenetics , and cognitive neuroscience , and will pose important questions that we must answer in future research . finally , impediments to progress in the field , and some recommendations for how to reduce them , are outlined . a few behavioral phenotypes have been examined in large , genome wide association studies ( gwas ) , and surprisingly , given reported high estimates of heritability , these studies initially revealed few replicable associations with specific genetic variants , and those variants identified accounted for only very small proportions of the phenotypic variability ( butcher et al . , however , more recent studies have applied novel methods that exploit gwas results to estimate heritability attributable to common genetic variants in aggregate ( so called chip heritability ) , and these suggest that substantial variability in behavioral phenotypes may indeed be explained by common variants , but the genetic architecture of these traits is likely to be highly polygenic , and extremely complex . a series of subsequent analyses of imaging data from this study examined more closely the genetic architecture of individual difference variability in cortical regionalization , that is , individual differences in the relative sizes of different cortical regions . work with rodent models has shown that arealization of the developing cortex is linked to gradients of secreted morphogens and expression of transcription factors in the neocortical proliferative zone during the embryonic period ( mallamaci and stoykova , 2006 ; o'leary et al . using data from 400 middle - aged male veterans from the vietnam era twin study cohort , chen et al . interestingly , this analysis yields a novel method for defining cortical parcels , one that is driven by analysis and simplification of the genetic architecture . after this analysis of cortical surface area variability was published , a similar analysis of genetic correlations for vertex - wise measures of cortical thickness was performed . , 2013 ) also revealed regional clustering of genetic inter - correlations , and the 12-cluster solution resembled the regional pattern for surface area expansion . however , the dominant patterns of genetic inter - correlation for thickness measures ( as expressed in the 24 cluster solutions ) differed substantially from those for surface area , and as previously reported for total surface area and mean cortical thickness ( panizzon et al . so far , however , these early reports suggest that , like heritable behavioral phenotypes , neuroimaging phenotypes may have highly complex genetic architecture , with few variants exerting large effects on the phenotypes , but perhaps many variants contributing strongly in aggregate . together these results suggest that substantial individual differences in the relative sizes of and average thickness in different functional regions of the cortex are genetically mediated to a significant degree , that across regions they are influenced by different sets of genetic factors , and that genetic factors influencing cortical regionalization show little overlap with those influencing thickness variability across the cortex . whatever the factors that give rise to them , genetic or otherwise , of one thing we can be certain : most behavioral phenotypes do not emerge at a particular point in time , but gradually , through the cumulative effects of factors influencing them over the life of the individual . 2 shows the long time course of developing calculation skills in children between ages 5 and 16 . again , there is evidence for some degree of genetic mediation of variability on both of these kinds of skills , but developing calculation skills are clearly modified by the instruction and learning experiences of children , and by other cultural factors , and even performance on a simple motor inhibitory task like this improves with practice . for simplicity 's sake , we might begin with this basic conceptual model ( fig . we might ask : to what degree , and via which biological mechanisms , does common genetic variation constrain or bias functions of maturing circuits in the human brain ? and to what degree , and under which circumstances , are there consequences ( of these gene effects ) for experience - dependent developmental processes , or for responses of the developing brain to neuroactive environmental factors ( such as trauma , toxicity , drug exposure , etc . ) only 2 decades ago it seemed unlikely to many that more knowledge about the biological development of the brain across the postnatal period would provide further insight into the nature of individual differences in part because the time course of the evolution of these complex behavioral phenotypes seemed much more protracted than what was thought of as a foreshortened period of the brain 's biological development . , 1991 ) ; not , certainly , because it is a particularly interesting paper by today 's standards , on the contrary , it will serve to illustrate how far we have come in those 2 decades ; but it is one of the earliest imaging papers to cite peter huttenlocher 's remarkable observations . the regions of interest ( rois ) for examining the cortex were essentially stereotactically - defined quadrants of the cerebrum , and are a long way back from the sophisticated surface - based methods we apply in our work today . measured cortical gray matter volumes in these large rois ; and in a modest sample of 39 young people ranging in age from 7 to 35 , observed highly significant , and linear , decreases in the cortical gray matter volumes ( adjusted for volumes of the supratentorial cranial vault ) in the two dorsal regions , with no real evidence of change in the ventral areas . obviously , we know a lot more about the age functions and anatomical pattern of these apparent changes in gray matter volume now , but at the time they were quite surprising to many people ; and the biological explanation for them was far from clear . since the one largely postnatal phenomenon of which we were all aware was the protracted course of myelination and oligodendrocyte maturation ( yakovlev and lecours , 1967 ) , we , and others , speculated that signal changes associated with the newly generated myelin sheaths of axons coursing beneath the cortex , or even intracortically , might contribute to these apparent volume reductions in cortical gray . we therefore speculated that our observations and those reported by huttenlocher might in both cases reflect some later process of maturation in the dorsal cortical regions during adolescence . before we move into the modern era of neuroimaging , we would emphasize one other result from this early paper , rarely mentioned in the subsequent literature , that seemed to suggest that the mechanisms underlying these apparent cortical volume changes probably involved both loss of tissue volume as well as changing signal contrast on mri . that was the observation that csf volume increased in the adjacent subarachnoid space in a very similar anatomical pattern to the cortical volume reductions , as though nearby tissue loss resulted in expansion of this space , ex vacuo . these early observations demonstrated that the neural architecture itself , like the behavioral phenotypes , continued to show dynamic age - related change over a similarly protracted period of development . since these early observations , much elegant imaging work has been done revealing robust indices of ongoing biological development of the brain that can be monitored noninvasively in children . though a number of studies have now examined age - effects on measures of cortical architecture during the postnatal years , and a few have included longitudinal data , details about the pattern of change have been inconsistent , probably in part because of modest sample sizes , different age - ranges examined , and variable imaging protocols and analysis methods . recently , investigators throughout the country collaborated on the large , multisite pediatric imaging , neurocognition , and genetics ( ping ) project in which well over 1000 children were studied . this imaging genetics study of children between the ages of 3 and 20 enrolled participants at 10 sites throughout the us . approved users can download the dataset for offline analysis and/or explore the data using advanced interactive statistical and visualization utilities in the data exploration module ( brown et al . , 2012 ; this dataset provides several advantages for defining postnatal changes on imaging phenotypes , including : the large number of participants studied with harmonized and standardized methods ; the wide age range of the participants ( and therefore the long developmental trajectories that can be estimated ) ; and the availability of genome - wide genotyping , which among other things made it possible to compute sensitive measures of genetic ancestry , in the form of 6 genetic ancestry factors ( alexander et al . application of extended freesurfer methods for computational morphometry produced a set of cortical biomarkers that , for example , isolated variability in surface area from variability in apparent cortical thickness . the effect of age on surface area is nonmonotonic ; surface area expands during early childhood years , and expansion decelerates during middle childhood giving way to gradual contraction during adolescence and thereafter . with other features of the portal one can estimate these effects at each vertex on the ( tessellated ) surface of the cortex , and create visualizations that code , for each vertex , an age - specific estimate of annualized rate of change . for each vertex a gam estimated a smooth function of age ( like the one shown above for total surface area ) , controlling for scanner , gender , ses ( family income and highest parental education ) , and a set of six genetic ancestry factors . the predicted values obtained from the age model were sampled at regular intervals ( 100 equally spaced intervals across the age range ) . note that the maps of change at ages 4 and 6 are coded differently than those at ages 8 and above to better visualize regional variability in the generally higher rates of expansion at these earlier ages . 6 are the trajectories for ( covariate - adjusted ) mean expansion coefficients for parcels in the dorsolateral prefrontal cortex ( blue ) , dorsome - dial frontal cortex ( red ) , and occipital cortex ( green ) ; labeled as parcels 2 , 3 , and 12 , respectively . comparing the models visually suggests that early to middle childhood expansion is more rapid in the dorsolateral prefrontal than in the occipital parcel . 8 are the trajectories for covariate - adjusted mean cortical thickness measures for the parcels in the dorsolateral prefrontal cortex ( blue ) , dorsomedial frontal cortex ( red ) , and occipital cortex ( green ) ; labeled as parcels 2 , 3 , and 12 , respectively , in fig . in summary , these results from ping illustrate the advantages of a more integrative , data - sharing approach to research that , in this case , yields more definitive estimates of parameters that relate imaging biomarkers to brain development . they highlight the distinct trajectories that relate age to cortical surface area and apparent cortical thickness , and they suggest that both the non - monotonic surface area function and the monotonic function for apparent thickness may exhibit some regional heterochronicity . however , while apparent differences among the regional trajectories are intriguing , the statistical reliability of these regional patterns is more difficult to assess and may require even larger samples , and certainly longitudinal data , for confirmation . these models describe the modal developmental course of surface area expansion ( and contraction ) and of apparent cortical thinning , but one might ask : what do we know about individual differences in these processes of cortical development and how might they relate to behavioral differences ? in recent years a number of observations in children have associated individual differences in the cortical architecture with individual differences in behavioral phenotypes , and a few suggest that trajectories of biological maturation in specific neural systems may themselves map onto emerging phenotypes . ( 2001 ) using early global regional measures , and more recently by investigators using surface - based methods ( tamnes et al . , 2013 ) , regressive changes ( volume loss or thinning ) of the cortex has been associated with better performance on memory , cognitive , and executive functions in developing children and adolescents . ( 2012 ) observed , in the 512 year old children , an association between greater ( relative ) cortical expansion in the anterior cingulate region and better performance on a flanker task , the latter measured as reduced effects of incongruent cues on reaction time . that the associations seemed to be absent in older individuals in the study is more consistent with the former than the latter interpretation , but these null effects in older participants could also be due to differences in measurement sensitivity or other factors . though these early clues about neural correlates of individual differences in children are intriguing , unfortunately , many important questions remain about what they mean and why they occur . even when genetic factors can be implicated , it is not clear whether this represents variable functional constraint within the circuitry of different individuals , or effects of functional biases rather than constraints per se . thus , we still have little understanding of the meaning of the associations , and therefore they provide little guidance toward promising routes for intervention . an important question , with clear translational implications , is whether the associations are mediated to a greater degree by direct effects of genetic variation on developing neural systems , by direct effects of experience through neuroadaptive , or activity - dependent , processes , or by indirect effects of genetic factors , mediated by experience , through gene - experience correlations ? information about possible gene - experience correlations is lacking , at least in part , because of the absence of large - scale , genetically - informed studies of behavioral phenotypes with sufficiently comprehensive assessments of putative experiential and environmental mediators . to answer these and a significant part of the problem stems from the fact that we do not yet know what the imaging biomarkers represent at a cellular level . focusing on the developmental signals we can monitor in maturing human cortex , we could ask : what differences in the architecture of the cortex attend early cortical surface expansion and later contraction ? could these changes reflect widening or narrowing of cortical columns as might attend expanding and contracting dendritic mass ? although investigators have often hypothesized that synaptic pruning might be related to cortical thinning during development , recent studies of human postmortem material suggest a different pattern of nonmonotonic change in synaptic density and dendritic extent , i.e. in summary , a critical priority for advancing human developmental science should be establishing which cellular changes in the neural architecture attend the changes in imaging biomarkers that allow us to monitor biological development of the brain noninvasively . however , longitudinal studies that include careful assessment of plausible experiential moderators of neurodevelopmental processes , and , ideally , prospective behavioral intervention studies , are also needed to examine the role of activity - dependent processes . further study is needed to determine which among the genetically mediated associations are due to effects of early patterning ( e.g. , that may be present throughout the lifespan ) , and which to genetic effects on the time course of brain development ; and of the latter , which exert temporary effects on brain and behavioral development and which exert persisting effects . , of practice , training , or emotion ) , it remains to determine how robust these effects are , how persistent they are , and which specific neural circuits they affect . we would argue that in order to answer these questions we need to take the research to a different level , and that a significant impediment to that aim is an unfortunate degree of fragmentation in developmental science generally , and in the science of the developing human mind and brain in particular . human developmental neuroscience , developmental psychology , child psychiatry , pediatric neurology , education research , and other relevant branches of developmental science are siloed , with few unifying or integrative structures . and unfortunately the behavioral disciplines are even further isolated from basic developmental neurobiology , in spite of the fact that both have much to gain from closer integration . above we posed the following as examples of big questions at the heart of our understanding of behavioral phenotypes : to what degree , and via which biological mechanisms , does common genetic variation constrain or bias functions of maturing circuits in the human brain ? and to what degree , and under which circumstances , are there consequences ( of these gene effects ) for experience - dependent developmental processes , or for responses of the developing brain to neuroactive environmental factors ( such as trauma , toxicity , drug exposure , etc . ) so how do we achieve the integration needed to answer big questions with this degree of complexity ? these studies must attempt to capture genetic and epigenetic variability , and include noninvasive imaging and other neurophysiological measures sensitive to differences in the neural architecture and to the status of the brain 's biological development . they should also include measures of the activity within maturing neural systems as well as behavioral and experiential assessments . these assessments must go beyond conventional forms of formal testing , observation , and parent and self - report methods to include innovative methods for sampling behavior and the environment with mobile devices and quantified life apps . it can not be emphasized enough that we must collect richer data detailing the behaviors and experiences of children , and the physical , social , and cultural contexts in which those experiences occur . whenever opportunities exist we should embed translational arms within these large - scale , longitudinal studies . translation can mean implementation of models derived from observational ( or theoretical ) work in applications that would intervene in the observed process , as well as transfer from basic to clinical , or from clinical to basic , research ; and for human developmental neuro - science all of these forms of translation are needed . first , we should consider implementing the prototyping , testing , and modification of novel interventions for mitigating risks in struggling or other high - risk groups , as well as interventions for enhancing learning and growth more generally , in high - information cohorts of longitudinal studies . thus the additional behavioral , developmental , neurobiological , and genetic information already available for these participants could be leveraged in models of the effects of the interventions . the availability of elective interventions , delivered , for example , in after - school or summer programs held in the same venues as ongoing longitudinal studies , or via web - based interactive applications , could also help to consolidate longer - term relationships with families , and would serve as a catalyst for more interdisciplinary work on translation , e.g. finally , a more integrative science would involve nested basic science programs explicitly designed to bridge gaps between the results from noninvasive studies in children and those from parallel studies of relevant cellular and molecular phenomena in animals , and in human postmortem and in vitro tissue . the aims of these studies would be to create new bridging behavioral paradigms for cross - species comparisons and to develop animal models of human developmental biomarkers by applying noninvasive methods parallel to those used in children . more comprehensive data from human pediatric postmortem material is a critical need , and should be given high priority ; but , again , this work would have larger impact if linked directly to noninvasive biomarkers , for example by retrieving relevant in vivo imaging or neurophysiological data or applying postmortem imaging protocols for bridging the gap . modeling of effects within these new cross - level , multimodality datasets will present significant challenges , and computational scientists with both the biostatistical expertise to implement appropriate mixed - models and sufficient domain knowledge to contribute to the interpretation of the effects must be trained and recruited to a new , more integrative developmental science . revising review criteria to incentivize : method harmonization across studies early data release and sharing integrative elements within programs method harmonization across studies early data release and sharing integrative elements within programs in conclusion , the remarkable progress made in developmental cognitive neuroscience in the last two decades is undeniable and very exciting but progress toward answering our big questions will languish unless we demand and create a more integrative science of the developing mind and brain . no societal campaign is more universally embraced than the pledge to ensure that each child can reach his or her own potential , but there is no consensus about how to accomplish this , and this situation reflects a sobering fact : insufficient evidence exists to reconcile contradictory assumptions and predictions . the answers to our questions are not beyond our reach , but they may be coming more slowly because we are not deploying our scientific assets in the most powerful way to answer the most important questions .

without much risk of controversy , one can safely assume that we all want more people in our society to learn , develop expertise , and innovate ; fewer people to fail to thrive or to become dependent on damaging substances or behaviors ; and for more people to experience a greater sense of well being . questions about the origins of differences among people are of intrinsic interest to many , but from a biomedical perspective , the incentive to study them comes from the well - documented observation that many seriously adverse outcomes evolve disproportionately in the context of specific behavioral phenotypes . one could say that from conception to the onset of these disorders , evolving risk phenotypes are simply forms of individuality , but a deeper understanding of the divergence of high - risk from low - risk phenotypes eludes us . this article will describe some lines of evidence relevant to these issues , including work from behavioral genetics , neurogenetics , and cognitive neuroscience , and will pose important questions that we must answer in future research . a few behavioral phenotypes have been examined in large , genome wide association studies ( gwas ) , and surprisingly , given reported high estimates of heritability , these studies initially revealed few replicable associations with specific genetic variants , and those variants identified accounted for only very small proportions of the phenotypic variability ( butcher et al . , however , more recent studies have applied novel methods that exploit gwas results to estimate heritability attributable to common genetic variants in aggregate ( so called chip heritability ) , and these suggest that substantial variability in behavioral phenotypes may indeed be explained by common variants , but the genetic architecture of these traits is likely to be highly polygenic , and extremely complex . so , in summary , genetic differences probably do play a large role in engendering behavioral individual differences , but it appears that thousands of variants may in some way influence these pheno - types , each with a very small additive effect ( purcell et al . interestingly , cortical surface area and mean thickness of the cortex , both with estimated heritability in the .8.9 range , nevertheless exhibited no evidence for genetic correlation in a study of middle - aged male twins ( panizzon et al . a series of subsequent analyses of imaging data from this study examined more closely the genetic architecture of individual difference variability in cortical regionalization , that is , individual differences in the relative sizes of different cortical regions . work with rodent models has shown that arealization of the developing cortex is linked to gradients of secreted morphogens and expression of transcription factors in the neocortical proliferative zone during the embryonic period ( mallamaci and stoykova , 2006 ; o'leary et al . this large matrix of genetic correlations was then further analyzed using a data - driven , fuzzy clustering method to identify 12 sets ( or clusters ) of vertices with relatively higher genetic inter - correlations and relatively lower genetic correlations with other sets ( or clusters ) . 1 and they represent regions of the cortex where the twin data suggest that individual differences in relative surface area expansion are related to distinct sets of underlying genetic factors . however , the dominant patterns of genetic inter - correlation for thickness measures ( as expressed in the 24 cluster solutions ) differed substantially from those for surface area , and as previously reported for total surface area and mean cortical thickness ( panizzon et al . these results suggest that genetic factors strongly influence both global area and thickness of the cortex as well as regional differences in these biomarkers ; however , the overlap in genetic factors contributing to the high heritability of area and thickness of the cortex is negligible . attempts to identify specific genetic variants that may contribute to the heritability of imaging phenotypes have been slow to appear because of the paucity of large studies with comparable brain imaging data . recently , the important enhancing neuro imaging genetics through meta - analysis ( enigma ) project has begun to aggregate imaging genomics data across many studies worldwide to facilitate the search for specific variants related to imaging phenotypes . so far , however , these early reports suggest that , like heritable behavioral phenotypes , neuroimaging phenotypes may have highly complex genetic architecture , with few variants exerting large effects on the phenotypes , but perhaps many variants contributing strongly in aggregate . together these results suggest that substantial individual differences in the relative sizes of and average thickness in different functional regions of the cortex are genetically mediated to a significant degree , that across regions they are influenced by different sets of genetic factors , and that genetic factors influencing cortical regionalization show little overlap with those influencing thickness variability across the cortex . in other words , the patterns suggest that the genetic architecture of the cortex is exceedingly complex , and each phenotype is likely to be mediated by a different large set of ( probably small additive ) genetic effects . whatever the factors that give rise to them , genetic or otherwise , of one thing we can be certain : most behavioral phenotypes do not emerge at a particular point in time , but gradually , through the cumulative effects of factors influencing them over the life of the individual . again , there is evidence for some degree of genetic mediation of variability on both of these kinds of skills , but developing calculation skills are clearly modified by the instruction and learning experiences of children , and by other cultural factors , and even performance on a simple motor inhibitory task like this improves with practice . 3 ) for an emerging behavioral phenotype in a developing child : that it represents some function of the effects exerted by ( 1 ) the hypothetical domain - relevant neural genotype ; and sometimes by ( 2 ) environmental effects on the neural apparatus relevant to the skill or domain ( e.g. , in cases in which damage or toxic exposure to the brain occurs ) ; but , more generally , also by ( 3 ) cumulative experiences throughout development encountering and manipulating material in the domain ; and finally by ( 4 ) interactions between these factors . however if we are ever to achieve our translational aims that is , to prevent adverse outcomes and lift the trajectories of well - being in at - risk individuals in our society then we must strive to answer some big questions that strike at the heart of the matter . and to what degree , and under which circumstances , are there consequences ( of these gene effects ) for experience - dependent developmental processes , or for responses of the developing brain to neuroactive environmental factors ( such as trauma , toxicity , drug exposure , etc . ) only 2 decades ago it seemed unlikely to many that more knowledge about the biological development of the brain across the postnatal period would provide further insight into the nature of individual differences in part because the time course of the evolution of these complex behavioral phenotypes seemed much more protracted than what was thought of as a foreshortened period of the brain 's biological development . , 1991 ) ; not , certainly , because it is a particularly interesting paper by today 's standards , on the contrary , it will serve to illustrate how far we have come in those 2 decades ; but it is one of the earliest imaging papers to cite peter huttenlocher 's remarkable observations . the regions of interest ( rois ) for examining the cortex were essentially stereotactically - defined quadrants of the cerebrum , and are a long way back from the sophisticated surface - based methods we apply in our work today . measured cortical gray matter volumes in these large rois ; and in a modest sample of 39 young people ranging in age from 7 to 35 , observed highly significant , and linear , decreases in the cortical gray matter volumes ( adjusted for volumes of the supratentorial cranial vault ) in the two dorsal regions , with no real evidence of change in the ventral areas . obviously , we know a lot more about the age functions and anatomical pattern of these apparent changes in gray matter volume now , but at the time they were quite surprising to many people ; and the biological explanation for them was far from clear . since the one largely postnatal phenomenon of which we were all aware was the protracted course of myelination and oligodendrocyte maturation ( yakovlev and lecours , 1967 ) , we , and others , speculated that signal changes associated with the newly generated myelin sheaths of axons coursing beneath the cortex , or even intracortically , might contribute to these apparent volume reductions in cortical gray . but we also had just become aware of huttenlocher 's observations suggesting synaptic pruning in what seemed to be an anatomically heterochronous pattern across the cortex ( huttenlocher and de courten , 1987 ; huttenlocher , 1990 ; huttenlocher and dabholkar , 1997 ) . before we move into the modern era of neuroimaging , we would emphasize one other result from this early paper , rarely mentioned in the subsequent literature , that seemed to suggest that the mechanisms underlying these apparent cortical volume changes probably involved both loss of tissue volume as well as changing signal contrast on mri . that was the observation that csf volume increased in the adjacent subarachnoid space in a very similar anatomical pattern to the cortical volume reductions , as though nearby tissue loss resulted in expansion of this space , ex vacuo . furthermore , imaging highlighted the considerable individual difference variability in brain morphology present across the entire age range , for example , the apparent differences in regionalization of the cortex , sometimes reflected in sulcal and gyral patterns , that were later the subject of the bakken et al . and however , little was known about whether the differences in brain morphology might be mirrored in any way in the behavioral variability , particularly in typically developing children . though a number of studies have now examined age - effects on measures of cortical architecture during the postnatal years , and a few have included longitudinal data , details about the pattern of change have been inconsistent , probably in part because of modest sample sizes , different age - ranges examined , and variable imaging protocols and analysis methods . the design was cross - sectional and involved only a limited number of developmental and cognitive phenotypes , but the dataset is now shared freely with the research community and has been accessed by people all around the world , through a web - based tool called the ping portal ( pingstudy.ucsd.edu ) . , 2012 ; this dataset provides several advantages for defining postnatal changes on imaging phenotypes , including : the large number of participants studied with harmonized and standardized methods ; the wide age range of the participants ( and therefore the long developmental trajectories that can be estimated ) ; and the availability of genome - wide genotyping , which among other things made it possible to compute sensitive measures of genetic ancestry , in the form of 6 genetic ancestry factors ( alexander et al . , thus in this dataset it has been possible to estimate age - differences and extrapolated trajectories while holding constant the scanner used , socioeconomic status of the family , and genetic ancestry , variables that could otherwise introduce cohort effects in a cross - sectional study . 4 shows plots produced with the data exploration module of the ping portal of age - differences ( and smooth functions of age ) for two global cortical phenotypes , total surface area and mean thickness ( across the entire cortical surface ) . with other features of the portal one can estimate these effects at each vertex on the ( tessellated ) surface of the cortex , and create visualizations that code , for each vertex , an age - specific estimate of annualized rate of change . 6 are the trajectories for ( covariate - adjusted ) mean expansion coefficients for parcels in the dorsolateral prefrontal cortex ( blue ) , dorsome - dial frontal cortex ( red ) , and occipital cortex ( green ) ; labeled as parcels 2 , 3 , and 12 , respectively . comparing the models visually suggests that early to middle childhood expansion is more rapid in the dorsolateral prefrontal than in the occipital parcel . thinning of the cortex appears to be present across the entire cortex and across the entire age range from 3 to 21 , contrasting with some previous reports of early childhood cortical thickening . again , the maps suggest some degree of regional heterochronicity in apparent thinning of the cortex , as highlighted by comparing the smooth age functions from the gams for the genetically - derived parcels ( note that mean thickness measures in rois derived from the surface area genetic parcels are used for consistency with fig . 8 are the trajectories for covariate - adjusted mean cortical thickness measures for the parcels in the dorsolateral prefrontal cortex ( blue ) , dorsomedial frontal cortex ( red ) , and occipital cortex ( green ) ; labeled as parcels 2 , 3 , and 12 , respectively , in fig . in summary , these results from ping illustrate the advantages of a more integrative , data - sharing approach to research that , in this case , yields more definitive estimates of parameters that relate imaging biomarkers to brain development . they highlight the distinct trajectories that relate age to cortical surface area and apparent cortical thickness , and they suggest that both the non - monotonic surface area function and the monotonic function for apparent thickness may exhibit some regional heterochronicity . these models describe the modal developmental course of surface area expansion ( and contraction ) and of apparent cortical thinning , but one might ask : what do we know about individual differences in these processes of cortical development and how might they relate to behavioral differences ? in recent years a number of observations in children have associated individual differences in the cortical architecture with individual differences in behavioral phenotypes , and a few suggest that trajectories of biological maturation in specific neural systems may themselves map onto emerging phenotypes . , 2013 ) , regressive changes ( volume loss or thinning ) of the cortex has been associated with better performance on memory , cognitive , and executive functions in developing children and adolescents . ( 2012 ) observed , in the 512 year old children , an association between greater ( relative ) cortical expansion in the anterior cingulate region and better performance on a flanker task , the latter measured as reduced effects of incongruent cues on reaction time . that the associations seemed to be absent in older individuals in the study is more consistent with the former than the latter interpretation , but these null effects in older participants could also be due to differences in measurement sensitivity or other factors . independent of age , gender , and genetic ancestry factors , anxiety was negatively associated with relative cortical surface area of the ventrome - dial prefrontal cortex , as well as with global cortical thickness , and these associations significantly diminished with age . these findings suggest that higher levels of anxiety , even in typically - developing children , may be characterized by both delayed expansion of the ventromedial prefrontal cortex and an altered trajectory of global cortical thinning . for example , for most of the associations reported here there is no direct evidence for or against a role of genetic factors in mediating them , though such effects are plausible given the findings in twin studies . thus , we still have little understanding of the meaning of the associations , and therefore they provide little guidance toward promising routes for intervention . an important question , with clear translational implications , is whether the associations are mediated to a greater degree by direct effects of genetic variation on developing neural systems , by direct effects of experience through neuroadaptive , or activity - dependent , processes , or by indirect effects of genetic factors , mediated by experience , through gene - experience correlations ? information about possible gene - experience correlations is lacking , at least in part , because of the absence of large - scale , genetically - informed studies of behavioral phenotypes with sufficiently comprehensive assessments of putative experiential and environmental mediators . focusing on the developmental signals we can monitor in maturing human cortex , we could ask : what differences in the architecture of the cortex attend early cortical surface expansion and later contraction ? in summary , a critical priority for advancing human developmental science should be establishing which cellular changes in the neural architecture attend the changes in imaging biomarkers that allow us to monitor biological development of the brain noninvasively . , that may be present throughout the lifespan ) , and which to genetic effects on the time course of brain development ; and of the latter , which exert temporary effects on brain and behavioral development and which exert persisting effects . we would argue that in order to answer these questions we need to take the research to a different level , and that a significant impediment to that aim is an unfortunate degree of fragmentation in developmental science generally , and in the science of the developing human mind and brain in particular . above we posed the following as examples of big questions at the heart of our understanding of behavioral phenotypes : to what degree , and via which biological mechanisms , does common genetic variation constrain or bias functions of maturing circuits in the human brain ? and to what degree , and under which circumstances , are there consequences ( of these gene effects ) for experience - dependent developmental processes , or for responses of the developing brain to neuroactive environmental factors ( such as trauma , toxicity , drug exposure , etc . ) these studies must attempt to capture genetic and epigenetic variability , and include noninvasive imaging and other neurophysiological measures sensitive to differences in the neural architecture and to the status of the brain 's biological development . translation can mean implementation of models derived from observational ( or theoretical ) work in applications that would intervene in the observed process , as well as transfer from basic to clinical , or from clinical to basic , research ; and for human developmental neuro - science all of these forms of translation are needed . first , we should consider implementing the prototyping , testing , and modification of novel interventions for mitigating risks in struggling or other high - risk groups , as well as interventions for enhancing learning and growth more generally , in high - information cohorts of longitudinal studies . the availability of elective interventions , delivered , for example , in after - school or summer programs held in the same venues as ongoing longitudinal studies , or via web - based interactive applications , could also help to consolidate longer - term relationships with families , and would serve as a catalyst for more interdisciplinary work on translation , e.g. finally , a more integrative science would involve nested basic science programs explicitly designed to bridge gaps between the results from noninvasive studies in children and those from parallel studies of relevant cellular and molecular phenomena in animals , and in human postmortem and in vitro tissue . the aims of these studies would be to create new bridging behavioral paradigms for cross - species comparisons and to develop animal models of human developmental biomarkers by applying noninvasive methods parallel to those used in children . developmental neurobiologists , working more directly and closely with human developmental neuroscientists , should be encouraged to generate novel hypotheses about the neurobiological sources of many robust developmental signals that we now monitor with noninvasive assessments . modeling of effects within these new cross - level , multimodality datasets will present significant challenges , and computational scientists with both the biostatistical expertise to implement appropriate mixed - models and sufficient domain knowledge to contribute to the interpretation of the effects must be trained and recruited to a new , more integrative developmental science . in order to achieve these goals , our sponsors also have a role to play , by : creating structures for integrating and leveraging resources within nih and through collaboration with nsf , department of education , and private sponsors.offering new mechanisms for funding of multi - pi , multidisciplinary collaborations ( cross - level , cross species , observations + interventions).providing support for infrastructure and informatics specifically designed for developmental data.revising review criteria to incentivize : method harmonization across studies early data release and sharing integrative elements within programs creating structures for integrating and leveraging resources within nih and through collaboration with nsf , department of education , and private sponsors . revising review criteria to incentivize : method harmonization across studies early data release and sharing integrative elements within programs method harmonization across studies early data release and sharing integrative elements within programs in conclusion , the remarkable progress made in developmental cognitive neuroscience in the last two decades is undeniable and very exciting but progress toward answering our big questions will languish unless we demand and create a more integrative science of the developing mind and brain . certainly the most daunting challenge we face is the enormous complexity of the subject , but this challenge is all the greater when we are hamstrung by an unnecessary degree of fragmentation in our field .

following the discovery of the lysosome by the nobel prize winner christian de duve in 1955 ( de duve et al . 1955 ) , research into the biochemistry , molecular biology and pathology of the lysosomal system unravelled many of its functions . nowadays , genetic lysosomal storage disorders ( lsds ) represent a group of about 50 disorders ( scriver et al . all of them are characterized by intralysosomal accumulation of compound(s ) , e.g. , enzyme substrates or products , mishandled bioconjugates , or segregated cytoplasmic components , in a variety of cell types and tissues . most lsds result from a deficient enzyme activity caused by a mutation in the gene encoding either the relevant enzyme , protecting protein , protein activators , or the endoplasmic reticulum however , some are caused by dysfunction of the lysosomal system itself due to mutations in genes encoding lysosomal membrane components or components closely associated with the lysosomal system ( saftig 2005 ; jalanko and braulke 2009 ) . the number of lysosomal enzymes and non - catalytic proteins reported in the literature has steadily increased and , therefore , more defects in the lysosomal system are expected to be discovered . the progress in the research into limp-2 deficiency serves as a recent example ( balreira et al . overall frequencies of occurrence of lsds have been reported for several countries ( meikle et al . 2004 ) , while other reports focused on lsd groups ( e.g. , mucopolysaccharidoses , ( mps ) : nelson et al . 2005 ; malm et al . 2008 ; lipidoses : ozkara and topcu 2004 ; neuronal ceroid lipofuscinoses , ncl : claussen et al . 1992 ) , or on particular lsds ( e.g. , mps i : moore et al . the frequency of lsds as a group varies among populations from 7.6 to 25 per 100,000 ( meikle et al . the aim of this study was to calculate the birth prevalence and carrier frequencies of lsds in the czech republic population , and to compare our results with reported epidemiologic data from other populations . we analyzed data from individuals who had been diagnosed with a lsd at the institute of inherited metabolic disorders in prague between 1975 and 2008 . our institute is the only laboratory providing diagnostic testing for lsds , including prenatal testing , in the czech republic . the patients had been referred to , or their blood samples had been sent to , the institute by departments of paediatrics , neurology , haematology , cardiology , nephrology , dermatology , gastroenterology , ophthalmology , pathology , and clinical genetics from hospitals all over the country . the initial diagnosis of most lsds was based on demonstration of accumulated substrates ( e.g. , sphingolipids , oligosaccharides , or glycosaminoglycans ) in tissues and/or body fluids using chromatographic or electrophoretic methods , or on histological proof of storage . the definite diagnosis was made by demonstrating the deficiency of the relevant enzyme and/or presence of pathogenic mutation , and/or by detection of undegraded substrate by loading tests in cell cultures . mucolipidosis ii was confirmed by detecting elevated activities of multiple lysosomal enzymes in serum and by molecular analysis . patients with unspecified types of mps or ncl , who had died before the availability of confirmatory tests , were also included in the birth prevalence calculation . their diagnosis was based on biochemical analyses of storage compound , evaluation of clinical data , and histochemical and/or ultrastructural examinations of samples obtained at biopsy or autopsy . for the calculation of the prevalence the prevalence was calculated as the total number of patients diagnosed with a particular lsd , divided by the total number of live births in the same period , as was the birth period of the diagnosed cases ( i.e. , the birth period is the interval between the year of the birth of the oldest patient and the year of the birth of the youngest patient ) the total number of live births in particular years was calculated using birth rates taken from the czech statistical office web page . to avoid underestimation , some patients were not included into the prevalence calculation ( e.g. , if there was only one patient diagnosed with the disease , the prevalence was calculated using the number of live births ( i.e. , 4,261,897 ) in the examined period ( i.e. , between 1975 and 2008 ) , as used previously by pinto et al . familial cases and 11 affected fetuses [ fabry disease ( 1 ) , niemann - pick disease type c ( 1 ) , mps i ( 3 ) , mps iii a ( 3 ) and mps iv a ( 3 ) ] were also included in the calculation of birth prevalence . for some specific lsds , the patient group consisted of subgroups with distinct clinical phenotypes ( e.g. , mld , gaucher disease ) . using the method mentioned above , we calculated the prevalence of the subgroups separately , and the overall prevalence of the specific lsd was estimated as a sum of the prevalences in the subgroups ( poorthuis et al . calculation of the frequency of fabry disease was done using two approaches , i.e. , excluding and including female heterozygotes . exclusion of heterozygotes ( n = 78 ) enabled us to compare the prevalence to other reported male - specific prevalence data ( meikle et al . 1999 ; poorthuis et al . 1999 ; ozkara and topcu 2004 ; pinto et al . fabry heterozygotes , who can be as severely affected as male patients , are nowadays classified as patients instead of carriers of the defective gene ( dobrovolny et al . 2005 ; wang et al . 2007 ; gibas et al . confidence intervals ( 95% ) for the birth prevalences were estimated from the poisson distribution using sisa ( simple interactive statistical analysis ) software ( uitenbroek 1997 ) . carrier frequency was calculated using the hardy weinberg equation ( strachan and read 2004 ) . in the past three decades , a total of 478 patients have been diagnosed with one of 34 specific types of lsds in the czech republic ( fig . 1 ) . the number of diagnosed patients gradually increased during the study period , from less than 10 cases per year in the 1970s to 2030 new cases diagnosed annually in recent years ( fig . 2 ) . more than half of all lsd patients had a lipidosis , about one - quarter a mucopolysaccharidosis , and the remaining quarter suffered from ncl , gsd ii , mucolipidoses , or glycoproteinoses ( fig . 1 ) . the combined birth prevalence for all lsds is 12.25 ( 95% ci 6.2524.1 ) per 100,000 live births . data on patients with lipidoses , mucopolysaccharidoses , and other lsds detected during the study period , including carrier frequencies in the czech population , are summarized in tables 1 , 2 and 3 , respectively . detailed data on the birth prevalence of specific disorders and their subtypes can be found in tables 4 , 5 and 6 . mps mucopolysaccharidosis , ncl neuronal ceroidlipofuscinosis , gsd ii glycogenosis type ii , ml mucolipidosis , gp glycoproteinosis , npa / b / c niemann - pick a / b / c , mld metachromatic leukodystrophy , cesd cholesterol ester storage disease , msd multiple sulphatase deficiency , gm1 gm1 gangliosidosis , gm2 gm2 gangliosidosis ( tay - sachs and sandhoff diseases ) , -man -mannosidosis , -man -mannosidosis , issd infantile sialic acid storage diseasefig . 2number of live births ( ) and number of lsd diagnoses ( ) in the czech population from 1975 to 2008 . in 1985 , electrophoretic analysis of glycosaminoglycans ( elfo gag ) and thin layer chromatography of oligosaccharides ( tlc ols ) in urine were introduced . from 1990 laboratory diagnostic methods for 45 lsds are available ( enzyme and dna analyses , loading assays)table 1lipidoses : comparison of data in different populationsdiseaseczech republicother countriesno . of patients 10nlportugalaustraliaturkeyprevalence per 100,000 live birthsgaucher ( all types)491.130.611.956.711.161.351.750.45niemann - pick a / b230.330.160.613.650.530.60.4niemann - pick c540.910.671.216.020.352.20.47fabry male490.52 ( 1.0)0.380.690.010.210.120.860.015fabry female780.770.600.98krabbe150.400.210.684.01.351.210.711mld ( all types)250.690.291.385.261.421.851.091.43cesd , wolman180.270.150.453.310.19gm1 gangliosidosis130.260.140.463.250.410.620.260.54gm2 tay - sachs100.300.140.553.450.413.130.50.23gm2 sandhoff30.190.040.552.740.341.490.260.95lipidoses all types259/3375.0/5.772.79 ( 3.39)8.53 ( 9.51)6.212.66.64.6nl netherlands , mld metachromatic leukodystrophy , cesd cholesteryl ester storage diseasetotal number of patients diagnosed between 1975 and 2008data for prevalence calculations are in table 4carrier per 1 live birthmale live births onlyfabry females not includedfabry females includedtable 2mucopolysaccharidoses : comparison of data in different populationsdiseaseczech republicother countriesno . of patients 10nlportugalaustraliagermanyprevalence per 100,000 live birthsmps i200.720.441.115.381.191.331.140.69mps ii220.43 ( 0.83)0.260.650.00850.671.090.740.64mps iii a180.470.270.754.311.1600.88mps iii b10.020.00.130.970.420.720.47mps iii c50.420.140.994.120.210.120.07mps iii d000.10.10mps iii ( all types)240.910.411.871.890.841.421.57mps iv a140.710.381.225.350.220.60.59mps iv b10.020.00.130.970.1400mps iv ( a + b)150.730.381.350.38mps vi20.050.010.181.400.150.420.430.23mps vii10.020.00.130.970.2400.05mps unspecified320.600.390.87msd30.260.050.773.240.050.480.07mps all types1193.721.946.934.54.84.443.53nl netherlands , mps mucopolysaccharidosis , msd multiple sulphatase deficiencytotal number of patients diagnosed between 1975 and 2008data for prevalence calculations are in table 5carrier per 1 live birthmale live births onlyunspecified cases of mps were diagnosed on the basis of analysis of glycosaminoglycan excreted in urine combined with the evaluation of clinical data . no material was available for enzyme and dna analysistable 3glycoproteinoses , mucolipidoses , glycogenosis type ii and neuronal ceroid lipofuscinoses : comparison of data in different populationsdiseaseczech republicother countriesno . of patients 19752008prevalence per 100,000poisson 95% confidence intervalcarrier frequency ii / iii30.220.050.652.980.240.810.31mucolipidosis iv10.020.00.130.97glycogenosis type ii ( all)120.370.130.813.8620.170.69ncl 120.190.020.702.790.17ncl 2280.360.240.523.790.07ncl 320.270.030.963.270.48ncl 410.020.00.130.97ncl 520.210.030.772.92ncl 610.020.00.130.971.43ncl 7180.850.491.365.81ncl unspecified200.370.220.58ncl ( all types)742.291.035.152.14nl netherlands , issd infantile sialic acid storage disease , ncl neuronal ceroid lipofuscinosistotal number of patients diagnosed between 1975 and 2008data for prevalence calculations are in table 6carrier per 1 live birthunspecified ncl were diagnosed on the basis of histochemical and ultrastructural examination of biopsy samples combined with the evaluation of clinical data . no material was available for enzyme and dna analysistable 4lipidoses : data for calculation of birth prevalencediseaseno . of patients 19752008years of birthno . of live birthsno . of patientsprevalence ( 1 per numbers of live births)prevalence per 100,000carrier frequencygaucher type i , early12 ( 10)197419963 090 90212257 5750.39254gaucher type i , late29 ( 28)194519805 956 02824248 1680.40249gaucher type ii and iii8 ( 8)198019982 352 4928294 0620.34271gaucher ( all types)494488 3971.13149niemann - pick a10 ( 8)196320025 480 88510548 0890.18370niemann - pick b + atyp13 ( 11)194819967 362 14611669 2860.15409niemann - pick a / b2321301 3270.33274niemann - pick c54 ( 45)196520035 271 31048109 8190.91166fabry49 ( 24)194520028 518 60144193 6050.5296 803male4 382 0684499 5921.00fabry female78 ( 24)194520058 812 16168129 5910.77krabbe15 ( 15)197720023 249 15013249 9350.40250mld infantile13 ( 11)197019994 195 56413322 7360.31284 juvenile5 ( 4)197020044 661 3245932 2650.11483 adult7 ( 7)196819802 188 0046364 6670.27302mld ( all types)2524144 6470.69190cesd , wolman18 ( 15)196320025 480 88515365 3920.27302gm1 gangliosidosis13 ( 13)197020034 563 66012380 3050.26308gm2 tay - sachs10 ( 10)197119923 362 88910336 2890.30290gm2 sandhoff3 ( 3)199220071 594 9493531 6500.19365lipidoses all types259/337234/30220 0005.0/5.7771mld metachromatic leukodystrophy , cesd cholesteryl ester storage diseasetotal number of patients diagnosed between 1975 and 2008number of families with particular disorder in parenthesesnumber of patients taken for the calculation of birth prevalence . to avoid underestimation , some patients were not included into the prevalence calculation ( e.g. , patients born before 1945 see patients and methodsexpressed as 1 carrier per number of shown live birthsone stillborn casenp type b in 6 slowly progressive visceral cases and atypical protracted neurovisceral phenotype in 7 cases [ pavl - pereira h , et al . j inherit metab dis 2005;28:203227]including one case of np type c2 [ elleder m. et al . virchows arch 2001;439:206211]classical phenotype in 43 male patients and cardiac variant in 6 male patients ( two families ) [ elleder m , et al . cas lek cesk 1990;129:368372]infantile form in 12 patients , late infantile in 2 patients , and adult form in one patient [ kostalova e , et al . ceska slov neurol neurochir 2006;69/102:200210]one infantile patient ( wolman disease ) and 17 juvenile and adult cases [ elleder m , et al . cas lek cesk 1999;138:719724]including two adult patientsfabry females not includedfabry females includedmale live birthscarrier frequency of lipidoses except for fabry diseasetable 5mucopolysaccharidoses : data for calculation of birth prevalencediseaseno . of patients 19752008years of birthno . of live birthsno . of patientsprevalence ( 1 per number of live births)prevalence per 100,000carrier frequencymps i20 ( 17)198420082,772,55920138,6280.72186mps ii22 ( 19)196920054,906,70021233,6520.43116,826male2,520,37321120,0180.83mps iii a18 ( 14)197720063,648,54117214,6200.47232mps iii b1197520084,261,89714,261,8970.021,032mps iii c5 ( 4)198419921,179,7455235,9490.42243mps iii ( all types)2423109,5020.91165mps iv a14 ( 9)198720031,826,47313140,4980.71187mps iv b1197520084,261,89714,261,8970.021,032mps iv ( a + b)1514136,9860.73185mps vi2 ( 2)196819954,115,05722,057,5290.05717mps vii1197520084,261,89714,261,8970.021,032mps i vii848134,9092.8693mps - unspecified32 ( 27)196019894,533,49627167,9070.60205msd3 ( 1)196219691,144,4673381,4890.26309mps all types11911126,8653.7282mps mucopolysaccharidosis , msd multiple sulphatase deficiencytotal number of patients diagnosed between 1975 and 2008number of families with particular disorder in parenthesesnumber of patients taken for the calculation of birth prevalence . to avoid underestimation , some patients were not included into the prevalence calculation ( e.g. , patients born before 1945 were excluded ) . for prevalence definition , see patients and methodsexpressed as 1 carrier per number of shown live birthshurler syndrom in 16 patients , hurler / scheie in 2 patients and scheie syndrom in 2 patientsmale live birthscarrier frequency of mps excluding mps iitable 6glycoproteinoses , mucolipidoses , glycogenosis type ii and neuronal ceroidlipofuscinoses : data for calculation of birth prevalencediseaseno . of patients 19752008years of birthno . ( 1 per numbers of live births)prevalence per 100,000carrier frequency-mannosidosis4 ( 3)198519921,042,8044260,7010.38255-mannosidosis2 ( 1)195019561,223,3862611,6930.16391issd1197520084,261,89714,261,8970.021,032mucolipidosis i3 ( 2)196919994,338,72931,446,2430.07601mucolipidosis ii / iii3 ( 3)199420071,352,2193450,7400.22336mucolipidosis iv1197520084,261,89714,261,8970.021,032gp + ml ( all types)1414112,9950.87170glycogenosis type ii infantile6 ( 5)197919992,614,0756435,6790.23330glycogenosis type ii juvenile6 ( 5)197220024,167,9306694,6550.14417glycogenosis type ii ( all)1212267,7500.37259ncl 12 ( 2)197019751,033,4472516,7240.19359ncl 228 ( 25)194920037,803,06828278,6810.36264ncl 32 ( 2)19791983749,5202374,7600.27306ncl 41197520084,261,89714,261,8970.021,032ncl 52 ( 1)19831989935,5532467,7770.21342ncl 61197520084,261,89714,261,8970.021,032ncl 718 ( 16)198520022,002,02517117,7660.85172ncl - unspecified20 ( 17)196519994,903,21418272,4010.37261ncl ( all types)747143,5512.29104issd infantile sialic acid storage disease , gp glycoproteinosis , ml mucolipidosis , ncl neuronal ceroid lipofuscinosistotal number of patients diagnosed between 1975 and 2008number of families with particular disorder in parenthesesnumber of patients taken for the calculation of birth prevalence . to avoid underestimation , some patients were not included into the prevalence calculation ( e.g. patients born before 1945 were excluded ) . for prevalence definition see patients and methodsexpressed as 1 carrier per number of shown live birthsone infantile patient and two juvenile siblings with cherry - red spot myoclonus syndrome [ ledvinova j , et al . j inherit metab dis 1994;17:118119]atypical case of ml iv with ocular restricted phenotype [ dobrovolny r , et al . am j ophthalmol 2007 ; 143:663671 ] relative rate of lysosomal storage disorders in the czech republic . mps mucopolysaccharidosis , ncl neuronal ceroidlipofuscinosis , gsd ii glycogenosis type ii , ml mucolipidosis , gp glycoproteinosis , npa / b / c niemann - pick a / b / c , mld metachromatic leukodystrophy , cesd cholesterol ester storage disease , msd multiple sulphatase deficiency , gm1 gm1 gangliosidosis , gm2 gm2 gangliosidosis ( tay - sachs and sandhoff diseases ) , -man -mannosidosis , -man -mannosidosis , issd infantile sialic acid storage disease number of live births ( ) and number of lsd diagnoses ( ) in the czech population from 1975 to 2008 . in 1985 , electrophoretic analysis of glycosaminoglycans ( elfo gag ) and thin layer chromatography of oligosaccharides ( tlc ols ) in urine were introduced . from 1990 nowadays , laboratory diagnostic methods for 45 lsds are available ( enzyme and dna analyses , loading assays ) lipidoses : comparison of data in different populations nl netherlands , mld metachromatic leukodystrophy , cesd cholesteryl ester storage disease total number of patients diagnosed between 1975 and 2008 data for prevalence calculations are in table 4 carrier per 1 live birth male live births only fabry females not included fabry females included mucopolysaccharidoses : comparison of data in different populations nl netherlands , mps mucopolysaccharidosis , msd multiple sulphatase deficiency total number of patients diagnosed between 1975 and 2008 data for prevalence calculations are in table 5 carrier per 1 live birth male live births only unspecified cases of mps were diagnosed on the basis of analysis of glycosaminoglycan excreted in urine combined with the evaluation of clinical data . no material was available for enzyme and dna analysis glycoproteinoses , mucolipidoses , glycogenosis type ii and neuronal ceroid lipofuscinoses : comparison of data in different populations nl netherlands , issd infantile sialic acid storage disease , ncl neuronal ceroid lipofuscinosis total number of patients diagnosed between 1975 and 2008 data for prevalence calculations are in table 6 carrier per 1 live birth unspecified ncl were diagnosed on the basis of histochemical and ultrastructural examination of biopsy samples combined with the evaluation of clinical data . no material was available for enzyme and dna analysis lipidoses : data for calculation of birth prevalence mld metachromatic leukodystrophy , cesd cholesteryl ester storage disease total number of patients diagnosed between 1975 and 2008 number of families with particular disorder in parentheses number of patients taken for the calculation of birth prevalence . to avoid underestimation , some patients were not included into the prevalence calculation ( e.g. , patients born before 1945 were excluded ) . for prevalence definition , see patients and methods expressed as 1 carrier per number of shown live births np type b in 6 slowly progressive visceral cases and atypical protracted neurovisceral phenotype in 7 cases [ pavl - pereira h , et al . j inherit metab dis 2005;28:203227 ] including one case of np type c2 [ elleder m. et al . virchows arch 2001;439:206211 ] classical phenotype in 43 male patients and cardiac variant in 6 male patients ( two families ) [ elleder m , et al . cas lek cesk 1990;129:368372 ] infantile form in 12 patients , late infantile in 2 patients , and adult form in one patient [ kostalova e , et al . ceska slov neurol neurochir 2006;69/102:200210 ] one infantile patient ( wolman disease ) and 17 juvenile and adult cases [ elleder m , et al . cas lek cesk 1999;138:719724 ] including two adult patients fabry females not included fabry females included carrier frequency of lipidoses except for fabry disease mucopolysaccharidoses : data for calculation of birth prevalence mps mucopolysaccharidosis , msd multiple sulphatase deficiency total number of patients diagnosed between 1975 and 2008 number of families with particular disorder in parentheses number of patients taken for the calculation of birth prevalence . to avoid underestimation , some patients were not included into the prevalence calculation ( e.g. , patients born before 1945 were excluded ) . for prevalence definition , see patients and methods expressed as 1 carrier per number of shown live births hurler syndrom in 16 patients , hurler / scheie in 2 patients and scheie syndrom in 2 patients carrier frequency of mps excluding mps ii glycoproteinoses , mucolipidoses , glycogenosis type ii and neuronal ceroidlipofuscinoses : data for calculation of birth prevalence issd infantile sialic acid storage disease , gp glycoproteinosis , ml mucolipidosis , ncl neuronal ceroid lipofuscinosis total number of patients diagnosed between 1975 and 2008 number of families with particular disorder in parentheses number of patients taken for the calculation of birth prevalence . to avoid underestimation , some patients were not included into the prevalence calculation ( e.g. patients born before 1945 were excluded ) . for prevalence definition see patients and methods expressed as 1 carrier per number of shown live births one infantile patient and two juvenile siblings with cherry - red spot myoclonus syndrome [ ledvinova j , et al . j inherit metab dis 1994;17:118119 ] atypical case of ml iv with ocular restricted phenotype [ dobrovolny r , et al . am j ophthalmol 2007 ; 143:663671 ] to date , no individuals in the czech population have been conclusively diagnosed with farber disease , schindler disease , danon disease , sanfilippo d disease , galactosialidosis , fucosidosis , aspartylglucosaminuria , or prosaposin , saposins , or gm2 activator protein deficiencies . the overall prevalence of lsds in the czech population ( 12.25 per 100,000 ) , which represents a typical central european population , is comparable to prevalences reported for australia ( 12.9 per 100,000 ) , the netherlands ( 14 per 100,000 ) and italy ( 12.1 per 100,000 ) ( meikle et al . . the higher prevalence of lsds reported for the northern portuguese population ( 25 per 100,000 ) might reflect the relative geographic isolation and more extensive studying of that particular population of this region ( pinto et al . the most frequent single lsd is gaucher disease in the czech republic , italy and australia , gm2 gangliosidosis in portugal , and pompe disease in the netherlands . in the czech population , lipidoses were most frequently diagnosed ( 5.0 per 100,000 live births ) , followed by mps ( 3.72 ) and ncl ( 2.29 ) . in addition to the lsd diagnoses in czech patients , 96 cases from slovakia were confirmed at our institute during the studied period ; 41% with lipidoses , 39% with mps , and 20% with gsd ii or ncls . these slovakian patients have not been included in our prevalence calculations as former czechoslovakia was divided into two independent states , the czech republic and the slovak republic , in 1993 . interestingly , the four cases of prosaposin deficiency so far diagnosed at our institute came from neighboring populations ( slovak , german ) ( hulkova et al . 2001 ; elleder et al . the birth prevalence of the most frequent lsd in the czech republic , gaucher disease , is 1.13 per 100,000 live births , followed by niemann - pick c ( 0.91 ) , mps iii ( 0.91 ) , mps iv ( 0.73 ) , and mps i ( 0.72 ) . comparison of these prevalences to those in selected foreign populations ( tables 1 , 2 and 3 ) is complicated by the differences in methods used for calculation . when we compared the calculations of prevalence in the czech population based on the method of poorthuis et al . ( 1999 ) used in this study ( see patients and methods ) to the method reported by meikle et al . ( 1999 ) ( the prevalence calculated as the number of patients divided by the total number of live births during the study period ) , the calculations resulted in underestimation or overestimations of some individual disorders . this is the case for disorders with delayed onset ( e.g. , frequencies of fabry disease were 0.52 and 1.14 per 100,000 , and frequencies of cholesterol ester storage disease were 0.27 and 0.42 , if calculated using the methods of poorthuis et al . also , unavailability of some diagnostic methods during the study period ( some methods were introduced as late as the 1990s ) may explain some discrepancies found in our calculations using the two methods . although prevalence values for some disorders calculated using either of the methods were within the confidence intervals ( data not shown ) , for other disorders prevalence values fell outside this interval ( e.g. , the frequencies differed for mps iii c ( 0.42 and 0.11 per 100,000 ) , mps iva ( 0.71 and 0.32 ) , ncl 2 ( 0.36 and 0.65 ) and ncl 7 ( 0.85 and 0.42 ) , if calculated according to the methods used by poorthuis et al . thus , establishment of standard rules for calculation of birth prevalence - a defined term in clinical epidemiology - is highly recommended . based on our experience , the calculation of the ratio of the number of patients to the number of live births during the patients births period ( poorthuis et al . underestimation of the birth prevalence may result from lack of recognition of early clinical signs and symptoms or phenotypic diversity , including adult forms of the disease ( e.g. , fabry disease , adult krabbe disease , adult mld , cholesteryl ester storage disease , and adult gsd ii ) . thus , the calculated birth prevalence is probably the lowest estimate as the diagnosis is commonly delayed for years . screening studies have shown that certain groups of patients , for example individuals with renal failure on haemodialysis , may harbor missed fabry patients ( nakao et al . the nationwide screening study among 3,370 chronic haemodialysis patients in the czech republic disclosed four previously unrecognized fabry males and one female from five unrelated families . subsequent family screening ascertained fabry disease in another 12 individuals , including 8 females ( merta et al . patients with unexplained cardiomyopathy represent another group at risk of having fabry disease where screening is justified ( monserrat et al . 2007 ) . such lsd screening initiatives for selected lsds will contribute to the identification of the true prevalence of these disorders ( spada et al . fabry heterozygotes have not been included in the calculation of birth prevalence in previous reports ( meikle et al . however , they commonly develop clinical symptoms and severe complications , generally a few years later , as in affected men ( deegan et al . 2006 ; wilcox et al . 2008 ) . when we included fabry heterozygotes in the calculations , the prevalence of fabry disease increased by 2.5 times to 1.29 per 100,000 live births , that of lipidoses from 5.0 to 5.77 per 100,000 and the overall prevalence of lsds reached 13.02 per 100,000 ( table 4 ) therefore , we propose that fabry heterozygotes ought to be included in the calculation of the birth prevalence of fabry disease . reliable calculation of the birth prevalence is dependant on a correctly defined pathogenic cause underlying the condition . recent molecular studies have provided evidence for at least ten separate genetic loci ( cln1cln10 ) for ncl ( peltonen et al . these new findings necessitated reclassification of our 37 cases that had formerly been provisionally classified as ncl6 into ncl5 , ncl6 , and ncl7 subgroups . the latter turned out to be of high prevalence in the roma gypsy population ( kousi et al . 2009 ) . about a quarter of the ncl cases remain unspecified due to the lack of material suitable for mutation analysis . athough specific lsds are generally reckoned to be rare disorders , as a group of inherited metabolic disorders they are quite common , as seen from published and our data . the information on birth prevalencies of lsds is available from countries with a long tradition of diagnosis of lsds ; however , for most populations , these date are missing . epidemiological data on these disorders are important in genetic counselling for calculation of the risk for the disorders in the other members of affected families and subsequently for the public health care systems . importantly , awareness of the clinical aspects of these life - threatening and progressively disabling genetic diseases among the medical community should increase , allowing earlier diagnosis . dependant on the diagnosed disorder , such intervention may include enzyme replacement therapy , bone marrow transplantation , or other current or future therapies . last but not least , it should be borne in mind that genetic counselling and prenatal diagnosis in families affected by a genetic metabolic disorder still form the basic strategy for lowering the number of patients with these severe disorders .

the aim of this retrospective study was to determine the prevalence of lysosomal storage disorders ( lsds ) in the czech republic . the data on cases diagnosed between 1975 and 2008 were collected and analyzed . the overall prevalence of lsds in the czech population ( 12.25 per 100,000 ) is comparable to that reported for the countries with well - established and advanced diagnostics of lsds such as the netherlands ( 14 per 100,000 ) , australia ( 12.9 per 100,000 ) and italy ( 12.1 per 100,000 ) . relatively higher prevalence of lsds was reported in the north of portugal ( 25 per 100,000 ) . thirty - four different lsds were diagnosed in a total of 478 individuals . gaucher disease was the most frequent lsd with a birth prevalence of 1.13 per 100,000 births . the most frequent lsd groups were lipidoses , mucopolysaccharidoses , and neuronal ceroid lipofuscinoses , with combined prevalences of 5.0 , 3.72 , and 2.29 per 100,000 live births , respectively . glycoproteinoses ( 0.57 per 100,000 live births ) , glycogenosis type ii ( 0.37 ) , and mucolipidoses ( 0.31 ) rarely occur in the czech population , and a range of other lsds have not been detected at all over the past three decades . knowledge of the birth prevalence and carrier frequency of particular disorders is important in genetic counselling for calculation of the risk for the disorder in the other members of affected families . earlier diagnosis of these disorders will permit timely intervention and may also result in lowering of the number of newborns with lsds .

Introduction Patients and methods Results Discussion

the aim of this study was to calculate the birth prevalence and carrier frequencies of lsds in the czech republic population , and to compare our results with reported epidemiologic data from other populations . for the calculation of the prevalence the prevalence was calculated as the total number of patients diagnosed with a particular lsd , divided by the total number of live births in the same period , as was the birth period of the diagnosed cases ( i.e. using the method mentioned above , we calculated the prevalence of the subgroups separately , and the overall prevalence of the specific lsd was estimated as a sum of the prevalences in the subgroups ( poorthuis et al . in the past three decades , a total of 478 patients have been diagnosed with one of 34 specific types of lsds in the czech republic ( fig . data on patients with lipidoses , mucopolysaccharidoses , and other lsds detected during the study period , including carrier frequencies in the czech population , are summarized in tables 1 , 2 and 3 , respectively . 2number of live births ( ) and number of lsd diagnoses ( ) in the czech population from 1975 to 2008 . of patients 10nlportugalaustraliaturkeyprevalence per 100,000 live birthsgaucher ( all types)491.130.611.956.711.161.351.750.45niemann - pick a / b230.330.160.613.650.530.60.4niemann - pick c540.910.671.216.020.352.20.47fabry male490.52 ( 1.0)0.380.690.010.210.120.860.015fabry female780.770.600.98krabbe150.400.210.684.01.351.210.711mld ( all types)250.690.291.385.261.421.851.091.43cesd , wolman180.270.150.453.310.19gm1 gangliosidosis130.260.140.463.250.410.620.260.54gm2 tay - sachs100.300.140.553.450.413.130.50.23gm2 sandhoff30.190.040.552.740.341.490.260.95lipidoses all types259/3375.0/5.772.79 ( 3.39)8.53 ( 9.51)6.212.66.64.6nl netherlands , mld metachromatic leukodystrophy , cesd cholesteryl ester storage diseasetotal number of patients diagnosed between 1975 and 2008data for prevalence calculations are in table 4carrier per 1 live birthmale live births onlyfabry females not includedfabry females includedtable 2mucopolysaccharidoses : comparison of data in different populationsdiseaseczech republicother countriesno . of patients 10nlportugalaustraliagermanyprevalence per 100,000 live birthsmps i200.720.441.115.381.191.331.140.69mps ii220.43 ( 0.83)0.260.650.00850.671.090.740.64mps iii a180.470.270.754.311.1600.88mps iii b10.020.00.130.970.420.720.47mps iii c50.420.140.994.120.210.120.07mps iii d000.10.10mps iii ( all types)240.910.411.871.890.841.421.57mps iv a140.710.381.225.350.220.60.59mps iv b10.020.00.130.970.1400mps iv ( a + b)150.730.381.350.38mps vi20.050.010.181.400.150.420.430.23mps vii10.020.00.130.970.2400.05mps unspecified320.600.390.87msd30.260.050.773.240.050.480.07mps all types1193.721.946.934.54.84.443.53nl netherlands , mps mucopolysaccharidosis , msd multiple sulphatase deficiencytotal number of patients diagnosed between 1975 and 2008data for prevalence calculations are in table 5carrier per 1 live birthmale live births onlyunspecified cases of mps were diagnosed on the basis of analysis of glycosaminoglycan excreted in urine combined with the evaluation of clinical data . no material was available for enzyme and dna analysistable 3glycoproteinoses , mucolipidoses , glycogenosis type ii and neuronal ceroid lipofuscinoses : comparison of data in different populationsdiseaseczech republicother countriesno . of patients 19752008prevalence per 100,000poisson 95% confidence intervalcarrier frequency ii / iii30.220.050.652.980.240.810.31mucolipidosis iv10.020.00.130.97glycogenosis type ii ( all)120.370.130.813.8620.170.69ncl 120.190.020.702.790.17ncl 2280.360.240.523.790.07ncl 320.270.030.963.270.48ncl 410.020.00.130.97ncl 520.210.030.772.92ncl 610.020.00.130.971.43ncl 7180.850.491.365.81ncl unspecified200.370.220.58ncl ( all types)742.291.035.152.14nl netherlands , issd infantile sialic acid storage disease , ncl neuronal ceroid lipofuscinosistotal number of patients diagnosed between 1975 and 2008data for prevalence calculations are in table 6carrier per 1 live birthunspecified ncl were diagnosed on the basis of histochemical and ultrastructural examination of biopsy samples combined with the evaluation of clinical data . of patientsprevalence ( 1 per numbers of live births)prevalence per 100,000carrier frequencygaucher type i , early12 ( 10)197419963 090 90212257 5750.39254gaucher type i , late29 ( 28)194519805 956 02824248 1680.40249gaucher type ii and iii8 ( 8)198019982 352 4928294 0620.34271gaucher ( all types)494488 3971.13149niemann - pick a10 ( 8)196320025 480 88510548 0890.18370niemann - pick b + atyp13 ( 11)194819967 362 14611669 2860.15409niemann - pick a / b2321301 3270.33274niemann - pick c54 ( 45)196520035 271 31048109 8190.91166fabry49 ( 24)194520028 518 60144193 6050.5296 803male4 382 0684499 5921.00fabry female78 ( 24)194520058 812 16168129 5910.77krabbe15 ( 15)197720023 249 15013249 9350.40250mld infantile13 ( 11)197019994 195 56413322 7360.31284 juvenile5 ( 4)197020044 661 3245932 2650.11483 adult7 ( 7)196819802 188 0046364 6670.27302mld ( all types)2524144 6470.69190cesd , wolman18 ( 15)196320025 480 88515365 3920.27302gm1 gangliosidosis13 ( 13)197020034 563 66012380 3050.26308gm2 tay - sachs10 ( 10)197119923 362 88910336 2890.30290gm2 sandhoff3 ( 3)199220071 594 9493531 6500.19365lipidoses all types259/337234/30220 0005.0/5.7771mld metachromatic leukodystrophy , cesd cholesteryl ester storage diseasetotal number of patients diagnosed between 1975 and 2008number of families with particular disorder in parenthesesnumber of patients taken for the calculation of birth prevalence . of patientsprevalence ( 1 per number of live births)prevalence per 100,000carrier frequencymps i20 ( 17)198420082,772,55920138,6280.72186mps ii22 ( 19)196920054,906,70021233,6520.43116,826male2,520,37321120,0180.83mps iii a18 ( 14)197720063,648,54117214,6200.47232mps iii b1197520084,261,89714,261,8970.021,032mps iii c5 ( 4)198419921,179,7455235,9490.42243mps iii ( all types)2423109,5020.91165mps iv a14 ( 9)198720031,826,47313140,4980.71187mps iv b1197520084,261,89714,261,8970.021,032mps iv ( a + b)1514136,9860.73185mps vi2 ( 2)196819954,115,05722,057,5290.05717mps vii1197520084,261,89714,261,8970.021,032mps i vii848134,9092.8693mps - unspecified32 ( 27)196019894,533,49627167,9070.60205msd3 ( 1)196219691,144,4673381,4890.26309mps all types11911126,8653.7282mps mucopolysaccharidosis , msd multiple sulphatase deficiencytotal number of patients diagnosed between 1975 and 2008number of families with particular disorder in parenthesesnumber of patients taken for the calculation of birth prevalence . for prevalence definition , see patients and methodsexpressed as 1 carrier per number of shown live birthshurler syndrom in 16 patients , hurler / scheie in 2 patients and scheie syndrom in 2 patientsmale live birthscarrier frequency of mps excluding mps iitable 6glycoproteinoses , mucolipidoses , glycogenosis type ii and neuronal ceroidlipofuscinoses : data for calculation of birth prevalencediseaseno . ( 1 per numbers of live births)prevalence per 100,000carrier frequency-mannosidosis4 ( 3)198519921,042,8044260,7010.38255-mannosidosis2 ( 1)195019561,223,3862611,6930.16391issd1197520084,261,89714,261,8970.021,032mucolipidosis i3 ( 2)196919994,338,72931,446,2430.07601mucolipidosis ii / iii3 ( 3)199420071,352,2193450,7400.22336mucolipidosis iv1197520084,261,89714,261,8970.021,032gp + ml ( all types)1414112,9950.87170glycogenosis type ii infantile6 ( 5)197919992,614,0756435,6790.23330glycogenosis type ii juvenile6 ( 5)197220024,167,9306694,6550.14417glycogenosis type ii ( all)1212267,7500.37259ncl 12 ( 2)197019751,033,4472516,7240.19359ncl 228 ( 25)194920037,803,06828278,6810.36264ncl 32 ( 2)19791983749,5202374,7600.27306ncl 41197520084,261,89714,261,8970.021,032ncl 52 ( 1)19831989935,5532467,7770.21342ncl 61197520084,261,89714,261,8970.021,032ncl 718 ( 16)198520022,002,02517117,7660.85172ncl - unspecified20 ( 17)196519994,903,21418272,4010.37261ncl ( all types)747143,5512.29104issd infantile sialic acid storage disease , gp glycoproteinosis , ml mucolipidosis , ncl neuronal ceroid lipofuscinosistotal number of patients diagnosed between 1975 and 2008number of families with particular disorder in parenthesesnumber of patients taken for the calculation of birth prevalence . am j ophthalmol 2007 ; 143:663671 ] relative rate of lysosomal storage disorders in the czech republic . mps mucopolysaccharidosis , ncl neuronal ceroidlipofuscinosis , gsd ii glycogenosis type ii , ml mucolipidosis , gp glycoproteinosis , npa / b / c niemann - pick a / b / c , mld metachromatic leukodystrophy , cesd cholesterol ester storage disease , msd multiple sulphatase deficiency , gm1 gm1 gangliosidosis , gm2 gm2 gangliosidosis ( tay - sachs and sandhoff diseases ) , -man -mannosidosis , -man -mannosidosis , issd infantile sialic acid storage disease number of live births ( ) and number of lsd diagnoses ( ) in the czech population from 1975 to 2008 . from 1990 nowadays , laboratory diagnostic methods for 45 lsds are available ( enzyme and dna analyses , loading assays ) lipidoses : comparison of data in different populations nl netherlands , mld metachromatic leukodystrophy , cesd cholesteryl ester storage disease total number of patients diagnosed between 1975 and 2008 data for prevalence calculations are in table 4 carrier per 1 live birth male live births only fabry females not included fabry females included mucopolysaccharidoses : comparison of data in different populations nl netherlands , mps mucopolysaccharidosis , msd multiple sulphatase deficiency total number of patients diagnosed between 1975 and 2008 data for prevalence calculations are in table 5 carrier per 1 live birth male live births only unspecified cases of mps were diagnosed on the basis of analysis of glycosaminoglycan excreted in urine combined with the evaluation of clinical data . no material was available for enzyme and dna analysis glycoproteinoses , mucolipidoses , glycogenosis type ii and neuronal ceroid lipofuscinoses : comparison of data in different populations nl netherlands , issd infantile sialic acid storage disease , ncl neuronal ceroid lipofuscinosis total number of patients diagnosed between 1975 and 2008 data for prevalence calculations are in table 6 carrier per 1 live birth unspecified ncl were diagnosed on the basis of histochemical and ultrastructural examination of biopsy samples combined with the evaluation of clinical data . no material was available for enzyme and dna analysis lipidoses : data for calculation of birth prevalence mld metachromatic leukodystrophy , cesd cholesteryl ester storage disease total number of patients diagnosed between 1975 and 2008 number of families with particular disorder in parentheses number of patients taken for the calculation of birth prevalence . cas lek cesk 1999;138:719724 ] including two adult patients fabry females not included fabry females included carrier frequency of lipidoses except for fabry disease mucopolysaccharidoses : data for calculation of birth prevalence mps mucopolysaccharidosis , msd multiple sulphatase deficiency total number of patients diagnosed between 1975 and 2008 number of families with particular disorder in parentheses number of patients taken for the calculation of birth prevalence . for prevalence definition , see patients and methods expressed as 1 carrier per number of shown live births hurler syndrom in 16 patients , hurler / scheie in 2 patients and scheie syndrom in 2 patients carrier frequency of mps excluding mps ii glycoproteinoses , mucolipidoses , glycogenosis type ii and neuronal ceroidlipofuscinoses : data for calculation of birth prevalence issd infantile sialic acid storage disease , gp glycoproteinosis , ml mucolipidosis , ncl neuronal ceroid lipofuscinosis total number of patients diagnosed between 1975 and 2008 number of families with particular disorder in parentheses number of patients taken for the calculation of birth prevalence . the overall prevalence of lsds in the czech population ( 12.25 per 100,000 ) , which represents a typical central european population , is comparable to prevalences reported for australia ( 12.9 per 100,000 ) , the netherlands ( 14 per 100,000 ) and italy ( 12.1 per 100,000 ) ( meikle et al . the higher prevalence of lsds reported for the northern portuguese population ( 25 per 100,000 ) might reflect the relative geographic isolation and more extensive studying of that particular population of this region ( pinto et al . the most frequent single lsd is gaucher disease in the czech republic , italy and australia , gm2 gangliosidosis in portugal , and pompe disease in the netherlands . in the czech population , lipidoses were most frequently diagnosed ( 5.0 per 100,000 live births ) , followed by mps ( 3.72 ) and ncl ( 2.29 ) . the birth prevalence of the most frequent lsd in the czech republic , gaucher disease , is 1.13 per 100,000 live births , followed by niemann - pick c ( 0.91 ) , mps iii ( 0.91 ) , mps iv ( 0.73 ) , and mps i ( 0.72 ) . when we included fabry heterozygotes in the calculations , the prevalence of fabry disease increased by 2.5 times to 1.29 per 100,000 live births , that of lipidoses from 5.0 to 5.77 per 100,000 and the overall prevalence of lsds reached 13.02 per 100,000 ( table 4 ) therefore , we propose that fabry heterozygotes ought to be included in the calculation of the birth prevalence of fabry disease . epidemiological data on these disorders are important in genetic counselling for calculation of the risk for the disorders in the other members of affected families and subsequently for the public health care systems .

1955 ) , research into the biochemistry , molecular biology and pathology of the lysosomal system unravelled many of its functions . most lsds result from a deficient enzyme activity caused by a mutation in the gene encoding either the relevant enzyme , protecting protein , protein activators , or the endoplasmic reticulum however , some are caused by dysfunction of the lysosomal system itself due to mutations in genes encoding lysosomal membrane components or components closely associated with the lysosomal system ( saftig 2005 ; jalanko and braulke 2009 ) . the number of lysosomal enzymes and non - catalytic proteins reported in the literature has steadily increased and , therefore , more defects in the lysosomal system are expected to be discovered . the aim of this study was to calculate the birth prevalence and carrier frequencies of lsds in the czech republic population , and to compare our results with reported epidemiologic data from other populations . we analyzed data from individuals who had been diagnosed with a lsd at the institute of inherited metabolic disorders in prague between 1975 and 2008 . the patients had been referred to , or their blood samples had been sent to , the institute by departments of paediatrics , neurology , haematology , cardiology , nephrology , dermatology , gastroenterology , ophthalmology , pathology , and clinical genetics from hospitals all over the country . the definite diagnosis was made by demonstrating the deficiency of the relevant enzyme and/or presence of pathogenic mutation , and/or by detection of undegraded substrate by loading tests in cell cultures . mucolipidosis ii was confirmed by detecting elevated activities of multiple lysosomal enzymes in serum and by molecular analysis . patients with unspecified types of mps or ncl , who had died before the availability of confirmatory tests , were also included in the birth prevalence calculation . their diagnosis was based on biochemical analyses of storage compound , evaluation of clinical data , and histochemical and/or ultrastructural examinations of samples obtained at biopsy or autopsy . for the calculation of the prevalence the prevalence was calculated as the total number of patients diagnosed with a particular lsd , divided by the total number of live births in the same period , as was the birth period of the diagnosed cases ( i.e. , the birth period is the interval between the year of the birth of the oldest patient and the year of the birth of the youngest patient ) the total number of live births in particular years was calculated using birth rates taken from the czech statistical office web page . , if there was only one patient diagnosed with the disease , the prevalence was calculated using the number of live births ( i.e. familial cases and 11 affected fetuses [ fabry disease ( 1 ) , niemann - pick disease type c ( 1 ) , mps i ( 3 ) , mps iii a ( 3 ) and mps iv a ( 3 ) ] were also included in the calculation of birth prevalence . for some specific lsds , the patient group consisted of subgroups with distinct clinical phenotypes ( e.g. using the method mentioned above , we calculated the prevalence of the subgroups separately , and the overall prevalence of the specific lsd was estimated as a sum of the prevalences in the subgroups ( poorthuis et al . exclusion of heterozygotes ( n = 78 ) enabled us to compare the prevalence to other reported male - specific prevalence data ( meikle et al . fabry heterozygotes , who can be as severely affected as male patients , are nowadays classified as patients instead of carriers of the defective gene ( dobrovolny et al . in the past three decades , a total of 478 patients have been diagnosed with one of 34 specific types of lsds in the czech republic ( fig . the number of diagnosed patients gradually increased during the study period , from less than 10 cases per year in the 1970s to 2030 new cases diagnosed annually in recent years ( fig . more than half of all lsd patients had a lipidosis , about one - quarter a mucopolysaccharidosis , and the remaining quarter suffered from ncl , gsd ii , mucolipidoses , or glycoproteinoses ( fig . data on patients with lipidoses , mucopolysaccharidoses , and other lsds detected during the study period , including carrier frequencies in the czech population , are summarized in tables 1 , 2 and 3 , respectively . detailed data on the birth prevalence of specific disorders and their subtypes can be found in tables 4 , 5 and 6 . mps mucopolysaccharidosis , ncl neuronal ceroidlipofuscinosis , gsd ii glycogenosis type ii , ml mucolipidosis , gp glycoproteinosis , npa / b / c niemann - pick a / b / c , mld metachromatic leukodystrophy , cesd cholesterol ester storage disease , msd multiple sulphatase deficiency , gm1 gm1 gangliosidosis , gm2 gm2 gangliosidosis ( tay - sachs and sandhoff diseases ) , -man -mannosidosis , -man -mannosidosis , issd infantile sialic acid storage diseasefig . of patients 10nlportugalaustraliaturkeyprevalence per 100,000 live birthsgaucher ( all types)491.130.611.956.711.161.351.750.45niemann - pick a / b230.330.160.613.650.530.60.4niemann - pick c540.910.671.216.020.352.20.47fabry male490.52 ( 1.0)0.380.690.010.210.120.860.015fabry female780.770.600.98krabbe150.400.210.684.01.351.210.711mld ( all types)250.690.291.385.261.421.851.091.43cesd , wolman180.270.150.453.310.19gm1 gangliosidosis130.260.140.463.250.410.620.260.54gm2 tay - sachs100.300.140.553.450.413.130.50.23gm2 sandhoff30.190.040.552.740.341.490.260.95lipidoses all types259/3375.0/5.772.79 ( 3.39)8.53 ( 9.51)6.212.66.64.6nl netherlands , mld metachromatic leukodystrophy , cesd cholesteryl ester storage diseasetotal number of patients diagnosed between 1975 and 2008data for prevalence calculations are in table 4carrier per 1 live birthmale live births onlyfabry females not includedfabry females includedtable 2mucopolysaccharidoses : comparison of data in different populationsdiseaseczech republicother countriesno . of patients 10nlportugalaustraliagermanyprevalence per 100,000 live birthsmps i200.720.441.115.381.191.331.140.69mps ii220.43 ( 0.83)0.260.650.00850.671.090.740.64mps iii a180.470.270.754.311.1600.88mps iii b10.020.00.130.970.420.720.47mps iii c50.420.140.994.120.210.120.07mps iii d000.10.10mps iii ( all types)240.910.411.871.890.841.421.57mps iv a140.710.381.225.350.220.60.59mps iv b10.020.00.130.970.1400mps iv ( a + b)150.730.381.350.38mps vi20.050.010.181.400.150.420.430.23mps vii10.020.00.130.970.2400.05mps unspecified320.600.390.87msd30.260.050.773.240.050.480.07mps all types1193.721.946.934.54.84.443.53nl netherlands , mps mucopolysaccharidosis , msd multiple sulphatase deficiencytotal number of patients diagnosed between 1975 and 2008data for prevalence calculations are in table 5carrier per 1 live birthmale live births onlyunspecified cases of mps were diagnosed on the basis of analysis of glycosaminoglycan excreted in urine combined with the evaluation of clinical data . of patients 19752008prevalence per 100,000poisson 95% confidence intervalcarrier frequency ii / iii30.220.050.652.980.240.810.31mucolipidosis iv10.020.00.130.97glycogenosis type ii ( all)120.370.130.813.8620.170.69ncl 120.190.020.702.790.17ncl 2280.360.240.523.790.07ncl 320.270.030.963.270.48ncl 410.020.00.130.97ncl 520.210.030.772.92ncl 610.020.00.130.971.43ncl 7180.850.491.365.81ncl unspecified200.370.220.58ncl ( all types)742.291.035.152.14nl netherlands , issd infantile sialic acid storage disease , ncl neuronal ceroid lipofuscinosistotal number of patients diagnosed between 1975 and 2008data for prevalence calculations are in table 6carrier per 1 live birthunspecified ncl were diagnosed on the basis of histochemical and ultrastructural examination of biopsy samples combined with the evaluation of clinical data . , patients born before 1945 see patients and methodsexpressed as 1 carrier per number of shown live birthsone stillborn casenp type b in 6 slowly progressive visceral cases and atypical protracted neurovisceral phenotype in 7 cases [ pavl - pereira h , et al . for prevalence definition , see patients and methodsexpressed as 1 carrier per number of shown live birthshurler syndrom in 16 patients , hurler / scheie in 2 patients and scheie syndrom in 2 patientsmale live birthscarrier frequency of mps excluding mps iitable 6glycoproteinoses , mucolipidoses , glycogenosis type ii and neuronal ceroidlipofuscinoses : data for calculation of birth prevalencediseaseno . mps mucopolysaccharidosis , ncl neuronal ceroidlipofuscinosis , gsd ii glycogenosis type ii , ml mucolipidosis , gp glycoproteinosis , npa / b / c niemann - pick a / b / c , mld metachromatic leukodystrophy , cesd cholesterol ester storage disease , msd multiple sulphatase deficiency , gm1 gm1 gangliosidosis , gm2 gm2 gangliosidosis ( tay - sachs and sandhoff diseases ) , -man -mannosidosis , -man -mannosidosis , issd infantile sialic acid storage disease number of live births ( ) and number of lsd diagnoses ( ) in the czech population from 1975 to 2008 . from 1990 nowadays , laboratory diagnostic methods for 45 lsds are available ( enzyme and dna analyses , loading assays ) lipidoses : comparison of data in different populations nl netherlands , mld metachromatic leukodystrophy , cesd cholesteryl ester storage disease total number of patients diagnosed between 1975 and 2008 data for prevalence calculations are in table 4 carrier per 1 live birth male live births only fabry females not included fabry females included mucopolysaccharidoses : comparison of data in different populations nl netherlands , mps mucopolysaccharidosis , msd multiple sulphatase deficiency total number of patients diagnosed between 1975 and 2008 data for prevalence calculations are in table 5 carrier per 1 live birth male live births only unspecified cases of mps were diagnosed on the basis of analysis of glycosaminoglycan excreted in urine combined with the evaluation of clinical data . no material was available for enzyme and dna analysis glycoproteinoses , mucolipidoses , glycogenosis type ii and neuronal ceroid lipofuscinoses : comparison of data in different populations nl netherlands , issd infantile sialic acid storage disease , ncl neuronal ceroid lipofuscinosis total number of patients diagnosed between 1975 and 2008 data for prevalence calculations are in table 6 carrier per 1 live birth unspecified ncl were diagnosed on the basis of histochemical and ultrastructural examination of biopsy samples combined with the evaluation of clinical data . no material was available for enzyme and dna analysis lipidoses : data for calculation of birth prevalence mld metachromatic leukodystrophy , cesd cholesteryl ester storage disease total number of patients diagnosed between 1975 and 2008 number of families with particular disorder in parentheses number of patients taken for the calculation of birth prevalence . for prevalence definition , see patients and methods expressed as 1 carrier per number of shown live births np type b in 6 slowly progressive visceral cases and atypical protracted neurovisceral phenotype in 7 cases [ pavl - pereira h , et al . cas lek cesk 1999;138:719724 ] including two adult patients fabry females not included fabry females included carrier frequency of lipidoses except for fabry disease mucopolysaccharidoses : data for calculation of birth prevalence mps mucopolysaccharidosis , msd multiple sulphatase deficiency total number of patients diagnosed between 1975 and 2008 number of families with particular disorder in parentheses number of patients taken for the calculation of birth prevalence . for prevalence definition , see patients and methods expressed as 1 carrier per number of shown live births hurler syndrom in 16 patients , hurler / scheie in 2 patients and scheie syndrom in 2 patients carrier frequency of mps excluding mps ii glycoproteinoses , mucolipidoses , glycogenosis type ii and neuronal ceroidlipofuscinoses : data for calculation of birth prevalence issd infantile sialic acid storage disease , gp glycoproteinosis , ml mucolipidosis , ncl neuronal ceroid lipofuscinosis total number of patients diagnosed between 1975 and 2008 number of families with particular disorder in parentheses number of patients taken for the calculation of birth prevalence . am j ophthalmol 2007 ; 143:663671 ] to date , no individuals in the czech population have been conclusively diagnosed with farber disease , schindler disease , danon disease , sanfilippo d disease , galactosialidosis , fucosidosis , aspartylglucosaminuria , or prosaposin , saposins , or gm2 activator protein deficiencies . the overall prevalence of lsds in the czech population ( 12.25 per 100,000 ) , which represents a typical central european population , is comparable to prevalences reported for australia ( 12.9 per 100,000 ) , the netherlands ( 14 per 100,000 ) and italy ( 12.1 per 100,000 ) ( meikle et al . the higher prevalence of lsds reported for the northern portuguese population ( 25 per 100,000 ) might reflect the relative geographic isolation and more extensive studying of that particular population of this region ( pinto et al . the most frequent single lsd is gaucher disease in the czech republic , italy and australia , gm2 gangliosidosis in portugal , and pompe disease in the netherlands . in addition to the lsd diagnoses in czech patients , 96 cases from slovakia were confirmed at our institute during the studied period ; 41% with lipidoses , 39% with mps , and 20% with gsd ii or ncls . these slovakian patients have not been included in our prevalence calculations as former czechoslovakia was divided into two independent states , the czech republic and the slovak republic , in 1993 . the birth prevalence of the most frequent lsd in the czech republic , gaucher disease , is 1.13 per 100,000 live births , followed by niemann - pick c ( 0.91 ) , mps iii ( 0.91 ) , mps iv ( 0.73 ) , and mps i ( 0.72 ) . when we compared the calculations of prevalence in the czech population based on the method of poorthuis et al . ( 1999 ) ( the prevalence calculated as the number of patients divided by the total number of live births during the study period ) , the calculations resulted in underestimation or overestimations of some individual disorders . , the frequencies differed for mps iii c ( 0.42 and 0.11 per 100,000 ) , mps iva ( 0.71 and 0.32 ) , ncl 2 ( 0.36 and 0.65 ) and ncl 7 ( 0.85 and 0.42 ) , if calculated according to the methods used by poorthuis et al . thus , establishment of standard rules for calculation of birth prevalence - a defined term in clinical epidemiology - is highly recommended . based on our experience , the calculation of the ratio of the number of patients to the number of live births during the patients births period ( poorthuis et al . underestimation of the birth prevalence may result from lack of recognition of early clinical signs and symptoms or phenotypic diversity , including adult forms of the disease ( e.g. the nationwide screening study among 3,370 chronic haemodialysis patients in the czech republic disclosed four previously unrecognized fabry males and one female from five unrelated families . such lsd screening initiatives for selected lsds will contribute to the identification of the true prevalence of these disorders ( spada et al . when we included fabry heterozygotes in the calculations , the prevalence of fabry disease increased by 2.5 times to 1.29 per 100,000 live births , that of lipidoses from 5.0 to 5.77 per 100,000 and the overall prevalence of lsds reached 13.02 per 100,000 ( table 4 ) therefore , we propose that fabry heterozygotes ought to be included in the calculation of the birth prevalence of fabry disease . epidemiological data on these disorders are important in genetic counselling for calculation of the risk for the disorders in the other members of affected families and subsequently for the public health care systems . importantly , awareness of the clinical aspects of these life - threatening and progressively disabling genetic diseases among the medical community should increase , allowing earlier diagnosis . last but not least , it should be borne in mind that genetic counselling and prenatal diagnosis in families affected by a genetic metabolic disorder still form the basic strategy for lowering the number of patients with these severe disorders .

advanced chronic renal failure is a progressive and irreversible disorder in which the kidneys ability to excrete metabolic waste products and to maintain fluid and electrolytes is lost and can lead to uremia . chronic renal failure and end - stage renal diseases ( esrds ) are progressive and irreversible disorders and one of the major problems around the world . the incidence of ckd in the usa , taiwan , and some regions in mexico is approaching 400 cases per million . in iran , the number of patients beginning maintenance renal replacement therapy ( rrt ) increased by 130% from 2000 to 2006 . the incidence of esrd linearly increased from 13.82 per million population in 1997 to 49.9 per million population in 2000 and to 63.8 per million population in 2006 . widespread and easy access of patients requiring hemodialysis treatment has resulted in increase in longevity of thousands of patients with esrds . hemodialysis is the most common treatment for the patients suffering from the esrds or irreversible progressive renal failure . changes in living patterns and its limitations impose complex and changed lives on the patients and their families and , finally , reduce their quality of life . in such situations , optimizing their quality of life will be one of the most important objectives of health care . nowadays , hemodialysis patients , in addition to increasing the lifetime thanks to hemodialysis , want to improve their quality of life . the results of studies on the health - related quality of life in the patients requiring hemodialysis have shown that related diseases have undesirable effects on patients physical , mental , and social functions , and measuring the quality of life in such patients has special importance . despite advances in the treatment of hemodialysis patients , their quality of life is affected by various factors that may make their physical and mental performances difficult . one of the ways of improving the patients quality of life is through self - care education . developing and using theories and models in nursing is one of the available training methods . development of strategic plans for hemodialysis patients for them to achieve efficient care is essential . those patients who have received self - care education , in comparison with those who have never received such training , have higher quality of life . hemodialysis patients in their own self - care do not have high self - efficacy . therefore , in order to improve their quality of life , training them in self - care programs has been taken into consideration as a strategy . several factors have effects on increasing the hemodialysis patients quality of life , one of which is self - care education . self - care is the patients ongoing efforts to promote their health and welfare and to make their lives better . the results of some studies have shown that there are positive and significant relationships between self - care ability and different aspects of quality of life . the results of a study conducted in taiwan in 2002 indicated that the quality of life in hemodialysis patients was lower than that in kidney transplant patients , and breast and colon cancer patients . several studies have been conducted in iran to assess the quality of life in hemodialysis patients . concluded in their study that more than half of hemodialysis patients had undesirable quality of life . 's study showed that self - care educational program had effects on decreasing the hemodialysis patients problems and improving their quality of life . 's study , the health - related quality of life in hemodialysis patients in iran was lower than that in european and asian countries due to the differences in lifestyle , socioeconomic status , the general level of education of the patients , as well as physician patient communication . in another study conducted by moattari et al . in iran , the results showed a positive effect of empowerment on hemodialysis patients self - efficacy and quality of life . controlling the problems and complications and improving quality of life requires patients participation in treatment and care . patients treated with hemodialysis need special and ongoing training as they have multiple drug treatments and a special diet , as well as for acquiring the required skills to cope with physical and mental disabilities . there are several models and approaches for educating and training patients . considering the chronic problems of these patients , hemodialysis inefficiency and inadequacy in addressing these problems , as well as the need for continuous care , education and training should provide active and informed participation of patients in their own self - care . according to the problems mentioned above , including the type , extent , and frequency of such patients physical and psychological problems and , on the other hand , the need for providing education to the patients so that they are qualified for their continuous self - care , this study aimed to determine how self - care education in these patients can be effective in reducing their problems and improving their quality of life , in order to provide more evidences in this area . the use of a nursing model helps to evaluate patients health status , establish good communications between patients and nurses , set goals of care , and improve the quality of care . the main goal of the self - care models is to improve the patients quality of life . the partnership care model focuses on the participation of the patient , his / her family , nurses , physicians , and other healthcare providers in the treatment processes and , based on the partnership approach , provides programs to ensure the continuity of this participation in order to promote and maintain the health of the patient . this model , in addition to explaining the collaborative approach , examines and explains the relationship between care and this participation , and in this regard , the assumptions , structure , basic concepts , and objectives are defined . this model pays special attention to the care and emphasizes two major dimensions including : skills , techniques , and special carethe caring relationship developed among patients , physicians , and nurses to promote and maintain patients health . skills , techniques , and special care the caring relationship developed among patients , physicians , and nurses to promote and maintain patients health . however , the second one is a software dimension , does not have a fixed and defined nature , and has a dynamic status . although certain skills and activities are necessary in the care processes , caring relationship plays an important role in the quality of care and promoting and maintaining the health . this model has several objectives , the most important of which is paying attention to the patients quality of life . in other words , this model provides an overall strategy to utilize all the capabilities of those involved in the care and treatment , including physicians , nurses , and the patients , and based on this model , providing care is only possible when the relationship among physicians , nurses , patients , and their families is continued and all these people have the same common objectives and understanding of patient care activities . this model includes four stages : ( 1 ) motivating , ( 2 ) preparing , ( 3 ) involving , and ( 4 ) evaluating . the results of some studies have shown that using partnership care model in patients with cardiovascular disease and hypertension has had positive effects on the patients quality of life , and given that using this model is not limited to a particular disease , implementing it for patients with diseases which reduce their quality of life has been considered as a necessity by researchers . therefore , this study aimed to determine and evaluate the effects of using this model on the quality of life in patients treated with hemodialysis . it was assumed that the implementation of the partnership care model has a positive impact on improving the quality of life in hemodialysis patients . in iran , the researchers of this study had used this model for the first time for hemodialysis patients in a military hospital to determine how self - care education based on partnership care model could be effective in reducing problems and improving the quality of life of these patients . this was a quasi - experimental study conducted in 2012 on patients who were referred to a military hospital in tehran , iran to be treated with hemodialysis . inclusion criteria were : willingness to participate in this study , loss of function of both kidneys based on clinical evidence , laboratory tests , and expert opinions , glomerular filtration rate ( gfr ) < 10% , having passed at least 6 months from starting hemodialysis , performing hemodialysis two times per week , and not suffering from any underlying acute or chronic diseases . the first section was related to the demographic data , including age , sex , marital status , employment status , education level , and duration of hemodialysis . the second section included data on weight , blood pressure , sleep , itching , and doing exercise by patients . the second questionnaire was the short form-36 ( sf-36 ) standardized questionnaire consisting of eight dimensions , including general health , vitality , physical function , physical role , bodily pain , mental health , social function , and emotional role . generally , this questionnaire has two components , including physical and mental components . its scales are scored from 0 to 100 in which 0 represents the worst quality of life and 100 represent the best quality of life . the content validity of the first questionnaire was approved using the opinions of 15 faculty members of nursing and midwifery school of tehran university of medical sciences , and its reliability was confirmed using test retest reliability coefficient ( r = 0.85 ) . validity and reliability of sf-36 questionnaire have been confirmed in previous studies . the model used in this study as one of the self - care models was partnership care model . this model consists of four stages : ( 1 ) motivating , ( 2 ) preparing , ( 3 ) involving , and ( 4 ) evaluating . during the motivating stage , patients became aware of their disease and its outcomes and consequences by attending a training session . at the preparing stage , the objectives , schedules , and time tables of partnership education programs , as well as how to track them were determined . during the involving stage , the educational meetings were held and , finally , at the evaluating stage , achieving the objectives of the model was evaluated . individuals should attend such a system based on their desire and motivation . on the other hand , care is also a voluntary relationship initiated and formed by choosing a physician , a nurse , or even the type of care and treatment by the patient . because all care and treatments are considered as activities invading patients privacy , giving informed choices to the patients is the prerequisite for providing care and treatment . explanation of what should be done , how , why , who , where , and when is essential . the second stage of the model is developing an appropriate plan to engage all members of the partnership team , including patients , nurses , and physicians , because the best ideas and programs , even with the strongest and most well - intentioned people and with the best working facilities , can not be implemented without members involvement or they will lead to very poor results . to create appropriate opportunities for involving and engaging all members , because care and treatments involve a series of specialized activities , it is necessary to define the required activities and their relationship with the members duties and responsibilities according to their abilities and skills . therefore , at this stage , it is required to do proper planning before any other action . one other important step at the preparing stage is planning for educational partnership visits , as well as following the achievement of this model 's objective , i.e. , ensuring the participation and compliance . at the next stage , time and content planning of collaborative visits will be done as follows : these visits are conducted according to prior planning . therefore , the objectives of the educational content and the teaching methods and tools required are determined . this plan can be classically developed according to the patients problems and diagnoses , and in collaboration with the physician , nurse , and the patients . however , it should be noted that it is not essential to implement the education plan classically and formallythe implementation of the education plan should be based on the philosophy and approach of participation and partnership , so that both patients and other participants are active . in other words , only the nurse and the physician are not responsible for trainingthe methods of undertaking the visits and providing education can be according to the patients conditions and by applying appropriate methods of explaining , question and answer methods , or based on problem solving and problem - based learning modelcollaborative visits are undertaken with the presence of those patients who have similar conditions . after the planning stage , care and treatments are provided according to the defined objectives . if the partnership plan is implemented without applying an effective management , the concept and principles of participation will be forgotten over time or will be filed and archived in the patients charts as a few slips of useless instructions . since a manager or coordinator should be selected for implementing each plan in the groups , the following steps should also be taken in the partnership plan : determining the head and manager of the partnership teamaccording to the nurses capabilities , it is better to give the responsibility of the team to its nurse based on members consensusteam members consensus on delegating the responsibility of the team to its head and manager , as well as cooperating with him / herimplementing the plan and undertaking the educational visits and required follow - ups . therefore , the objectives of the educational content and the teaching methods and tools required are determined . this plan can be classically developed according to the patients problems and diagnoses , and in collaboration with the physician , nurse , and the patients . however , it should be noted that it is not essential to implement the education plan classically and formally the implementation of the education plan should be based on the philosophy and approach of participation and partnership , so that both patients and other participants are active . in other words , only the nurse and the physician are not responsible for training the methods of undertaking the visits and providing education can be according to the patients conditions and by applying appropriate methods of explaining , question and answer methods , or based on problem solving and problem - based learning model collaborative visits are undertaken with the presence of those patients who have similar conditions . after the planning stage , care and treatments are provided according to the defined objectives . if the partnership plan is implemented without applying an effective management , the concept and principles of participation will be forgotten over time or will be filed and archived in the patients charts as a few slips of useless instructions . since a manager or coordinator should be selected for implementing each plan in the groups , the following steps should also be taken in the partnership plan : determining the head and manager of the partnership teamaccording to the nurses capabilities , it is better to give the responsibility of the team to its nurse based on members consensusteam members consensus on delegating the responsibility of the team to its head and manager , as well as cooperating with him / herimplementing the plan and undertaking the educational visits and required follow - ups . determining the head and manager of the partnership team according to the nurses capabilities , it is better to give the responsibility of the team to its nurse based on members consensus team members consensus on delegating the responsibility of the team to its head and manager , as well as cooperating with him / her implementing the plan and undertaking the educational visits and required follow - ups . to identify patients problems , the measurement of biochemical compositions of the blood , weight gain between two hemodialysis sessions , edema , and hypertension was used . the patients were weighed before and after hemodialysis with predetermined dress and with a scale that was calibrated and was constant during the intervention . for measuring edema , the patients edema in the wrist , leg , and around the eyes the edema in the hip and abdominal circumference was also measured by the meter . to gain more confidence in the data collected , blood biochemical compositions were measured within 2 months before training and other variables were measured two to four times with 2-week intervals before training and their mean values were considered as the data before training . also , after training , the blood biochemical compositions were measured within 2 months and other variables were measured for two to four times with 2-week intervals and their mean values were considered as the mean after training . quality of life was measured after training for two times , at 6 and 8 weeks after training , and their mean was calculated as the data after training . the study patients blood pressure was measured after 5 min of rest , in sitting mode , from the arm which did not have any fistula , in a position at heart level , and in a position in which the patient had an appropriate support and backrest . the education was provided to the patients through a training manual which had been prepared based on valid and scientific articles about exercise , sleep , nutrition , stress , hemodialysis equipment , hemodialysis methods , and psychology . patients companions were also trained for 2 h in a class , and then , practical and additional training was provided for them during hemodialysis . the study patients were trained in all educational topics including the topics of nutrition , exercise , sleep , ways to prevent itching , etc . for example , for training the patients in the topic of exercise , a physiotherapist and the researcher gave theoretical and practical training to all the patients of a work shift ( eight patients ) for 2 h. afterward , in the next session , the researcher evaluated those patients , responded to their related questions , and explained that topic again to them ( if needed ) so that they learned the topic completely . then , the researcher started training the other topic . between the 1 and 2 weeks , the topic that was dealt with in the 1 week was evaluated . in all stages of the research , , chicago , il , usa ) and some statistical tests including paired samples t - test , wilcoxon and mcnemar tests . paired samples t - test was used for analyzing and comparing the data collected on weight and blood pressure before and after the intervention . in order to assess insomnia , edema , and the itching status before and after the intervention , required data were collected through another part of the questionnaire with yes / no items and analyzed using mcnemar test . individuals should attend such a system based on their desire and motivation . on the other hand , care is also a voluntary relationship initiated and formed by choosing a physician , a nurse , or even the type of care and treatment by the patient . because all care and treatments are considered as activities invading patients privacy , giving informed choices to the patients is the prerequisite for providing care and treatment . explanation of what should be done , how , why , who , where , and when is essential . the second stage of the model is developing an appropriate plan to engage all members of the partnership team , including patients , nurses , and physicians , because the best ideas and programs , even with the strongest and most well - intentioned people and with the best working facilities , can not be implemented without members involvement or they will lead to very poor results . to create appropriate opportunities for involving and engaging all members , because care and treatments involve a series of specialized activities , it is necessary to define the required activities and their relationship with the members duties and responsibilities according to their abilities and skills . therefore , at this stage , it is required to do proper planning before any other action . one other important step at the preparing stage is planning for educational partnership visits , as well as following the achievement of this model 's objective , i.e. , ensuring the participation and compliance . at the next stage , time and content planning of collaborative visits will be done as follows : these visits are conducted according to prior planning . therefore , the objectives of the educational content and the teaching methods and tools required are determined . this plan can be classically developed according to the patients problems and diagnoses , and in collaboration with the physician , nurse , and the patients . however , it should be noted that it is not essential to implement the education plan classically and formallythe implementation of the education plan should be based on the philosophy and approach of participation and partnership , so that both patients and other participants are active . in other words , only the nurse and the physician are not responsible for trainingthe methods of undertaking the visits and providing education can be according to the patients conditions and by applying appropriate methods of explaining , question and answer methods , or based on problem solving and problem - based learning modelcollaborative visits are undertaken with the presence of those patients who have similar conditions . after the planning stage , care and treatments are provided according to the defined objectives . if the partnership plan is implemented without applying an effective management , the concept and principles of participation will be forgotten over time or will be filed and archived in the patients charts as a few slips of useless instructions . since a manager or coordinator should be selected for implementing each plan in the groups , the following steps should also be taken in the partnership plan : determining the head and manager of the partnership teamaccording to the nurses capabilities , it is better to give the responsibility of the team to its nurse based on members consensusteam members consensus on delegating the responsibility of the team to its head and manager , as well as cooperating with him / herimplementing the plan and undertaking the educational visits and required follow - ups . therefore , the objectives of the educational content and the teaching methods and tools required are determined . this plan can be classically developed according to the patients problems and diagnoses , and in collaboration with the physician , nurse , and the patients . however , it should be noted that it is not essential to implement the education plan classically and formally the implementation of the education plan should be based on the philosophy and approach of participation and partnership , so that both patients and other participants are active . in other words , only the nurse and the physician are not responsible for training the methods of undertaking the visits and providing education can be according to the patients conditions and by applying appropriate methods of explaining , question and answer methods , or based on problem solving and problem - based learning model collaborative visits are undertaken with the presence of those patients who have similar conditions . after the planning stage , care and treatments are provided according to the defined objectives . if the partnership plan is implemented without applying an effective management , the concept and principles of participation will be forgotten over time or will be filed and archived in the patients charts as a few slips of useless instructions . since a manager or coordinator should be selected for implementing each plan in the groups , the following steps should also be taken in the partnership plan : determining the head and manager of the partnership teamaccording to the nurses capabilities , it is better to give the responsibility of the team to its nurse based on members consensusteam members consensus on delegating the responsibility of the team to its head and manager , as well as cooperating with him / herimplementing the plan and undertaking the educational visits and required follow - ups . determining the head and manager of the partnership team according to the nurses capabilities , it is better to give the responsibility of the team to its nurse based on members consensus team members consensus on delegating the responsibility of the team to its head and manager , as well as cooperating with him / her implementing the plan and undertaking the educational visits and required follow - ups . to identify patients problems , the measurement of biochemical compositions of the blood , weight gain between two hemodialysis sessions , edema , and hypertension was used . the patients were weighed before and after hemodialysis with predetermined dress and with a scale that was calibrated and was constant during the intervention . for measuring edema , the patients edema in the wrist , leg , and around the eyes was measured using the pitting edema grading scale . the edema in the hip and abdominal circumference was also measured by the meter . to gain more confidence in the data collected , blood biochemical compositions were measured within 2 months before training and other variables were measured two to four times with 2-week intervals before training and their mean values were considered as the data before training . also , after training , the blood biochemical compositions were measured within 2 months and other variables were measured for two to four times with 2-week intervals and their mean values were considered as the mean after training . quality of life was measured after training for two times , at 6 and 8 weeks after training , and their mean was calculated as the data after training . the study patients blood pressure was measured after 5 min of rest , in sitting mode , from the arm which did not have any fistula , in a position at heart level , and in a position in which the patient had an appropriate support and backrest . the education was provided to the patients through a training manual which had been prepared based on valid and scientific articles about exercise , sleep , nutrition , stress , hemodialysis equipment , hemodialysis methods , and psychology . educating patients patients companions were also trained for 2 h in a class , and then , practical and additional training was provided for them during hemodialysis . the study patients were trained in all educational topics including the topics of nutrition , exercise , sleep , ways to prevent itching , etc . , for training the patients in the topic of exercise , a physiotherapist and the researcher gave theoretical and practical training to all the patients of a work shift ( eight patients ) for 2 h. afterward , in the next session , the researcher evaluated those patients , responded to their related questions , and explained that topic again to them ( if needed ) so that they learned the topic completely . then , the researcher started training the other topic . between the 1 and 2 weeks , the topic that was dealt with in the 1 week was evaluated . in all stages of the research , chicago , il , usa ) and some statistical tests including paired samples t - test , wilcoxon and mcnemar tests . paired samples t - test was used for analyzing and comparing the data collected on weight and blood pressure before and after the intervention . in order to assess insomnia , edema , and the itching status before and after the intervention , required data were collected through another part of the questionnaire with yes / no items and analyzed using mcnemar test . the results showed that most of the study patients were men ( n = 17 , 53.1% ) , in the 56 - 65 years age group , illiterate ( n = 9 , 28.1% ) , married ( n = 23 , 71.9% ) , employees ( n = 18 , 56.3% ) , and being treated with hemodialysis for 1 - 3 years ( n = 13 , 40.6% ) [ table 1 ] . also , the results showed that the mean and standard deviation ( sd ) of patients parameters including weight and blood pressure improved significantly after the educational intervention compared to before the intervention ( p < 0.001 ) . in addition , the mean of systolic and diastolic blood pressure decreased after the intervention ( p < 0.001 ) [ table 2 ] . demographic characteristics of the patients referred to the hospital for hemodialysis mean and standard deviation of the parameters considered ( weight and blood pressure ) in the study patients before and after the intervention while 46.8% of the study patients did exercise rarely before the intervention , 59.4% of them started doing exercise after the educational intervention . before the intervention , all study patients were complaining of insomnia . however , after the educational intervention , only 59.4% patients had this problem ( p < 0.001 ) . also , the number of study patients with edema and itching before and after the educational intervention had significant differences ( p < 0.001 ) [ table 3 ] . comparison of the percentages of the study patients based on changes in their exercise , sleep , edema , and itching before and after the educational intervention table 4 shows that the differences between the mean values of the study patients laboratory tests before and after the intervention were statistically significant ( p < 0.001 ) . mean and standard deviation of the study patients laboratory test results before and after the intervention the mean and sd of the quality of life dimensions ( including general health , vitality , physical function , role physical , bodily pain , mental health , social function , and role emotional ) had been improved after the educational intervention . these differences were statistically significant before and after the intervention ( p < 0.001 ) [ table 5 ] . mean and standard deviation of the dimensions of the study patients quality of life before and after the educational intervention due to the low quality of life in hemodialysis patients , taking some measures to improve the quality of life of these patients is essential . the results of studies show that the quality of life indicates the quality of health care provided and is a part of hemodialysis patient care program . measuring these patients quality of life will provide more information for the health team . in the field of nursing , conducting studies on the patients quality of life and forming interventions that would result in improved quality of life are growing rapidly , which leads to improvements in the lives of people with such chronic diseases . in the present study , in baraz et al . 's study , it was 46.69 . in another study which was conducted by pakpour et al . in their study , the performance of mental component was more than that of physical component . according to the results of rostami et al . 's study , the mean of hemodialysis patients quality of life was 44.29 . therefore , the results of the present study and other studies conducted in iran show that the quality of life in hemodialysis patients in iran is not at a desirable level . in other words , it can be concluded that the psychological and physical quality of life in the patients treated by hemodialysis for a long period has been poor . 's study showed that the mean of all eight dimensions of the quality of life was higher than 50% and the overall mean of quality of life was 68.38 . in diaz - buxom et al . 's study , the mean of hemodialysis patients quality of life was more than that in the present study and other similar studies conducted in iran . overall , the mean of quality of life in iranian hemodialysis patients compared to that in other countries is at a lower level , and the difference may be related to the factors such as study patients lifestyle , socioeconomic status , low level of awareness , and lack of appropriate self - care . despite the lower quality of life of the patients in the present study , the mean of quality of life after implementing the educational plan significantly increased and indicated that the provided education was effective . in baraz 's study , the mean of quality of life had increased from 46.69 to 54.64 after education , indicating its lower effectiveness compared to that in the present study results . although these two studies have been conducted with the same methodology , their results are slightly different because of the differences in the factors among the study patients , such as sex , education level , and socioeconomic status . if they do not pay careful attention to their daily fluid intake , they are faced with fluid retention and problems such as general body swelling , shortness of breath , cardiac and pulmonary disorders , weight gain , etc . the present study results show that using the partnership care model had positive effects on some parameters in the study patients , such as their sleep , exercise , weight , and blood pressure . for instance , all these patients had sleep problem before the educational intervention ; however , this problem decreased significantly and only 40.6% of them had it after the intervention . . 's study results showed that insomnia was a common problem in hemodialysis patients which had significant relationship with their low quality of life . results of another study showed that implementing continuous care model had a positive effect on the quality of sleep in the hemodialysis patients . braz found in his study that self - care education decreased the amount of edema significantly in hemodialysis patients . in the present study , the results of lugon 's study showed that itching is an annoying sensation which is the common problem in most of the hemodialysis patients and can affect their quality of life and cause depression . in the current study , while the study patients were not exercising before the intervention , a large number of them ( 59.4% ) started exercising after the intervention . the results of jennen and uhlenbruck 's study showed that exercising increased the sense of satisfaction and the scores of quality of life dimensions . the mean of blood pressure and weight loss had significant difference before and after the intervention , as they improved after the intervention . the results of salehi 's study showed that education had a positive effect on weight loss between two hemodialysis sessions . in the present study , that the mean of serum urea decreased significantly after training , which is similar to the present study results . uremia causes irritability , loss of appetite , insomnia , drowsiness , fatigue , memory loss , confusion , making mistakes in judgments , and lack of focus , each of which has effects on the patients quality of life . the results of salehi 's study showed that unlike the present study results , the mean of potassium levels did not show any statistically significant decrease after training . advanced chronic renal failure is the main cause of increased blood potassium because the kidneys are unable to excrete excess potassium in the blood . however , in the present study , the blood potassium levels of the study patients decreased because of following correct nutrition and diet patterns . the results of the current study showed a statistically significant decrease in the mean of serum phosphorus levels after training . the study results of shichiri et al . also showed that the serum phosphorus levels of the study patients decreased significantly after 5 weeks of training . one of the major complications of renal failure is renal osteodystrophy which occurs as a result of decrease in blood calcium and increase in blood phosphorus levels . since hemodialysis can not remove excess blood phosphorus , choosing the proper diet is important in reducing this type of disorder . in the present study , 's study showed that following a low - protein diet reduced the serum uric acid levels significantly . in the current study , systolic and the results of sarafi 's study also indicated the positive effects of the training program on decreasing the blood pressures in hemodialysis patients . in the present study , the weight gain between two hemodialysis sessions increased the patients blood pressures by 3 mm hg / kg at the same time . the results of oka et al . 's study showed that the control of feeding behavior had significant negative correlations with blood urea nitrogen , potassium , phosphorus , as well as the weight gain between two hemodialysis sessions . 's study indicated that training hemodialysis patients in nutrition and diet , as well as in fluid intake can result in fluid intake restrictions and , subsequently , weight loss . the present study results show that the use of self - care model had a positive and significant effect on the study patients , as all quality of life dimensions improved after the educational intervention . rahimi 's study results showed that using continuous care model had positive effects on several parameters and indicators of the hemodialysis patients , such as their quality of life . found in their study that after a 12-week exercise program , the psychological status , quality of life , and work capacity in hemodialysis patients significantly improved , which confirms the present study results . the results of tsay and lee 's study showed that implementing an adaptation training program for patients with esrd decreased their stress and increased the physical and mental dimensions of their quality of life significantly , which is similar to the results of the current study . learning rate of the study patients was different due to their individual and motivational differences and could not be controlled . use of care models , especially those models which are compatible with the culture of our society , can increase the quality of life in the hemodialysis patients . assisting the hemodialysis patients the results of the present study showed that using self - care model had significant effects on all quality of life dimensions , including social and physical function , general health , etc . , therefore , matrons and nursing managers in their planning for applying different education methods can use the self - care model in their units to improve the inpatients overall health status . in order to show the positive effects of using the self - care model and develop strategies for better implementation of this model , it is recommended to compare the effects of using this model with those of providing traditional physician visits and also conduct similar studies on hemodialysis patients in other hospitals with larger sample sizes . also , conducting a study on the problems of and barriers to using this model is suggested . it is also suggested conducting similar studies using different educational methods and models to determine and compare their effectiveness .

background : hemodialysis patients have lower quality of life and one of the ways to improve their quality of life is providing self - care education to them using some models including self - care model . this study aimed to determine and evaluate the effects of using self - care model on health and quality of life outcomes in hemodialysis patients.materials and methods : this was a quasi - experimental study conducted in 2012 on the patients who were referred to a military hospital in tehran , iran to be treated with hemodialysis . all 32 patients referred to this hospital in 2012 were selected and studied . required data were collected using the short form-36 ( sf-36 ) standard questionnaire and a researcher - made questionnaire . the educational intervention was implemented using self - care model . collected data were analyzed using spss for windows version 18.0 and some statistical tests including paired samples t - test , wilcoxon and mcnemar tests.results:the results showed that the mean and standard deviation ( sd ) of patients parameters including weight and blood pressure improved significantly after the educational intervention compared to before the intervention ( p < 0.001 ) . also , all dimensions of the quality of life of hemodialysis patients , including physical function , role physical , bodily pain , general health , vitality , social function , mental health , and role emotional improved compared to those before the intervention ( p < 0.001).conclusion : implementing the self - care model increased the quality of life of hemodialysis patients . therefore , the use of this model in hemodialysis patients is recommended .

INTRODUCTION MATERIALS AND METHODS Detailed description of the partnership care model RESULTS DISCUSSION CONCLUSION

chronic renal failure and end - stage renal diseases ( esrds ) are progressive and irreversible disorders and one of the major problems around the world . changes in living patterns and its limitations impose complex and changed lives on the patients and their families and , finally , reduce their quality of life . in such situations , optimizing their quality of life will be one of the most important objectives of health care . nowadays , hemodialysis patients , in addition to increasing the lifetime thanks to hemodialysis , want to improve their quality of life . the results of studies on the health - related quality of life in the patients requiring hemodialysis have shown that related diseases have undesirable effects on patients physical , mental , and social functions , and measuring the quality of life in such patients has special importance . despite advances in the treatment of hemodialysis patients , their quality of life is affected by various factors that may make their physical and mental performances difficult . one of the ways of improving the patients quality of life is through self - care education . those patients who have received self - care education , in comparison with those who have never received such training , have higher quality of life . hemodialysis patients in their own self - care do not have high self - efficacy . therefore , in order to improve their quality of life , training them in self - care programs has been taken into consideration as a strategy . several factors have effects on increasing the hemodialysis patients quality of life , one of which is self - care education . self - care is the patients ongoing efforts to promote their health and welfare and to make their lives better . the results of some studies have shown that there are positive and significant relationships between self - care ability and different aspects of quality of life . the results of a study conducted in taiwan in 2002 indicated that the quality of life in hemodialysis patients was lower than that in kidney transplant patients , and breast and colon cancer patients . several studies have been conducted in iran to assess the quality of life in hemodialysis patients . concluded in their study that more than half of hemodialysis patients had undesirable quality of life . 's study showed that self - care educational program had effects on decreasing the hemodialysis patients problems and improving their quality of life . 's study , the health - related quality of life in hemodialysis patients in iran was lower than that in european and asian countries due to the differences in lifestyle , socioeconomic status , the general level of education of the patients , as well as physician patient communication . in iran , the results showed a positive effect of empowerment on hemodialysis patients self - efficacy and quality of life . considering the chronic problems of these patients , hemodialysis inefficiency and inadequacy in addressing these problems , as well as the need for continuous care , education and training should provide active and informed participation of patients in their own self - care . according to the problems mentioned above , including the type , extent , and frequency of such patients physical and psychological problems and , on the other hand , the need for providing education to the patients so that they are qualified for their continuous self - care , this study aimed to determine how self - care education in these patients can be effective in reducing their problems and improving their quality of life , in order to provide more evidences in this area . the use of a nursing model helps to evaluate patients health status , establish good communications between patients and nurses , set goals of care , and improve the quality of care . the main goal of the self - care models is to improve the patients quality of life . the partnership care model focuses on the participation of the patient , his / her family , nurses , physicians , and other healthcare providers in the treatment processes and , based on the partnership approach , provides programs to ensure the continuity of this participation in order to promote and maintain the health of the patient . this model , in addition to explaining the collaborative approach , examines and explains the relationship between care and this participation , and in this regard , the assumptions , structure , basic concepts , and objectives are defined . this model pays special attention to the care and emphasizes two major dimensions including : skills , techniques , and special carethe caring relationship developed among patients , physicians , and nurses to promote and maintain patients health . this model has several objectives , the most important of which is paying attention to the patients quality of life . in other words , this model provides an overall strategy to utilize all the capabilities of those involved in the care and treatment , including physicians , nurses , and the patients , and based on this model , providing care is only possible when the relationship among physicians , nurses , patients , and their families is continued and all these people have the same common objectives and understanding of patient care activities . the results of some studies have shown that using partnership care model in patients with cardiovascular disease and hypertension has had positive effects on the patients quality of life , and given that using this model is not limited to a particular disease , implementing it for patients with diseases which reduce their quality of life has been considered as a necessity by researchers . therefore , this study aimed to determine and evaluate the effects of using this model on the quality of life in patients treated with hemodialysis . it was assumed that the implementation of the partnership care model has a positive impact on improving the quality of life in hemodialysis patients . in iran , the researchers of this study had used this model for the first time for hemodialysis patients in a military hospital to determine how self - care education based on partnership care model could be effective in reducing problems and improving the quality of life of these patients . this was a quasi - experimental study conducted in 2012 on patients who were referred to a military hospital in tehran , iran to be treated with hemodialysis . the first section was related to the demographic data , including age , sex , marital status , employment status , education level , and duration of hemodialysis . the second questionnaire was the short form-36 ( sf-36 ) standardized questionnaire consisting of eight dimensions , including general health , vitality , physical function , physical role , bodily pain , mental health , social function , and emotional role . its scales are scored from 0 to 100 in which 0 represents the worst quality of life and 100 represent the best quality of life . the content validity of the first questionnaire was approved using the opinions of 15 faculty members of nursing and midwifery school of tehran university of medical sciences , and its reliability was confirmed using test retest reliability coefficient ( r = 0.85 ) . the model used in this study as one of the self - care models was partnership care model . during the involving stage , the educational meetings were held and , finally , at the evaluating stage , achieving the objectives of the model was evaluated . the second stage of the model is developing an appropriate plan to engage all members of the partnership team , including patients , nurses , and physicians , because the best ideas and programs , even with the strongest and most well - intentioned people and with the best working facilities , can not be implemented without members involvement or they will lead to very poor results . therefore , the objectives of the educational content and the teaching methods and tools required are determined . therefore , the objectives of the educational content and the teaching methods and tools required are determined . since a manager or coordinator should be selected for implementing each plan in the groups , the following steps should also be taken in the partnership plan : determining the head and manager of the partnership teamaccording to the nurses capabilities , it is better to give the responsibility of the team to its nurse based on members consensusteam members consensus on delegating the responsibility of the team to its head and manager , as well as cooperating with him / herimplementing the plan and undertaking the educational visits and required follow - ups . to identify patients problems , the measurement of biochemical compositions of the blood , weight gain between two hemodialysis sessions , edema , and hypertension was used . also , after training , the blood biochemical compositions were measured within 2 months and other variables were measured for two to four times with 2-week intervals and their mean values were considered as the mean after training . quality of life was measured after training for two times , at 6 and 8 weeks after training , and their mean was calculated as the data after training . for example , for training the patients in the topic of exercise , a physiotherapist and the researcher gave theoretical and practical training to all the patients of a work shift ( eight patients ) for 2 h. afterward , in the next session , the researcher evaluated those patients , responded to their related questions , and explained that topic again to them ( if needed ) so that they learned the topic completely . in all stages of the research , , chicago , il , usa ) and some statistical tests including paired samples t - test , wilcoxon and mcnemar tests . paired samples t - test was used for analyzing and comparing the data collected on weight and blood pressure before and after the intervention . in order to assess insomnia , edema , and the itching status before and after the intervention , required data were collected through another part of the questionnaire with yes / no items and analyzed using mcnemar test . the second stage of the model is developing an appropriate plan to engage all members of the partnership team , including patients , nurses , and physicians , because the best ideas and programs , even with the strongest and most well - intentioned people and with the best working facilities , can not be implemented without members involvement or they will lead to very poor results . therefore , the objectives of the educational content and the teaching methods and tools required are determined . therefore , the objectives of the educational content and the teaching methods and tools required are determined . determining the head and manager of the partnership team according to the nurses capabilities , it is better to give the responsibility of the team to its nurse based on members consensus team members consensus on delegating the responsibility of the team to its head and manager , as well as cooperating with him / her implementing the plan and undertaking the educational visits and required follow - ups . to identify patients problems , the measurement of biochemical compositions of the blood , weight gain between two hemodialysis sessions , edema , and hypertension was used . for measuring edema , the patients edema in the wrist , leg , and around the eyes was measured using the pitting edema grading scale . also , after training , the blood biochemical compositions were measured within 2 months and other variables were measured for two to four times with 2-week intervals and their mean values were considered as the mean after training . , for training the patients in the topic of exercise , a physiotherapist and the researcher gave theoretical and practical training to all the patients of a work shift ( eight patients ) for 2 h. afterward , in the next session , the researcher evaluated those patients , responded to their related questions , and explained that topic again to them ( if needed ) so that they learned the topic completely . in all stages of the research , chicago , il , usa ) and some statistical tests including paired samples t - test , wilcoxon and mcnemar tests . paired samples t - test was used for analyzing and comparing the data collected on weight and blood pressure before and after the intervention . in order to assess insomnia , edema , and the itching status before and after the intervention , required data were collected through another part of the questionnaire with yes / no items and analyzed using mcnemar test . the results showed that most of the study patients were men ( n = 17 , 53.1% ) , in the 56 - 65 years age group , illiterate ( n = 9 , 28.1% ) , married ( n = 23 , 71.9% ) , employees ( n = 18 , 56.3% ) , and being treated with hemodialysis for 1 - 3 years ( n = 13 , 40.6% ) [ table 1 ] . also , the results showed that the mean and standard deviation ( sd ) of patients parameters including weight and blood pressure improved significantly after the educational intervention compared to before the intervention ( p < 0.001 ) . in addition , the mean of systolic and diastolic blood pressure decreased after the intervention ( p < 0.001 ) [ table 2 ] . demographic characteristics of the patients referred to the hospital for hemodialysis mean and standard deviation of the parameters considered ( weight and blood pressure ) in the study patients before and after the intervention while 46.8% of the study patients did exercise rarely before the intervention , 59.4% of them started doing exercise after the educational intervention . before the intervention , all study patients were complaining of insomnia . however , after the educational intervention , only 59.4% patients had this problem ( p < 0.001 ) . also , the number of study patients with edema and itching before and after the educational intervention had significant differences ( p < 0.001 ) [ table 3 ] . comparison of the percentages of the study patients based on changes in their exercise , sleep , edema , and itching before and after the educational intervention table 4 shows that the differences between the mean values of the study patients laboratory tests before and after the intervention were statistically significant ( p < 0.001 ) . mean and standard deviation of the study patients laboratory test results before and after the intervention the mean and sd of the quality of life dimensions ( including general health , vitality , physical function , role physical , bodily pain , mental health , social function , and role emotional ) had been improved after the educational intervention . these differences were statistically significant before and after the intervention ( p < 0.001 ) [ table 5 ] . mean and standard deviation of the dimensions of the study patients quality of life before and after the educational intervention due to the low quality of life in hemodialysis patients , taking some measures to improve the quality of life of these patients is essential . the results of studies show that the quality of life indicates the quality of health care provided and is a part of hemodialysis patient care program . in the field of nursing , conducting studies on the patients quality of life and forming interventions that would result in improved quality of life are growing rapidly , which leads to improvements in the lives of people with such chronic diseases . 's study , the mean of hemodialysis patients quality of life was 44.29 . therefore , the results of the present study and other studies conducted in iran show that the quality of life in hemodialysis patients in iran is not at a desirable level . in other words , it can be concluded that the psychological and physical quality of life in the patients treated by hemodialysis for a long period has been poor . 's study showed that the mean of all eight dimensions of the quality of life was higher than 50% and the overall mean of quality of life was 68.38 . 's study , the mean of hemodialysis patients quality of life was more than that in the present study and other similar studies conducted in iran . overall , the mean of quality of life in iranian hemodialysis patients compared to that in other countries is at a lower level , and the difference may be related to the factors such as study patients lifestyle , socioeconomic status , low level of awareness , and lack of appropriate self - care . despite the lower quality of life of the patients in the present study , the mean of quality of life after implementing the educational plan significantly increased and indicated that the provided education was effective . in baraz 's study , the mean of quality of life had increased from 46.69 to 54.64 after education , indicating its lower effectiveness compared to that in the present study results . although these two studies have been conducted with the same methodology , their results are slightly different because of the differences in the factors among the study patients , such as sex , education level , and socioeconomic status . the present study results show that using the partnership care model had positive effects on some parameters in the study patients , such as their sleep , exercise , weight , and blood pressure . for instance , all these patients had sleep problem before the educational intervention ; however , this problem decreased significantly and only 40.6% of them had it after the intervention . 's study results showed that insomnia was a common problem in hemodialysis patients which had significant relationship with their low quality of life . results of another study showed that implementing continuous care model had a positive effect on the quality of sleep in the hemodialysis patients . braz found in his study that self - care education decreased the amount of edema significantly in hemodialysis patients . in the present study , the results of lugon 's study showed that itching is an annoying sensation which is the common problem in most of the hemodialysis patients and can affect their quality of life and cause depression . in the current study , while the study patients were not exercising before the intervention , a large number of them ( 59.4% ) started exercising after the intervention . the results of jennen and uhlenbruck 's study showed that exercising increased the sense of satisfaction and the scores of quality of life dimensions . the mean of blood pressure and weight loss had significant difference before and after the intervention , as they improved after the intervention . in the present study , that the mean of serum urea decreased significantly after training , which is similar to the present study results . uremia causes irritability , loss of appetite , insomnia , drowsiness , fatigue , memory loss , confusion , making mistakes in judgments , and lack of focus , each of which has effects on the patients quality of life . the results of salehi 's study showed that unlike the present study results , the mean of potassium levels did not show any statistically significant decrease after training . also showed that the serum phosphorus levels of the study patients decreased significantly after 5 weeks of training . in the current study , systolic and the results of sarafi 's study also indicated the positive effects of the training program on decreasing the blood pressures in hemodialysis patients . in the present study , the weight gain between two hemodialysis sessions increased the patients blood pressures by 3 mm hg / kg at the same time . the present study results show that the use of self - care model had a positive and significant effect on the study patients , as all quality of life dimensions improved after the educational intervention . rahimi 's study results showed that using continuous care model had positive effects on several parameters and indicators of the hemodialysis patients , such as their quality of life . found in their study that after a 12-week exercise program , the psychological status , quality of life , and work capacity in hemodialysis patients significantly improved , which confirms the present study results . the results of tsay and lee 's study showed that implementing an adaptation training program for patients with esrd decreased their stress and increased the physical and mental dimensions of their quality of life significantly , which is similar to the results of the current study . use of care models , especially those models which are compatible with the culture of our society , can increase the quality of life in the hemodialysis patients . assisting the hemodialysis patients the results of the present study showed that using self - care model had significant effects on all quality of life dimensions , including social and physical function , general health , etc . , therefore , matrons and nursing managers in their planning for applying different education methods can use the self - care model in their units to improve the inpatients overall health status . in order to show the positive effects of using the self - care model and develop strategies for better implementation of this model , it is recommended to compare the effects of using this model with those of providing traditional physician visits and also conduct similar studies on hemodialysis patients in other hospitals with larger sample sizes . also , conducting a study on the problems of and barriers to using this model is suggested .

advanced chronic renal failure is a progressive and irreversible disorder in which the kidneys ability to excrete metabolic waste products and to maintain fluid and electrolytes is lost and can lead to uremia . the results of studies on the health - related quality of life in the patients requiring hemodialysis have shown that related diseases have undesirable effects on patients physical , mental , and social functions , and measuring the quality of life in such patients has special importance . despite advances in the treatment of hemodialysis patients , their quality of life is affected by various factors that may make their physical and mental performances difficult . therefore , in order to improve their quality of life , training them in self - care programs has been taken into consideration as a strategy . the results of a study conducted in taiwan in 2002 indicated that the quality of life in hemodialysis patients was lower than that in kidney transplant patients , and breast and colon cancer patients . 's study , the health - related quality of life in hemodialysis patients in iran was lower than that in european and asian countries due to the differences in lifestyle , socioeconomic status , the general level of education of the patients , as well as physician patient communication . in iran , the results showed a positive effect of empowerment on hemodialysis patients self - efficacy and quality of life . considering the chronic problems of these patients , hemodialysis inefficiency and inadequacy in addressing these problems , as well as the need for continuous care , education and training should provide active and informed participation of patients in their own self - care . according to the problems mentioned above , including the type , extent , and frequency of such patients physical and psychological problems and , on the other hand , the need for providing education to the patients so that they are qualified for their continuous self - care , this study aimed to determine how self - care education in these patients can be effective in reducing their problems and improving their quality of life , in order to provide more evidences in this area . the use of a nursing model helps to evaluate patients health status , establish good communications between patients and nurses , set goals of care , and improve the quality of care . the partnership care model focuses on the participation of the patient , his / her family , nurses , physicians , and other healthcare providers in the treatment processes and , based on the partnership approach , provides programs to ensure the continuity of this participation in order to promote and maintain the health of the patient . this model , in addition to explaining the collaborative approach , examines and explains the relationship between care and this participation , and in this regard , the assumptions , structure , basic concepts , and objectives are defined . this model pays special attention to the care and emphasizes two major dimensions including : skills , techniques , and special carethe caring relationship developed among patients , physicians , and nurses to promote and maintain patients health . although certain skills and activities are necessary in the care processes , caring relationship plays an important role in the quality of care and promoting and maintaining the health . in other words , this model provides an overall strategy to utilize all the capabilities of those involved in the care and treatment , including physicians , nurses , and the patients , and based on this model , providing care is only possible when the relationship among physicians , nurses , patients , and their families is continued and all these people have the same common objectives and understanding of patient care activities . the results of some studies have shown that using partnership care model in patients with cardiovascular disease and hypertension has had positive effects on the patients quality of life , and given that using this model is not limited to a particular disease , implementing it for patients with diseases which reduce their quality of life has been considered as a necessity by researchers . therefore , this study aimed to determine and evaluate the effects of using this model on the quality of life in patients treated with hemodialysis . in iran , the researchers of this study had used this model for the first time for hemodialysis patients in a military hospital to determine how self - care education based on partnership care model could be effective in reducing problems and improving the quality of life of these patients . this was a quasi - experimental study conducted in 2012 on patients who were referred to a military hospital in tehran , iran to be treated with hemodialysis . inclusion criteria were : willingness to participate in this study , loss of function of both kidneys based on clinical evidence , laboratory tests , and expert opinions , glomerular filtration rate ( gfr ) < 10% , having passed at least 6 months from starting hemodialysis , performing hemodialysis two times per week , and not suffering from any underlying acute or chronic diseases . the second questionnaire was the short form-36 ( sf-36 ) standardized questionnaire consisting of eight dimensions , including general health , vitality , physical function , physical role , bodily pain , mental health , social function , and emotional role . the content validity of the first questionnaire was approved using the opinions of 15 faculty members of nursing and midwifery school of tehran university of medical sciences , and its reliability was confirmed using test retest reliability coefficient ( r = 0.85 ) . the second stage of the model is developing an appropriate plan to engage all members of the partnership team , including patients , nurses , and physicians , because the best ideas and programs , even with the strongest and most well - intentioned people and with the best working facilities , can not be implemented without members involvement or they will lead to very poor results . to create appropriate opportunities for involving and engaging all members , because care and treatments involve a series of specialized activities , it is necessary to define the required activities and their relationship with the members duties and responsibilities according to their abilities and skills . however , it should be noted that it is not essential to implement the education plan classically and formallythe implementation of the education plan should be based on the philosophy and approach of participation and partnership , so that both patients and other participants are active . in other words , only the nurse and the physician are not responsible for trainingthe methods of undertaking the visits and providing education can be according to the patients conditions and by applying appropriate methods of explaining , question and answer methods , or based on problem solving and problem - based learning modelcollaborative visits are undertaken with the presence of those patients who have similar conditions . if the partnership plan is implemented without applying an effective management , the concept and principles of participation will be forgotten over time or will be filed and archived in the patients charts as a few slips of useless instructions . since a manager or coordinator should be selected for implementing each plan in the groups , the following steps should also be taken in the partnership plan : determining the head and manager of the partnership teamaccording to the nurses capabilities , it is better to give the responsibility of the team to its nurse based on members consensusteam members consensus on delegating the responsibility of the team to its head and manager , as well as cooperating with him / herimplementing the plan and undertaking the educational visits and required follow - ups . however , it should be noted that it is not essential to implement the education plan classically and formally the implementation of the education plan should be based on the philosophy and approach of participation and partnership , so that both patients and other participants are active . in other words , only the nurse and the physician are not responsible for training the methods of undertaking the visits and providing education can be according to the patients conditions and by applying appropriate methods of explaining , question and answer methods , or based on problem solving and problem - based learning model collaborative visits are undertaken with the presence of those patients who have similar conditions . if the partnership plan is implemented without applying an effective management , the concept and principles of participation will be forgotten over time or will be filed and archived in the patients charts as a few slips of useless instructions . since a manager or coordinator should be selected for implementing each plan in the groups , the following steps should also be taken in the partnership plan : determining the head and manager of the partnership teamaccording to the nurses capabilities , it is better to give the responsibility of the team to its nurse based on members consensusteam members consensus on delegating the responsibility of the team to its head and manager , as well as cooperating with him / herimplementing the plan and undertaking the educational visits and required follow - ups . determining the head and manager of the partnership team according to the nurses capabilities , it is better to give the responsibility of the team to its nurse based on members consensus team members consensus on delegating the responsibility of the team to its head and manager , as well as cooperating with him / her implementing the plan and undertaking the educational visits and required follow - ups . to identify patients problems , the measurement of biochemical compositions of the blood , weight gain between two hemodialysis sessions , edema , and hypertension was used . for measuring edema , the patients edema in the wrist , leg , and around the eyes the edema in the hip and abdominal circumference was also measured by the meter . to gain more confidence in the data collected , blood biochemical compositions were measured within 2 months before training and other variables were measured two to four times with 2-week intervals before training and their mean values were considered as the data before training . the study patients blood pressure was measured after 5 min of rest , in sitting mode , from the arm which did not have any fistula , in a position at heart level , and in a position in which the patient had an appropriate support and backrest . the education was provided to the patients through a training manual which had been prepared based on valid and scientific articles about exercise , sleep , nutrition , stress , hemodialysis equipment , hemodialysis methods , and psychology . for example , for training the patients in the topic of exercise , a physiotherapist and the researcher gave theoretical and practical training to all the patients of a work shift ( eight patients ) for 2 h. afterward , in the next session , the researcher evaluated those patients , responded to their related questions , and explained that topic again to them ( if needed ) so that they learned the topic completely . in order to assess insomnia , edema , and the itching status before and after the intervention , required data were collected through another part of the questionnaire with yes / no items and analyzed using mcnemar test . the second stage of the model is developing an appropriate plan to engage all members of the partnership team , including patients , nurses , and physicians , because the best ideas and programs , even with the strongest and most well - intentioned people and with the best working facilities , can not be implemented without members involvement or they will lead to very poor results . to create appropriate opportunities for involving and engaging all members , because care and treatments involve a series of specialized activities , it is necessary to define the required activities and their relationship with the members duties and responsibilities according to their abilities and skills . however , it should be noted that it is not essential to implement the education plan classically and formallythe implementation of the education plan should be based on the philosophy and approach of participation and partnership , so that both patients and other participants are active . in other words , only the nurse and the physician are not responsible for trainingthe methods of undertaking the visits and providing education can be according to the patients conditions and by applying appropriate methods of explaining , question and answer methods , or based on problem solving and problem - based learning modelcollaborative visits are undertaken with the presence of those patients who have similar conditions . if the partnership plan is implemented without applying an effective management , the concept and principles of participation will be forgotten over time or will be filed and archived in the patients charts as a few slips of useless instructions . since a manager or coordinator should be selected for implementing each plan in the groups , the following steps should also be taken in the partnership plan : determining the head and manager of the partnership teamaccording to the nurses capabilities , it is better to give the responsibility of the team to its nurse based on members consensusteam members consensus on delegating the responsibility of the team to its head and manager , as well as cooperating with him / herimplementing the plan and undertaking the educational visits and required follow - ups . however , it should be noted that it is not essential to implement the education plan classically and formally the implementation of the education plan should be based on the philosophy and approach of participation and partnership , so that both patients and other participants are active . in other words , only the nurse and the physician are not responsible for training the methods of undertaking the visits and providing education can be according to the patients conditions and by applying appropriate methods of explaining , question and answer methods , or based on problem solving and problem - based learning model collaborative visits are undertaken with the presence of those patients who have similar conditions . if the partnership plan is implemented without applying an effective management , the concept and principles of participation will be forgotten over time or will be filed and archived in the patients charts as a few slips of useless instructions . since a manager or coordinator should be selected for implementing each plan in the groups , the following steps should also be taken in the partnership plan : determining the head and manager of the partnership teamaccording to the nurses capabilities , it is better to give the responsibility of the team to its nurse based on members consensusteam members consensus on delegating the responsibility of the team to its head and manager , as well as cooperating with him / herimplementing the plan and undertaking the educational visits and required follow - ups . determining the head and manager of the partnership team according to the nurses capabilities , it is better to give the responsibility of the team to its nurse based on members consensus team members consensus on delegating the responsibility of the team to its head and manager , as well as cooperating with him / her implementing the plan and undertaking the educational visits and required follow - ups . to identify patients problems , the measurement of biochemical compositions of the blood , weight gain between two hemodialysis sessions , edema , and hypertension was used . the study patients blood pressure was measured after 5 min of rest , in sitting mode , from the arm which did not have any fistula , in a position at heart level , and in a position in which the patient had an appropriate support and backrest . the education was provided to the patients through a training manual which had been prepared based on valid and scientific articles about exercise , sleep , nutrition , stress , hemodialysis equipment , hemodialysis methods , and psychology . , for training the patients in the topic of exercise , a physiotherapist and the researcher gave theoretical and practical training to all the patients of a work shift ( eight patients ) for 2 h. afterward , in the next session , the researcher evaluated those patients , responded to their related questions , and explained that topic again to them ( if needed ) so that they learned the topic completely . in order to assess insomnia , edema , and the itching status before and after the intervention , required data were collected through another part of the questionnaire with yes / no items and analyzed using mcnemar test . the results showed that most of the study patients were men ( n = 17 , 53.1% ) , in the 56 - 65 years age group , illiterate ( n = 9 , 28.1% ) , married ( n = 23 , 71.9% ) , employees ( n = 18 , 56.3% ) , and being treated with hemodialysis for 1 - 3 years ( n = 13 , 40.6% ) [ table 1 ] . also , the results showed that the mean and standard deviation ( sd ) of patients parameters including weight and blood pressure improved significantly after the educational intervention compared to before the intervention ( p < 0.001 ) . demographic characteristics of the patients referred to the hospital for hemodialysis mean and standard deviation of the parameters considered ( weight and blood pressure ) in the study patients before and after the intervention while 46.8% of the study patients did exercise rarely before the intervention , 59.4% of them started doing exercise after the educational intervention . comparison of the percentages of the study patients based on changes in their exercise , sleep , edema , and itching before and after the educational intervention table 4 shows that the differences between the mean values of the study patients laboratory tests before and after the intervention were statistically significant ( p < 0.001 ) . mean and standard deviation of the study patients laboratory test results before and after the intervention the mean and sd of the quality of life dimensions ( including general health , vitality , physical function , role physical , bodily pain , mental health , social function , and role emotional ) had been improved after the educational intervention . mean and standard deviation of the dimensions of the study patients quality of life before and after the educational intervention due to the low quality of life in hemodialysis patients , taking some measures to improve the quality of life of these patients is essential . the results of studies show that the quality of life indicates the quality of health care provided and is a part of hemodialysis patient care program . in the field of nursing , conducting studies on the patients quality of life and forming interventions that would result in improved quality of life are growing rapidly , which leads to improvements in the lives of people with such chronic diseases . therefore , the results of the present study and other studies conducted in iran show that the quality of life in hemodialysis patients in iran is not at a desirable level . 's study showed that the mean of all eight dimensions of the quality of life was higher than 50% and the overall mean of quality of life was 68.38 . overall , the mean of quality of life in iranian hemodialysis patients compared to that in other countries is at a lower level , and the difference may be related to the factors such as study patients lifestyle , socioeconomic status , low level of awareness , and lack of appropriate self - care . despite the lower quality of life of the patients in the present study , the mean of quality of life after implementing the educational plan significantly increased and indicated that the provided education was effective . in baraz 's study , the mean of quality of life had increased from 46.69 to 54.64 after education , indicating its lower effectiveness compared to that in the present study results . in the present study , the results of lugon 's study showed that itching is an annoying sensation which is the common problem in most of the hemodialysis patients and can affect their quality of life and cause depression . uremia causes irritability , loss of appetite , insomnia , drowsiness , fatigue , memory loss , confusion , making mistakes in judgments , and lack of focus , each of which has effects on the patients quality of life . in the current study , systolic and the results of sarafi 's study also indicated the positive effects of the training program on decreasing the blood pressures in hemodialysis patients . 's study showed that the control of feeding behavior had significant negative correlations with blood urea nitrogen , potassium , phosphorus , as well as the weight gain between two hemodialysis sessions . the present study results show that the use of self - care model had a positive and significant effect on the study patients , as all quality of life dimensions improved after the educational intervention . the results of tsay and lee 's study showed that implementing an adaptation training program for patients with esrd decreased their stress and increased the physical and mental dimensions of their quality of life significantly , which is similar to the results of the current study . assisting the hemodialysis patients the results of the present study showed that using self - care model had significant effects on all quality of life dimensions , including social and physical function , general health , etc . in order to show the positive effects of using the self - care model and develop strategies for better implementation of this model , it is recommended to compare the effects of using this model with those of providing traditional physician visits and also conduct similar studies on hemodialysis patients in other hospitals with larger sample sizes .

peripheral nerve injuries produce acute pain and often induce neuropathic pain , a severe clinical problem and chronic debilitating condition that affects the nervous system . neuropathic pain is relatively common and impairs the quality of life of sufferers . in the past few decades , neuropathic pain animal models have been used to study pain mechanisms and analgesia effects . chronic constrictive injury ( cci ) has been the common neuropathic pain model since 1988 [ 1 , 2 ] . the hippocampus , a part of the limbic system , has the function of learning , memory , emotion , and affect and also has relationships with chronic and acute pain . it has been reported that hippocampal formation plays an important role in pain information processing , including anatomical features , behavioral experiments , electrophysiology , functional imaging , and other molecular research . hippocampal n - acetylaspartate / creatine decreased in elderly patients suffering from acute pain [ 4 , 5 ] . neuropathic pain induced hippocampal interleukin-1 beta ( il-1 ) mrna levels upregulation , and the changes of il-1 mrna expression correlated with the injured side of the hippocampus . xiao et al . , yang et al . , and li et al . reported in a series of studies that acetylcholine ( ach ) influences the pain - induced discharge frequency and the electric activities of pain - related neurons in the hippocampus of rats . acupuncture has been widely applied in china and other asian countries for thousands of years to ameliorate a number of diseases , including acute and chronic pain . acupuncture analgesia has been gradually accepted by people due to its advantages , but its mechanism has not yet been clarified in detail . mounting evidence from many laboratories over the past years suggests that acupuncture has effects on the hippocampus . traditional acupuncture treatments significantly decreased functional magnetic resonance imaging ( fmri ) signals and the metabolism in the hippocampus ; 2 hz electrical acupoint stimulation - induced analgesia has negative correlations with the averaged fmri activation levels of the bilateral hippocampus . electroacupuncture ( ea ) could modulate the function of interneurons in the hippocampus , significantly enhancing long - term potentiation ( ltp ) . in some of our previous studies , chronic pain had an effect on the hippocampal cholinergic system , and ea treatment relieved pain by regulating hippocampal cholinergic neurons . ea had an obvious analgesic effect and in cci rats significantly diminished the injury - induced increase in synaptic cleft width and thinning of the postsynaptic density [ 15 , 16 ] . it also activated the extracellular regulated protein kinases ( erk ) signaling pathway in the hippocampus . previous studies suggested that erk / mitogen - activated protein kinase ( mek ) play an essential role in neuropathic pain . in the spinal cord , cci - induced neuropathic pain activated the erk- and camp - response element binding protein ( creb ) signaling pathway . however , it remains largely unknown how the erk is involved in pain modulation in the hippocampus . on the basis of these studies , the objective of this research is to investigate the effects of ea and the erk signaling pathway on the acute pain - induced responses of pain - related neurons in the hippocampus of wistar rats under both control and neuropathic pain conditions . adult male wistar rats , weighing from 220 g to 280 g ( n = 43 ) , purchased from beijing union medical college , were acclimatized to standard laboratory conditions ( 12-hour light and dark cycle ) at the beijing acupuncture and moxibustion institute for a week and given free access to standard chow pellet diet and water . the rats were randomly divided into three groups : ( 1 ) sham / control group : n = 15 ( sham - operated rats ) ; ( 2 ) cci group , n = 13 ; ( 3 ) u0126 ( erk1/2 inhibitor ) group , n = 15 ; u0126 ( 2.26 l , 10 g ) was injected to the rats . with the automatic injector , all surgical interventions and postoperative animal care were performed in accordance with the guidelines for declaration of the national institutes of health guide for the care and use of laboratory animals ( publication number 80 - 23 , revised 1996 ) . the rats were anesthetized by a mixture of a solution of urethane ( 28 mg/100 g ) plus chloralose ( 3.3 mg/100 g ) . rectal body temperature was monitored throughout the experiment , and a heating pad was used to maintain the temperature of the animals at 37.0c 0.5c . the rats were fixed on the back and the hair on the neck was shaved . the trachea was exposed by blunt dissection through neck muscles after cutting off the neck skin above the manubrium . a t - shaped incision was sheared at the bottom of the thyroid isthmus , and then tracheal intubation was performed using special intubation designed by the laboratory of the institute of acupuncture and moxibustion . in the prone position , the left sciatic nerve was isolated ( the chronic constrictive injured nerve in the cci group ) and covered with liquid paraffin . as described by bennett and xie , cci was used as the neuropathic pain model . local application of antibiotics ( sodium penicillin , 900010000 u / rat ) was used to avoid postoperative infection . the rats were anesthetized by a mixture of urethane ( 28 mg/100 g ) and chloralose ( 3.3 mg/100 g ) . the head of the rat was fixed on the stereotaxic apparatus ( sr-6r , nihon kohden , tokyo , japan ) . with the aid of the stereotaxic atlas of the rat brain , two small skull windows were opened and covered with warm liquid paraffin at the positions for inserting recording electrodes and microsyringe ( kds-310-plus , kd scientific , holliston , ma , usa ) . a glass microelectrode ( 5 m , filled with 3 mol / l kcl ) was inserted into the right hippocampal ca1 area ( ap : 3.23.6 mm ; ml : 2.53.0 mm ; dv : 2.53.2 mm ) by a micromanipulator ( sm-21 , nihon kohden , tokyo , japan ) as recording electrode . another micromanipulator was used to insert the microsyringe into the lateral ventricle ( ap : 1.0 mm ; ml : 1.32.0 mm ; dv : 3.0 mm ) to inject the experimental drug ( mek1/2 inhibitor ) . the neuronal discharges were monitored by an oscilloscope ( vc-10 , nihon kohden , tokyo , japan ) at the same time . after recording spontaneous neural discharge for 5 min as control , the sciatic nerve was stimulated by a double stainless electrode ( delay 0 , interval 50 msec , duration 0.3 msec , and current intensity 5 ma ) for 10 s as noxious stimulation . if there was no change in the neural discharge after noxious stimulation , the neural discharges were not recorded anymore . when the discharge frequency returned to control level ( about 10 min after giving the noxious stimulation ) , the sciatic nerve was given another noxious stimulation for 10 s. at the end of noxious stimulation , bilateral acupoints were punctured with stainless - steel acupuncture needles ( gauge 28 , 0.20 mm in diameter ) to a depth of about 4 mm , respectively , and stimulated electrically using a hans ea stimulator for 1 min , and u0126 was administered intracerebroventricularly for 2 min at the same time . zusanli ( st36 ) and yanglingquan ( gb34 ) are the main points for treating sciatica and other types of leg pain according to the theory of traditional chinese medicine . ( st36 ) and yanglingquan ( gb34 ) has cumulative analgesic effects for neuropathic pain [ 1417 ] . the response of the neurons to noxious stimuli can have three forms : excitement , inhibition , and no response . neurons that respond to nociceptive stimulation are defined as pain - related neurons , neurons excited after noxious stimuli are called pain - excited neurons ( pens ) , and the inhibited neurons are defined as pain - inhibited neurons ( pins ) . the discharge frequencies before the noxious stimulus served as control ( corresponding to 100% ) to observe the changes of discharge frequencies of pain - related neurons . we analyzed the electrical activities of pain - related neurons whose discharge frequencies increased or decreased by more than 20% after noxious stimuli . the experimental data were scanned on a computer with spike ii ( ced instruments , cambridge , united kingdom ) after management and analyzed with spike ii software . all data were expressed as mean sem ( standard error of the mean ) . 29 pain - related neurons were recorded in the control group ( n = 15 ) . the electric activities of 17 pens ( 58.6% ) were increased while those of 12 pins ( 41.4% ) were decreased after noxious stimuli . in the cci group ( n = 13 ) , we recorded 18 pain - related neurons , 14 of which were pens ( 78% ) and 4 were pins ( 22% ) , so the number of recorded pins in the control group was larger than in the cci group . the u0126 group ( n = 15 ) showed 15 pain - related neurons , among which there were 9 pens ( 60% ) and 6 pins ( 40% ) . in sham rats , brief sciatic nerve stimulation significantly increased the electrical activities of 17 hippocampal pens in the control group ( 134.53 18.50% ) and ea group ( 126.1 8.97% ) , and there was no difference between the two groups ( p > 0.05 , figure 1(c ) ) . ea reduced the excitatory effects of brief sciatic nerve stimulation on the firing rates of 17 pens . at 2 min after the noxious stimuli , the discharge frequency changes of hippocampal pens in the ea group ( 121.18 7.45% ) were markedly lower than those in the control group ( 168.68 10.64% , p < 0.05 ) . at 4 min after the noxious stimuli , although the firing rates of hippocampal pens in the control group ( 145.08 7.22% ) were still increased , the firing rates of hippocampal pens in the ea group ( 108.65 4.48% ) had almost returned to normal level . no significant difference was found between the control group ( 116.86 8.21% , 103.22 2.13% ) and the ea group ( 102.14 6.03% , 97.65 4.12% ) at 8 and 10 min after the noxious stimuli . the electric activities of pens in the control group almost returned to normal level at 8 min after the noxious stimuli . it is suggested that ea played an inhibiting role in regulating excitatory effects of the acute noxious stimulus on the electrical activity of pens in sham rats . the electric activities of 12 hippocampal pins were decreased by the brief sciatic nerve stimulation in sham rats ( figure 1(d ) ) , and the frequency changes in the control group were larger than those in the ea group . at 0 minutes ( immediately ) after brief sciatic nerve stimulation , the firing rates in the control group ( 67.88% 6.00 ) were lower than those in the ea group ( 93.35% 6.46 , p < 0.05 ) . the frequencies of pins in the control group were still decreased at 2 min ( 54.35% 4.97 ) , 4 min ( 53.01% 6.12 ) , and 6 min ( 67.93% 7.64 ) and returned to normal level at 10 min ( 94.96% 6.11 ) after the stimulation . the frequencies of pins in the ea group were still at a low level at 2 min ( 74.36% 6.28 ) and 4 min ( 87.37% 3.78 ) and returned to normal level at 6 min ( 100.59% 8.80 ) , earlier than in the control group . in comparison with the control group , the frequency changes of pins in the ea group were higher ( p < 0.05 ) at 2 , 4 , and 6 min after giving the noxious stimuli . those findings suggested that ea played an exciting role in regulating inhibitory effects of the acute noxious stimulus on the electrical activity of pins in sham rats . the electric activities of 14 hippocampal pens reached 216.46 25.40% in the cci group and 219.57 44.15% in the cci + ea group after the noxious stimulus . there was no significant difference between the two groups ( p > 0.05 , figure 2(c ) ) of cci rats . similar to the sham rats , the discharge frequencies of 14 pens were decreased after ea treatment . the frequency changes of hippocampal pens in the cci group ( 193.54 21.50% ) were markedly higher than those in the cci + ea group ( 139.40% 12.78 , p < 0.05 ) at 2 min after the noxious stimuli . the firing rates of pens in the cci + ea group ( 116.99% 20.90 ) returned to normal level and were observably lower than those in the cci group ( 188.82% 16.16 , p < 0.05 ) at 6 min . the frequency changes of pens in the cci + ea group were 105.87% 13.26 and 99.58% 9.06 at 8 min and 10 min after the brief sciatic nerve stimulation , which are lower than those in the cci group ( 167.27% 14.86 , 148.82% 20.71 , p < 0.05 ) . it is suggested that ea treatment also reduced the excitatory effects of brief nerve stimulation on the firing rate of pens in cci rats . the discharge frequencies of 4 hippocampal pins were decreased significantly in both the cci ( 56.44% 8.68 ) and the cci + ea ( 68.27% 7.96 ) group after brief sciatic nerve stimulation ( figure 2(d ) ) . there was no significant difference between the two groups at 0 , 2 , and 4 min . at 6 min after giving the noxious stimuli , the frequencies of pins in the cci group ( 59.82% 9.57 ) were still inhibited , and the frequency of pins in the cci + ea group ( 81.40% 16.29 ) had almost returned to normal level . at 8 and 10 min after giving the noxious stimuli , the frequency changes of hippocampal pins reached 70.65 10.31% and 77.94 2.67% , respectively , in the cci group ; however , the frequencies in the cci + ea group were still at the normal level ( 100.30% 15.36 , 96.69% 5.66 ) . the discharge frequency changes of the cci and the cci + ea group showed no significant differences at 10 min . although the number of recorded pins was small , it is also suggested that ea treatment reduced the effect of brief sciatic nerve stimulation on the frequency of pins in cci rats . the discharges of 17 pens and 12 pins were recorded in the hippocampus of 15 sham rats , and those of 14 pens and 4 pins were recorded in the cci rats . at 2 min , there was no difference between sham and cci rats in the frequency changes ( p > 0.05 , figure 3(a ) ) ; however , brief sciatic nerve stimulation induced bigger frequency changes of pens in the cci rats than in the sham rats ( p < 0.05 ) from 4 min to 8 min . the discharge frequencies of pens in the cci rats were still increased at 10 min , while the discharge frequencies of pens in the sham rats had almost returned to normal level at 8 min after the acute noxious stimuli . the discharge frequency changes of pins in the cci group were slightly higher than in the control group at 2 and 10 min after the noxious stimuli ( p > 0.05 , figure 3(b ) ) . this might be associated with the small number of pins recorded in the cci group . compared with the ea group , the discharge frequency changes of pens in the cci + ea group were slightly increased ( p > 0.05 , figure 3(c ) ) , and those of pins in the cci + ea group were mildly decreased ( p > 0.05 , figure 3(d ) ) from 2 to 10 min after the noxious stimuli . ea suppressed the excitatory and inhibitory effects of the acute noxious stimulus on the electric activities of pens and pins in both sham and cci rats . compared with the ea group , the discharge frequency changes of 9 hippocampal pens in the u0126 group were not significant ( p > 0.05 , figure 4(b ) ) at 0 min . compared with the ea group , the discharge frequency changes of hippocampal pens in the u0126 group ( 195.20 22.98% ) increased significantly ( p < 0.05 ) at 2 min after the noxious stimuli ( after injection of u0126 ) . from 4 to 8 min after giving the noxious stimuli , the discharge frequency changes of pens in the u0126 group ( 165.80 39.32% , 188.32 25.35% , and 181.14 43.41% ) were still higher than those in the ea group ( p < 0.05 ) . at 10 min after giving the noxious stimuli , no significant differences were found between the u0126 group ( 118.34% 20.80 ) and the ea group . there was no significant difference between the u0126 group and the ea group in the discharge frequency changes of 6 hippocampal pins ( p > 0.05 ) at 0 min . the discharge frequency changes of hippocampal pins in the u0126 group ( 55.6% 13.27 ) were bigger compared to those in the ea group at 2 min after the noxious stimuli ( and after injection of u0126 ) . during a period of 6 to 10 min , the discharge frequency changes of pins in the u0126 group exhibited obvious differences when compared with the same period in the ea group ( p < 0.05 ) . the electrical activities of pins gradually returned to baseline levels at 6 min after the noxious stimuli in the ea group , while those in the u0126 group did not . combination of u0126 + ea in sham rats produced effects markedly greater than those observed when brief sciatic nerve stimulation was performed alone ( without drugs ) , so it may be assumed that u0126 increased the effect of noxious stimulation through a pathway other than that of the ea pathway , thus masking the effect of ea . it is suggested that the mek1/2 inhibitor u0126 blocked the ea effect on acute noxious stimulation . pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage , or described in terms of such damage . acute pain is very common in clinical practice , including pain in the perioperative setting , pain in patients with severe or concurrent medical illnesses ( such as arthritis ) , pain related to cancer or cancer treatment , and labor pain . tens of thousands of people are affected by acute pain , and the treatment costs amount to billions of dollars every year . to enhance the quality of life and maintain the patient 's function ability , clinical medicine is used to treat acute pain , but these medications have substantial side effects . acupuncture analgesia has its advantages , such as an obvious analgesic effect and little side effects , but its mechanism of action is not clarified at the moment . as a part of the limbic system , the hippocampus is associated with memory , but also with pain and acupuncture analgesia . the dorsal hippocampal dopamine receptors exert an analgesic effect during the orofacial pain test ; microinjections of nonsteroidal anti - inflammatory drugs or 2-ag into the hippocampus induce antinociception . a recent study indicates that persistent peripheral nociception induced by subcutaneous injection of bee venom upregulated mtor target p70 s6 kinase signaling and facilitated long - term potentiation which could be reversed by mtor inhibitor in the hippocampus . in our experiment , we observed that the discharge frequencies of hippocampal ca1 pain - related neurons ( pens or pins ) were changed after brief electrical impulses applied to the sciatic nerve . the electric activities of pens and pins returned to normal level at 8 and 10 min after the noxious stimuli , respectively . focused on electric activities of pain - related neurons in the hippocampus after noxious stimuli for many years . in the hippocampal ca1 and ca3 area and dentate gyrus , cholinergic neurons and muscarinic receptors have effects on the electric activities of pens and pins , so that they are involved in pain modulation [ 79 , 32 ] . glutamate and its receptors , noradrenaline ( ne ) , phentolamine , and alpha - adrenoceptors also have effects on pain modulation by regulating electric activities of pens and pins in the hippocampal ca3 region [ 33 , 34 ] . these studies reported the effects of pain - induced discharges of hippocampal neurons , but few research papers pay attention to electric activities of hippocampal neurons in a chronic pain state . hains et al . reported that after spinal cord contusion injury the changes of the spontaneous discharge and afterdischarge of extracellular neurons in the thalamus are related to an upregulated sodium channel expression . in cci rats , the acute noxious stimulation evoked greater changes of electric activities of pens and pins , and the recorded number of hippocampal pens was obviously bigger and that of pins smaller compared with sham rats . it took more time for the pain - related neurons to return to normal level in the cci rats than in sham rats . this might be because the effect of brief sciatic nerve stimulation on firing rates of pens and pins was enhanced under the advanced cci situation . the cci rats may have experienced more pain than the sham rats after the short noxious stimulation . the cci rats were in a state of hyperalgesia , an increased response to noxious stimuli , which means the same intensity of electrical stimulation causes more pain intensity and finally a prolonged time of recovery to normal . shi et al . reported that pain - related neurons were involved in the modulation of ea analgesia , and ea stimulation resulted in an inhibiting effect on the electrical activity of pens and an activating effect on the electrical activity of pins . after ea at the acupoint hegu , or dolantin given intravenously , gao et al . stimulated the head of the caudate nucleus , eliciting an inhibitory effect on pens and a reduction of inhibition or release on pins . ea has been shown to suppress pens and excite pins , which can be taken as an electrophysiological index for ea analgesia [ 38 , 39 ] . yang et al . also reported that ea treatment could play an inhibiting role in mediating the evoked discharge of pens and an activating role in that of pins , and cholecystokinin-8 's ( cck-8 ) b receptor antagonist could be antagonistic to the effect of cck on ea analgesia . figure 1 ) showed that , in the hippocampal ca1 area , ea inhibited the excitatory effect of brief sciatic nerve stimulation on the electric activities of pens and activated the inhibitory effect of brief sciatic nerve stimulation on electric activities of pins in sham rats , which caused the firing rates of pens and pins to return to normal level at 6 min after the noxious stimuli , earlier than in the control group . these findings suggest that the analgesic effect of ea is related to the electric activities of pain - related neurons in the ca1 area , which were affected by noxious stimulation . ea reduced the effect of acute noxious stimulation on the electric activities of pens and pins . the electric activities of pens and pins play an important role in mediating ea analgesia , as reported before . in cci rats , ea also had an effect on the firing rate of pens and pins similar to that evoked by acute noxious stimuli , but the electric activities returned to normal level at 6 or 8 min after administration of the noxious stimulus . it is suggested that the effect of ea treatment is closely related to the severity of pain . intrathecal administration of the inhibitor of the mapk family members mek1/2 , such as u0126 , pd198306 , and pd98059 , had analgesic effects and significantly potentiated the effectiveness of opioids in neuropathy in the spinal cord [ 4143 ] . u0126 downregulated the increased late responses and afterdischarge induced by melittin ( a pain - related peptidergic component ) in wide - dynamic - range neurons of the spinal cord . accumulating evidence showed that erk expression increased at the peripheral nerve and spinal cord horn [ 4547 ] . phospho - erk ( perk ) in the spinal cord is activated immediately in neurons ( < 6 h ) , then in microglia on days 1 and 3 and both in astrocytes and in microglia on day 10 , and finally in satellite cells on days 10 and 21 after spinal nerve ligation , and this activation contributes to mechanical allodynia . pd 98059 may modulate the nociceptive factors and antinociceptive factors that are released by glial cells , which have a close relationship with reduced symptoms of neuropathy [ 43 , 46 ] . these reports suggested that mek1/2 inhibitors have an analgesic effect on neuropathic models in the primary injury and that neuropathic pain is associated with the activity of the mapk / erk signal pathway within the spinal cord . few studies focused on the relationship between the expression of erk and the hippocampus in chronic neuropathic pain , and an agreement has not been reached [ 48 , 49 ] . the phosphorylation of the erk signal transduction pathway was enhanced by ea in depression rat tissue ; however , there was no report on the study of ea analgesia on the hippocampus erk signal pathway . our previous results showed that in the hippocampus the erk signal pathway was inhibited in chronic neuropathic rats , and after acupuncture treatment the erk signaling pathway was activated . our experimental results showed that , compared with the ea group , brief sciatic nerve stimulation produced a greater excitatory and inhibitory effect on the firing rate of pens and pins in u0126 + ea group , suggesting that u0126 suppressed the effect of ea on the analgesic pathway . it is suggested that involvement of the activation of erk signaling pathway in the hippocampal ca1 region in ea treatment induced pain relief . in cci rats , acute noxious stimulation required a longer time for the firing rate of pain - related neurons to return to normal level ; ea treatment could suppress the effect of the noxious stimulus on pens and pins in both sham and cci rats , which suggests a close relationship with the ea analgesic effect ; and the erk signal pathway is probably involved in pain and ea analgesia .

to study the effects of acupuncture analgesia on the hippocampus , we observed the effects of electroacupuncture ( ea ) and mitogen - activated protein kinase ( mek ) inhibitor on pain - excited neurons ( pens ) and pain - inhibited neurons ( pins ) in the hippocampal area ca1 of sham or chronic constrictive injury ( cci ) rats . the animals were randomly divided into a control , a cci , and a u0126 ( mek1/2 inhibitor ) group . in all experiments , we briefly ( 10-second duration ) stimulated the sciatic nerve electrically and recorded the firing rates of pens and pins . the results showed that in both sham and cci rats brief sciatic nerve stimulation significantly increased the electrical activity of pens and markedly decreased the electrical activity of pins . these effects were significantly greater in cci rats compared to sham rats . ea treatment reduced the effects of the noxious stimulus on pens and pins in both sham and cci rats . the effects of ea treatment could be inhibited by u0126 in sham - operated rats . the results suggest that ea reduces effects of acute sciatic nerve stimulation on pens and pins in the ca1 region of the hippocampus of both sham and cci rats and that the erk ( extracellular regulated kinase ) signaling pathway is involved in the modulation of ea analgesia .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

chronic constrictive injury ( cci ) has been the common neuropathic pain model since 1988 [ 1 , 2 ] . the hippocampus , a part of the limbic system , has the function of learning , memory , emotion , and affect and also has relationships with chronic and acute pain . neuropathic pain induced hippocampal interleukin-1 beta ( il-1 ) mrna levels upregulation , and the changes of il-1 mrna expression correlated with the injured side of the hippocampus . reported in a series of studies that acetylcholine ( ach ) influences the pain - induced discharge frequency and the electric activities of pain - related neurons in the hippocampus of rats . traditional acupuncture treatments significantly decreased functional magnetic resonance imaging ( fmri ) signals and the metabolism in the hippocampus ; 2 hz electrical acupoint stimulation - induced analgesia has negative correlations with the averaged fmri activation levels of the bilateral hippocampus . electroacupuncture ( ea ) could modulate the function of interneurons in the hippocampus , significantly enhancing long - term potentiation ( ltp ) . in some of our previous studies , chronic pain had an effect on the hippocampal cholinergic system , and ea treatment relieved pain by regulating hippocampal cholinergic neurons . ea had an obvious analgesic effect and in cci rats significantly diminished the injury - induced increase in synaptic cleft width and thinning of the postsynaptic density [ 15 , 16 ] . it also activated the extracellular regulated protein kinases ( erk ) signaling pathway in the hippocampus . previous studies suggested that erk / mitogen - activated protein kinase ( mek ) play an essential role in neuropathic pain . in the spinal cord , cci - induced neuropathic pain activated the erk- and camp - response element binding protein ( creb ) signaling pathway . however , it remains largely unknown how the erk is involved in pain modulation in the hippocampus . on the basis of these studies , the objective of this research is to investigate the effects of ea and the erk signaling pathway on the acute pain - induced responses of pain - related neurons in the hippocampus of wistar rats under both control and neuropathic pain conditions . the rats were randomly divided into three groups : ( 1 ) sham / control group : n = 15 ( sham - operated rats ) ; ( 2 ) cci group , n = 13 ; ( 3 ) u0126 ( erk1/2 inhibitor ) group , n = 15 ; u0126 ( 2.26 l , 10 g ) was injected to the rats . rectal body temperature was monitored throughout the experiment , and a heating pad was used to maintain the temperature of the animals at 37.0c 0.5c . a t - shaped incision was sheared at the bottom of the thyroid isthmus , and then tracheal intubation was performed using special intubation designed by the laboratory of the institute of acupuncture and moxibustion . in the prone position , the left sciatic nerve was isolated ( the chronic constrictive injured nerve in the cci group ) and covered with liquid paraffin . another micromanipulator was used to insert the microsyringe into the lateral ventricle ( ap : 1.0 mm ; ml : 1.32.0 mm ; dv : 3.0 mm ) to inject the experimental drug ( mek1/2 inhibitor ) . after recording spontaneous neural discharge for 5 min as control , the sciatic nerve was stimulated by a double stainless electrode ( delay 0 , interval 50 msec , duration 0.3 msec , and current intensity 5 ma ) for 10 s as noxious stimulation . when the discharge frequency returned to control level ( about 10 min after giving the noxious stimulation ) , the sciatic nerve was given another noxious stimulation for 10 s. at the end of noxious stimulation , bilateral acupoints were punctured with stainless - steel acupuncture needles ( gauge 28 , 0.20 mm in diameter ) to a depth of about 4 mm , respectively , and stimulated electrically using a hans ea stimulator for 1 min , and u0126 was administered intracerebroventricularly for 2 min at the same time . neurons that respond to nociceptive stimulation are defined as pain - related neurons , neurons excited after noxious stimuli are called pain - excited neurons ( pens ) , and the inhibited neurons are defined as pain - inhibited neurons ( pins ) . the discharge frequencies before the noxious stimulus served as control ( corresponding to 100% ) to observe the changes of discharge frequencies of pain - related neurons . we analyzed the electrical activities of pain - related neurons whose discharge frequencies increased or decreased by more than 20% after noxious stimuli . in the cci group ( n = 13 ) , we recorded 18 pain - related neurons , 14 of which were pens ( 78% ) and 4 were pins ( 22% ) , so the number of recorded pins in the control group was larger than in the cci group . in sham rats , brief sciatic nerve stimulation significantly increased the electrical activities of 17 hippocampal pens in the control group ( 134.53 18.50% ) and ea group ( 126.1 8.97% ) , and there was no difference between the two groups ( p > 0.05 , figure 1(c ) ) . ea reduced the excitatory effects of brief sciatic nerve stimulation on the firing rates of 17 pens . at 4 min after the noxious stimuli , although the firing rates of hippocampal pens in the control group ( 145.08 7.22% ) were still increased , the firing rates of hippocampal pens in the ea group ( 108.65 4.48% ) had almost returned to normal level . no significant difference was found between the control group ( 116.86 8.21% , 103.22 2.13% ) and the ea group ( 102.14 6.03% , 97.65 4.12% ) at 8 and 10 min after the noxious stimuli . the electric activities of pens in the control group almost returned to normal level at 8 min after the noxious stimuli . it is suggested that ea played an inhibiting role in regulating excitatory effects of the acute noxious stimulus on the electrical activity of pens in sham rats . the electric activities of 12 hippocampal pins were decreased by the brief sciatic nerve stimulation in sham rats ( figure 1(d ) ) , and the frequency changes in the control group were larger than those in the ea group . at 0 minutes ( immediately ) after brief sciatic nerve stimulation , the firing rates in the control group ( 67.88% 6.00 ) were lower than those in the ea group ( 93.35% 6.46 , p < 0.05 ) . the frequencies of pins in the control group were still decreased at 2 min ( 54.35% 4.97 ) , 4 min ( 53.01% 6.12 ) , and 6 min ( 67.93% 7.64 ) and returned to normal level at 10 min ( 94.96% 6.11 ) after the stimulation . the frequencies of pins in the ea group were still at a low level at 2 min ( 74.36% 6.28 ) and 4 min ( 87.37% 3.78 ) and returned to normal level at 6 min ( 100.59% 8.80 ) , earlier than in the control group . in comparison with the control group , the frequency changes of pins in the ea group were higher ( p < 0.05 ) at 2 , 4 , and 6 min after giving the noxious stimuli . those findings suggested that ea played an exciting role in regulating inhibitory effects of the acute noxious stimulus on the electrical activity of pins in sham rats . the electric activities of 14 hippocampal pens reached 216.46 25.40% in the cci group and 219.57 44.15% in the cci + ea group after the noxious stimulus . the firing rates of pens in the cci + ea group ( 116.99% 20.90 ) returned to normal level and were observably lower than those in the cci group ( 188.82% 16.16 , p < 0.05 ) at 6 min . the frequency changes of pens in the cci + ea group were 105.87% 13.26 and 99.58% 9.06 at 8 min and 10 min after the brief sciatic nerve stimulation , which are lower than those in the cci group ( 167.27% 14.86 , 148.82% 20.71 , p < 0.05 ) . it is suggested that ea treatment also reduced the excitatory effects of brief nerve stimulation on the firing rate of pens in cci rats . the discharge frequencies of 4 hippocampal pins were decreased significantly in both the cci ( 56.44% 8.68 ) and the cci + ea ( 68.27% 7.96 ) group after brief sciatic nerve stimulation ( figure 2(d ) ) . at 6 min after giving the noxious stimuli , the frequencies of pins in the cci group ( 59.82% 9.57 ) were still inhibited , and the frequency of pins in the cci + ea group ( 81.40% 16.29 ) had almost returned to normal level . at 8 and 10 min after giving the noxious stimuli , the frequency changes of hippocampal pins reached 70.65 10.31% and 77.94 2.67% , respectively , in the cci group ; however , the frequencies in the cci + ea group were still at the normal level ( 100.30% 15.36 , 96.69% 5.66 ) . although the number of recorded pins was small , it is also suggested that ea treatment reduced the effect of brief sciatic nerve stimulation on the frequency of pins in cci rats . the discharges of 17 pens and 12 pins were recorded in the hippocampus of 15 sham rats , and those of 14 pens and 4 pins were recorded in the cci rats . at 2 min , there was no difference between sham and cci rats in the frequency changes ( p > 0.05 , figure 3(a ) ) ; however , brief sciatic nerve stimulation induced bigger frequency changes of pens in the cci rats than in the sham rats ( p < 0.05 ) from 4 min to 8 min . the discharge frequencies of pens in the cci rats were still increased at 10 min , while the discharge frequencies of pens in the sham rats had almost returned to normal level at 8 min after the acute noxious stimuli . the discharge frequency changes of pins in the cci group were slightly higher than in the control group at 2 and 10 min after the noxious stimuli ( p > 0.05 , figure 3(b ) ) . this might be associated with the small number of pins recorded in the cci group . compared with the ea group , the discharge frequency changes of pens in the cci + ea group were slightly increased ( p > 0.05 , figure 3(c ) ) , and those of pins in the cci + ea group were mildly decreased ( p > 0.05 , figure 3(d ) ) from 2 to 10 min after the noxious stimuli . ea suppressed the excitatory and inhibitory effects of the acute noxious stimulus on the electric activities of pens and pins in both sham and cci rats . from 4 to 8 min after giving the noxious stimuli , the discharge frequency changes of pens in the u0126 group ( 165.80 39.32% , 188.32 25.35% , and 181.14 43.41% ) were still higher than those in the ea group ( p < 0.05 ) . at 10 min after giving the noxious stimuli , no significant differences were found between the u0126 group ( 118.34% 20.80 ) and the ea group . there was no significant difference between the u0126 group and the ea group in the discharge frequency changes of 6 hippocampal pins ( p > 0.05 ) at 0 min . the discharge frequency changes of hippocampal pins in the u0126 group ( 55.6% 13.27 ) were bigger compared to those in the ea group at 2 min after the noxious stimuli ( and after injection of u0126 ) . during a period of 6 to 10 min , the discharge frequency changes of pins in the u0126 group exhibited obvious differences when compared with the same period in the ea group ( p < 0.05 ) . the electrical activities of pins gradually returned to baseline levels at 6 min after the noxious stimuli in the ea group , while those in the u0126 group did not . combination of u0126 + ea in sham rats produced effects markedly greater than those observed when brief sciatic nerve stimulation was performed alone ( without drugs ) , so it may be assumed that u0126 increased the effect of noxious stimulation through a pathway other than that of the ea pathway , thus masking the effect of ea . as a part of the limbic system , the hippocampus is associated with memory , but also with pain and acupuncture analgesia . a recent study indicates that persistent peripheral nociception induced by subcutaneous injection of bee venom upregulated mtor target p70 s6 kinase signaling and facilitated long - term potentiation which could be reversed by mtor inhibitor in the hippocampus . in our experiment , we observed that the discharge frequencies of hippocampal ca1 pain - related neurons ( pens or pins ) were changed after brief electrical impulses applied to the sciatic nerve . the electric activities of pens and pins returned to normal level at 8 and 10 min after the noxious stimuli , respectively . focused on electric activities of pain - related neurons in the hippocampus after noxious stimuli for many years . in the hippocampal ca1 and ca3 area and dentate gyrus , cholinergic neurons and muscarinic receptors have effects on the electric activities of pens and pins , so that they are involved in pain modulation [ 79 , 32 ] . glutamate and its receptors , noradrenaline ( ne ) , phentolamine , and alpha - adrenoceptors also have effects on pain modulation by regulating electric activities of pens and pins in the hippocampal ca3 region [ 33 , 34 ] . these studies reported the effects of pain - induced discharges of hippocampal neurons , but few research papers pay attention to electric activities of hippocampal neurons in a chronic pain state . reported that after spinal cord contusion injury the changes of the spontaneous discharge and afterdischarge of extracellular neurons in the thalamus are related to an upregulated sodium channel expression . in cci rats , the acute noxious stimulation evoked greater changes of electric activities of pens and pins , and the recorded number of hippocampal pens was obviously bigger and that of pins smaller compared with sham rats . it took more time for the pain - related neurons to return to normal level in the cci rats than in sham rats . this might be because the effect of brief sciatic nerve stimulation on firing rates of pens and pins was enhanced under the advanced cci situation . reported that pain - related neurons were involved in the modulation of ea analgesia , and ea stimulation resulted in an inhibiting effect on the electrical activity of pens and an activating effect on the electrical activity of pins . stimulated the head of the caudate nucleus , eliciting an inhibitory effect on pens and a reduction of inhibition or release on pins . also reported that ea treatment could play an inhibiting role in mediating the evoked discharge of pens and an activating role in that of pins , and cholecystokinin-8 's ( cck-8 ) b receptor antagonist could be antagonistic to the effect of cck on ea analgesia . figure 1 ) showed that , in the hippocampal ca1 area , ea inhibited the excitatory effect of brief sciatic nerve stimulation on the electric activities of pens and activated the inhibitory effect of brief sciatic nerve stimulation on electric activities of pins in sham rats , which caused the firing rates of pens and pins to return to normal level at 6 min after the noxious stimuli , earlier than in the control group . these findings suggest that the analgesic effect of ea is related to the electric activities of pain - related neurons in the ca1 area , which were affected by noxious stimulation . ea reduced the effect of acute noxious stimulation on the electric activities of pens and pins . the electric activities of pens and pins play an important role in mediating ea analgesia , as reported before . in cci rats , ea also had an effect on the firing rate of pens and pins similar to that evoked by acute noxious stimuli , but the electric activities returned to normal level at 6 or 8 min after administration of the noxious stimulus . it is suggested that the effect of ea treatment is closely related to the severity of pain . intrathecal administration of the inhibitor of the mapk family members mek1/2 , such as u0126 , pd198306 , and pd98059 , had analgesic effects and significantly potentiated the effectiveness of opioids in neuropathy in the spinal cord [ 4143 ] . u0126 downregulated the increased late responses and afterdischarge induced by melittin ( a pain - related peptidergic component ) in wide - dynamic - range neurons of the spinal cord . phospho - erk ( perk ) in the spinal cord is activated immediately in neurons ( < 6 h ) , then in microglia on days 1 and 3 and both in astrocytes and in microglia on day 10 , and finally in satellite cells on days 10 and 21 after spinal nerve ligation , and this activation contributes to mechanical allodynia . these reports suggested that mek1/2 inhibitors have an analgesic effect on neuropathic models in the primary injury and that neuropathic pain is associated with the activity of the mapk / erk signal pathway within the spinal cord . few studies focused on the relationship between the expression of erk and the hippocampus in chronic neuropathic pain , and an agreement has not been reached [ 48 , 49 ] . the phosphorylation of the erk signal transduction pathway was enhanced by ea in depression rat tissue ; however , there was no report on the study of ea analgesia on the hippocampus erk signal pathway . our previous results showed that in the hippocampus the erk signal pathway was inhibited in chronic neuropathic rats , and after acupuncture treatment the erk signaling pathway was activated . our experimental results showed that , compared with the ea group , brief sciatic nerve stimulation produced a greater excitatory and inhibitory effect on the firing rate of pens and pins in u0126 + ea group , suggesting that u0126 suppressed the effect of ea on the analgesic pathway . it is suggested that involvement of the activation of erk signaling pathway in the hippocampal ca1 region in ea treatment induced pain relief . in cci rats , acute noxious stimulation required a longer time for the firing rate of pain - related neurons to return to normal level ; ea treatment could suppress the effect of the noxious stimulus on pens and pins in both sham and cci rats , which suggests a close relationship with the ea analgesic effect ; and the erk signal pathway is probably involved in pain and ea analgesia .

the hippocampus , a part of the limbic system , has the function of learning , memory , emotion , and affect and also has relationships with chronic and acute pain . it has been reported that hippocampal formation plays an important role in pain information processing , including anatomical features , behavioral experiments , electrophysiology , functional imaging , and other molecular research . neuropathic pain induced hippocampal interleukin-1 beta ( il-1 ) mrna levels upregulation , and the changes of il-1 mrna expression correlated with the injured side of the hippocampus . reported in a series of studies that acetylcholine ( ach ) influences the pain - induced discharge frequency and the electric activities of pain - related neurons in the hippocampus of rats . traditional acupuncture treatments significantly decreased functional magnetic resonance imaging ( fmri ) signals and the metabolism in the hippocampus ; 2 hz electrical acupoint stimulation - induced analgesia has negative correlations with the averaged fmri activation levels of the bilateral hippocampus . electroacupuncture ( ea ) could modulate the function of interneurons in the hippocampus , significantly enhancing long - term potentiation ( ltp ) . in some of our previous studies , chronic pain had an effect on the hippocampal cholinergic system , and ea treatment relieved pain by regulating hippocampal cholinergic neurons . ea had an obvious analgesic effect and in cci rats significantly diminished the injury - induced increase in synaptic cleft width and thinning of the postsynaptic density [ 15 , 16 ] . in the spinal cord , cci - induced neuropathic pain activated the erk- and camp - response element binding protein ( creb ) signaling pathway . however , it remains largely unknown how the erk is involved in pain modulation in the hippocampus . on the basis of these studies , the objective of this research is to investigate the effects of ea and the erk signaling pathway on the acute pain - induced responses of pain - related neurons in the hippocampus of wistar rats under both control and neuropathic pain conditions . adult male wistar rats , weighing from 220 g to 280 g ( n = 43 ) , purchased from beijing union medical college , were acclimatized to standard laboratory conditions ( 12-hour light and dark cycle ) at the beijing acupuncture and moxibustion institute for a week and given free access to standard chow pellet diet and water . the rats were randomly divided into three groups : ( 1 ) sham / control group : n = 15 ( sham - operated rats ) ; ( 2 ) cci group , n = 13 ; ( 3 ) u0126 ( erk1/2 inhibitor ) group , n = 15 ; u0126 ( 2.26 l , 10 g ) was injected to the rats . with the automatic injector , all surgical interventions and postoperative animal care were performed in accordance with the guidelines for declaration of the national institutes of health guide for the care and use of laboratory animals ( publication number 80 - 23 , revised 1996 ) . a t - shaped incision was sheared at the bottom of the thyroid isthmus , and then tracheal intubation was performed using special intubation designed by the laboratory of the institute of acupuncture and moxibustion . in the prone position , the left sciatic nerve was isolated ( the chronic constrictive injured nerve in the cci group ) and covered with liquid paraffin . with the aid of the stereotaxic atlas of the rat brain , two small skull windows were opened and covered with warm liquid paraffin at the positions for inserting recording electrodes and microsyringe ( kds-310-plus , kd scientific , holliston , ma , usa ) . when the discharge frequency returned to control level ( about 10 min after giving the noxious stimulation ) , the sciatic nerve was given another noxious stimulation for 10 s. at the end of noxious stimulation , bilateral acupoints were punctured with stainless - steel acupuncture needles ( gauge 28 , 0.20 mm in diameter ) to a depth of about 4 mm , respectively , and stimulated electrically using a hans ea stimulator for 1 min , and u0126 was administered intracerebroventricularly for 2 min at the same time . zusanli ( st36 ) and yanglingquan ( gb34 ) are the main points for treating sciatica and other types of leg pain according to the theory of traditional chinese medicine . neurons that respond to nociceptive stimulation are defined as pain - related neurons , neurons excited after noxious stimuli are called pain - excited neurons ( pens ) , and the inhibited neurons are defined as pain - inhibited neurons ( pins ) . the discharge frequencies before the noxious stimulus served as control ( corresponding to 100% ) to observe the changes of discharge frequencies of pain - related neurons . we analyzed the electrical activities of pain - related neurons whose discharge frequencies increased or decreased by more than 20% after noxious stimuli . in the cci group ( n = 13 ) , we recorded 18 pain - related neurons , 14 of which were pens ( 78% ) and 4 were pins ( 22% ) , so the number of recorded pins in the control group was larger than in the cci group . the u0126 group ( n = 15 ) showed 15 pain - related neurons , among which there were 9 pens ( 60% ) and 6 pins ( 40% ) . in sham rats , brief sciatic nerve stimulation significantly increased the electrical activities of 17 hippocampal pens in the control group ( 134.53 18.50% ) and ea group ( 126.1 8.97% ) , and there was no difference between the two groups ( p > 0.05 , figure 1(c ) ) . at 2 min after the noxious stimuli , the discharge frequency changes of hippocampal pens in the ea group ( 121.18 7.45% ) were markedly lower than those in the control group ( 168.68 10.64% , p < 0.05 ) . at 4 min after the noxious stimuli , although the firing rates of hippocampal pens in the control group ( 145.08 7.22% ) were still increased , the firing rates of hippocampal pens in the ea group ( 108.65 4.48% ) had almost returned to normal level . no significant difference was found between the control group ( 116.86 8.21% , 103.22 2.13% ) and the ea group ( 102.14 6.03% , 97.65 4.12% ) at 8 and 10 min after the noxious stimuli . the electric activities of 12 hippocampal pins were decreased by the brief sciatic nerve stimulation in sham rats ( figure 1(d ) ) , and the frequency changes in the control group were larger than those in the ea group . at 0 minutes ( immediately ) after brief sciatic nerve stimulation , the firing rates in the control group ( 67.88% 6.00 ) were lower than those in the ea group ( 93.35% 6.46 , p < 0.05 ) . the frequencies of pins in the control group were still decreased at 2 min ( 54.35% 4.97 ) , 4 min ( 53.01% 6.12 ) , and 6 min ( 67.93% 7.64 ) and returned to normal level at 10 min ( 94.96% 6.11 ) after the stimulation . the frequencies of pins in the ea group were still at a low level at 2 min ( 74.36% 6.28 ) and 4 min ( 87.37% 3.78 ) and returned to normal level at 6 min ( 100.59% 8.80 ) , earlier than in the control group . in comparison with the control group , the frequency changes of pins in the ea group were higher ( p < 0.05 ) at 2 , 4 , and 6 min after giving the noxious stimuli . those findings suggested that ea played an exciting role in regulating inhibitory effects of the acute noxious stimulus on the electrical activity of pins in sham rats . the electric activities of 14 hippocampal pens reached 216.46 25.40% in the cci group and 219.57 44.15% in the cci + ea group after the noxious stimulus . the frequency changes of hippocampal pens in the cci group ( 193.54 21.50% ) were markedly higher than those in the cci + ea group ( 139.40% 12.78 , p < 0.05 ) at 2 min after the noxious stimuli . the firing rates of pens in the cci + ea group ( 116.99% 20.90 ) returned to normal level and were observably lower than those in the cci group ( 188.82% 16.16 , p < 0.05 ) at 6 min . the frequency changes of pens in the cci + ea group were 105.87% 13.26 and 99.58% 9.06 at 8 min and 10 min after the brief sciatic nerve stimulation , which are lower than those in the cci group ( 167.27% 14.86 , 148.82% 20.71 , p < 0.05 ) . the discharge frequencies of 4 hippocampal pins were decreased significantly in both the cci ( 56.44% 8.68 ) and the cci + ea ( 68.27% 7.96 ) group after brief sciatic nerve stimulation ( figure 2(d ) ) . at 6 min after giving the noxious stimuli , the frequencies of pins in the cci group ( 59.82% 9.57 ) were still inhibited , and the frequency of pins in the cci + ea group ( 81.40% 16.29 ) had almost returned to normal level . at 8 and 10 min after giving the noxious stimuli , the frequency changes of hippocampal pins reached 70.65 10.31% and 77.94 2.67% , respectively , in the cci group ; however , the frequencies in the cci + ea group were still at the normal level ( 100.30% 15.36 , 96.69% 5.66 ) . the discharges of 17 pens and 12 pins were recorded in the hippocampus of 15 sham rats , and those of 14 pens and 4 pins were recorded in the cci rats . at 2 min , there was no difference between sham and cci rats in the frequency changes ( p > 0.05 , figure 3(a ) ) ; however , brief sciatic nerve stimulation induced bigger frequency changes of pens in the cci rats than in the sham rats ( p < 0.05 ) from 4 min to 8 min . the discharge frequencies of pens in the cci rats were still increased at 10 min , while the discharge frequencies of pens in the sham rats had almost returned to normal level at 8 min after the acute noxious stimuli . the discharge frequency changes of pins in the cci group were slightly higher than in the control group at 2 and 10 min after the noxious stimuli ( p > 0.05 , figure 3(b ) ) . compared with the ea group , the discharge frequency changes of pens in the cci + ea group were slightly increased ( p > 0.05 , figure 3(c ) ) , and those of pins in the cci + ea group were mildly decreased ( p > 0.05 , figure 3(d ) ) from 2 to 10 min after the noxious stimuli . ea suppressed the excitatory and inhibitory effects of the acute noxious stimulus on the electric activities of pens and pins in both sham and cci rats . compared with the ea group , the discharge frequency changes of 9 hippocampal pens in the u0126 group were not significant ( p > 0.05 , figure 4(b ) ) at 0 min . compared with the ea group , the discharge frequency changes of hippocampal pens in the u0126 group ( 195.20 22.98% ) increased significantly ( p < 0.05 ) at 2 min after the noxious stimuli ( after injection of u0126 ) . from 4 to 8 min after giving the noxious stimuli , the discharge frequency changes of pens in the u0126 group ( 165.80 39.32% , 188.32 25.35% , and 181.14 43.41% ) were still higher than those in the ea group ( p < 0.05 ) . there was no significant difference between the u0126 group and the ea group in the discharge frequency changes of 6 hippocampal pins ( p > 0.05 ) at 0 min . the discharge frequency changes of hippocampal pins in the u0126 group ( 55.6% 13.27 ) were bigger compared to those in the ea group at 2 min after the noxious stimuli ( and after injection of u0126 ) . during a period of 6 to 10 min , the discharge frequency changes of pins in the u0126 group exhibited obvious differences when compared with the same period in the ea group ( p < 0.05 ) . the electrical activities of pins gradually returned to baseline levels at 6 min after the noxious stimuli in the ea group , while those in the u0126 group did not . combination of u0126 + ea in sham rats produced effects markedly greater than those observed when brief sciatic nerve stimulation was performed alone ( without drugs ) , so it may be assumed that u0126 increased the effect of noxious stimulation through a pathway other than that of the ea pathway , thus masking the effect of ea . acute pain is very common in clinical practice , including pain in the perioperative setting , pain in patients with severe or concurrent medical illnesses ( such as arthritis ) , pain related to cancer or cancer treatment , and labor pain . a recent study indicates that persistent peripheral nociception induced by subcutaneous injection of bee venom upregulated mtor target p70 s6 kinase signaling and facilitated long - term potentiation which could be reversed by mtor inhibitor in the hippocampus . in our experiment , we observed that the discharge frequencies of hippocampal ca1 pain - related neurons ( pens or pins ) were changed after brief electrical impulses applied to the sciatic nerve . in the hippocampal ca1 and ca3 area and dentate gyrus , cholinergic neurons and muscarinic receptors have effects on the electric activities of pens and pins , so that they are involved in pain modulation [ 79 , 32 ] . glutamate and its receptors , noradrenaline ( ne ) , phentolamine , and alpha - adrenoceptors also have effects on pain modulation by regulating electric activities of pens and pins in the hippocampal ca3 region [ 33 , 34 ] . these studies reported the effects of pain - induced discharges of hippocampal neurons , but few research papers pay attention to electric activities of hippocampal neurons in a chronic pain state . reported that after spinal cord contusion injury the changes of the spontaneous discharge and afterdischarge of extracellular neurons in the thalamus are related to an upregulated sodium channel expression . in cci rats , the acute noxious stimulation evoked greater changes of electric activities of pens and pins , and the recorded number of hippocampal pens was obviously bigger and that of pins smaller compared with sham rats . the cci rats were in a state of hyperalgesia , an increased response to noxious stimuli , which means the same intensity of electrical stimulation causes more pain intensity and finally a prolonged time of recovery to normal . reported that pain - related neurons were involved in the modulation of ea analgesia , and ea stimulation resulted in an inhibiting effect on the electrical activity of pens and an activating effect on the electrical activity of pins . also reported that ea treatment could play an inhibiting role in mediating the evoked discharge of pens and an activating role in that of pins , and cholecystokinin-8 's ( cck-8 ) b receptor antagonist could be antagonistic to the effect of cck on ea analgesia . figure 1 ) showed that , in the hippocampal ca1 area , ea inhibited the excitatory effect of brief sciatic nerve stimulation on the electric activities of pens and activated the inhibitory effect of brief sciatic nerve stimulation on electric activities of pins in sham rats , which caused the firing rates of pens and pins to return to normal level at 6 min after the noxious stimuli , earlier than in the control group . these findings suggest that the analgesic effect of ea is related to the electric activities of pain - related neurons in the ca1 area , which were affected by noxious stimulation . in cci rats , ea also had an effect on the firing rate of pens and pins similar to that evoked by acute noxious stimuli , but the electric activities returned to normal level at 6 or 8 min after administration of the noxious stimulus . intrathecal administration of the inhibitor of the mapk family members mek1/2 , such as u0126 , pd198306 , and pd98059 , had analgesic effects and significantly potentiated the effectiveness of opioids in neuropathy in the spinal cord [ 4143 ] . u0126 downregulated the increased late responses and afterdischarge induced by melittin ( a pain - related peptidergic component ) in wide - dynamic - range neurons of the spinal cord . phospho - erk ( perk ) in the spinal cord is activated immediately in neurons ( < 6 h ) , then in microglia on days 1 and 3 and both in astrocytes and in microglia on day 10 , and finally in satellite cells on days 10 and 21 after spinal nerve ligation , and this activation contributes to mechanical allodynia . these reports suggested that mek1/2 inhibitors have an analgesic effect on neuropathic models in the primary injury and that neuropathic pain is associated with the activity of the mapk / erk signal pathway within the spinal cord . few studies focused on the relationship between the expression of erk and the hippocampus in chronic neuropathic pain , and an agreement has not been reached [ 48 , 49 ] . the phosphorylation of the erk signal transduction pathway was enhanced by ea in depression rat tissue ; however , there was no report on the study of ea analgesia on the hippocampus erk signal pathway . our previous results showed that in the hippocampus the erk signal pathway was inhibited in chronic neuropathic rats , and after acupuncture treatment the erk signaling pathway was activated . our experimental results showed that , compared with the ea group , brief sciatic nerve stimulation produced a greater excitatory and inhibitory effect on the firing rate of pens and pins in u0126 + ea group , suggesting that u0126 suppressed the effect of ea on the analgesic pathway . it is suggested that involvement of the activation of erk signaling pathway in the hippocampal ca1 region in ea treatment induced pain relief . in cci rats , acute noxious stimulation required a longer time for the firing rate of pain - related neurons to return to normal level ; ea treatment could suppress the effect of the noxious stimulus on pens and pins in both sham and cci rats , which suggests a close relationship with the ea analgesic effect ; and the erk signal pathway is probably involved in pain and ea analgesia .

enhancing the binding potency of carbohydrate inhibitors of protein carbohydrate interactions is an important step towards improved medicinal applications.1 this is mostly the case because of the relatively weak interactions between proteins and carbohydrate ligands . ensuring multivalency of ligands is a proven method to achieve this.2 leca is an important virulence factor of pseudomonas aeruginosa and is involved in bacterial adhesion and biofilm formation.3 the protein has recently become a popular target for the design of multivalent inhibitors.4 the tetrameric protein promotes the adhesion of the bacteria to tissue cell surfaces , thus facilitating subsequent steps such as cell invasion and biofilm formation . inhibiting bacterial adhesion proteins has the potential to become a mild and less resistance - prone method to treat and prevent bacterial infections.5 two of the four binding sites , with specificity for galactosides , are relatively close together with a separation of about 26 .6 this arrangement has led us to design and synthesize divalent galactoside ligands with well - defined and rigid spacers that should allow a chelation - type divalent binding mode.7 flexible spacers are commonly used as they are forgiving of imperfect design and usually yield sizeable potency enhancements in multivalent systems.7 they are , however , not optimal as there will be a significant entropy loss upon binding and , moreover , achieving selectivity will be less likely . in our search for an optimal spacer we found compound 1 ( figure 1 a ) to be a highly potent divalent ligand with nanomolar inhibitory potency.8a the spacer of this structure contains direct linkages between the glucose moieties and the connected triazoles.8 this arrangement leads to a relatively rigid structure , in which rotations of the components can take place , but the overall geometry remains mostly linear . a ) structure of potent divalent leca inhibitor 1 with the relatively rigid glucose - triazole - based spacer ; b ) schematic divalent binding mode of a divalent ligand to two leca subunits ; c ) x - ray structure of leca with bound galactose moieties . the two asp 47 carboxylates in the spacer path are shown explicitly.6 most importantly , good solubility in water was observed . we found that three glucose - triazole units was the optimal length for leca inhibition , while divalent ligands with two and four units showed far inferior inhibition . all data were consistent with a chelating binding mode ; especially convincing was the stoichiometry derived from isothermal titration calorimetry ( itc ) binding experiments . furthermore , the short linkage of just a single carbon between the galactoside ligand and the triazole proved to be a major contributing factor to the success of this compound . in order to further optimize the potency of the compound and to explore the principle of protein spacer interactions , we now report on our functionalization of the spacer of 1 with various functional groups . the presence of the two carboxylates ( asp 47 in each subunit ) is apparent when looking at the path between the two bound galactosides , which the spacer is likely to span on the leca protein surface when chelating bivalent binding occurs ( figure 1 c ) . these two carboxylates are likely to be in close proximity to the 6-oh group on the terminal spacer glucoside units , depending on the rotational state of the molecule . molecular modeling was used in order to gauge whether positively charged functional groups at the c-6 position on the terminal spacer glucoside units would be able to interact with the asp 47 residues . firstly , ammonium groups were used ( as derived from 12 , see below ) . creating a stabilized conformation with the positive charges in close proximity to the asp 47 carboxylates was possible , and this orientation was used as the starting point of additional simulations . when running an unrestrained nanosecond molecular dynamics ( md ) simulation with explicit water molecules , the stabilized geometry persisted throughout the simulation ( see supporting information ) . especially , the two hydrogen - bonded salt bridges , between the asp 47 side chains and the ammonium groups of the protonated form of 12 , remained within 3 and were compatible with a geometry that included two fully bound galactoside ligand units . such a bound geometry was also possible for a compound that included a pyridinium group ( as in 13 ) , but resulted in longer distances between the carboxylate oxygens and the pyridinium nitrogens , due to bigger steric requirements of the pyridinium units . furthermore , while performing a similar md simulation , this structure was not maintained , indicating indeed the larger steric requirements of the pyridinium group when facing the protein surface . these experiments lead to the conclusion that the introduction of ammonium groups would be most promising . we chose to prepare a series of derivatives with various new substituents in the spacer in hopes of further enhancing the leca inhibitory potency of the molecules . positively charged substituents were included , as reasoned above . also , negatively charged and more lipophilic groups were included as these could possibly benefit from interacting with other proximate parts of the protein that would not have been previously obvious . the synthesis of the modified spacers started with two previously prepared building blocks 2 and 3.8 these building blocks were coupled by a double copper(i)-catalyzed azide alkyne cycloaddition ( cuaac ) reaction . next , the two galactosyl axial 4-oh groups of the resulting product 4 were turned into triflates , thus enabling the subsequent substitution by azide with inversion to give 5 . the terminal azides were subsequently linked to the protected galactosyl ligand 6 a by cuaac yielding 7 . selective removal of the tert - butyldimethylsilyl ( tbdms ) groups then gave 8 which is ready for further functionalization . reagents and conditions : a ) cuso45 h2o , naasc , dmf with 10 % h2o , 80 c , 30 min , 85 % ; b ) 1 ) tf2o , 10 % pyridine in ch2cl2 , 0 c , 3 h , 2 ) nan3 , acetone / h2o ( 4:1 ) , rt , o / n , 98 % ; c ) 6 a , cuso45 h2o , naasc , dmf with 10 % h2o , 80 c , 30 min , 64 % ; d ) p - tsoh , ch3cn / h2o ( 7:1 ) , rt , 6 h , 83 % . the two hydroxymethylene groups of compound 8 were oxidized to carboxylic acids using 2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl ( tempo ) ( scheme 2 ) . reaction of 8 with triflic anhydride in the presence of pyridine followed by zempln deprotection gave the bis pyridinium compound 10 . tosylation of the primary hydroxy groups of 8 followed by reaction with sodium azide gave the intermediate 11 . zempln deprotection of 11 , followed by the hydrogenation of the azido groups gave the diamine 12 . cuaac coupling of phenylacetylene to 11 and subsequent zempln deprotection gave the bis - triazole 13 . all final products were purified by preparative high - performance liquid chromatography ( hplc ) . reagents and conditions : a ) 1 ) tempo , naocl , nabr , bu4nbr , nahco3/na2co3 ph 9.5 , 0 c , 2 h , 2 ) naome , meoh , rt , o / n , 38 % ; b ) 1 ) tf2o , 10 % pyridine in ch2cl2 , 0crt , 3 h , 2 ) naome , meoh , rt , o / n , 44 % , c ) 1 ) tscl , dabco , ch2cl2 , rt , o / n , 2 ) nan3 , dmf , 95 c , o / n , 34 % , d ) 1 ) naome , meoh , rt , o / n , 2 ) h2 , pd / c , rt , 60 % , e ) 1 ) phenylacetylene , cuso45 h2o , naasc , dmf with 10 % h2o , 80 c , 30 min , 2 ) naome , meoh , rt , o / n , 24% . the compounds were tested on an array chip in an assay similar to an enzyme - linked immunosorbent assay ( elisa ) , as previously reported.8 fluorescein - labeled leca was incubated with the inhibitors and exposed to a galactoside - functionalized chip surface . monovalent ligand 6 b was previously determined to have an ic50 in this assay of 22 m.8a clearly all divalent compounds were far more potent and showed major multivalency effects ( table 1 ) . the previous best inhibitor , 1 , still remained the most potent compound in the present series with an ic50 in the 2 - 3 nm range , as before . the pyridinium - functionalized 10 and the phenylacetylene - derived 13 showed only a minor drop in potency , with ic50 values in the 5 nm range . larger potency drops of about an order of magnitude were observed for both the negatively charged bis - carboxylate 9 and the positively charged bis - amine 12 . subsequently , itc experiments were conducted , which confirmed the divalent binding mode in all cases , with the stoichiometry n values being close to 0.5 . as before8a the dissociation constants ( kd ) were somewhat higher than the ic50 values from the chip - based elisa - like assay . furthermore the small potency differences of the chip - based assay were not seen in the itc assay . interestingly , when looking at the enthalpic and entropic components of the binding event , the lipophilic and noncharged compound 13 , showed enthalpy entropy compensation , with a lower beneficial binding enthalpy and , at the same time , a lowered entropy loss upon binding . the latter is understandable as an example of the hydrophobic effect where water molecules in an ice - like structure are liberated from the lipophilic surfaces . inhibitory potencies ( ic50 ) of the divalent inhibitors on leca binding[a ] and dissociation constants ( kd ) , stoichiometry , and thermodynamic binding parameters[b ] [ a ] chip - based elisa - like assay : fitc - labeled leca ( 5 g ml ) binding to a galactoside functionalized chip surface . [ b ] obtained from isothermal titration microcalorimetry ( itc ) : [ leca]=2040 m . it proved possible to use selectively protected 6-oh groups to build up the ligand in its protected form . subsequently , the selectively deprotectable silyl groups were removed , and the resulting primary hydroxy groups were converted to carboxylate groups by oxidation , to tosylates and subsequently to azides by substitution , and to pyridiniums via their corresponding triflates . the azides allowed cuaac coupling and also further reduction to the corresponding amino groups . while it was anticipated that some of these groups would be able to take advantage of additional beneficial interactions with the nearby protein surface , we did not observe this through enhanced inhibitory or binding potencies . this was true even for the positively charged groups that could possibly take advantage of the nearby positioned carboxylate groups of asp 47 . however , a possible alternative scenario , where the newly added groups point into the solution and thus away from the protein surface , is apparently more favorable in this case . in future designs nevertheless , the functionalization of the spacers will also be important in order to fine - tune other properties of this type of ligand , such as toxicity and absorption , distribution , metabolism , and excretion ( adme ) , which are important for drug development . general : chemicals were obtained from commercial sources and were used without further purification unless noted otherwise . compounds 2 , 3 , and 6 were synthesized following literature procedures.7 solvents were purchased from biosolve ( valkenswaard , the netherlands ) . anhydrous solvents were dried over molecular sieves of 4 or 3 . thin - layer chromatography ( tlc ) was performed on merck precoated silica 60 plates . spots were visualized by uv light and also by 10 % h2so4 in meoh . microwave reactions were carried out in a microwave initiator system ( biotage , uppsala , sweden ) . analytical hplc runs were performed on an automated hplc system ( shimadzu , kyoto , japan ) with a reversed - phase column ( reprospher 100 , c4 , 5 m , 2504.6 mm , dr . maisch gmbh , ammerbuch - entringen , germany ) which was equipped with an evaporative light scattering detector ( plels 1000 , polymer laboratories , amherst , usa ) and a uv / vis detector operating at 220 nm and 250 nm . preparative hplc runs were performed on an applied biosystems ( waltham , usa ) workstation . elution was effected by using a linear gradient of 5 % ch3cn/0.1 % trifluoroacetic acid ( tfa ) in h2o to 5 % h2o/0.1 % tfa in ch3cn or by a gradient of h2o to 30 % ch3cn in h2o . h and c nmr spectroscopy was carried on an 400-mr spectrometer ( agilent , santa clara , usa ) operating at 400 mhz for h and 100 mhz for c. heteronuclear single quantum coherence ( hsqc ) and total correlated spectroscopy ( tocsy ) nmr ( 500 mhz ) were performed with an inova 500 instrument ( varian , palo alto , usa ) . electrospray ionization mass spectrometry ( esi - ms ) experiments were performed with a shimadzu lcms qp-8000 . high - resolution mass spectrometry ( hrms ) analysis was recorded using an esi - q - tof ii spectrometer ( bruker , billerica , usa ) . the proton numbering scheme of all compounds can be found in the supporting information and is used in the assignments of the signals for the nmr spectra below . isothermal titration microcalorimetry ( itc ) : the lectin leca was obtained from sigma aldrich and was dissolved in buffer ( 0.1 m tris - hcl , 6 mm cacl2 , ph 7.5 ) and degassed . protein concentration ( between 20 and 40 m depending on the ligand affinity ) was checked by measurement of optical density by using a theoretical molar extinction coefficient of 28 000 . carbohydrate ligands were dissolved directly into the same buffer , degassed , and placed in the injection syringe ( concentration range : 0.10.2 mm ) . itc was performed using a microcal auto itc200 ( malvern , worcestershire , uk ) . leca ( 0.020.04 mm ) was placed into the 200 l sample cell at 25 c . titration was performed with injections of carbohydrate ligands ( 2.5 l ) every 120 s. data were fitted using the one - site model using microcal origin 7 software according to standard procedures . fitted data yielded the stoichiometry ( n ) , the association constant ( ka ) , the enthalpy ( h ) and the entropy of binding . the kd value was calculated as 1/ka , and t is 298 k. each ligand test was performed in duplicate . leca inhibiton assay : lectin leca was labeled with fluorescein isothiocyanate ( fitc ) according to a literature procedure.9 microarray experiments were performed by using a pamchip array run on a pamstation 12 instrument ( pam - gene , s - hertogenbosch , the netherlands ) . data were obtained by real - time imaging of the fluorescence signal by a ccd camera . the fluorescence intensities were expressed in arbitrary units , and the relative intensities were the average of the two duplicate spots . aliquots of a solution of fitc - labeled leca ( 5 g ml for all tested compounds ) in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid / bovine serum albumin ( hepes / bsa ) buffer ( 10 mm hepes , 100 mm nacl , 0.1 % bsa . ph 7.4 ) , containing different concentrations of the inhibitors were incubated for 1 h at rt and subsequently added to the galactoside - functionalized chip . the binding process was monitored for 2 h , and the end values of the fluorescence detection were taken for the determination of the ic50 by using prism 5 ( graphpad software , inc . , la jolla , usa ) . molecular modeling : all molecular modeling studies were performed using the molecular graphics , modeling , and simulation program yasara version 13.9.8 ( yasara biosciences , vienna , austria ) . the bivalent ligands were first constructed in yasara as isolated molecules . subsequently , the complex with leca was built by superposition of one of the galactose units of the ligand with a bound galactose of one of the subunits of the leca crystal structure ( pdb i d : 1oko).3a the other galactose unit of the divalent ligand was then pulled into the adjacent galactose binding site of leca by restraint md using distance restraints based on the position of the bound galactose present in the x - ray structure with respect to a number of leca residues . possible electrostatic interactions between two positively charged amino and pyridinium substitutions in the linker region with two asp 47s of leca were investigated in more detail . in order to induce these interactions , the subunits comprising the substituted moieties were first rotated , and subsequently , the nitrogen atoms of respectively the amino and the pyridinium group were restrained to bring them in close proximity to the carbon atoms of the carboxylic acid moieties of asp 47 . after restrained md , the molecules were subjected to a 1000 ps free md simulation in water . general procedure of microwave - assisted click reaction : to a solution of the azide and alkyne compounds in dimethylformamide ( dmf ) with 10 % h2o , was added cuso45 the mixture was then heated by microwave irradiation at 80 c for 30 min . when the mixture cooled to rt,0 the copper salts were removed by a resin ( cuprisorb ) , and the solvents were removed under reduced pressure . compound 4 : a click reaction of a mixture of compound 2 ( 0.253 g , 0.71 mmol ) , compound 3 ( 0.83 g , 1.63 mmol ) , cuso45 h2o ( 0.06 g , 0.24 mmol ) , and naasc ( 0.096 g , 0.48 mmol ) in dmf ( 5 ml ) containing 10 % h2o was performed following the general procedure described above to afford compound 4 ( 0.84 g , 85 % ) ; h nmr ( 400 mhz , cdcl3 ) : = 8.037.89 ( m , 5 h , 4ch benzoyl , h-1 ) , 7.867.76 ( m , 5 h , 4ch benzoyl , h-1 ) , 7.527.26 ( m , 12 h , 12ch benzoyl ) , 6.08 ( dd , j4,3=j4,5=10 hz , 1 h , h-4 ) , 6.025.90 ( m , 2 h , h-4 , h-3 ) , 5.81 ( dd , j5,4=j5,6=9 hz , 1 h , h-5 ) , 5.60 ( dd , j4,3=j4,5=9 hz , 1 h , h-4 ) , 5.515.41 ( m , 2 h , 2h-5 ) , 5.024.92 ( m , 2 h , 2h-3 ) , 4.81 ( dd , j6,5=j6,7=10 hz , 1 h , h-6 ) , 4.674.54 ( m , 3 h , h-7 , 2 h-6 ) , 4.164.07 ( m , 1 h h-8a ) , 4.073.93 ( m , 2 h , 2h-8 ) , 3.913.82 ( m , 2h-7 ) , 3.703.60 ( m , 1 h , h-8b ) , 3.573.51 ( m , 1 h , 6-oh ) , 3.353.29 ( 1 h , 6-oh ) , 2.02 , 1.65 , 1.58 ( s , 9 h , 3ch3 , acetyl ) , 0.90 ( s , 18 h , 2sic(ch3)3 ) , 0.07 ppm ( 2 s , 12 h , 2si(ch3)2 ) ; c nmr ( 100 mhz , cdcl3 ) : =69.97 , 169.10 , 168.95 ( 3c = o acetyl ) , 166.14 , 166.05 , 165.63 , 165.55 ( 4c = o benzoyl ) , 145.61 , 145.58 ( 2c-2 ) , 133.44133.21 ( ch benzoyl ) , 129.99128.41 ( ch benzoyl ) , 129.39129.20 ( c benzoyl ) 123.43 , 122.13 ( 2c-1 ) , 85.55 ( c-3 ) , 77.97 ( 2c-7 ) , 75.75 ( c-5 ) , 75.48 ( c-5 ) , 74.84 ( c-7 ) , 74.03 , 73.77 ( 2c-3 ) , 72.41 ( c-5 ) , 70.85 ( c-4 ) , 69.94 , 69.59 ( 2c-4 ) , 68.92 , 68.53 ( 2c-6 ) , 63.65 , 63.13 ( 2c-8 ) , 61.61 ( c-8 ) , 59.84 ( c-6 ) , 25.99 ( sic(ch3)3 ) , 20.72 , 19.98 , 19.89 ( 3ch3 acetyl ) , 18.44 ( sic(ch3)3 ) , 5.31 ppm ( si(ch3)2 ) ; ms ( esi ) m / z [ m+h ] calcd for c68h84n6o21si2 : 1378.59 , found 1378.00 ; hrms ( q - tof ) m / z [ m+h ] calcd for c68h84n6o21si2 : 1377.5228 , found 1377.5326 . compound 5 : compound 4 ( 0.84 g , 0.61 mmol ) in anhydrous ch2cl2 ( 30 ml ) containing pyridine ( 4.22 ml ) was treated with triflic anhydride ( 12.2 ml of a 1 m solution in ch2cl2 , 12.2 mmol ) . the mixture was stirred at 0 c for 3 h , after which cold 1 n khso4 ( 20 ml ) was added . the organic layer was washed with cold h2o ( 220 ml ) and , once with cold brine ( 20 ml ) , dried over na2so4 , filtered , and concentrated . the residue was dissolved in an acetone / h2o mixture ( 15 ml , 4:1 ) , and nan3 ( 0.397 g , 6.1 mmol ) was added . the mixture was stirred at rt overnight and diluted with a cold h2o / ch2cl2 mixture ( 50 ml , 4:1 ) . the water layer was separated and extracted once with ch2cl2 ( 10 ml ) . the combined organic layers were dried on na2so4 , filtered , and concentrated to afford compound 5 as a yellowish solid ( 0.86 g , 0.60 mmol , 98 % ) ; h nmr ( 400 mhz , cdcl3 ) : =8.007.93 ( m , 4 h , 4ch benzoyl ) , 7.857.72 ( m , 5 h , 4ch benzoyl , h-1 ) , 7.65 ( s , 1 h , h-1 ) , 7.587.26 ( m , 12 h , 12ch benzoyl ) , 5.90 ( d , j3,4=9 hz , 1 h , h-3 ) , 5.865.65 ( m , 3 h , h-4 , 2h-5 ) , 5.57 ( m , 2 h , h-4 , h-5 ) , 5.42 ( dd , j4,3=j45=10 hz , 1 h , h-4 ) , 4.974.88 ( m , 2 h , 2h-3 ) , 4.81 ( dd , j6,5=j6,7=10 hz , 1 h , h-6 ) , 4.614.51 ( m , 1 h , h-7 ) , 4.174.03 ( m , 3 h , h-8a , 2h-6 ) , 4.033.93 ( m , 2 h , 2h-8 ) , 3.683.55 ( m , 3 h , h-8b , 2h-7 ) , 2.01 , 1.69 , 1.62 ( s , 9 h , 3ch3 , acetyl ) , 0.95 ( s , 18 h , 2sic(ch3)3 ) , 0.180.04 ppm ( m , 12 h , 2si(ch3)2 ) ; c nmr ( 100 mhz , cdcl3 ) : =169.85 , 169.01 , 168.96 ( 3c = o acetyl ) , 165.85 , 165.78 , 165.56 , 165.47 ( 4c = o benzoyl ) , 145.18 ( 2c-2 ) , 133.57133.39 ( ch benzoyl ) , 129.96128.44 ( ch benzoyl ) , 129.39129.20 ( c benzoyl ) 123.08 , 121.61 ( 2c-1 ) , 85.64 ( c-3 ) , 79.88 , 79.86 ( 2c-7 ) , 75.01 , 74.96 ( 2c-5 ) , 74.69 ( c-7 ) , 73.60 , 73.35 ( 2c-3 ) , 72.60 ( c-4 ) , 72.17 , 72.12 ( 2c-4 ) , 70.73 ( c-5 ) , 62.50 , 62.40 ( 2c-8 ) , 61.50 ( c-8 ) , 60.43 ( 2c-6 ) , 59.84 ( c-6 ) , 26.04 ( sic(ch3)3 ) , 20.67 , 20.01 , 19.94 ( 3ch3 acetyl ) , 18.57 ( sic(ch3)3 ) , 4.97(5.26 ) ppm ( si(ch3)2 ) ; ms ( esi ) m / z [ m+h ] calcd for c68h82n12o19si2 : 1427.54 , found 1427.75 ; hrms ( q - tof ) m / z [ m+h ] calcd for c68h82n12o19si2 : 1427.5358 , found 1427.5389 . compound 7 : a click reaction of a mixture of compound 5 ( 0.88 g , 0.62 mmol ) and compound 6 a ( 0.570 g , 1.48 mmol ) with cuso45 h2o ( 28 mg , 0.11 mmol ) and naasc ( 0.0443 g , 0.22 mmol ) in dmf ( 15 ml ) containing 10 % h2o was performed following the general procedure described above to afford compound 7 ( 0.87 g , 64 % ) ; h nmr ( 400 mhz , cdcl3 ) : =7.88 ( s , 1 h , h-1 ) , 7.827.68 ( m , 9 h , 8ch benzoyl , h-1 ) , 7.687.59 ( 2 s , 2 h , 2h-1 ) , 7.517.39 ( m , 4 h , 4ch benzoyl ) , 7.367.26 ( m , 9 h , 8ch benzoyl ) , 6.356.22 ( m , 2 h , 2h-5 ) , 5.94 ( d , j3,4=9 hz , 1 h , h-3 ) , 5.885.73 ( m , 2 h , 2h-4 ) , 5.675.52 ( m , 2 h , h-5 , h-4 ) , 5.435.33 ( m , 2 h , 2h-13 ) , 5.275.07 ( m , 6 h , 2h-3 , 2h-11 , 2h-6 ) , 5.034.71 ( m , 7 h , 2 h-12 , 2h-9a , h-6 , 2h-9b ) , 4.634.53 ( m , 1 h , h-7 ) , 4.484.37 ( m , 4 h , 2h-7 , 2h-10 ) , 4.294.04 ( m , 5 h , 2h-15a , h-8a , 2h-15b ) , 3.943.85 ( m , 2 h , 2h-14 ) , 3.80 ( d , j8a,8b=12 hz , 2 h , 2h-8a ) , 3.65 ( dd , j8b,8a=12 hz , j8b,7=3 hz , 1 h , h-8b ) , 3.453.32 ( m , 2 h , 2 h-8b ) , 2.201.88 ( m , 21 h , 7ch3 , acetyl ) , 1.771.63 ( m , 12 h , 4ch3 , acetyl ) , 0.940.84 ( m , 18 h , 2sic(ch3)3 ) , 0.05(0.07 ) ppm ( m , 12 h , 2si(ch3)2 ) ; c nmr ( 100 mhz , cdcl3 ) : =170.83168.96 ( c = o acetyl ) , 165.51 ( c = o benzoyl ) , 165.39 ( c = o benzoyl ) , 165.07 ( c = o benzoyl ) , 144.90 ( 2c-2 ) , 143.54 ( 2c-2 ) , 133.64 , 129.81128.53 ( ch benzoyl ) , 128.81 ( c benzoyl ) 123.66 , 123.57 , 123.14 , 121.67 ( 4c-1 ) , 99.29 , 99.26 ( 2c-10 ) , 85.70 ( c-3 ) , 79.40 , 79.33 ( 2c-7 ) , 75.02 ( c-7 ) , 74.20 , 73.85 ( 2c-5 ) , 73.67 , 73.42 ( 2c-3 ) , 72.70 ( c-5 ) , 72.22 , 72.15 ( 2c-4 ) , 70.99 ( 2c-12 ) , 70.78 ( 2c-14 ) , 70.73 ( c-4 ) , 68.74 ( 2c-11 ) , 67.25 ( 2c-13 ) , 61.84 ( 2c-9 , 2c-8 ) , 61.43 ( 2c-15 , c-8 ) , 60.17 , 60.12 ( 2c-6 ) , 59.81 ( c-6 ) , 25.96 ( sic(ch3)3 ) , 20.9719.92 ( ch3 acetyl ) , 18.52 ( sic(ch3)3 ) , 5.11(5.34 ) ppm ( si(ch3)2 ) ; ms ( esi ) m / z [ m+2 h]calcd for c102h126n12o39si2 : 1100.89 , found 1100.60 ; hrms ( q - tof ) m / z [ m+2 h ] calcd for c102h126n12o39si2 : 1100.3892 , found 1100.8980 . compound 8 : compound 7 ( 0.87 g , 0.40 mmol ) was dissolved in ch3cn / h2o ( 24 ml , 7:1 ) , and p - tsoh ( 0.114 g , 0.60 mmol ) was added . the mixture was stirred at rt for 6 h and was then diluted with ch2cl2 ( 50 ml ) , which was followed by the addition of 10 % nahco3 ( 20 ml ) . the organic layer was washed once with brine ( 20 ml ) and dried over na2so4 , filtered , and concentrated . the residue was purified by silica gel chromatography to afford compound 8 ( 0.65 g , 0.33 mmol , 83 % ) ; h nmr ( 400 mhz , cdcl3 ) : =8.22 ( s , 1 h , h-1 ) , 8.12 ( s , 1 h , h-1 ) , 7.92 ( s , 1 h , h-1 ) , 7.87 ( s , 1 h , h-1 ) , 7.81 ( d , j=8 hz , 4 h , 4ch benzoyl ) , 7.767.69 ( m , 4 h , 4ch benzoyl ) , 7.477.35 ( m , 4 h , 4ch benzoyl ) , 7.317.16 ( m , 8 h , 8ch benzoyl ) , 6.456.33 ( m , 2 h , 2h-5 ) , 6.30 ( d , j3,4=8 hz , 1 h , h-3 ) , 6.015.88 ( m , 2 h , 2h-4 ) , 5.81 ( dd , j5,4=j5,6=10 hz , 1 h , h-5 ) , 5.68 ( dd , j4,3=j4,5=10 hz 1 h , h-4 ) , 5.535.37 ( m , 4 h , 2 h-13 , 2h-6 ) , 5.32 ( d , j3,4=12 hz , 2 h , 2h-3 ) , 5.225.09 ( m , 3 h , 2h-11 , h-6 ) , 5.044.80 ( m , 6 h , 2h-12 , 2h-9a , 2h-9b ) , 4.734.65 ( m , 1 h , h-7 ) , 4.514.40 ( m , 4 h , 2h-7 , 2h-10 ) , 4.334.23 ( m , 2 h , 2h-15a ) , 4.174.07 ( m , 2 h , 2h-15b ) , 4.043.93 ( m , 3 h , 2h-14 , h-8a ) , 3.80 ( d , j8a,8b=12 hz , 2 h , 2h-8a ) , 3.56 ( dd , j8b,8a=12 hz , j8b,7=4 hz , 1 h , h-8b ) , 3.29 ( d , j8b,8a=12 hz , 2 h , 2h-8b ) , 2.201.93 ( m , 21 h , 7ch3 , acetyl ) , 1.861.48 ppm ( m , 12 h , 4ch3 , acetyl ) ; c nmr ( 100 mhz , cdcl3 ) : =170.88169.10 ( c = o acetyl ) , 165.51 ( c = o benzoyl ) , 165.26 ( c = o benzoyl ) , 165.11 ( c = o benzoyl ) , 144.59 , 144.40 , 143.75 , 143.71 ( 4c-2 ) , 133.60 , 129.91128.44 ( ch benzoyl ) , 128.77 , 128.37 ( c benzoyl ) 123.79 ( 2c-1 ) , 123.63 ( 2c-1 ) , 99.20 , 99.10 ( 2c-10 ) , 85.29 ( c-3 ) , 79.35 , 79.15 ( 2c-7 ) , 74.85 ( c-7 ) , 73.99 ( 2c-5 ) , 73.68 , 73.33 ( 2c-3 ) , 72.72 ( c-5 ) , 72.12 ( 2c-4 ) , 70.94 ( 2c-12 ) , 70.83 ( 2c-14 ) , 70.81 ( c-4 ) , 68.84 ( 2c-11 ) , 67.27 ( 2c-13 ) , 61.64 ( 2c-9 ) , 61.45 ( 2c-15 ) , 61.31 ( c-8 ) , 60.64 , 60.56 ( 2c-8 ) , 59.92 , 59.88 ( 2c-6 ) , 59.84 ( c-6 ) , 20.9619.83 ppm ( ch3 acetyl ) ; hrms ( q - tof ) m / z [ m+2 h ] calcd for c90h98n12o39 : 986.3027 , found 986.8078 . compound 9 : a solution of compound 8 ( 51.6 mg , 0.026 mmol ) and tempo ( 0.164 mg , 1.05 mol ) in ch2cl2 ( 1 ml ) was added to a solution of nabr ( 1.1 mg , 10.5 mol ) and bu4nbr ( 3.4 mg , 10.5 mol ) in nahco3/na2co3 buffer ( 1 ml , ph was adjusted to 9.5 with saturated na2co3 ) . the mixture was cooled to 0 c , and naocl ( 614 % , 105 l ) was added dropwise . the mixture was stirred vigorously at 0 c for 2 h and then quenched by the addition of na2s2o3 ( sat . , 0.5 ml ) , after which h2o ( 1.5 ml ) and ch2cl2 ( 4 ml ) were added , and the ph was adjusted to ph 3 with aqueous hcl ( 6 n ) . the organic phase was washed once with brine ( 1 ml ) , dried over na2so4 , and concentrated in vacuo . the resulting oxidized compound was exposed to zmpln conditions , followed by h resin , and was concentrated . the residue was subjected to preparative hplc purification , which gave compound 9 ( 11.1 mg , 0.010 mmol , 38 % ) ; h nmr ( 400 mhz , d2o ) : =8.56 ( s , 1 h , h-1 ) , 8.43 ( s , 1 h , h-1 ) , 8.26 ( s , 2 h , 2h-1 ) , 6.10 ( d , j3,4=12 hz , 1 h , h-3 ) , 5.06 ( d , j9a,9b=12 hz , 2 h , 2h-9a ) , 5.014.91 ( m , 5 h , 2h-9b , h-6 , 2h-3 ) , 4.894.72 ( m , 4 h , 2h-7 , 2h-6 ) , 4.584.41 ( m , 6 h , h-7 , 2h-10 , h-5 , 2h-5 ) , 4.30 ( dd , j4,3=j4,5=9.2 hz , 1 h , h-4 ) , 4.08 ( dd , j4,3=j4,5=9.2 hz , 2 h , 2h-4 ) , 3.973.92 ( m , 2 h , 2h-13 ) , 3.883.70 ( m , 6 h , 2h-15ab , 2h-14 ) , 3.693.61 ( m , 3 h , 2h-12 , h-8a ) , 3.56 ( dd , j11,10=j11,12=8 hz , 2 h , 2h-11 ) , 3.40 ppm ( dd , j8b,8a=12 hz , j8b,7=4 hz , 1 h , h-8b ) ; c nmr ( 100 mhz , d2o ) : =172.26 ( 2c-8 ) , 145.12 ( c-2 ) , 145.01 ( c-2 ) , 144.38 ( 2c-2 ) , 126.65 ( 2c-1 ) , 126.51 ( c-1 ) , 125.47 ( c-1 ) , 102.56 ( 2c-10 ) , 88.28 ( c-3 ) , 78.82 ( 2c-7 ) , 77.71 ( c-7 ) , 75.95 ( 2c-14 ) , 75.04 ( c-5 ) , 75.00 ( c-5 ) , 74.40 ( c-3 ) , 74.38 ( c-3 ) , 74.28 ( c-5 ) , 73.64 ( c-4 ) , 73.59 ( c-4 ) , 73.42 ( 2c-12 ) , 73.30 ( c-4 ) , 71.39 ( 2c-11 ) , 69.33 ( 2c-13 ) , 64.60 ( 2 c-6 ) , 62.41 ( 2c-9 ) , 62.33 ( c-6 ) , 61.68 ( 2c-15 ) , 60.32 ppm ( c-8 ) ; ms ( esi ) m / z [ m+h ] calcd for c40h56n12o26 : 1121.34 , found 1121.05 ; hrms ( q - tof ) m / z [ m+h ] calcd for c40h56n12o26 : 1121.3429 , found 1121.3465 , [ m+na ] 1143.3282 . compound 10 : compound 8 ( 49.2 mg , 0.025 mmol ) was dissolved in dry ch2cl2 ( 2 ml ) with pyridine ( 200 l ) . the mixture was cooled down to 0 c , after which triflic anhydride ( 203 l , 0.203 mmol ) was added dropwise to the above solution . the reaction was allowed to warm up to rt and stirred for 3 h after which 1 n khso4 ( 2 ml ) and ch2cl2 ( 15 ml ) were added . the organic layer was washed once with h2o ( 5 ml ) and once with brine ( 5 ml ) , dried on na2so4 , filtered , and concentrated . the resulting material was then treated with 0.5 m naome in meoh ( 5 ml ) , stirred at rt overnight , after which 1 n hcl was added to adjust to ph6 , and the solvents were evaporated in vacuo . the residue was purified by preparative hplc , which gave compound 10 ( 13.3 mg , 0.011 mmol , 44 % ) ; h nmr ( 400 mhz , d2o ) : =8.69 ( d , j16,17=8 hz , 4 h , 4h-16 ) , 8.56 ( dd , j18,17=j18,17=8 hz , 2 h , 2h-18 ) , 8.42 ( s , 1 h , h-1 ) , 8.38 ( s , 2 h , 2h-1 ) , 8.28 ( s , 2 h , h-1 ) , 8.057.98 ( m , 4 h , 4h-17 ) , 6.06 ( d , j3,4=8 hz , 1 h , h-3 ) , 5.12 ( d , j9a,9b=12 hz , 2 h , 2h-9a ) , 5.004.92 ( m , 3 h , 2h-9b , h-6 ) , 4.924.73 ( m , 8 h , 2h-7 , 2h-6 , 2h-3 , 2h-8a ) , 4.624.42 ( m , 6 h , 2h-10 , 2h-8b , h-7 , h-5 ) , 4.344.23 ( m , 3 h , 2h-5 , h-4 ) , 4.073.98 ( m , 2 h , 2h-4 ) , 3.983.93 ( m , 2 h , 2h-13 ) , 3.883.73 ( m , 6 h , 2h-15ab , 2h-14 ) , 3.713.63 ( m , 3 h , 2h-12 , h-8a ) , 3.633.54 ( m , 2 h , 2h-11 ) , 3.35 ppm ( dd , j8b,8a=12 hz , j8b,7=4 hz , 1 h , h-8b ) ; c nmr ( 100 mhz , d2o ) : =146.77 ( 2 c-18 ) , 145.32 ( 4c-16 ) 144.63 ( 2c-2 ) , 144.37 ( c-2 ) , 144.23 ( c-2 ) , 128.21 ( 4c-17 ) , 125.96 ( 2c-1 ) , 125.56 ( c-1 ) , 124.75 ( c-1 ) , 102.42 ( 2c-10 ) , 87.61 ( c-3 ) , 77.08 ( c-7 ) , 75.87 ( 2c-7 ) , 75.49 ( 2c-14 ) , 74.68(2c-5 ) , 73.84 ( 2c-3 ) , 73.73 ( c-5 ) , 72.96(2c-4 ) , 72.89 ( 2c-12 ) , 72.72 ( c-4 ) , 70.84 ( 2c-11 ) , 68.76 ( 2c-13 ) , 63.61 ( 2c-6 ) , 62.07 ( 2c-9 ) , 61.71 ( c-6 ) , 61.43 ( 2c-8 ) , 61.20 ( 2c-15 ) , 59.76 ppm ( c-8 ) ; ms ( esi ) m / z [ m ] calcd for c50h68n14o22 : 608.23 , found 608.30 ; hrms ( q - tof ) m / z [ m ] calcd for c50h68n14o22 : 608.2311 , found 608.2356 . compound 11 : compound 8 ( 246 mg , 0.125 mmol ) was dissolved in dry ch2cl2 ( 5 ml ) , and tosyl chloride ( 238.4 mg , 1.25 mmol ) and 1,4-diazabicyclo[2.2.2]octane ( dabco ) ( 38 mg , 0.34 mmol ) were added . the residue was redissolved in ch2cl2 ( 15 ml ) and washed once with h2o ( 5 ml ) and once with brine ( 5 ml ) , dried on na2so4 , filtered , and concentrated . the resulting material was purified by silica gel chromatography to give the tosylated compound ( 220 mg , 77.2 % ) . the residue was then dissolved in dry dmf ( 8 ml ) to which nan3 ( 33.6 mg , 0.52 mmol ) was added . the mixture was stirred at 95 c overnight after which the solvent was removed . the residue was dissolved in ch2cl2 ( 15 ml ) , washed once with 1 n khso4 ( 5 ml ) and once with h2o ( 5 ml ) , dried on na2so4 , filtered , and concentrated . the residue was purified by silica gel chromatography to afford compound 11 ( 86.8 mg , 0.043 mmol , 34 % ) ; h nmr ( 400 mhz , cdcl3 ) : =7.95 ( s , 1 h , h-1 ) , 7.827.765 ( m , 11 h , 4ch benzoyl , 3h-1 ) , 7.517.39 ( m , 4 h , 4ch benzoyl ) , 7.367.22 ( m , 8 h , 8ch benzoyl ) , 6.366.23 ( m , 2 h , 2h-5 ) , 5.98 ( d , j3,4=8 hz , 1 h , h-3 ) , 5.925.76 ( m , 2 h , 2h-4 ) , 5.65 ( t , j5,4=j5,6=8 hz , 1 h , h-5 ) , 5.57 ( t , j4,3=j4,5=8 hz , 1 h , h-4 ) , 5.435.35 ( m , 2 h , 2h-13 ) , 5.29 ( d , j3,4=8 hz , 2 h , 2h-3 ) , 5.225.09 ( m , 4 h , 2h-11 , 2h-6 ) , 5.024.83 ( m , 5 h , 2h-12 , 2h-9a , h-6 ) , 4.77 ( d , j9b,9a=12 hz , 2 h , 2h-9b ) , 4.714.56 ( m , 3 h , 2h-7 , h-7 ) , 4.44 ( d , j10,11=8 hz , 2 h , 2h-10 ) , 4.284.18 ( m , 2 h , 2h-15a ) , 4.184.05 ( m , 3 h , 2h-15b , h-8a ) , 3.943.86 ( m , 2 h , 2h-14 ) , 3.723.62 ( m , 1 h , h-8b ) , 3.56 ( t , j8a,8b=12 hz , 2 h , 2 h-8a ) , 2.95 , 2.88 ( 2dd , j8b,8a=12 hz , j8b,7=4 hz , 2 h , 2h-8b ) , 2.211.92 ( 21 h , 7ch3 , acetyl ) , 1.831.59 ppm ( 12 h , 4ch3 , acetyl ) ; c nmr ( 100 mhz , cdcl3 ) : =170.88169.06 ( c = o acetyl ) , 165.36 ( c = o benzoyl ) , 165.28 ( c = o benzoyl ) , 164.99 ( c = o benzoyl ) , 144.72 , 144.65 , 144.25 , 144.24 ( 4c-2 ) , 133.75133.54 , 129.85128.49 ( ch benzoyl ) , 128.72 , 128.29 , 128.26 ( c benzoyl ) 123.49 , 123.40 , 123.30 ( 3c-1 ) , 121.72 ( 2c-1 ) , 99.66 , 99.60 ( 2c-10 ) , 85.68 ( c-3 ) , 77.93 , 77.72 ( 2c-7 ) , 75.00 ( c-7 ) , 73.76 , 73.71 ( 2c-5 ) , 73.50 , 73.28 ( 2c-3 ) , 72.66 ( c-5 ) , 72.07 , 72.03 ( 2c-4 ) , 70.91 ( 2 c-12 , c-4 ) , 70.85 ( 2c-14 ) , 70.81 ( c-4 ) , 68.78 ( 2c-11 ) , 67.23 ( 2 c-13 ) , 62.17 , 62.13 ( 2c-9 ) , 61.43 ( 2c-15 , c-8 ) , 60.81 ( 2c-6 ) , 59.72 ( c-6 ) , 50.58 , 50.37 ( 2c-8 ) , 20.9419.92 ppm ( ch3 acetyl ) ; ms ( esi ) m / z calcd for c90h96n18o37 ( m+2 h ) 1011.91 , found 1012.35 ; hrms ( q - tof ) m / z [ m+2 h ] calcd for c90h96n18o37 : 1011.3092 , found 1011.3091 , [ m+h+na ] 1022.8011 , [ m+2na ] 1033.2923 . compound 12 : compound 11 ( 22.6 mg , 0.020 mmol ) was treated with 0.5 m naome in meoh ( 2.5 ml ) at rt overnight and briefly with h resin . the residue was concentrated and dissolved in h2o ( 2 ml ) , and pd / c ( 15 mg , 10 % pd ) was added . the ph was adjusted to ph 1 by 6 n hcl , and the mixture was stirred at rt under an h2 atmosphere until the hydrogenation was complete . the filtrate was concentrated under reduced pressure and subjected to preparative hplc purification , which gave compound 12 ( 12.9 mg , 0.012 mmol , 60 % ) ; h nmr ( 400 mhz , d2o ) : =8.56 ( s , 1 h , h-1 ) , 8.43 ( s , 1 h , h-1 ) , 8.33 ( s , 2 h , 2h-1 ) , 6.13 ( d , j3,4=8 hz , 1 h , h-3 ) , 5.09 ( d , j9a,9b=10 hz , 2 h , 2h-9a ) , 5.034.89 ( m , 5 h , 2h-3 , h-6 , 2h-9b ) , 4.894.70 ( m , 2 h , 2h-6 ) , 4.664.47 ( m , 6 h , 2h-7 , 2h-10 , h-7 , h-5 ) , 4.414.28 ( m , 3 h , 2h-5 , h-4 ) , 4.083.99 ( m , 2 h , 2h-4 ) , 3.993.93 ( m , 2 h , 2h-13 ) , 3.893.73 ( m , 6 h , 2h-15ab , 2h-14 ) , 3.713.64 ( m , 3 h , 2h-12 , h-8a ) , 3.57 ( dd , j11,10=j11,12=8 hz , 2 h , 2h-11 ) , 3.40 ( dd , j8b,8a=12 hz , j8b,7=4 hz , 1 h , h-8b ) , 3.243.11 ( m , 2 h , 2h-8a ) , 2.86 ppm ( d , j8b,8a=12 hz , 2 h , 2h-8b ) ; c nmr ( 100 mhz , d2o ) : =144.94 , 144.82 ( 2c-2 ) , 144.69 ( 2c-2 ) , 126.18 ( 2c-1 ) , 126.13 ( c-1 ) , 125.07 ( c-1 ) , 102.63 ( 2c-10 ) , 87.97 ( c-3 ) , 77.46 ( c-7 ) , 75.76 ( 2c-14 ) , 74.90 ( 2c-5 ) , 74.51 ( 2c-7 ) , 74.33 ( 2c-3 ) , 74.07 ( c-5 ) , 73.45 ( 2c-4 ) , 73.18 ( 2c-12 ) , 73.13 ( c-4 ) , 71.14 ( 2c-11 ) , 69.09 ( 2c-13 ) , 64.27 ( 2c-6 ) , 62.39 ( 2c-9 ) , 62.08 ( c-6 ) , 61.48 ( 2c-15 ) , 60.12 ( c-8 ) , 40.59 ppm ( 2c-8 ) ; ms ( esi ) m / z [ m+2 h ] calcd for c40h62n14o22 : 546.21 , found 546.90 ; hrms ( q - tof ) m / z [ m+h ] calcd for c40h62n14o22 : 1091.4163 , found 1091.4282 , [ m+na ] 1113.4132 . compound 13 : a click reaction of a mixture of compound 11 ( 61.8 mg , 0.031 mmol ) , phenylacetylene ( 11.1 mg , 0.109 mmol ) , naasc ( 6.5 mg , 0.033 mmol ) , and cuso45 h2o ( 4.1 mg , 0.016 mmol ) was performed following the general procedure described above to afford the coupling product compound ( 54 mg , 80 % ) , which was then treated with naome in meoh ( 0.5 m , 5 ml ) . the mixture was stirred at rt overnight , treated with h resin , concentrated , and subjected to preparative hplc purification , which gave compound 13 ( 9.8 mg , 7.3 mol , 24 % ) ; h nmr ( 400 mhz , d2o with 30 % cd3cn ) : =8.62 ( s , 1 h , h-1 ) , 8.50 , 8.49 ( 2 s , 2 h , 2h-1 ) , 8.46 ( s , 1 h , h-1 ) , 8.42 , 8.41 ( 2 s , 2 h , 2h-1 ) , 8.057.98 ( m , 4 h , 4c-17 ) , 7.75 ( t , j18,17=j18,19=8 hz , 4 h , 4c-18 ) , 7.717.62 ( m , 2 h , 2c-19 ) , 6.21 ( d , j34=9 hz , 1 h , h-3 ) , 5.27 ( d , j9a,9b=12 hz , 2 h , 2h-9a ) , 5.164.99 ( m , 7 h , 2h-9b , 2h-3 , h-6 , 2h-7 ) , 4.924.83 ( m , 2 h , 2h-6 ) , 4.834.70 ( m , 4 h , 2h-8a , 2h-10 ) , 4.704.58 ( m , 4 h , 2h-8b , h-5 , h-7 ) , 4.574.48 ( m , 2 h , 2h-5 ) , 4.43 ( t , j4,3=j4,5=8 hz , 1 h , h-4 ) , 4.214.11 ( m , 4 h , 2h-4 , 2h-13 ) , 4.083.90 ( m , 6 h , 2h-15ab , 2h-14 ) , 3.893.82 ( m , 2 h , 2h-12 ) , 3.823.73 ( m , 3 h , 2h-11 , h-8a ) , 3.46 ppm ( dd , j8b,8a=13 hz , j8b,7=4 hz , 1 h , h-8b ) ; c nmr ( 100 mhz , d2o with 30 % cd3cn ) : =147.90 ( 4c-2 ) , 145.44 ( 2c-2 ) , 130.82129.48 ( 4c-18 , 2c-19 ) , 130.00 ( 2c-16 ) , 126.40 ( 2c-1 ) , 126.21 ( 4c-17 ) , 125.78 ( c-1 ) , 124.76 ( c-1 ) , 123.66 ( 2c-1 ) , 102.73 ( 2c-10 ) , 88.11 ( c-3 ) , 77.554 ( c-7 ) , 76.27 ( 2c-7 ) , 75.81 ( 2c-14 ) , 75.31 ( 2c-5 ) , 74.50 ( 2c-3 ) , 74.20 ( c-5 ) , 73.62 ( 2c-4 ) , 73.38 ( 2c-12 ) , 73.25 ( c-4 ) , 71.29 ( 2c-11 ) , 69.18 ( 2 c-13 ) , 64.29 ( 2c-6 ) , 62.37 ( 2c-9 ) , 62.07 ( c-6 ) , 61.55 ( 2c-15 ) , 60.22 ( c-8 ) , 51.44 ppm ( 2c-8 ) ; ms ( esi ) m / z [ m+2 h ] calcd for c56h70n18o22 : 674.63 , found 674.95 ; hrms ( q - tof ) as a service to our authors and readers , this journal provides supporting information supplied by the authors . such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors .

the bacterial adhesion lectin leca is an attractive target for interference with the infectivity of its producer p. aeruginosa . divalent ligands with two terminal galactoside moieties connected by an alternating glucose - triazole spacer were previously shown to be very potent inhibitors . in this study , we chose to prepare a series of derivatives with various new substituents in the spacer in hopes of further enhancing the leca inhibitory potency of the molecules . based on the binding mode , modifications were made to the spacer to enable additional spacer protein interactions . the introduction of positively charged , negatively charged , and also lipophilic functional groups was successful . the compounds were good leca ligands , but no improved binding was seen , even though altered thermodynamic parameters were observed by isothermal titration calorimetry ( itc ) .

Introduction Results and Discussion Conclusion Experimental Section Supporting Information

enhancing the binding potency of carbohydrate inhibitors of protein carbohydrate interactions is an important step towards improved medicinal applications.1 this is mostly the case because of the relatively weak interactions between proteins and carbohydrate ligands . ensuring multivalency of ligands is a proven method to achieve this.2 leca is an important virulence factor of pseudomonas aeruginosa and is involved in bacterial adhesion and biofilm formation.3 the protein has recently become a popular target for the design of multivalent inhibitors.4 the tetrameric protein promotes the adhesion of the bacteria to tissue cell surfaces , thus facilitating subsequent steps such as cell invasion and biofilm formation . inhibiting bacterial adhesion proteins has the potential to become a mild and less resistance - prone method to treat and prevent bacterial infections.5 two of the four binding sites , with specificity for galactosides , are relatively close together with a separation of about 26 .6 this arrangement has led us to design and synthesize divalent galactoside ligands with well - defined and rigid spacers that should allow a chelation - type divalent binding mode.7 flexible spacers are commonly used as they are forgiving of imperfect design and usually yield sizeable potency enhancements in multivalent systems.7 they are , however , not optimal as there will be a significant entropy loss upon binding and , moreover , achieving selectivity will be less likely . in our search for an optimal spacer we found compound 1 ( figure 1 a ) to be a highly potent divalent ligand with nanomolar inhibitory potency.8a the spacer of this structure contains direct linkages between the glucose moieties and the connected triazoles.8 this arrangement leads to a relatively rigid structure , in which rotations of the components can take place , but the overall geometry remains mostly linear . a ) structure of potent divalent leca inhibitor 1 with the relatively rigid glucose - triazole - based spacer ; b ) schematic divalent binding mode of a divalent ligand to two leca subunits ; c ) x - ray structure of leca with bound galactose moieties . the two asp 47 carboxylates in the spacer path are shown explicitly.6 most importantly , good solubility in water was observed . we found that three glucose - triazole units was the optimal length for leca inhibition , while divalent ligands with two and four units showed far inferior inhibition . all data were consistent with a chelating binding mode ; especially convincing was the stoichiometry derived from isothermal titration calorimetry ( itc ) binding experiments . furthermore , the short linkage of just a single carbon between the galactoside ligand and the triazole proved to be a major contributing factor to the success of this compound . in order to further optimize the potency of the compound and to explore the principle of protein spacer interactions , we now report on our functionalization of the spacer of 1 with various functional groups . the presence of the two carboxylates ( asp 47 in each subunit ) is apparent when looking at the path between the two bound galactosides , which the spacer is likely to span on the leca protein surface when chelating bivalent binding occurs ( figure 1 c ) . these two carboxylates are likely to be in close proximity to the 6-oh group on the terminal spacer glucoside units , depending on the rotational state of the molecule . molecular modeling was used in order to gauge whether positively charged functional groups at the c-6 position on the terminal spacer glucoside units would be able to interact with the asp 47 residues . creating a stabilized conformation with the positive charges in close proximity to the asp 47 carboxylates was possible , and this orientation was used as the starting point of additional simulations . especially , the two hydrogen - bonded salt bridges , between the asp 47 side chains and the ammonium groups of the protonated form of 12 , remained within 3 and were compatible with a geometry that included two fully bound galactoside ligand units . such a bound geometry was also possible for a compound that included a pyridinium group ( as in 13 ) , but resulted in longer distances between the carboxylate oxygens and the pyridinium nitrogens , due to bigger steric requirements of the pyridinium units . these experiments lead to the conclusion that the introduction of ammonium groups would be most promising . we chose to prepare a series of derivatives with various new substituents in the spacer in hopes of further enhancing the leca inhibitory potency of the molecules . also , negatively charged and more lipophilic groups were included as these could possibly benefit from interacting with other proximate parts of the protein that would not have been previously obvious . the synthesis of the modified spacers started with two previously prepared building blocks 2 and 3.8 these building blocks were coupled by a double copper(i)-catalyzed azide alkyne cycloaddition ( cuaac ) reaction . next , the two galactosyl axial 4-oh groups of the resulting product 4 were turned into triflates , thus enabling the subsequent substitution by azide with inversion to give 5 . reaction of 8 with triflic anhydride in the presence of pyridine followed by zempln deprotection gave the bis pyridinium compound 10 . cuaac coupling of phenylacetylene to 11 and subsequent zempln deprotection gave the bis - triazole 13 . the compounds were tested on an array chip in an assay similar to an enzyme - linked immunosorbent assay ( elisa ) , as previously reported.8 fluorescein - labeled leca was incubated with the inhibitors and exposed to a galactoside - functionalized chip surface . monovalent ligand 6 b was previously determined to have an ic50 in this assay of 22 m.8a clearly all divalent compounds were far more potent and showed major multivalency effects ( table 1 ) . larger potency drops of about an order of magnitude were observed for both the negatively charged bis - carboxylate 9 and the positively charged bis - amine 12 . subsequently , itc experiments were conducted , which confirmed the divalent binding mode in all cases , with the stoichiometry n values being close to 0.5 . furthermore the small potency differences of the chip - based assay were not seen in the itc assay . interestingly , when looking at the enthalpic and entropic components of the binding event , the lipophilic and noncharged compound 13 , showed enthalpy entropy compensation , with a lower beneficial binding enthalpy and , at the same time , a lowered entropy loss upon binding . the latter is understandable as an example of the hydrophobic effect where water molecules in an ice - like structure are liberated from the lipophilic surfaces . inhibitory potencies ( ic50 ) of the divalent inhibitors on leca binding[a ] and dissociation constants ( kd ) , stoichiometry , and thermodynamic binding parameters[b ] [ a ] chip - based elisa - like assay : fitc - labeled leca ( 5 g ml ) binding to a galactoside functionalized chip surface . [ b ] obtained from isothermal titration microcalorimetry ( itc ) : [ leca]=2040 m . subsequently , the selectively deprotectable silyl groups were removed , and the resulting primary hydroxy groups were converted to carboxylate groups by oxidation , to tosylates and subsequently to azides by substitution , and to pyridiniums via their corresponding triflates . the azides allowed cuaac coupling and also further reduction to the corresponding amino groups . while it was anticipated that some of these groups would be able to take advantage of additional beneficial interactions with the nearby protein surface , we did not observe this through enhanced inhibitory or binding potencies . this was true even for the positively charged groups that could possibly take advantage of the nearby positioned carboxylate groups of asp 47 . in future designs nevertheless , the functionalization of the spacers will also be important in order to fine - tune other properties of this type of ligand , such as toxicity and absorption , distribution , metabolism , and excretion ( adme ) , which are important for drug development . spots were visualized by uv light and also by 10 % h2so4 in meoh . the proton numbering scheme of all compounds can be found in the supporting information and is used in the assignments of the signals for the nmr spectra below . isothermal titration microcalorimetry ( itc ) : the lectin leca was obtained from sigma aldrich and was dissolved in buffer ( 0.1 m tris - hcl , 6 mm cacl2 , ph 7.5 ) and degassed . protein concentration ( between 20 and 40 m depending on the ligand affinity ) was checked by measurement of optical density by using a theoretical molar extinction coefficient of 28 000 . carbohydrate ligands were dissolved directly into the same buffer , degassed , and placed in the injection syringe ( concentration range : 0.10.2 mm ) . the kd value was calculated as 1/ka , and t is 298 k. each ligand test was performed in duplicate . the fluorescence intensities were expressed in arbitrary units , and the relative intensities were the average of the two duplicate spots . ph 7.4 ) , containing different concentrations of the inhibitors were incubated for 1 h at rt and subsequently added to the galactoside - functionalized chip . the binding process was monitored for 2 h , and the end values of the fluorescence detection were taken for the determination of the ic50 by using prism 5 ( graphpad software , inc . subsequently , the complex with leca was built by superposition of one of the galactose units of the ligand with a bound galactose of one of the subunits of the leca crystal structure ( pdb i d : 1oko).3a the other galactose unit of the divalent ligand was then pulled into the adjacent galactose binding site of leca by restraint md using distance restraints based on the position of the bound galactose present in the x - ray structure with respect to a number of leca residues . possible electrostatic interactions between two positively charged amino and pyridinium substitutions in the linker region with two asp 47s of leca were investigated in more detail . in order to induce these interactions , the subunits comprising the substituted moieties were first rotated , and subsequently , the nitrogen atoms of respectively the amino and the pyridinium group were restrained to bring them in close proximity to the carbon atoms of the carboxylic acid moieties of asp 47 . compound 4 : a click reaction of a mixture of compound 2 ( 0.253 g , 0.71 mmol ) , compound 3 ( 0.83 g , 1.63 mmol ) , cuso45 h2o ( 0.06 g , 0.24 mmol ) , and naasc ( 0.096 g , 0.48 mmol ) in dmf ( 5 ml ) containing 10 % h2o was performed following the general procedure described above to afford compound 4 ( 0.84 g , 85 % ) ; h nmr ( 400 mhz , cdcl3 ) : = 8.037.89 ( m , 5 h , 4ch benzoyl , h-1 ) , 7.867.76 ( m , 5 h , 4ch benzoyl , h-1 ) , 7.527.26 ( m , 12 h , 12ch benzoyl ) , 6.08 ( dd , j4,3=j4,5=10 hz , 1 h , h-4 ) , 6.025.90 ( m , 2 h , h-4 , h-3 ) , 5.81 ( dd , j5,4=j5,6=9 hz , 1 h , h-5 ) , 5.60 ( dd , j4,3=j4,5=9 hz , 1 h , h-4 ) , 5.515.41 ( m , 2 h , 2h-5 ) , 5.024.92 ( m , 2 h , 2h-3 ) , 4.81 ( dd , j6,5=j6,7=10 hz , 1 h , h-6 ) , 4.674.54 ( m , 3 h , h-7 , 2 h-6 ) , 4.164.07 ( m , 1 h h-8a ) , 4.073.93 ( m , 2 h , 2h-8 ) , 3.913.82 ( m , 2h-7 ) , 3.703.60 ( m , 1 h , h-8b ) , 3.573.51 ( m , 1 h , 6-oh ) , 3.353.29 ( 1 h , 6-oh ) , 2.02 , 1.65 , 1.58 ( s , 9 h , 3ch3 , acetyl ) , 0.90 ( s , 18 h , 2sic(ch3)3 ) , 0.07 ppm ( 2 s , 12 h , 2si(ch3)2 ) ; c nmr ( 100 mhz , cdcl3 ) : =69.97 , 169.10 , 168.95 ( 3c = o acetyl ) , 166.14 , 166.05 , 165.63 , 165.55 ( 4c = o benzoyl ) , 145.61 , 145.58 ( 2c-2 ) , 133.44133.21 ( ch benzoyl ) , 129.99128.41 ( ch benzoyl ) , 129.39129.20 ( c benzoyl ) 123.43 , 122.13 ( 2c-1 ) , 85.55 ( c-3 ) , 77.97 ( 2c-7 ) , 75.75 ( c-5 ) , 75.48 ( c-5 ) , 74.84 ( c-7 ) , 74.03 , 73.77 ( 2c-3 ) , 72.41 ( c-5 ) , 70.85 ( c-4 ) , 69.94 , 69.59 ( 2c-4 ) , 68.92 , 68.53 ( 2c-6 ) , 63.65 , 63.13 ( 2c-8 ) , 61.61 ( c-8 ) , 59.84 ( c-6 ) , 25.99 ( sic(ch3)3 ) , 20.72 , 19.98 , 19.89 ( 3ch3 acetyl ) , 18.44 ( sic(ch3)3 ) , 5.31 ppm ( si(ch3)2 ) ; ms ( esi ) m / z [ m+h ] calcd for c68h84n6o21si2 : 1378.59 , found 1378.00 ; hrms ( q - tof ) m / z [ m+h ] calcd for c68h84n6o21si2 : 1377.5228 , found 1377.5326 . the residue was dissolved in an acetone / h2o mixture ( 15 ml , 4:1 ) , and nan3 ( 0.397 g , 6.1 mmol ) was added . compound 8 : compound 7 ( 0.87 g , 0.40 mmol ) was dissolved in ch3cn / h2o ( 24 ml , 7:1 ) , and p - tsoh ( 0.114 g , 0.60 mmol ) was added . the organic phase was washed once with brine ( 1 ml ) , dried over na2so4 , and concentrated in vacuo . the mixture was stirred at rt overnight , treated with h resin , concentrated , and subjected to preparative hplc purification , which gave compound 13 ( 9.8 mg , 7.3 mol , 24 % ) ; h nmr ( 400 mhz , d2o with 30 % cd3cn ) : =8.62 ( s , 1 h , h-1 ) , 8.50 , 8.49 ( 2 s , 2 h , 2h-1 ) , 8.46 ( s , 1 h , h-1 ) , 8.42 , 8.41 ( 2 s , 2 h , 2h-1 ) , 8.057.98 ( m , 4 h , 4c-17 ) , 7.75 ( t , j18,17=j18,19=8 hz , 4 h , 4c-18 ) , 7.717.62 ( m , 2 h , 2c-19 ) , 6.21 ( d , j34=9 hz , 1 h , h-3 ) , 5.27 ( d , j9a,9b=12 hz , 2 h , 2h-9a ) , 5.164.99 ( m , 7 h , 2h-9b , 2h-3 , h-6 , 2h-7 ) , 4.924.83 ( m , 2 h , 2h-6 ) , 4.834.70 ( m , 4 h , 2h-8a , 2h-10 ) , 4.704.58 ( m , 4 h , 2h-8b , h-5 , h-7 ) , 4.574.48 ( m , 2 h , 2h-5 ) , 4.43 ( t , j4,3=j4,5=8 hz , 1 h , h-4 ) , 4.214.11 ( m , 4 h , 2h-4 , 2h-13 ) , 4.083.90 ( m , 6 h , 2h-15ab , 2h-14 ) , 3.893.82 ( m , 2 h , 2h-12 ) , 3.823.73 ( m , 3 h , 2h-11 , h-8a ) , 3.46 ppm ( dd , j8b,8a=13 hz , j8b,7=4 hz , 1 h , h-8b ) ; c nmr ( 100 mhz , d2o with 30 % cd3cn ) : =147.90 ( 4c-2 ) , 145.44 ( 2c-2 ) , 130.82129.48 ( 4c-18 , 2c-19 ) , 130.00 ( 2c-16 ) , 126.40 ( 2c-1 ) , 126.21 ( 4c-17 ) , 125.78 ( c-1 ) , 124.76 ( c-1 ) , 123.66 ( 2c-1 ) , 102.73 ( 2c-10 ) , 88.11 ( c-3 ) , 77.554 ( c-7 ) , 76.27 ( 2c-7 ) , 75.81 ( 2c-14 ) , 75.31 ( 2c-5 ) , 74.50 ( 2c-3 ) , 74.20 ( c-5 ) , 73.62 ( 2c-4 ) , 73.38 ( 2c-12 ) , 73.25 ( c-4 ) , 71.29 ( 2c-11 ) , 69.18 ( 2 c-13 ) , 64.29 ( 2c-6 ) , 62.37 ( 2c-9 ) , 62.07 ( c-6 ) , 61.55 ( 2c-15 ) , 60.22 ( c-8 ) , 51.44 ppm ( 2c-8 ) ; ms ( esi ) m / z [ m+2 h ] calcd for c56h70n18o22 : 674.63 , found 674.95 ; hrms ( q - tof ) as a service to our authors and readers , this journal provides supporting information supplied by the authors . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors .

ensuring multivalency of ligands is a proven method to achieve this.2 leca is an important virulence factor of pseudomonas aeruginosa and is involved in bacterial adhesion and biofilm formation.3 the protein has recently become a popular target for the design of multivalent inhibitors.4 the tetrameric protein promotes the adhesion of the bacteria to tissue cell surfaces , thus facilitating subsequent steps such as cell invasion and biofilm formation . inhibiting bacterial adhesion proteins has the potential to become a mild and less resistance - prone method to treat and prevent bacterial infections.5 two of the four binding sites , with specificity for galactosides , are relatively close together with a separation of about 26 .6 this arrangement has led us to design and synthesize divalent galactoside ligands with well - defined and rigid spacers that should allow a chelation - type divalent binding mode.7 flexible spacers are commonly used as they are forgiving of imperfect design and usually yield sizeable potency enhancements in multivalent systems.7 they are , however , not optimal as there will be a significant entropy loss upon binding and , moreover , achieving selectivity will be less likely . in our search for an optimal spacer we found compound 1 ( figure 1 a ) to be a highly potent divalent ligand with nanomolar inhibitory potency.8a the spacer of this structure contains direct linkages between the glucose moieties and the connected triazoles.8 this arrangement leads to a relatively rigid structure , in which rotations of the components can take place , but the overall geometry remains mostly linear . a ) structure of potent divalent leca inhibitor 1 with the relatively rigid glucose - triazole - based spacer ; b ) schematic divalent binding mode of a divalent ligand to two leca subunits ; c ) x - ray structure of leca with bound galactose moieties . in order to further optimize the potency of the compound and to explore the principle of protein spacer interactions , we now report on our functionalization of the spacer of 1 with various functional groups . the presence of the two carboxylates ( asp 47 in each subunit ) is apparent when looking at the path between the two bound galactosides , which the spacer is likely to span on the leca protein surface when chelating bivalent binding occurs ( figure 1 c ) . especially , the two hydrogen - bonded salt bridges , between the asp 47 side chains and the ammonium groups of the protonated form of 12 , remained within 3 and were compatible with a geometry that included two fully bound galactoside ligand units . such a bound geometry was also possible for a compound that included a pyridinium group ( as in 13 ) , but resulted in longer distances between the carboxylate oxygens and the pyridinium nitrogens , due to bigger steric requirements of the pyridinium units . reagents and conditions : a ) cuso45 h2o , naasc , dmf with 10 % h2o , 80 c , 30 min , 85 % ; b ) 1 ) tf2o , 10 % pyridine in ch2cl2 , 0 c , 3 h , 2 ) nan3 , acetone / h2o ( 4:1 ) , rt , o / n , 98 % ; c ) 6 a , cuso45 h2o , naasc , dmf with 10 % h2o , 80 c , 30 min , 64 % ; d ) p - tsoh , ch3cn / h2o ( 7:1 ) , rt , 6 h , 83 % . reagents and conditions : a ) 1 ) tempo , naocl , nabr , bu4nbr , nahco3/na2co3 ph 9.5 , 0 c , 2 h , 2 ) naome , meoh , rt , o / n , 38 % ; b ) 1 ) tf2o , 10 % pyridine in ch2cl2 , 0crt , 3 h , 2 ) naome , meoh , rt , o / n , 44 % , c ) 1 ) tscl , dabco , ch2cl2 , rt , o / n , 2 ) nan3 , dmf , 95 c , o / n , 34 % , d ) 1 ) naome , meoh , rt , o / n , 2 ) h2 , pd / c , rt , 60 % , e ) 1 ) phenylacetylene , cuso45 h2o , naasc , dmf with 10 % h2o , 80 c , 30 min , 2 ) naome , meoh , rt , o / n , 24% . interestingly , when looking at the enthalpic and entropic components of the binding event , the lipophilic and noncharged compound 13 , showed enthalpy entropy compensation , with a lower beneficial binding enthalpy and , at the same time , a lowered entropy loss upon binding . inhibitory potencies ( ic50 ) of the divalent inhibitors on leca binding[a ] and dissociation constants ( kd ) , stoichiometry , and thermodynamic binding parameters[b ] [ a ] chip - based elisa - like assay : fitc - labeled leca ( 5 g ml ) binding to a galactoside functionalized chip surface . in future designs nevertheless , the functionalization of the spacers will also be important in order to fine - tune other properties of this type of ligand , such as toxicity and absorption , distribution , metabolism , and excretion ( adme ) , which are important for drug development . subsequently , the complex with leca was built by superposition of one of the galactose units of the ligand with a bound galactose of one of the subunits of the leca crystal structure ( pdb i d : 1oko).3a the other galactose unit of the divalent ligand was then pulled into the adjacent galactose binding site of leca by restraint md using distance restraints based on the position of the bound galactose present in the x - ray structure with respect to a number of leca residues . in order to induce these interactions , the subunits comprising the substituted moieties were first rotated , and subsequently , the nitrogen atoms of respectively the amino and the pyridinium group were restrained to bring them in close proximity to the carbon atoms of the carboxylic acid moieties of asp 47 . general procedure of microwave - assisted click reaction : to a solution of the azide and alkyne compounds in dimethylformamide ( dmf ) with 10 % h2o , was added cuso45 the mixture was then heated by microwave irradiation at 80 c for 30 min . compound 4 : a click reaction of a mixture of compound 2 ( 0.253 g , 0.71 mmol ) , compound 3 ( 0.83 g , 1.63 mmol ) , cuso45 h2o ( 0.06 g , 0.24 mmol ) , and naasc ( 0.096 g , 0.48 mmol ) in dmf ( 5 ml ) containing 10 % h2o was performed following the general procedure described above to afford compound 4 ( 0.84 g , 85 % ) ; h nmr ( 400 mhz , cdcl3 ) : = 8.037.89 ( m , 5 h , 4ch benzoyl , h-1 ) , 7.867.76 ( m , 5 h , 4ch benzoyl , h-1 ) , 7.527.26 ( m , 12 h , 12ch benzoyl ) , 6.08 ( dd , j4,3=j4,5=10 hz , 1 h , h-4 ) , 6.025.90 ( m , 2 h , h-4 , h-3 ) , 5.81 ( dd , j5,4=j5,6=9 hz , 1 h , h-5 ) , 5.60 ( dd , j4,3=j4,5=9 hz , 1 h , h-4 ) , 5.515.41 ( m , 2 h , 2h-5 ) , 5.024.92 ( m , 2 h , 2h-3 ) , 4.81 ( dd , j6,5=j6,7=10 hz , 1 h , h-6 ) , 4.674.54 ( m , 3 h , h-7 , 2 h-6 ) , 4.164.07 ( m , 1 h h-8a ) , 4.073.93 ( m , 2 h , 2h-8 ) , 3.913.82 ( m , 2h-7 ) , 3.703.60 ( m , 1 h , h-8b ) , 3.573.51 ( m , 1 h , 6-oh ) , 3.353.29 ( 1 h , 6-oh ) , 2.02 , 1.65 , 1.58 ( s , 9 h , 3ch3 , acetyl ) , 0.90 ( s , 18 h , 2sic(ch3)3 ) , 0.07 ppm ( 2 s , 12 h , 2si(ch3)2 ) ; c nmr ( 100 mhz , cdcl3 ) : =69.97 , 169.10 , 168.95 ( 3c = o acetyl ) , 166.14 , 166.05 , 165.63 , 165.55 ( 4c = o benzoyl ) , 145.61 , 145.58 ( 2c-2 ) , 133.44133.21 ( ch benzoyl ) , 129.99128.41 ( ch benzoyl ) , 129.39129.20 ( c benzoyl ) 123.43 , 122.13 ( 2c-1 ) , 85.55 ( c-3 ) , 77.97 ( 2c-7 ) , 75.75 ( c-5 ) , 75.48 ( c-5 ) , 74.84 ( c-7 ) , 74.03 , 73.77 ( 2c-3 ) , 72.41 ( c-5 ) , 70.85 ( c-4 ) , 69.94 , 69.59 ( 2c-4 ) , 68.92 , 68.53 ( 2c-6 ) , 63.65 , 63.13 ( 2c-8 ) , 61.61 ( c-8 ) , 59.84 ( c-6 ) , 25.99 ( sic(ch3)3 ) , 20.72 , 19.98 , 19.89 ( 3ch3 acetyl ) , 18.44 ( sic(ch3)3 ) , 5.31 ppm ( si(ch3)2 ) ; ms ( esi ) m / z [ m+h ] calcd for c68h84n6o21si2 : 1378.59 , found 1378.00 ; hrms ( q - tof ) m / z [ m+h ] calcd for c68h84n6o21si2 : 1377.5228 , found 1377.5326 . compound 7 : a click reaction of a mixture of compound 5 ( 0.88 g , 0.62 mmol ) and compound 6 a ( 0.570 g , 1.48 mmol ) with cuso45 h2o ( 28 mg , 0.11 mmol ) and naasc ( 0.0443 g , 0.22 mmol ) in dmf ( 15 ml ) containing 10 % h2o was performed following the general procedure described above to afford compound 7 ( 0.87 g , 64 % ) ; h nmr ( 400 mhz , cdcl3 ) : =7.88 ( s , 1 h , h-1 ) , 7.827.68 ( m , 9 h , 8ch benzoyl , h-1 ) , 7.687.59 ( 2 s , 2 h , 2h-1 ) , 7.517.39 ( m , 4 h , 4ch benzoyl ) , 7.367.26 ( m , 9 h , 8ch benzoyl ) , 6.356.22 ( m , 2 h , 2h-5 ) , 5.94 ( d , j3,4=9 hz , 1 h , h-3 ) , 5.885.73 ( m , 2 h , 2h-4 ) , 5.675.52 ( m , 2 h , h-5 , h-4 ) , 5.435.33 ( m , 2 h , 2h-13 ) , 5.275.07 ( m , 6 h , 2h-3 , 2h-11 , 2h-6 ) , 5.034.71 ( m , 7 h , 2 h-12 , 2h-9a , h-6 , 2h-9b ) , 4.634.53 ( m , 1 h , h-7 ) , 4.484.37 ( m , 4 h , 2h-7 , 2h-10 ) , 4.294.04 ( m , 5 h , 2h-15a , h-8a , 2h-15b ) , 3.943.85 ( m , 2 h , 2h-14 ) , 3.80 ( d , j8a,8b=12 hz , 2 h , 2h-8a ) , 3.65 ( dd , j8b,8a=12 hz , j8b,7=3 hz , 1 h , h-8b ) , 3.453.32 ( m , 2 h , 2 h-8b ) , 2.201.88 ( m , 21 h , 7ch3 , acetyl ) , 1.771.63 ( m , 12 h , 4ch3 , acetyl ) , 0.940.84 ( m , 18 h , 2sic(ch3)3 ) , 0.05(0.07 ) ppm ( m , 12 h , 2si(ch3)2 ) ; c nmr ( 100 mhz , cdcl3 ) : =170.83168.96 ( c = o acetyl ) , 165.51 ( c = o benzoyl ) , 165.39 ( c = o benzoyl ) , 165.07 ( c = o benzoyl ) , 144.90 ( 2c-2 ) , 143.54 ( 2c-2 ) , 133.64 , 129.81128.53 ( ch benzoyl ) , 128.81 ( c benzoyl ) 123.66 , 123.57 , 123.14 , 121.67 ( 4c-1 ) , 99.29 , 99.26 ( 2c-10 ) , 85.70 ( c-3 ) , 79.40 , 79.33 ( 2c-7 ) , 75.02 ( c-7 ) , 74.20 , 73.85 ( 2c-5 ) , 73.67 , 73.42 ( 2c-3 ) , 72.70 ( c-5 ) , 72.22 , 72.15 ( 2c-4 ) , 70.99 ( 2c-12 ) , 70.78 ( 2c-14 ) , 70.73 ( c-4 ) , 68.74 ( 2c-11 ) , 67.25 ( 2c-13 ) , 61.84 ( 2c-9 , 2c-8 ) , 61.43 ( 2c-15 , c-8 ) , 60.17 , 60.12 ( 2c-6 ) , 59.81 ( c-6 ) , 25.96 ( sic(ch3)3 ) , 20.9719.92 ( ch3 acetyl ) , 18.52 ( sic(ch3)3 ) , 5.11(5.34 ) ppm ( si(ch3)2 ) ; ms ( esi ) m / z [ m+2 h]calcd for c102h126n12o39si2 : 1100.89 , found 1100.60 ; hrms ( q - tof ) m / z [ m+2 h ] calcd for c102h126n12o39si2 : 1100.3892 , found 1100.8980 . compound 13 : a click reaction of a mixture of compound 11 ( 61.8 mg , 0.031 mmol ) , phenylacetylene ( 11.1 mg , 0.109 mmol ) , naasc ( 6.5 mg , 0.033 mmol ) , and cuso45 h2o ( 4.1 mg , 0.016 mmol ) was performed following the general procedure described above to afford the coupling product compound ( 54 mg , 80 % ) , which was then treated with naome in meoh ( 0.5 m , 5 ml ) . the mixture was stirred at rt overnight , treated with h resin , concentrated , and subjected to preparative hplc purification , which gave compound 13 ( 9.8 mg , 7.3 mol , 24 % ) ; h nmr ( 400 mhz , d2o with 30 % cd3cn ) : =8.62 ( s , 1 h , h-1 ) , 8.50 , 8.49 ( 2 s , 2 h , 2h-1 ) , 8.46 ( s , 1 h , h-1 ) , 8.42 , 8.41 ( 2 s , 2 h , 2h-1 ) , 8.057.98 ( m , 4 h , 4c-17 ) , 7.75 ( t , j18,17=j18,19=8 hz , 4 h , 4c-18 ) , 7.717.62 ( m , 2 h , 2c-19 ) , 6.21 ( d , j34=9 hz , 1 h , h-3 ) , 5.27 ( d , j9a,9b=12 hz , 2 h , 2h-9a ) , 5.164.99 ( m , 7 h , 2h-9b , 2h-3 , h-6 , 2h-7 ) , 4.924.83 ( m , 2 h , 2h-6 ) , 4.834.70 ( m , 4 h , 2h-8a , 2h-10 ) , 4.704.58 ( m , 4 h , 2h-8b , h-5 , h-7 ) , 4.574.48 ( m , 2 h , 2h-5 ) , 4.43 ( t , j4,3=j4,5=8 hz , 1 h , h-4 ) , 4.214.11 ( m , 4 h , 2h-4 , 2h-13 ) , 4.083.90 ( m , 6 h , 2h-15ab , 2h-14 ) , 3.893.82 ( m , 2 h , 2h-12 ) , 3.823.73 ( m , 3 h , 2h-11 , h-8a ) , 3.46 ppm ( dd , j8b,8a=13 hz , j8b,7=4 hz , 1 h , h-8b ) ; c nmr ( 100 mhz , d2o with 30 % cd3cn ) : =147.90 ( 4c-2 ) , 145.44 ( 2c-2 ) , 130.82129.48 ( 4c-18 , 2c-19 ) , 130.00 ( 2c-16 ) , 126.40 ( 2c-1 ) , 126.21 ( 4c-17 ) , 125.78 ( c-1 ) , 124.76 ( c-1 ) , 123.66 ( 2c-1 ) , 102.73 ( 2c-10 ) , 88.11 ( c-3 ) , 77.554 ( c-7 ) , 76.27 ( 2c-7 ) , 75.81 ( 2c-14 ) , 75.31 ( 2c-5 ) , 74.50 ( 2c-3 ) , 74.20 ( c-5 ) , 73.62 ( 2c-4 ) , 73.38 ( 2c-12 ) , 73.25 ( c-4 ) , 71.29 ( 2c-11 ) , 69.18 ( 2 c-13 ) , 64.29 ( 2c-6 ) , 62.37 ( 2c-9 ) , 62.07 ( c-6 ) , 61.55 ( 2c-15 ) , 60.22 ( c-8 ) , 51.44 ppm ( 2c-8 ) ; ms ( esi ) m / z [ m+2 h ] calcd for c56h70n18o22 : 674.63 , found 674.95 ; hrms ( q - tof ) as a service to our authors and readers , this journal provides supporting information supplied by the authors .

to study bacteria at the micrometer scale or single - cell resolution ( kreft et al . this can be achieved by microscopy in conjunction with fluorescent markers , e.g. dyes such as propidium iodide and syto9 allowing to determine the membrane integrity of bacterial cells ( berney et al . 2007 ) , fluorescence in situ hybridization ( remus - emsermann et al . 2014 ) , or by labeling bacteria using fluorescent proteins ( fps ) ( ledermann et al . 2015 ) . often , fps were used under the control of inducible promoters as bioreporters , which allow the assessment of the availability of inducing agents , such as fructose ( leveau and lindow 2001a ) , or under the control of constitutive promoters to identify bacteria in situ ( bloemberg et al . the use of minitn7-transposon delivery plasmids is widespread in molecular and environmental microbiology , e.g. to chromosomally integrate promoter - reporter gene constructs ( choi and schweizer 2006 , mckenzie and craig 2006 , lagendijk et al . 2010 ) or for the complementation of knockout mutants ( crepin , harel and dozois 2012 ) . the bacterial tn7 transposon provides an excellent non - destructive tool for several reasons : ( i ) it integrates with high affinity at the atttn7 site within a wide range of gram - negative bacteria ; ( ii ) it does not disrupt genes , omitting potential pleiotropic effect ; ( iii ) and antibiotics are not required to maintain the inserted transposons ( choi and schweizer 2006 , choi and kim 2009 ) . most tn7-transposon delivery systems have in common that they employ a suicide plasmid - based approach using vectors carrying a puc origin of replication ( lambertsen , sternberg and molin 2004 , choi and schweizer 2006 , lagendijk et al . plasmids featuring this origin of replication do not replicate in bacterial recipients and classically , the transposon machinery is provided on a helper plasmid . in enterobacteriaceae , however , the used puc origin of replication works efficiently , thereby excluding escherichia coli and salmonella spp . as possible recipients . using a temperature - sensitive tn7-delivery system constructed by mckenzie and craig ( 2006 ) , we provide a ready - to - use alternative for fluorescent labeling of enterobacteriaceae . we provide two different promoters , the synthetic , constitutive , laci - repressible tac promoter ( ptac ) ( de boer , comstock and vasser 1983 ) and the constitutive nptii promoter ( pnptii ) ( ledermann et al . 2015 ) in combination with fluorescent marker genes , encoding blue , cyan , yellow or red fps . in combination with two - parental mating employing e. coli s17 - 1 , which provides the tra operon necessary for the mobilization of plasmids as a chromosomal insertion , we overcome the often tedious low efficiency of transposition and preparation of competent recipient cells . the here - proposed sets of plasmids offer a convenient alternative to generate multicolored sets of stably fluorescently labeled bacteria . the fps allow for multichannel fluorescent microscopy analysis with little to no overlap of fp emissions to study multistrain synthetic communities ( fig . s2 , supporting information ) . employing the repressability of the ptac by laci , e.g. by coexpressing laci , it is possible to construct so - called reproductive success , or cusper , bioreporter strains ( reproductive success backwards = cusper ) ( remus - emsermann and leveau 2010 , remus - emsermann et al . cusper bioreporters are equipped with a ptac - controlled fp and express laci , thereby the expression of the fp is repressed . when derepressing the expression , i.e. by adding lactose or isopropyl -d-1-thiogalactopyranoside to growing cells , cells can be loaded with fp ( leveau and lindow 2001b ) . if the derepressor is subsequently removed , no de novo production of fp occurs and the preformed fp is then diluted from growing cells ( remus - emsermann and leveau 2010 ) . the fluorescence intensity of growing cells relative to their ancestors thereby becomes a proxy for the number of divisions individual cells underwent ( remus - emsermann and leveau 2010 ) . cusper bioreporters were used to study the probability of successful colonization of bacterial cells on plant leaves , the heterogeneity of microhabitats on plant leaf surface , the probability of secondary bacterial colonization on precolonized leaves ( remus - emsermann and leveau 2010 , remus - emsermann et al . 2012 , remus - emsermann , kowalchuk and leveau 2013 ) , and to estimate reproductive success in a spatial context in mice spleens and plant leaves ( helaine et al . 2010 , tecon and leveau 2012 ) . using the model strain e. coli o157:h7 stx , we demonstrate the transposon delivery of the promoter fluorescence reporter constructs and resulting fluorescence at the population and single - cell resolution . escherichia coli neb 5-alpha ( new england biolabs , ipswich , ma , usa ) , e. coli s17 - 1 and e. coli o157:h7 stx ( nctc 12900 ) were routinely grown on lysogeny broth ( lb ) or lb agar at 37c . to prevent plasmids loss , for counterselection of auxotroph e. coli s17 - 1 after mating , mm2 medium agar ( 4 g l l - asparagine , 2 g l k2hpo4 , 0.2 g l mgso4 , 3 g l nacl , 10 g l sorbitol , 15 g l agar ) was used . where necessary , media were supplemented with 100 g ml ampicillin , 50 g ml kanamycin or 20 g ml tetracycline . for microtiter plate reader experiments , cells were grown in m9 minimal medium ( 20 ml l 20% ( w / v ) casamino acids ( amresco ) , 40 ml l 10% ( w / v ) carbon source , 2 ml l 1 m mgso4 , 1 ml l 0.1 m cacl2 , 100 ml l 10 m9 salts ( 85.1 g l na2hpo4 2 h2o , 30 g l kh2po4 , 5 g l nacl , 10 g l nh4cl , ph 7 ) ) containing either glucose or lactose as sole carbon source . all plasmids used in this study are given in table 1 , a generic map of all produced plasmids is given in fig . 1 . all pcrs were conducted using phusion polymerase ( new england biolabs , ipswich , ma , usa ) following the manufacturer 's recommendations and annealing temperatures were chosen based on the respective melting temperature of the primers ( table 2 ) . all plasmids that were used for pcr amplification were isolated using the nucleospin plasmid extraction kit ( macherey - nagel , oensingen , switzerland ) following the manufacturer 's recommendations . the designated plasmid backbone of the herein constructed plasmids , pgrg36 ( a gift from nancy craig ( addgene plasmid # 16666 ) ) , was isolated using the nucleobond xtra plus midiprep kit ( macherey - nagel , oensingen , switzerland ) following the manufacturer 's recommendations . the map shows pgrg36 including the insertion site of the different promoter fp constructs and other relevant features . arabad promoter = arabinose - inducible promoter , tnsa - d = transposase genes , ampr = ampicillin resistance conferring beta - lactamase , orit = origin of transfer , psc101 = origin of replication , tn7 left end = left border of tn7 transposon , mcs = multiple cloning site , tn7 right end = right border of tn7 transposon . tm specific to the overlap to the specified targets , overlaps for isothermal assembly were designed to anneal at 50c ; abr = antibiotic resistance : amp = ampicillin , kan = kanamycin , zeo = zeocin , tet = tetracycline . as source of ptac mcherry gene fragment , plasmid pmp7607 was used . the fragment was amplified and equipped with restriction sites xhoi and noti using primers xho_ptac_mche_for and noti_mche_rev . the resulting amplicon was noti / xhoi digested and ligated into equally digested pgrg36 to generate pmre100-ptac - mche . the ligation reaction was transformed into e. coli neb 5-alpha chemically competent cells following the manufacturer 's recommendations and selected on lb agar containing ampicillin . to construct pmre101-ptac - ecfp and pmre102-ptac - eyfp , ecfp and eyfp were amplified from puc18t - minitn7-zeo - ecfp and puc18t - minitn7-zeo - eyfp , respectively , using primers ptac_c / yfp.for , which contains ptac , and ptac_c / yfp.rev . the resulting amplicons were amplified using primers gib_ptac+c / yfp.fwd and gib_ptac+c / yfp.rev to generate amplicons with overlaps to smai - digested pgrg36 . each amplicon was fused into pgrg36 using isothermal assembly as described by gibson et al . briefly , amplicons were mixed in equimolar ratios with 100 ng smai - linearized pgrg36 each in a total of 5 l before 15 l isothermal assembly reaction mix was added ( for 1.2 ml isothermal assembly mix , combine 320 l isothermal assembly buffer ( 25% peg-8000 ( amresco , cleveland , oh , usa ) , 500 mm tris - hcl ph 7.5 ( sigma , saint louise , mo , usa ) , 50 mm mgcl2 ( rockland , limerick , pa , usa ) , 50 mm dtt ( amresco , cleveland , oh , usa ) , 1 mm each of the four dntps ( amresco , cleveland , oh , usa ) and 5 mm nad ) with 1.2 l t5 exonuclease ( new england biolabs , ipswich , ma , usa ) , 20 l phusion polymerase ( new england biolabs , ipswich , ma , usa ) , 160 l taq dna ligase ( new england biolabs , ipswich , ma , usa ) and 700 l ddh2o ) . after incubation at 50c for 15 min , 10 l of the reactions were used to transform e. coli neb 5-alpha chemically competent cells . to construct pmre103-pnptii - mche , pnptii was amplified from pfru97 using primers gib_pnptii.fwd and gib_pnptii.rev , yielding an amplicon with 5 overlap to smai - digested pgrg36 and 3 overlap to the 5 end of gib_mche.fwd and gib_mche.rev amplified mcherry . the two amplicons and smai - digested pgrg36 were isothermal assembled as described above , yielding pmre103-pnptii - mche . pmre104-pnptii - ecfp and pmre105-pnptii - eyfp were constructed by amplifying pnptii from pfru97 using primers gib_pnptii.for and gib_c / yfp_pnptii.rev and amplifying ecfp and eyfp from pmre101-ptac - ecfp and pmre102-ptac - eyfp , respectively , using primers gib_pnptii_c / yfp.for and gib_pnptii_c / yfp.rev . plasmids pmre106-pnptii - ebfp2 and pmre107-pnptii - mtq2 were constructed by amplifying the respective fluorophore genes and pnptii using primers gib_pnptii_ngfps.for and gib_ngfps.rev from prjaph_ebfp2 or prjaph_mtq2 . all generated plasmids were purified from e. coli neb 5-alpha , verified by pcr sequencing and transformed into e. coli s17 - 1 to allow two - parental mating of the mobilizable pmre - series plasmids . plasmid nucleotide sequences were deposited under genbank accession numbers : ku973693ku973700 . for two - parental mating , donor e. coli s17 - 1 containing minitn7-delivery plasmids were grown overnight as a lawn on lb agar containing ampicillin at 32c and recipient e. coli o157:h7 were grown overnight as a lawn on lb agar at 37c . freshly grown lawns of donor and recipient were harvested using inoculation loops , resuspended in 1pbs ( 8 g l nacl , 0.24 g l kcl , 1.42 g l na2hpo4 , 0.24 g l kh2po4 ) , washed twice by centrifugation at 3500 g and resuspension in 1pbs . donor and recipient were mixed to reach the same number of cells before they were harvested by centrifugation at 3500 g. the donor / recipient mixture was resuspended in 1pbs to reach a final od600 nm of 20 . 100 l of this suspension was spotted onto lb containing no antibiotics and left to dry in a laminar flow . afterwards , spotted cell mixes were incubated at 32c overnight before they were harvested using an inoculation loop . harvested cells were resuspended in 1 pbs and plated on mm2 containing ampicillin where they were incubated at 32c , preventing auxotrophic e. coli s17 - 1 to grow . briefly , e. coli were cultivated in lb containing ampicillin at 220 rpm and 32c overnight to promote transposition ( in e. coli leaky expression of the arabinose promoter - driven tnsabcd , transposase genes were sufficient for transposition into atttn7 locus , for other species and strains it might be advantageous to add 0.1% arabinose to the medium to induce the transposons promoter ) . overnight cultures were plated onto lb agar containing no ampicillin and were cultivated at 42c to block replication of the heat - sensitive plasmid . colonies growing on non - selective agar were screened for their ability to fluoresce on a fluorescence microscope . to verify that the ampicillin resistance conferring plasmid was lost , 10 individual colonies were streaked onto lb agar containing the antibiotic as well as onto lb agar plates without . colonies that were not able to grow on ampicillin but grew on lb were screened once more for their ability to fluoresce . bacterial strains containing chromosomally inserted fps under the control of ptac were cultivated in m9 containing lactose and casamino acids or in m9 containing glucose and casamino acids . strains containing chromosomally inserted fps under the control of pnptii were cultivated in m9 containing glucose and casamino acids . strains were cultivated in triplets of 300 l at 28c and 3 s of orbital shaking every 10 min in a polystyrene , 96-well , flat bottom microtiter plate ( greiner bio - one , frickenhausen , germany ) in an infinite m200 microtiter plate reader ( tecan , mnnedorf , switzerland ) . the initial density of the cultures was between 0.001 and 0.005 od600 as measured by the plate reader . fluorescence was determined by exciting the culture at 383 9 nm , 420 9 nm , 497 9 or 560 9 nm and measuring emission at 448 20 nm , 481 20 nm , 533 20 nm or 610 20 nm for ebfp2 , cyan fps ( ecfp and mturquoise2 ) , eyfp or mcherry , respectively . fluorescence and absorbance were determined every 10 min for a total of 48 h or 288 cycles , respectively . the determined fluorescence intensity was background subtracted using the respective t0 fluorescence intensity . microscopy was performed on a zeiss axioimager.z2 microscope equipped with a zeiss axiocam mrm for image acquisition . ebfp2 was visualized using zeiss filter set 49 ( g 365 nm / ft 395 nm / bp 445/50 nm ) , ecfp and mturquoise2 were visualized using zeiss filter set 47 he ( bp 436/25 nm / ft 455 nm / bp 480/40 nm ) , eyfp was visualized using zeiss filter set 46 he ( bp 500/25 nm / ft 515 nm / bp 535/30 nm ) and mcherry was visualized using zeiss filter set 43 he ( bp 550/25 nm / ft 570 nm / bp 605/70 nm ) . to visualize fluorescence of individual bacterial cells , an ec plan - neofluar 100 objective ( 1.30 na , oil , ph3 ) or an ec plan - neofluar 40 objective ( 0.75 na , air , ph2 ) was used . individual colonies were investigated using a plan - apochromat 5 objective ( 0.16 na ) . to measure fluorescence intensity of individual cells , freshly grown colonies were harvested using an inoculation loop and resuspended in 1 pbs . serial dilutions of the suspensions were spotted onto gelatin coated 10-well microscope slides ( thermo scientific , dreieich , germany ) and dried for 15 min to bind cells to the surface of the slide . dilutions of appropriated densities , e.g. containing several well - separated individual cells per field of view at 100 magnification , were further investigated . single - cell fluorescence intensity was determined of at least 100 cells per strain by acquiring multichannel images of respective fluorescence signals and phase contrast signals . cells were separated from the background based on their phase contrast using the fiji thresholding command and standard settings . cells were added to the fiji region of interest manager using the analyze particles command . the multimeasure command of the region of interest manager was then used to determine the average fluorescence of individual cells . when grown in media lacking lactose , escherichia coli o157:h7 cells harboring : : mre100 , : : mre101 or : : mre102 exhibited weak fluorescence ( fig . 2 ) due to the presence of the ptac repressor gene laci in e. coli o157:h7 . addition of lactose to minimal medium led to the induction of fps in e. coli o157:h7 harboring : : mre - ptac constructs causing an accumulation of fp in cells compared to their repressed controls as shown by microscopy and microtiter plate reader ( fig . 2 ) . chromosomally inserted mcherry in e. coli o157:h7::mre100 exhibited a significantly stronger signal after induction and the strongest observed signal of the here - introduced reporter set ( fig . escherichia coli o157:h7::mre101 expressing ecfp exhibited a slightly stronger , however not significant , fluorescence signal after induction , which could be shown only using microscopy . possibly , ecfps short excitation wavelength led to a high autofluorescent signal of the medium and/or microtiter plate plastic in the plate reader which decreased the signal - to - noise ratio . lastly , e. coli o157:h7::mre102 expressing eyfp exhibited the second strongest fluorescence , however not significantly increased , signal after induction . analysis of chromosomally inserted ptac fluorescence protein gene labels using microscopy and microtiter plate reader . ( a ) phase contrast and fluorescence micrographs of e. coli o157:h7 carrying chromosomally integrated tn7-transposons containing mcherry , ecfp or eyfp encoding genes under the control of the lactose derepressible tac promoter . the left two columns show phase contrast and fluorescence images of non - induced cells , the right two columns show lactose - induced cells . for fair comparisons of fluorescence intensities and background , ( exposure times for the respectively measured fluorophores are given in fluorescence micrographs , scale bar = 5 m . ) ( b ) average single - cell fluorescence intensity per millisecond exposure after background subtraction . non - induced cells are represented as white bars , and induced cells as black bars . statistical differences in fluorescence intensity between treatments were assessed by performing a one - way anova . * * = p < 0.01 ; * * * * = p < 0.0001 . ( c ) background - subtracted fluorescence of e. coli o157:h7 carrying the tn7 insertions or wild - type cells cultivated under non - induced or induced conditions . lines reflect floating averages of three replicates ; stippled lines reflect the standard deviation of the mean . these results show that the repression of the ptac by the in e. coli endogenously present laci was not sufficient to tightly control the activity of ptac as also non - induced bacterial cells exhibited fluorescence , albeit at lower average intensities , which were , however , only significantly lower in the case of mcherry expressing e. coli o157:h7::mre100 ( fig . to show that the activity of ptac can be controlled more tightly , the ptac repressor laci was expressed from the multicopy plasmid pcpp39 ( leveau and lindow 2001b , remus - emsermann and leveau 2010 ) and transformed into e. coli o157:h7::mre100 . in e. coli o157:h7::mre100 ( pcpp39 ) , absolutely no fluorescence was detected in cells grown on minimal medium containing glucose as sole carbon , while cells grown on minimal medium containing lactose as sole carbon were fluorescent ( fig . s1 , supporting information ) . this tightly controlled gene expression system can be employed to generate cusper bioreporters ( remus - emsermann and leveau 2010 , remus - emsermann et al . cells can then be loaded with a fluorophore by adding the derepressor and after its removal , the constitutive repression abolishes the de novo production of the fp . the now fp - loaded cells can be used as cusper bioreporters , i.e. while growing , they will evenly dilute the fp to daughter cells , which will contain less fp than the mother cell . all fps that were placed under the control of pnptii were functionally expressed in e. coli o157:h7 while present on plasmids ( data not shown ) or after chromosomal insertion ( fig . the strongest discernable fluorescence , as assessed by microscopy and microtiter plate reader , was exhibited by mcherry expressing e. coli o157:h7::mre103 , followed by cells e. coli o157:h7::mre107 expressing mturquoise2 , e. coli o157:h7::mre105 expressing eyfp , e. coli o157:h7::mre104 expressing ecfp and finally e. coli o157:h7::mre106 expressing ebfp2 . notably , the fluorescence of chromosomally inserted ebfp2 was very low using the available fluorescence microscope and plate reader , with signals barely above the limit of detection . furthermore , the fluorescence of cells expressing ecfp was far inferior to cells expressing mturquoise2 . ( a ) phase contrast and fluorescence micrographs of e. coli o157:h7 carrying chromosomally integrated tn7 transposons containing mcherry , ecfp , eyfp , ebfp2 or mturquoise2 encoding genes under the control of the constitutive nptii promoter . the first column shows phase contrast images , the second corresponding fluorescence images ( exposure times are given in the fluorescent images , scale bars = 5 m ) . for fair comparisons of fluorescence intensities and background , ( b ) fluorescence intensity of e. coli o157:h7 carrying the tn7 insertions or wild - type cells . lines reflect the mean of three replicates ; stippled lines reflect the standard deviation of the mean . ( c ) average single - cell fluorescence intensity per millisecond exposure after background subtraction . statistical differences in fluorescence intensity between treatments were assessed by performing a one - way anova , and significant differences are indicated in the graph . the here - presented plasmids feature a narrow - host range origin of replication and should be functional in many relevant enterobacteriaceae including e. coli , salmonella spp . and shigella spp ( mckenzie and craig 2006 ) . the tn7-transposon delivery machinery and promoters are considered to be functional in a broad host range including all enterobacteriaceae ( miller , leveau and lindow 2000 , peters and craig 2001 ) . thereby the plasmids are of value for many different studies and allow to rapidly setup and perform experiments at single - cell resolution , for example , the investigation of multispecies biofilms and interactions within ( sternberg et al . 2014 ) and bacterial behavior in microbe microbe or microbe host interactions ( bloemberg et al . 2000 , remus - emsermann , kowalchuk and leveau 2013 , janissen et al . 2015 , ledermann et al . 2015 ) . as the promoter - reporter constructs are inserted chromosomally , furthermore , by avoiding the integration of antibiotic resistances it is possible to add additional genetic elements that require antibiotics pressure , such as plasmids or transposons , in the host bacteria . with non - optimized wide - field microscopy systems , it is possible to combine the following three fps to allow for overlap - free observations of subpopulations of bacteria : ecfp / mturquoise2 , eyfp and mcherry ( fig . 4 ; fig . s2 , supporting information ) . to add a fourth population , i.e. an ebfp2-labeled strain , it is necessary to optimize the detection system by using optimized fluorescence emission and excitation filters or a filter - free confocal microscopy system . the observation of several subpopulations at the same time allows for the application of spatial statistics ( daims , lcker and wagner 2006 , remus - emsermann et al . 2014 ) , which will give deep insights into the behavior of bacterial strains towards each other in a given environment , e.g. if they aggregate or segregate . artificial mixtures of e. coli o157:h7 containing different tn7-transposon inserted promoter fluorescent - protein labels . ( a ) a mix of induced e. coli o157:h7::mre100 , e. coli o157:h7::mre101 and e. coli o157:h7::mre102 . left image , phase contrast image , right image , false color overlay of mcherry ( b ) a mix of e. coli o157:h7::mre103 , e. coli o157:h7::mre105 and e. coli o157:h7::mre107 . left image , phase contrast image ; right image , false color overlay of mcherry ( red ) , eyfp ( green ) and mturquoise2 ( blue ) . with this novel set of fp labels harbored on minitn7-transposon delivery plasmids in combination with the conjugation strain e. coli s17 - 1 , we provide a convenient ready - to - go tool to generate fluorescently labeled enterobacterial strains for microbe microbe and microbe mre and dd conceived the study , mre planned the experiments , mre and pg performed the experiments , mre analyzed the data and mre wrote the manuscript with critical input from dd . this work was financially supported by the agroscope research program reduction and dynamics of antibiotic - resistant and persistent microorganisms along food chains ( redymo ) .

here we present the generation and function of two sets of bacterial plasmids that harbor fluorescent genes encoding either blue , cyan , yellow or red fluorescent proteins . in the first set , protein expression is controlled by the strong and constitutive nptii promoter whereas in the second set , the strong tac promoter was chosen that underlies laciq regulation . furthermore , the plasmids are mobilizable , contain tn7 transposons and a temperature - sensitive origin of replication . using escherichia coli s17 - 1 as donor strain , the plasmids allow fast and convenient tn7-transposon delivery into many enterobacterial hosts , such as the here - used e. coli o157:h7 . this procedure omits the need of preparing competent recipient cells and antibiotic resistances are only transiently conferred to the recipients . as the fluorescence proteins show little to no overlap in fluorescence emission , the constructs are well suited for the study of multicolored synthetic bacterial communities during biofilm production or in host colonization studies , e.g. of plant surfaces . furthermore , tac promoter - reporter constructs allow the generation of so - called reproductive success reporters , which allow to estimate past doublings of bacterial individuals after introduction into environments , emphasizing the role of individual cells during colonization .

INTRODUCTION MATERIAL AND METHODS RESULTS AND DISCUSSION CONCLUSION AUTHOR CONTRIBUTIONS SUPPLEMENTARY DATA FUNDING

this can be achieved by microscopy in conjunction with fluorescent markers , e.g. dyes such as propidium iodide and syto9 allowing to determine the membrane integrity of bacterial cells ( berney et al . 2014 ) , or by labeling bacteria using fluorescent proteins ( fps ) ( ledermann et al . often , fps were used under the control of inducible promoters as bioreporters , which allow the assessment of the availability of inducing agents , such as fructose ( leveau and lindow 2001a ) , or under the control of constitutive promoters to identify bacteria in situ ( bloemberg et al . the use of minitn7-transposon delivery plasmids is widespread in molecular and environmental microbiology , e.g. to chromosomally integrate promoter - reporter gene constructs ( choi and schweizer 2006 , mckenzie and craig 2006 , lagendijk et al . 2010 ) or for the complementation of knockout mutants ( crepin , harel and dozois 2012 ) . most tn7-transposon delivery systems have in common that they employ a suicide plasmid - based approach using vectors carrying a puc origin of replication ( lambertsen , sternberg and molin 2004 , choi and schweizer 2006 , lagendijk et al . plasmids featuring this origin of replication do not replicate in bacterial recipients and classically , the transposon machinery is provided on a helper plasmid . in enterobacteriaceae , however , the used puc origin of replication works efficiently , thereby excluding escherichia coli and salmonella spp . using a temperature - sensitive tn7-delivery system constructed by mckenzie and craig ( 2006 ) , we provide a ready - to - use alternative for fluorescent labeling of enterobacteriaceae . we provide two different promoters , the synthetic , constitutive , laci - repressible tac promoter ( ptac ) ( de boer , comstock and vasser 1983 ) and the constitutive nptii promoter ( pnptii ) ( ledermann et al . 2015 ) in combination with fluorescent marker genes , encoding blue , cyan , yellow or red fps . in combination with two - parental mating employing e. coli s17 - 1 , which provides the tra operon necessary for the mobilization of plasmids as a chromosomal insertion , we overcome the often tedious low efficiency of transposition and preparation of competent recipient cells . the here - proposed sets of plasmids offer a convenient alternative to generate multicolored sets of stably fluorescently labeled bacteria . the fps allow for multichannel fluorescent microscopy analysis with little to no overlap of fp emissions to study multistrain synthetic communities ( fig . employing the repressability of the ptac by laci , e.g. by coexpressing laci , it is possible to construct so - called reproductive success , or cusper , bioreporter strains ( reproductive success backwards = cusper ) ( remus - emsermann and leveau 2010 , remus - emsermann et al . the fluorescence intensity of growing cells relative to their ancestors thereby becomes a proxy for the number of divisions individual cells underwent ( remus - emsermann and leveau 2010 ) . cusper bioreporters were used to study the probability of successful colonization of bacterial cells on plant leaves , the heterogeneity of microhabitats on plant leaf surface , the probability of secondary bacterial colonization on precolonized leaves ( remus - emsermann and leveau 2010 , remus - emsermann et al . 2012 , remus - emsermann , kowalchuk and leveau 2013 ) , and to estimate reproductive success in a spatial context in mice spleens and plant leaves ( helaine et al . using the model strain e. coli o157:h7 stx , we demonstrate the transposon delivery of the promoter fluorescence reporter constructs and resulting fluorescence at the population and single - cell resolution . escherichia coli neb 5-alpha ( new england biolabs , ipswich , ma , usa ) , e. coli s17 - 1 and e. coli o157:h7 stx ( nctc 12900 ) were routinely grown on lysogeny broth ( lb ) or lb agar at 37c . to prevent plasmids loss , for counterselection of auxotroph e. coli s17 - 1 after mating , mm2 medium agar ( 4 g l l - asparagine , 2 g l k2hpo4 , 0.2 g l mgso4 , 3 g l nacl , 10 g l sorbitol , 15 g l agar ) was used . where necessary , media were supplemented with 100 g ml ampicillin , 50 g ml kanamycin or 20 g ml tetracycline . all plasmids used in this study are given in table 1 , a generic map of all produced plasmids is given in fig . all plasmids that were used for pcr amplification were isolated using the nucleospin plasmid extraction kit ( macherey - nagel , oensingen , switzerland ) following the manufacturer 's recommendations . arabad promoter = arabinose - inducible promoter , tnsa - d = transposase genes , ampr = ampicillin resistance conferring beta - lactamase , orit = origin of transfer , psc101 = origin of replication , tn7 left end = left border of tn7 transposon , mcs = multiple cloning site , tn7 right end = right border of tn7 transposon . tm specific to the overlap to the specified targets , overlaps for isothermal assembly were designed to anneal at 50c ; abr = antibiotic resistance : amp = ampicillin , kan = kanamycin , zeo = zeocin , tet = tetracycline . the ligation reaction was transformed into e. coli neb 5-alpha chemically competent cells following the manufacturer 's recommendations and selected on lb agar containing ampicillin . to construct pmre101-ptac - ecfp and pmre102-ptac - eyfp , ecfp and eyfp were amplified from puc18t - minitn7-zeo - ecfp and puc18t - minitn7-zeo - eyfp , respectively , using primers ptac_c / yfp.for , which contains ptac , and ptac_c / yfp.rev . after incubation at 50c for 15 min , 10 l of the reactions were used to transform e. coli neb 5-alpha chemically competent cells . to construct pmre103-pnptii - mche , pnptii was amplified from pfru97 using primers gib_pnptii.fwd and gib_pnptii.rev , yielding an amplicon with 5 overlap to smai - digested pgrg36 and 3 overlap to the 5 end of gib_mche.fwd and gib_mche.rev amplified mcherry . pmre104-pnptii - ecfp and pmre105-pnptii - eyfp were constructed by amplifying pnptii from pfru97 using primers gib_pnptii.for and gib_c / yfp_pnptii.rev and amplifying ecfp and eyfp from pmre101-ptac - ecfp and pmre102-ptac - eyfp , respectively , using primers gib_pnptii_c / yfp.for and gib_pnptii_c / yfp.rev . all generated plasmids were purified from e. coli neb 5-alpha , verified by pcr sequencing and transformed into e. coli s17 - 1 to allow two - parental mating of the mobilizable pmre - series plasmids . for two - parental mating , donor e. coli s17 - 1 containing minitn7-delivery plasmids were grown overnight as a lawn on lb agar containing ampicillin at 32c and recipient e. coli o157:h7 were grown overnight as a lawn on lb agar at 37c . harvested cells were resuspended in 1 pbs and plated on mm2 containing ampicillin where they were incubated at 32c , preventing auxotrophic e. coli s17 - 1 to grow . briefly , e. coli were cultivated in lb containing ampicillin at 220 rpm and 32c overnight to promote transposition ( in e. coli leaky expression of the arabinose promoter - driven tnsabcd , transposase genes were sufficient for transposition into atttn7 locus , for other species and strains it might be advantageous to add 0.1% arabinose to the medium to induce the transposons promoter ) . overnight cultures were plated onto lb agar containing no ampicillin and were cultivated at 42c to block replication of the heat - sensitive plasmid . bacterial strains containing chromosomally inserted fps under the control of ptac were cultivated in m9 containing lactose and casamino acids or in m9 containing glucose and casamino acids . the initial density of the cultures was between 0.001 and 0.005 od600 as measured by the plate reader . fluorescence was determined by exciting the culture at 383 9 nm , 420 9 nm , 497 9 or 560 9 nm and measuring emission at 448 20 nm , 481 20 nm , 533 20 nm or 610 20 nm for ebfp2 , cyan fps ( ecfp and mturquoise2 ) , eyfp or mcherry , respectively . microscopy was performed on a zeiss axioimager.z2 microscope equipped with a zeiss axiocam mrm for image acquisition . ebfp2 was visualized using zeiss filter set 49 ( g 365 nm / ft 395 nm / bp 445/50 nm ) , ecfp and mturquoise2 were visualized using zeiss filter set 47 he ( bp 436/25 nm / ft 455 nm / bp 480/40 nm ) , eyfp was visualized using zeiss filter set 46 he ( bp 500/25 nm / ft 515 nm / bp 535/30 nm ) and mcherry was visualized using zeiss filter set 43 he ( bp 550/25 nm / ft 570 nm / bp 605/70 nm ) . to visualize fluorescence of individual bacterial cells , an ec plan - neofluar 100 objective ( 1.30 na , oil , ph3 ) or an ec plan - neofluar 40 objective ( 0.75 na , air , ph2 ) was used . to measure fluorescence intensity of individual cells , freshly grown colonies were harvested using an inoculation loop and resuspended in 1 pbs . serial dilutions of the suspensions were spotted onto gelatin coated 10-well microscope slides ( thermo scientific , dreieich , germany ) and dried for 15 min to bind cells to the surface of the slide . dilutions of appropriated densities , e.g. containing several well - separated individual cells per field of view at 100 magnification , were further investigated . cells were added to the fiji region of interest manager using the analyze particles command . the multimeasure command of the region of interest manager was then used to determine the average fluorescence of individual cells . when grown in media lacking lactose , escherichia coli o157:h7 cells harboring : : mre100 , : : mre101 or : : mre102 exhibited weak fluorescence ( fig . 2 ) due to the presence of the ptac repressor gene laci in e. coli o157:h7 . addition of lactose to minimal medium led to the induction of fps in e. coli o157:h7 harboring : : mre - ptac constructs causing an accumulation of fp in cells compared to their repressed controls as shown by microscopy and microtiter plate reader ( fig . chromosomally inserted mcherry in e. coli o157:h7::mre100 exhibited a significantly stronger signal after induction and the strongest observed signal of the here - introduced reporter set ( fig . escherichia coli o157:h7::mre101 expressing ecfp exhibited a slightly stronger , however not significant , fluorescence signal after induction , which could be shown only using microscopy . possibly , ecfps short excitation wavelength led to a high autofluorescent signal of the medium and/or microtiter plate plastic in the plate reader which decreased the signal - to - noise ratio . lastly , e. coli o157:h7::mre102 expressing eyfp exhibited the second strongest fluorescence , however not significantly increased , signal after induction . ( a ) phase contrast and fluorescence micrographs of e. coli o157:h7 carrying chromosomally integrated tn7-transposons containing mcherry , ecfp or eyfp encoding genes under the control of the lactose derepressible tac promoter . the left two columns show phase contrast and fluorescence images of non - induced cells , the right two columns show lactose - induced cells . for fair comparisons of fluorescence intensities and background , ( exposure times for the respectively measured fluorophores are given in fluorescence micrographs , scale bar = 5 m . ) ( b ) average single - cell fluorescence intensity per millisecond exposure after background subtraction . non - induced cells are represented as white bars , and induced cells as black bars . statistical differences in fluorescence intensity between treatments were assessed by performing a one - way anova . ( c ) background - subtracted fluorescence of e. coli o157:h7 carrying the tn7 insertions or wild - type cells cultivated under non - induced or induced conditions . these results show that the repression of the ptac by the in e. coli endogenously present laci was not sufficient to tightly control the activity of ptac as also non - induced bacterial cells exhibited fluorescence , albeit at lower average intensities , which were , however , only significantly lower in the case of mcherry expressing e. coli o157:h7::mre100 ( fig . to show that the activity of ptac can be controlled more tightly , the ptac repressor laci was expressed from the multicopy plasmid pcpp39 ( leveau and lindow 2001b , remus - emsermann and leveau 2010 ) and transformed into e. coli o157:h7::mre100 . in e. coli o157:h7::mre100 ( pcpp39 ) , absolutely no fluorescence was detected in cells grown on minimal medium containing glucose as sole carbon , while cells grown on minimal medium containing lactose as sole carbon were fluorescent ( fig . cells can then be loaded with a fluorophore by adding the derepressor and after its removal , the constitutive repression abolishes the de novo production of the fp . the now fp - loaded cells can be used as cusper bioreporters , i.e. while growing , they will evenly dilute the fp to daughter cells , which will contain less fp than the mother cell . all fps that were placed under the control of pnptii were functionally expressed in e. coli o157:h7 while present on plasmids ( data not shown ) or after chromosomal insertion ( fig . the strongest discernable fluorescence , as assessed by microscopy and microtiter plate reader , was exhibited by mcherry expressing e. coli o157:h7::mre103 , followed by cells e. coli o157:h7::mre107 expressing mturquoise2 , e. coli o157:h7::mre105 expressing eyfp , e. coli o157:h7::mre104 expressing ecfp and finally e. coli o157:h7::mre106 expressing ebfp2 . notably , the fluorescence of chromosomally inserted ebfp2 was very low using the available fluorescence microscope and plate reader , with signals barely above the limit of detection . furthermore , the fluorescence of cells expressing ecfp was far inferior to cells expressing mturquoise2 . ( a ) phase contrast and fluorescence micrographs of e. coli o157:h7 carrying chromosomally integrated tn7 transposons containing mcherry , ecfp , eyfp , ebfp2 or mturquoise2 encoding genes under the control of the constitutive nptii promoter . the first column shows phase contrast images , the second corresponding fluorescence images ( exposure times are given in the fluorescent images , scale bars = 5 m ) . for fair comparisons of fluorescence intensities and background , ( b ) fluorescence intensity of e. coli o157:h7 carrying the tn7 insertions or wild - type cells . ( c ) average single - cell fluorescence intensity per millisecond exposure after background subtraction . statistical differences in fluorescence intensity between treatments were assessed by performing a one - way anova , and significant differences are indicated in the graph . the here - presented plasmids feature a narrow - host range origin of replication and should be functional in many relevant enterobacteriaceae including e. coli , salmonella spp . the tn7-transposon delivery machinery and promoters are considered to be functional in a broad host range including all enterobacteriaceae ( miller , leveau and lindow 2000 , peters and craig 2001 ) . thereby the plasmids are of value for many different studies and allow to rapidly setup and perform experiments at single - cell resolution , for example , the investigation of multispecies biofilms and interactions within ( sternberg et al . as the promoter - reporter constructs are inserted chromosomally , furthermore , by avoiding the integration of antibiotic resistances it is possible to add additional genetic elements that require antibiotics pressure , such as plasmids or transposons , in the host bacteria . an ebfp2-labeled strain , it is necessary to optimize the detection system by using optimized fluorescence emission and excitation filters or a filter - free confocal microscopy system . the observation of several subpopulations at the same time allows for the application of spatial statistics ( daims , lcker and wagner 2006 , remus - emsermann et al . 2014 ) , which will give deep insights into the behavior of bacterial strains towards each other in a given environment , e.g. artificial mixtures of e. coli o157:h7 containing different tn7-transposon inserted promoter fluorescent - protein labels . ( a ) a mix of induced e. coli o157:h7::mre100 , e. coli o157:h7::mre101 and e. coli o157:h7::mre102 . left image , phase contrast image , right image , false color overlay of mcherry ( b ) a mix of e. coli o157:h7::mre103 , e. coli o157:h7::mre105 and e. coli o157:h7::mre107 . with this novel set of fp labels harbored on minitn7-transposon delivery plasmids in combination with the conjugation strain e. coli s17 - 1 , we provide a convenient ready - to - go tool to generate fluorescently labeled enterobacterial strains for microbe microbe and microbe mre and dd conceived the study , mre planned the experiments , mre and pg performed the experiments , mre analyzed the data and mre wrote the manuscript with critical input from dd . this work was financially supported by the agroscope research program reduction and dynamics of antibiotic - resistant and persistent microorganisms along food chains ( redymo ) .

2007 ) , fluorescence in situ hybridization ( remus - emsermann et al . often , fps were used under the control of inducible promoters as bioreporters , which allow the assessment of the availability of inducing agents , such as fructose ( leveau and lindow 2001a ) , or under the control of constitutive promoters to identify bacteria in situ ( bloemberg et al . the use of minitn7-transposon delivery plasmids is widespread in molecular and environmental microbiology , e.g. to chromosomally integrate promoter - reporter gene constructs ( choi and schweizer 2006 , mckenzie and craig 2006 , lagendijk et al . 2010 ) or for the complementation of knockout mutants ( crepin , harel and dozois 2012 ) . the bacterial tn7 transposon provides an excellent non - destructive tool for several reasons : ( i ) it integrates with high affinity at the atttn7 site within a wide range of gram - negative bacteria ; ( ii ) it does not disrupt genes , omitting potential pleiotropic effect ; ( iii ) and antibiotics are not required to maintain the inserted transposons ( choi and schweizer 2006 , choi and kim 2009 ) . most tn7-transposon delivery systems have in common that they employ a suicide plasmid - based approach using vectors carrying a puc origin of replication ( lambertsen , sternberg and molin 2004 , choi and schweizer 2006 , lagendijk et al . plasmids featuring this origin of replication do not replicate in bacterial recipients and classically , the transposon machinery is provided on a helper plasmid . in enterobacteriaceae , however , the used puc origin of replication works efficiently , thereby excluding escherichia coli and salmonella spp . using a temperature - sensitive tn7-delivery system constructed by mckenzie and craig ( 2006 ) , we provide a ready - to - use alternative for fluorescent labeling of enterobacteriaceae . we provide two different promoters , the synthetic , constitutive , laci - repressible tac promoter ( ptac ) ( de boer , comstock and vasser 1983 ) and the constitutive nptii promoter ( pnptii ) ( ledermann et al . in combination with two - parental mating employing e. coli s17 - 1 , which provides the tra operon necessary for the mobilization of plasmids as a chromosomal insertion , we overcome the often tedious low efficiency of transposition and preparation of competent recipient cells . the here - proposed sets of plasmids offer a convenient alternative to generate multicolored sets of stably fluorescently labeled bacteria . the fps allow for multichannel fluorescent microscopy analysis with little to no overlap of fp emissions to study multistrain synthetic communities ( fig . by coexpressing laci , it is possible to construct so - called reproductive success , or cusper , bioreporter strains ( reproductive success backwards = cusper ) ( remus - emsermann and leveau 2010 , remus - emsermann et al . cusper bioreporters are equipped with a ptac - controlled fp and express laci , thereby the expression of the fp is repressed . by adding lactose or isopropyl -d-1-thiogalactopyranoside to growing cells , cells can be loaded with fp ( leveau and lindow 2001b ) . if the derepressor is subsequently removed , no de novo production of fp occurs and the preformed fp is then diluted from growing cells ( remus - emsermann and leveau 2010 ) . the fluorescence intensity of growing cells relative to their ancestors thereby becomes a proxy for the number of divisions individual cells underwent ( remus - emsermann and leveau 2010 ) . cusper bioreporters were used to study the probability of successful colonization of bacterial cells on plant leaves , the heterogeneity of microhabitats on plant leaf surface , the probability of secondary bacterial colonization on precolonized leaves ( remus - emsermann and leveau 2010 , remus - emsermann et al . 2012 , remus - emsermann , kowalchuk and leveau 2013 ) , and to estimate reproductive success in a spatial context in mice spleens and plant leaves ( helaine et al . using the model strain e. coli o157:h7 stx , we demonstrate the transposon delivery of the promoter fluorescence reporter constructs and resulting fluorescence at the population and single - cell resolution . escherichia coli neb 5-alpha ( new england biolabs , ipswich , ma , usa ) , e. coli s17 - 1 and e. coli o157:h7 stx ( nctc 12900 ) were routinely grown on lysogeny broth ( lb ) or lb agar at 37c . to prevent plasmids loss , for counterselection of auxotroph e. coli s17 - 1 after mating , mm2 medium agar ( 4 g l l - asparagine , 2 g l k2hpo4 , 0.2 g l mgso4 , 3 g l nacl , 10 g l sorbitol , 15 g l agar ) was used . for microtiter plate reader experiments , cells were grown in m9 minimal medium ( 20 ml l 20% ( w / v ) casamino acids ( amresco ) , 40 ml l 10% ( w / v ) carbon source , 2 ml l 1 m mgso4 , 1 ml l 0.1 m cacl2 , 100 ml l 10 m9 salts ( 85.1 g l na2hpo4 2 h2o , 30 g l kh2po4 , 5 g l nacl , 10 g l nh4cl , ph 7 ) ) containing either glucose or lactose as sole carbon source . all plasmids used in this study are given in table 1 , a generic map of all produced plasmids is given in fig . all pcrs were conducted using phusion polymerase ( new england biolabs , ipswich , ma , usa ) following the manufacturer 's recommendations and annealing temperatures were chosen based on the respective melting temperature of the primers ( table 2 ) . all plasmids that were used for pcr amplification were isolated using the nucleospin plasmid extraction kit ( macherey - nagel , oensingen , switzerland ) following the manufacturer 's recommendations . the designated plasmid backbone of the herein constructed plasmids , pgrg36 ( a gift from nancy craig ( addgene plasmid # 16666 ) ) , was isolated using the nucleobond xtra plus midiprep kit ( macherey - nagel , oensingen , switzerland ) following the manufacturer 's recommendations . the map shows pgrg36 including the insertion site of the different promoter fp constructs and other relevant features . arabad promoter = arabinose - inducible promoter , tnsa - d = transposase genes , ampr = ampicillin resistance conferring beta - lactamase , orit = origin of transfer , psc101 = origin of replication , tn7 left end = left border of tn7 transposon , mcs = multiple cloning site , tn7 right end = right border of tn7 transposon . tm specific to the overlap to the specified targets , overlaps for isothermal assembly were designed to anneal at 50c ; abr = antibiotic resistance : amp = ampicillin , kan = kanamycin , zeo = zeocin , tet = tetracycline . to construct pmre101-ptac - ecfp and pmre102-ptac - eyfp , ecfp and eyfp were amplified from puc18t - minitn7-zeo - ecfp and puc18t - minitn7-zeo - eyfp , respectively , using primers ptac_c / yfp.for , which contains ptac , and ptac_c / yfp.rev . briefly , amplicons were mixed in equimolar ratios with 100 ng smai - linearized pgrg36 each in a total of 5 l before 15 l isothermal assembly reaction mix was added ( for 1.2 ml isothermal assembly mix , combine 320 l isothermal assembly buffer ( 25% peg-8000 ( amresco , cleveland , oh , usa ) , 500 mm tris - hcl ph 7.5 ( sigma , saint louise , mo , usa ) , 50 mm mgcl2 ( rockland , limerick , pa , usa ) , 50 mm dtt ( amresco , cleveland , oh , usa ) , 1 mm each of the four dntps ( amresco , cleveland , oh , usa ) and 5 mm nad ) with 1.2 l t5 exonuclease ( new england biolabs , ipswich , ma , usa ) , 20 l phusion polymerase ( new england biolabs , ipswich , ma , usa ) , 160 l taq dna ligase ( new england biolabs , ipswich , ma , usa ) and 700 l ddh2o ) . to construct pmre103-pnptii - mche , pnptii was amplified from pfru97 using primers gib_pnptii.fwd and gib_pnptii.rev , yielding an amplicon with 5 overlap to smai - digested pgrg36 and 3 overlap to the 5 end of gib_mche.fwd and gib_mche.rev amplified mcherry . pmre104-pnptii - ecfp and pmre105-pnptii - eyfp were constructed by amplifying pnptii from pfru97 using primers gib_pnptii.for and gib_c / yfp_pnptii.rev and amplifying ecfp and eyfp from pmre101-ptac - ecfp and pmre102-ptac - eyfp , respectively , using primers gib_pnptii_c / yfp.for and gib_pnptii_c / yfp.rev . plasmids pmre106-pnptii - ebfp2 and pmre107-pnptii - mtq2 were constructed by amplifying the respective fluorophore genes and pnptii using primers gib_pnptii_ngfps.for and gib_ngfps.rev from prjaph_ebfp2 or prjaph_mtq2 . all generated plasmids were purified from e. coli neb 5-alpha , verified by pcr sequencing and transformed into e. coli s17 - 1 to allow two - parental mating of the mobilizable pmre - series plasmids . briefly , e. coli were cultivated in lb containing ampicillin at 220 rpm and 32c overnight to promote transposition ( in e. coli leaky expression of the arabinose promoter - driven tnsabcd , transposase genes were sufficient for transposition into atttn7 locus , for other species and strains it might be advantageous to add 0.1% arabinose to the medium to induce the transposons promoter ) . bacterial strains containing chromosomally inserted fps under the control of ptac were cultivated in m9 containing lactose and casamino acids or in m9 containing glucose and casamino acids . strains containing chromosomally inserted fps under the control of pnptii were cultivated in m9 containing glucose and casamino acids . strains were cultivated in triplets of 300 l at 28c and 3 s of orbital shaking every 10 min in a polystyrene , 96-well , flat bottom microtiter plate ( greiner bio - one , frickenhausen , germany ) in an infinite m200 microtiter plate reader ( tecan , mnnedorf , switzerland ) . the initial density of the cultures was between 0.001 and 0.005 od600 as measured by the plate reader . ebfp2 was visualized using zeiss filter set 49 ( g 365 nm / ft 395 nm / bp 445/50 nm ) , ecfp and mturquoise2 were visualized using zeiss filter set 47 he ( bp 436/25 nm / ft 455 nm / bp 480/40 nm ) , eyfp was visualized using zeiss filter set 46 he ( bp 500/25 nm / ft 515 nm / bp 535/30 nm ) and mcherry was visualized using zeiss filter set 43 he ( bp 550/25 nm / ft 570 nm / bp 605/70 nm ) . to visualize fluorescence of individual bacterial cells , an ec plan - neofluar 100 objective ( 1.30 na , oil , ph3 ) or an ec plan - neofluar 40 objective ( 0.75 na , air , ph2 ) was used . single - cell fluorescence intensity was determined of at least 100 cells per strain by acquiring multichannel images of respective fluorescence signals and phase contrast signals . the multimeasure command of the region of interest manager was then used to determine the average fluorescence of individual cells . when grown in media lacking lactose , escherichia coli o157:h7 cells harboring : : mre100 , : : mre101 or : : mre102 exhibited weak fluorescence ( fig . 2 ) due to the presence of the ptac repressor gene laci in e. coli o157:h7 . addition of lactose to minimal medium led to the induction of fps in e. coli o157:h7 harboring : : mre - ptac constructs causing an accumulation of fp in cells compared to their repressed controls as shown by microscopy and microtiter plate reader ( fig . chromosomally inserted mcherry in e. coli o157:h7::mre100 exhibited a significantly stronger signal after induction and the strongest observed signal of the here - introduced reporter set ( fig . possibly , ecfps short excitation wavelength led to a high autofluorescent signal of the medium and/or microtiter plate plastic in the plate reader which decreased the signal - to - noise ratio . ( a ) phase contrast and fluorescence micrographs of e. coli o157:h7 carrying chromosomally integrated tn7-transposons containing mcherry , ecfp or eyfp encoding genes under the control of the lactose derepressible tac promoter . the left two columns show phase contrast and fluorescence images of non - induced cells , the right two columns show lactose - induced cells . ( c ) background - subtracted fluorescence of e. coli o157:h7 carrying the tn7 insertions or wild - type cells cultivated under non - induced or induced conditions . these results show that the repression of the ptac by the in e. coli endogenously present laci was not sufficient to tightly control the activity of ptac as also non - induced bacterial cells exhibited fluorescence , albeit at lower average intensities , which were , however , only significantly lower in the case of mcherry expressing e. coli o157:h7::mre100 ( fig . to show that the activity of ptac can be controlled more tightly , the ptac repressor laci was expressed from the multicopy plasmid pcpp39 ( leveau and lindow 2001b , remus - emsermann and leveau 2010 ) and transformed into e. coli o157:h7::mre100 . in e. coli o157:h7::mre100 ( pcpp39 ) , absolutely no fluorescence was detected in cells grown on minimal medium containing glucose as sole carbon , while cells grown on minimal medium containing lactose as sole carbon were fluorescent ( fig . this tightly controlled gene expression system can be employed to generate cusper bioreporters ( remus - emsermann and leveau 2010 , remus - emsermann et al . cells can then be loaded with a fluorophore by adding the derepressor and after its removal , the constitutive repression abolishes the de novo production of the fp . all fps that were placed under the control of pnptii were functionally expressed in e. coli o157:h7 while present on plasmids ( data not shown ) or after chromosomal insertion ( fig . the strongest discernable fluorescence , as assessed by microscopy and microtiter plate reader , was exhibited by mcherry expressing e. coli o157:h7::mre103 , followed by cells e. coli o157:h7::mre107 expressing mturquoise2 , e. coli o157:h7::mre105 expressing eyfp , e. coli o157:h7::mre104 expressing ecfp and finally e. coli o157:h7::mre106 expressing ebfp2 . ( a ) phase contrast and fluorescence micrographs of e. coli o157:h7 carrying chromosomally integrated tn7 transposons containing mcherry , ecfp , eyfp , ebfp2 or mturquoise2 encoding genes under the control of the constitutive nptii promoter . for fair comparisons of fluorescence intensities and background , ( b ) fluorescence intensity of e. coli o157:h7 carrying the tn7 insertions or wild - type cells . the here - presented plasmids feature a narrow - host range origin of replication and should be functional in many relevant enterobacteriaceae including e. coli , salmonella spp . the tn7-transposon delivery machinery and promoters are considered to be functional in a broad host range including all enterobacteriaceae ( miller , leveau and lindow 2000 , peters and craig 2001 ) . thereby the plasmids are of value for many different studies and allow to rapidly setup and perform experiments at single - cell resolution , for example , the investigation of multispecies biofilms and interactions within ( sternberg et al . as the promoter - reporter constructs are inserted chromosomally , furthermore , by avoiding the integration of antibiotic resistances it is possible to add additional genetic elements that require antibiotics pressure , such as plasmids or transposons , in the host bacteria . with non - optimized wide - field microscopy systems , it is possible to combine the following three fps to allow for overlap - free observations of subpopulations of bacteria : ecfp / mturquoise2 , eyfp and mcherry ( fig . the observation of several subpopulations at the same time allows for the application of spatial statistics ( daims , lcker and wagner 2006 , remus - emsermann et al . with this novel set of fp labels harbored on minitn7-transposon delivery plasmids in combination with the conjugation strain e. coli s17 - 1 , we provide a convenient ready - to - go tool to generate fluorescently labeled enterobacterial strains for microbe microbe and microbe mre and dd conceived the study , mre planned the experiments , mre and pg performed the experiments , mre analyzed the data and mre wrote the manuscript with critical input from dd .

over the past two decades , the emergence of metal organic frameworks ( mofs ) and covalent organic frameworks ( cofs ) has reinvigorated the field of crystalline microporous materials , previously dominated by zeolites . however , with widespread applications in areas including , but not restricted to , heterogeneous catalysis , the adsorption of pollutants , and resistant coatings , zeolites are still regarded as the benchmark for functional porous materials . although the porosity of zeolites and related materials has been exploited for centuries , real insights into the behavior of guest molecules in confined environments , and the role of the host in which they reside , have only recently become possible through advanced crystallographic techniques . composed primarily of the naturally abundant elements o , si , and al , zeolites have remarkable thermal stability upon evacuation , regular pore sizes , and high acidity . however , tuning zeolite functionality is generally limited to controlling the ratio of si : al in the framework , while extensive structure modulation of these otherwise simple inorganic materials relies on the introduction of organic molecules , either as structure - directing agents ( sdas ) or as functional organic platforms incorporated within the zeolite scaffold itself . mof materials , including hybrid zeolite analogues , are born of remarkably sophisticated design principles , and display a richly diverse range of properties . synthetic control in mof chemistry is largely attributable to the modular nature of mof preparation , with secondary building units ( sbus ) utilized as geometrically well - defined components to access many of the great variety of framework topologies currently realized . despite rapid progress made in this field and the widespread application of mofs , there remains scope to explore and extend the thermal and chemical stability range of the compounds due to the nature of the organic constituents . therefore , the development of porous transition metal oxide - based molecular materials could be interesting due to the promise of flexibility and tunable redox , catalytic , and thermal properties . as such , pom - frameworks promise to combine the thermal stability and general applicability of zeolites , combined with the synthetic control and tunable properties of mofs . polyoxometalates ( poms ) are a diverse class of early transition metal oxide clusters , typically comprised of multiple w , mo , or v centers connected through shared oxygen atoms , and commonly incorporating additional heteroelements such as si , p , or ge . although by their definition poms are discrete ( 0-d ) molecules , the introduction of transition metals ( tms ) and/or organic moieties to pom solutions may lead to connectivity between clusters , and their extension into coordinatively linked 1-d chains , 2-d sheets , or 3-d networks . by application of the topological node and linker designation given to the structural components of a mof , in multidimensional inorganic pom assemblies the pom units may be considered as inorganic nodes , which extend into infinite lattice arrangements through the coordination of tm linkers . however , in contrast to mofs , it is primarily the versatility of the node , and not the linker , which generates the diversity of structures found in tm - linked pom framework materials . like mofs , these purely inorganic networks have been shown to exhibit tunable properties through rational synthetic strategies . furthermore , the recent trend for the incorporation of pom guests inside porous frameworks recognizes the acidic , electronic , and catalytic properties that these clusters may embellish on the overall framework structure . it is therefore clear that the direct incorporation of poms into porous framework scaffolds is a valid strategy for the modular synthesis of robust functional materials , without the need for an organic component . two main strategies are typically employed in the construction of modular frameworks with high porosity : ( 1 ) the selection of long , narrow linkers ( which may lead to the interpenetration of multiple frameworks ) , and ( 2 ) the pre - fabrication of a pore within one of the components . the latter approach is most commonly adopted to incorporate poms within porous framework scaffolds , since these clusters often assume cyclic configurations . in particular , the crown - type heteropolyanion , [ p8w48o184 ] ( hereafter referred to as { p8w48 } ) is notable for several properties , including its high - negative charge and remarkable electrochemistry . its intrinsic nanometer - sized cavity means { p8w48 } is an ideal candidate to be utilized as a network synthon , to prepare open framework materials with microporosity ( figure 1 ) . such structures have already been prepared by introducing first - row tms to aqueous solutions of { p8w48 } ; however , the desired control in preparing { p8w48}-based porous frameworks is yet to be realized . ( a ) nanoporous [ p8w48o184 ] structure , with simplified ring representation showing both ( b ) endocyclic ( red ) and ( c ) exocyclic ( green ) coordination of transition metals . dark green spheres , w ; red spheres , o ; light blue rings , [ p8w48o184 ] . detailed herein are the syntheses of three coordinatively linked framework structures , based on the minimal building block library of { p8w48 } nodes and tm linkers . in addition , the tm coordination sites of { p8w48 } are fully mapped , allowing a comprehensive analysis of ring connectivity within the known { p8w48}-based frameworks . the term pomzites is introduced to apply to this unified class of materials , alluding both to their pom - based constituents and to their zeolitic nature . despite the various similarities between pomzites and zeolites in terms of their composition and properties , the two compound families are accessed via contrasting routes of assembly . as described here , pomzites are prepared in a modular fashion , whereas the synthesis of zeolites typically follows a one - pot methodology . the modular nature of pomzite preparation affords a valuable synthetic handle to exert greater control over the eventual framework topologies , which could enable the precise tuning of these porous inorganic materials toward tailored applications . k28li5h7[p8w48o184]92h2o and li17(nh4)21h2[p8w48o184]85h2o were prepared according to the published syntheses with minor modifications for optimization . single - crystal xrd data sets were collected at 150 k on an oxford diffraction gemini ultra s instrument equipped with a graphite monochromator and atlas ccd detector , or a bruker apex ii quasar instrument with ccd detector ( mo k = 0.71073 ) . inductively coupled plasma optical emission spectroscopy ( icp - oes ) was carried out on a tja - iris - advantage spectrometer , with echelle optics and a cid detector used to observe in the 170900 nm wavelength range . a minimum of 10 mg of each compound was submitted to the institut fr festkrperforschung in jlich for analysis . where relevant , carbon , hydrogen , and nitrogen content were determined by the microanalysis services within the school of chemistry , university of glasgow , using an ea 1110 chns , ce-440 elemental analyzer . for fourier transform infrared ( ft - ir ) spectroscopy , the materials were prepared as kbr pellets , and ft - ir spectra were collected in transmission mode using a jasco ft / ir 4100 spectrometer . wavenumbers ( ) are given in cm ; intensities are denoted as wk = weak , sh = sharp , m = medium , br = broad , s = strong . thermogravimetric analysis ( tga ) was performed on a ta instruments q500 thermogravimetric analyzer . to 20 ml of 2 mol l lich3co2 buffer solution ( ph 4.0 ) in a 50 ml round - bottomed flask was added mn(clo4)2xh2o ( 102 mg , 0.40 mmol ) , followed 5 min later by k28li5h7[p8w48o184]92h2o ( 100 mg , 6.75 mol ) . after a further 5 min of rapid stirring , the resulting pale yellow solution was then heated to 80 c overnight on a hot plate with a reflux condenser attached ( approximately 20 h ) upon which it gained a more vibrant golden color . after cooling slowly back to room temperature while still on the hot plate and connected to the condenser , the solution was transferred to a 50 ml conical flask and covered , before being placed in the refrigerator ( 4 c ) for crystallization . large , rectangular yellow plate crystals formed overnight and were collected after 5 days . yield : 19.8 mg , 1.36 mol , 20.1% based on [ p8w48o184 ] . icp - oes analysis for the hydrated material , h182k8li17mn6.5o275.5p8w48.5 , mw = 14543.82 g mol . calculated values ( found values in parentheses ) : k 2.14 ( 2.18 ) , li 0.80 ( 0.79 ) , mn 2.45 ( 2.54 ) , p 1.70 ( 1.70 ) w 61.3 ( 63.1 ) . characteristic ftir bands : 1620 ( s ) , 1133 ( s , sh ) , 1086 ( s , sh ) , 1022 ( w ) , 933 ( s ) , 798 ( br ) , 698 ( br ) . tga water loss from 20 to 250 c , calculated ( found ) : 11.3 ( 11.3 ) % . to a 20 ml buffer solution of 2 mol l lich3co2 at ph 4.0 in a 50 ml round - bottomed flask was first added mn(clo4)2xh2o ( 102 mg , 0.40 mmol ) , followed 5 min later by li17(nh4)21h2[p8w48o184]85h2o ( 93 mg , 6.75 mol ) . after a further 5 min of rapid stirring , the white suspension was then heated to 90 c on a hot plate with a reflux condenser attached , giving a lightly colored , clear , golden solution . after stirring at 90 c overnight ( approximately 20 h ) , the reaction mixture was allowed to cool slowly back to room temperature while still on the hot plate and connected to the condenser , before being filtered into four narrow test tubes . the test tubes were plugged with cotton wool and placed in a container , which was roughly 10% filled with methanol , to facilitate crystal growth . well - formed , pale yellow , hexagonal block crystals appeared in solution after 5 days . yield : 22.0 mg , 1.49 mol , 22.1% based on [ p8w48o184 ] . icp - oes and chn analysis for the hydrated material , h250li18mn8n6o297p8w48 , mw = 14725.04 g mol . calculated values ( found values in parentheses ) : li 0.86 ( 0.92 ) , mn 2.98 ( 2.93 ) , n 0.57 ( 0 . ) , p 1.68 ( 1.71 ) , w 59.9 ( 62.9 ) . characteristic ftir bands : 1620 ( s ) , 1402 ( s ) , 1134 ( s , sh ) , 1084 ( s , sh ) , 1022 ( w ) , 932 ( s ) , 797 ( m ) , 673 ( br ) . tga water loss from 20 to 250 c , calculated ( found ) : 13.8 ( 13.8)% . to 20 ml of 2 mol l lich3co2 buffer solution at ph 4.0 in a 50 ml round - bottomed flask was added nicl26h2o ( 88 mg , 0.37 mmol ) , followed 5 min later by li12(nh4)21h7[p8w48o184]85h2o ( 102 mg , 6.75 mol ) . after a further 5 min of rapid stirring , the resulting apple - green solution was heated to 90 c for 25 h on a hot plate with a reflux condenser attached . after cooling slowly back to room temperature while still on the hot plate and connected to the condenser , the solution was syringe - filtered and divided between four narrow test tubes , which were plugged with cotton wool . each test tube was place in the same large container , which was roughly 10% filled with acetone . pale green plate crystals were observed in the test tubes after 5 days and collected 1 week later . yield : 11.5 mg , 0.80 mol , 11.9% based on [ p8w48o184 ] . icp - oes and chn analysis for the hydrated material , h200li22n5ni6o274.25p8w48.25 , mw = 14283.57 g mol . calculated values ( found values in parentheses ) : li 1.08 ( 1.12 ) , n 0.49 ( 0.49 ) , ni 2.43 ( 2.45 ) , p 1.73 ( 1.82 ) , w 62.1 ( 59.4 ) . characteristic ftir bands : 1635 ( br ) , 1399 ( sh ) , 1132 ( s , sh ) , 1083 ( s , sh ) , 1019 ( w ) , 934 ( s , br ) , 704 ( m ) , 663 ( br ) . tga water loss from 20 to 250 c , calculated ( found ) : 11.4 ( 11.4)% . by utilizing the minimal building block library of { p8w48 } nodes and tm linkers , three new coordinatively linked inorganic frameworks have been synthesized . each compound has been prepared in a modular fashion , with the intrinsically porous pom building block formed prior to the assembly of the frameworks . the same general synthetic system was employed in each case , based on a lithium acetate buffer solution at ph 4.0 , heated to just below boiling and stirred overnight . these compounds further expand the growing family of tm - linked { p8w48 } structures . k8li17[mn6.5{w0.5o0.5}p8w48o184]91h2o ( compound 1 , figure 2 ) was crystallized from lithium acetate buffer solution following the reaction of mn(clo4)2 with k28li5h7[p8w48o184]92h2o . each { p8w48 } ring in the structure is coordinated to 8.5 mn centers ( 4 mn centers are shared between adjacent rings , hence there are only 6.5 mn atoms in the formula ) . two mn atoms are positioned in the internal cavity of the ring , occupying hinge sites on opposite sides of the cluster , in octahedral coordination environments of two pom terminal oxygen ligands ( w = ot ) , and four water molecules . two mn atoms are coordinated on the outside of the remaining two hinge regions , also through two w = ot sites . these mn atoms , and another two mn atoms situated on the outside of the ring ( coordinated to only one w = ot ) , are shared between adjacent { p8w48 } rings . mn atoms occupy a further two equivalent sites on the external surface of the ring in a disordered fashion across roughly one in every four clusters in the crystal . as has been observed previously , the four - fold symmetry of the parent cluster is distorted , and an additional 49th tungsten center is accommodated within the hinge region of the { p8w48 } rings , occupationally disordered across half of the clusters throughout the crystal . ball - and - stick representation of the { p8w48 } sbu in compound 1 from ( a ) top - down and ( b ) side - on . dark green spheres , w ; red spheres , o ; orange spheres , mn ; dark blue spheres , p. the four shared mn atoms which are coordinated to the outer surface of { p8w48 } connect the rings into a one - dimensional chain . each ring is oriented in the same manner , but coordinated rings are displaced both horizontally and vertically , resulting in a diagonal , stepped , polymeric structure . in the crystal packing , potassium and lithium cations , as well as solvent water molecules , form a supramolecular bridge between chains in a disorder fashion . similarly , these alkali metal cations and water molecules are also situated inside the { p8w48 } ring cavities , and could potentially be exchanged by solid - state cation sorption experiments , as observed for similar structures . in accordance with compound 1 , li18mn8(nh4)6[p8w48o184]113h2o ( compound 2 , figure 3 ) was formed in a lithium acetate buffered reaction mixture of mn(clo4)2 and { p8w48}. however , in contrast to the preparation of compound 1 , the synthesis of compound 2 utilized the li17(nh4)21h2[p8w48o184]85h2o starting material instead of k28li5h7[p8w48o184]92h2o . the influence of specific alkali metal cations on the assembly of { p8w48}-based frameworks has previously been discussed ; however , until the recent preparation of a potassium - deficient salt of { p8w48 } , this effect could not be directly investigated . ball - and - stick representation of the { p8w48 } sbu in compound 2 from ( a ) top - down and ( b ) side - on . connectivity of pomzite-13(mn ) in three dimensions from ( c ) top - down and ( d ) side - on . dark green spheres , w ; red spheres , o ; orange spheres , mn ; dark blue spheres , p. compound 2 has eight mn centers situated on the outside of the { p8w48 } hinge regions , each coordinated to two pom w = ot sites ( these are all shared with adjacent rings , and so constitute only four mn atoms in the formula of the compound ) . incidentally , this position is also occupied in compound 1 , but only at two hinge sites instead of eight . the remaining coordinated mn centers in compound 2 reside in the center of the { p8w48 } cavity , disordered across the inside of each hinge site . additional mn content , identified by icp - oes but not observed crystallographically , is assumed not to be coordinated to { p8w48 } , but to reside in the pores of the framework structure as charge - balancing , and potentially exchangeable , [ mn(h2o)6 ] countercation . each ring in compound 2 retains the approximate d4h symmetry of the parent { p8w48 } structure , with no additional tungsten content in the hinge positions . { ( w o)2mn(o w)2 } bridges connect each ring to eight surrounding clusters . all rings are oriented in a uniform manner throughout the framework , creating a three - dimensional framework with layers stacked directly on top of each other in an abab fashion . this arrangement of { p8w48 } into parallel columns creates cylindrical channels of roughly 1 nm diameter , in which reside a mixture of the crystallographically unaccounted - for [ mn(h2o)6 ] cations , nh4 cations , li cations , and solvent water molecules . by recognizing the role played by nh4 cations in the synthesis of compound 2 , the potassium - free starting material , li17(nh4)21h2[p8w48o184]85h2o , was investigated for its effect on the synthesis of further { p8w48}-based compounds . similarly , the recent publication of the first ni - linked { p8w48 } framework indicated that ni could be a suitable tm to link { p8w48 } clusters and add further new compounds based on this system . consequently , li22(nh4)5ni6[{w0.25o0.25}p8w48o184]90h2o ( compound 3 , figure 4 ) , an ni - linked , potassium - free { p8w48}-based compound was formed under typical reaction conditions . in this compound , a total of six ni atoms have been identified by icp - oes ; however , only 4.2 ni centers have been located crystallographically . as in compound 1 , the inner hinge sites are filled in two positions , on opposite sides of the ring . the remaining ni atoms are located on the outer surface of the ring , with significant positional disorder preventing crystallographic resolution of their complete coordination environments . again similarly to compound 1 , the four - fold symmetry of the parent cluster is distorted , and an additional 49th tungsten center is accommodated within the hinge region of the rings . in compound 3 , this supplementary position is occupationally disordered over a quarter of the clusters throughout the crystal . ball - and - stick representation of the { p8w48 } sbu in compound 3 from ( a ) top - down and ( b ) side - on . dark green spheres , w ; red spheres , o ; light green spheres , mn ; dark blue spheres , p. compound 3 has a planar one - dimensional chain - type structure , and as with the two previously described compounds , each { p8w48 } cluster is oriented uniformly throughout the crystal . however , in contrast to the chains of compound 1 , each of the connected rings resides in the same plane . vacancies in the structure of compound 3 are occupied by a combination of nh4 and li cations , solvent water molecules , and the remaining [ ni(h2o)6 ] cations which are not coordinated directly to the ring via dative bonds . during the course of this work , a number of additional frameworks based on { p8w48 } and tms have been identified . however , many of the compounds currently suffer from a lack of reproducibility and are yet to be fully characterized . among the strategies which have shown promise so far include the introduction of secondary cations to the reaction mixture , the results of which approach will be reported in due course . however , the rapid expansion of this class of materials has highlighted the need for a greater understanding of the nature of tm coordination to { p8w48 } , and its subsequent extension into multidimensional frameworks . further , the establishment of a classification system to cover all one- , two- , and three - dimensional { p8w48}-based structures is needed to facilitate both the identification and discussion of new compounds . although the nanometer - sized cavity of { p8w48 } makes it an ideal building block for the modular construction of porous frameworks , its tm - mediated extension into multidimensional architectures has so far been plagued by unpredictability . this is primarily because there are several sites around the { p8w48 } ring to which tms may coordinate , causing rings which are connected through one or more common tm atoms to adopt a variety of different positions and orientations in relation to each other . crucially , the specific nanoscale arrangement of neighboring rings determines the long - range organization of { p8w48 } on the mesoscale , into coordinatively linked , one- , two- , or three - dimensional structures . such topological diversity arises despite the same minimal library of building blocks being employed in each case . in order to control the extension of the effectively 0-d { p8w48 } sbu into higher - dimensional structures , it is therefore crucial to gain a clearer insight into tm coordination to { p8w48 } by fully mapping the binding sites of the heteropolyanion , and using the wealth of information available from the tm - linked { p8w48 } structures which are currently known ( a complete list of the compounds considered in this study is given in the supporting information ) . in general , poms act as nucleophilic o - donor ligands for the coordination of first - row tms . more specifically , the { p8w48 } ring features a total of 64 reactive , coordinatively unsaturated terminal oxygen ( w = ot ) sites to which 3d tms may bind . despite this apparent complexity , however , in its idealized d4h configuration there are only four inequivalent groups of w = ot , each made up of 16 equiv sites around the ring , and labeled either a , b , c , or d according to their environment ( figure 5 ) . ( a ) { p8w48 } ring - shaped sbu node , with close - up of the smallest repeating [ p0.5w3o11.5 ] subunit , highlighting the three inequivalent w atoms and four inequivalent w = ot ( labeled a , b , c , and d ) . ( b ) octahedral { tmo6 } linker ( tm = mn , co , ni ) which can be used to connect adjacent { p8w48 } sbus . although tm binding is a prerequisite for the extension of { p8w48 } into a coordination polymer , it does not always result in ring connectivity . pendant metal coordination , in cases where tm cations are bound to only one { p8w48 } sbu , has been observed at each of the four binding sites around the ring ( figure 6 ) . in accordance with the labeling system for these sites , the binding modes of the four possible monodentate pendant positions can in turn be labeled a , b , c , or d according to the identity of the coordinated w = ot . four types of monodentate pendant coordination modes , labeled a , b , c , and d according to the identity of the coordinated terminal oxygen . in addition to the simple monodentate nonbridging modes of tm coordination to { p8w48 } , the two components may also be combined in bidentate fashion ( figure 7 ) . there are 10 potential bidentate modes of coordination based on the binary combination of recognized w = ot sites ; however , only aa and dd combinations have been observed for the first - row tms . in addition to these sites at the hinge region of { p8w48 } , a third mode is possible when { p8w48 } is reacted with f - block metals . labeled dd * , these binding sites are on the inner face of { p8w48 } , and are seemingly favored by bulkier cations , due to the typically larger spacing between the o - termini in comparison with the dd coordination mode . moreover , the three observed bidentate pendant modes of tm binding to { p8w48 } suggest that bidentate nonbridging coordination is forbidden for two different w = ot sites of the same { p8w48 } ring . three observed modes of bidentate pendant coordination , labeled aa , dd , and dd * according to the identity of the coordinated terminal oxygens . note that dd * coordination has only been observed for f - block transition metals . transition metals which are bound to { p8w48 } in pendant modes undoubtedly have a significant influence on the general properties of the compound . pendant tms typically have at least four coordination sites occupied by aqua ligands , which may be exchangeable in nonaqueous solvents . in mof chemistry , open metal sites are commonly attributed with enhancing catalytic activity , improving uptake , and increasing the specificity of interactions . we have also demonstrated that pendant sites in { p8w48}-based structures may be activated to form linkers , and in turn entirely new frameworks , which are only accessible through an intermediary , preorganized structure with nonbridging tms . although pendant sites must not be ignored in pomzite chemistry , the extension of { p8w48 } into multidimensional structures requires the coordination of tm centers which are common to two or more { p8w48 } sbus . as with the small number of observed bidentate pendant coordination modes , there are many potential combinations of pendant - type coordination modes which are not , or are only rarely , observed as linkers . of the common linker modes , three may be considered as homotopic i.e. , bridging between the same sites of two { p8w48 } sbus ( figure 8) . these are labeled aaaa , bb , and cc , where the prime indicates the coordination of a site from a second ring . further , there are four common heterotopic linker modes which bridge neighboring rings via two different binding sites ( figure 9 ) . following the same convention as for the labeling of homotopic linkers , the heterotopic linkers are designated as aab , aac , bc , and ddc. as the seven common linker modes are crucial in determining the positional and orientational relationships between neighboring rings , they play a crucial structure - directing role in the extension of { p8w48 } into coordinatively linked frameworks , and are responsible for the structural diversity seen for the pomzite family of materials . three main homotopic linker coordination modes observed in pomzites : a - type linkage , green ; b - type linkage , blue ; c - type linkage , yellow . four main heterotopic linker coordination modes observed in pomzites : a - type linkage , green ; b - type linkage , blue ; c - type linkage , yellow ; d - type linkage , red . four main structural types of pomzite have been observed so far , namely chain , column , herringbone , and cube ( figure 10 ) . the simplest structural type is the chain arrangement , seen for both compounds 1 and 3 , in which { p8w48 } rings are linked edge - to - edge in only one dimension . each ring in the chain structure is orientated in a uniform fashion , and 2-coordinated , i.e. , linked to two other rings . similarly , each ring in the column structure has the same orientation , but the rings are connected in a face - to - face manner . the coordination number is also 2 if the rings are connected in only one dimension ; however , column structures may also be linked into two or three dimensions by edge - to - edge connectivity . for two - dimensional structures , which may be considered as rows of columns , the coordination number is typically 6 , while for three - dimensional structures rings are 8-connected and reside in a columnar matrix . in both the herringbone and cube structures , there are two different ways in which the rings are orientated . in the two - dimensional herringbone structure , rings are related by an approximate 45 rotation , with edge - to - face connectivity . herringbone structures are typically 4-coordinated , but may also be linked edge - to - edge to become three - dimensional with higher coordination numbers . finally , the cube architecture is a three - dimensional construct of 8-coordinated rings in a stacked - box it seems highly likely that further structure types will be identified in future . in particular , a sheet structure based on the chain arrangement with additional edge - to - edge connectivity in the second dimension is easy to visualize . representative models for the four structural types of known pomzites : chain , column , cube , and herringbone . one of the major benefits of disentangling the complex connectivity of { p8w48 } rings in pomzite structures by defining a limited number of linker modes , is that it allows simplified representations of the structures to be presented . this gives the reader a much greater insight into the three - dimensional arrangements of { p8w48}-based compounds than with conventional ball - and - stick models , and enables facile comparisons between pomzites to be made . in total , 14 unique pomzite architectures have been observed so far , and are numbered chronologically according to their date of publication ( table 1 ) . if multiple compounds were presented in a single publication , the order of numbering within the article has been retained in the current system . additionally , if more than one compound has been identified with any given structure , it is the publication date of the first compound to be presented which determines its numbering here . for example , the first multidimensional { p8w48 } compound to be presented was the co - linked structure , k15li5[co10(h2o)34(p8w48o184)]54h2o , labeled compound 1 in a communication published in 2009 which presented two new compounds . pomzite-1(co ) to indicate that this was the first pomzite discovered , and that it incorporated co linkers . compound 1 , presented herein , is a mn - linked analogue of pomzite-1(co ) , appropriately termed isostructural pomzites have the same tm substitution patterns and connectivity , although they may differ in the actual occupancy of each tm coordination site . indeed , we have observed small variations in heterometal content between crystals from different batches of a number of pom framework materials , although the batches are still considered to be of the same compound . in the future , reports of new pomzites should indicate the range of tm incorporation with which a material can be prepared . as the chemical formulas of pomzites are typically complex , and often quite similar , this universal system of nomenclature should simplify discussions concerning all pomzites . for each pomzite the left - hand column details the pendant modes present and the right - hand column the linker modes ( as described in the main text ) . the figures in parentheses are the tm occupancies of such sites per { p8w48 } ring . for detailed composition of each pomzite architecture , it is apparent that a distinction has been made between several structures with similar substitution patterns and linker modes . in particular , pomzite-1 , pomzite-4 , and pomzite-9 all have chain structures with aab connectivity . in pomzite-1 , the presence of an external tm coordinated in the b position sets it apart from the two other compounds for two reasons : ( 1 ) pendant functionality is important to the general properties of the framework and can be activated by single crystal to single crystal transformations , and ( 2 ) the steric effect of a tm positioned externally causes spatial displacement of adjacent chains in the solid state . as the aab linkers present in both compounds are heterotopic , they also have direction ; i.e. , they connect two inequivalent termini . in pomzite-4 and pomzite-9 , the same positions are occupied in both compounds , but the direction of the aab linkers is reversed . notably , the authors of the latter publication did not refer to the prior - published compound when presenting pomzite-9(mn ) , despite its close similarity to pomzite-4(co ) . both the binding site analysis carried out here , and the structural model representations which this allowed , make compound comparisons much simpler . the differences between the three compounds discussed here can be much better understood using this system . three new compounds based on a minimal building block library of the cyclic polyoxometalate { p8w48 } and transition metal cations have been prepared . each material consists of a purely inorganic metal oxide scaffold , with intrinsic porosity resulting from the use of a crown - type building block . despite the apparent limitations of employing only two main structural components , this study expands the number of frameworks based on this system to 14 . furthermore , a comprehensive { p8w48 } binding site analysis of the literature has been performed to gain a greater understanding of how the various structure - types are accessed . as a result , a previously disparate group of compounds has been united and proposed to be named pomzite materials have tremendous potential to bridge the gap in porous framework materials between mof tunability and zeolite functionality . previously , the structural complexity of these materials , and a lack of understanding regarding the connectivity within them , has held back their development . due to their light nature , mofs hold a clear technological advantage over pom framework materials for such applications as gas storage . the most obvious advantage is in catalysis , where the benefit of poms is already realized by the dispersion of these clusters throughout mof architectures . pomzites are self - supported porous materials with pom functionality inherent in their structure . in the future , the use of organic sdas , a well - known strategy in zeolite synthesis , will be employed to access novel pomzite archetypes . the potential for further expanding this library is great , with possibilities to introduce other tms and employ other sdas representing just a couple of examples of how this might be achieved . it is highly likely that many more structures will arise from this fruitful chemical system through careful control of reaction conditions . work is currently underway in our research group to fully utilize the pore space within these materials to exploit their unique pore environments .

we describe why the cyclic heteropolyanion [ p8w48o184]40 ( abbreviated as { p8w48 } ) is an ideal building block for the construction of intrinsically porous framework materials by classifying and analyzing > 30 coordination polymers incorporating this polyoxometalate ( pom ) ligand . this analysis shows that the exocyclic coordination of first - row transition metals ( tms ) to { p8w48 } typically yields frameworks which extend through { w o tm o w } bridges in one , two , or three dimensions . however , despite the rich structural diversity of such compounds , the coordination of tms to the { p8w48 } ring is poorly understood , and therefore largely unpredictable , and had not until now been present with any structural classification that could allow rational design . herein , not only do we present a new approach to understand and classify this new class of materials , we also present three { p8w48}-based frameworks which complement those frameworks which have previously been described . these new compounds help us postulate a new taxonomy of these materials . this is possible because the tm coordination sites of the { p8w48 } ring are found , once fully mapped , to lead to well - defined classes of connectivity . together , analysis provides insight into the nature of the building block connectivity within each framework , to facilitate comparisons between related structures , and to fundamentally unite this family of compounds . hence we have tentatively named these compounds as pomzites to reflect the pom - based composition and zeolitic nature of each family member , although crucially , pomzites differ from zeolites in the modular manner of their preparation . as the synthesis of further pomzites is anticipated , the classification system and terminology introduced here will allow new compounds to be categorized and understood in the context of the established materials . a better understanding of tm coordination to the { p8w48 } ring may allow the targeted synthesis of new frameworks rather than the reliance on serendipity apparent in current methods .

Introduction Experimental Methods Results and Discussion Conclusions

however , with widespread applications in areas including , but not restricted to , heterogeneous catalysis , the adsorption of pollutants , and resistant coatings , zeolites are still regarded as the benchmark for functional porous materials . however , tuning zeolite functionality is generally limited to controlling the ratio of si : al in the framework , while extensive structure modulation of these otherwise simple inorganic materials relies on the introduction of organic molecules , either as structure - directing agents ( sdas ) or as functional organic platforms incorporated within the zeolite scaffold itself . synthetic control in mof chemistry is largely attributable to the modular nature of mof preparation , with secondary building units ( sbus ) utilized as geometrically well - defined components to access many of the great variety of framework topologies currently realized . despite rapid progress made in this field and the widespread application of mofs , there remains scope to explore and extend the thermal and chemical stability range of the compounds due to the nature of the organic constituents . therefore , the development of porous transition metal oxide - based molecular materials could be interesting due to the promise of flexibility and tunable redox , catalytic , and thermal properties . polyoxometalates ( poms ) are a diverse class of early transition metal oxide clusters , typically comprised of multiple w , mo , or v centers connected through shared oxygen atoms , and commonly incorporating additional heteroelements such as si , p , or ge . although by their definition poms are discrete ( 0-d ) molecules , the introduction of transition metals ( tms ) and/or organic moieties to pom solutions may lead to connectivity between clusters , and their extension into coordinatively linked 1-d chains , 2-d sheets , or 3-d networks . by application of the topological node and linker designation given to the structural components of a mof , in multidimensional inorganic pom assemblies the pom units may be considered as inorganic nodes , which extend into infinite lattice arrangements through the coordination of tm linkers . however , in contrast to mofs , it is primarily the versatility of the node , and not the linker , which generates the diversity of structures found in tm - linked pom framework materials . furthermore , the recent trend for the incorporation of pom guests inside porous frameworks recognizes the acidic , electronic , and catalytic properties that these clusters may embellish on the overall framework structure . it is therefore clear that the direct incorporation of poms into porous framework scaffolds is a valid strategy for the modular synthesis of robust functional materials , without the need for an organic component . two main strategies are typically employed in the construction of modular frameworks with high porosity : ( 1 ) the selection of long , narrow linkers ( which may lead to the interpenetration of multiple frameworks ) , and ( 2 ) the pre - fabrication of a pore within one of the components . in particular , the crown - type heteropolyanion , [ p8w48o184 ] ( hereafter referred to as { p8w48 } ) is notable for several properties , including its high - negative charge and remarkable electrochemistry . its intrinsic nanometer - sized cavity means { p8w48 } is an ideal candidate to be utilized as a network synthon , to prepare open framework materials with microporosity ( figure 1 ) . such structures have already been prepared by introducing first - row tms to aqueous solutions of { p8w48 } ; however , the desired control in preparing { p8w48}-based porous frameworks is yet to be realized . detailed herein are the syntheses of three coordinatively linked framework structures , based on the minimal building block library of { p8w48 } nodes and tm linkers . in addition , the tm coordination sites of { p8w48 } are fully mapped , allowing a comprehensive analysis of ring connectivity within the known { p8w48}-based frameworks . the term pomzites is introduced to apply to this unified class of materials , alluding both to their pom - based constituents and to their zeolitic nature . despite the various similarities between pomzites and zeolites in terms of their composition and properties , the two compound families are accessed via contrasting routes of assembly . by utilizing the minimal building block library of { p8w48 } nodes and tm linkers , three new coordinatively linked inorganic frameworks have been synthesized . each compound has been prepared in a modular fashion , with the intrinsically porous pom building block formed prior to the assembly of the frameworks . these compounds further expand the growing family of tm - linked { p8w48 } structures . each { p8w48 } ring in the structure is coordinated to 8.5 mn centers ( 4 mn centers are shared between adjacent rings , hence there are only 6.5 mn atoms in the formula ) . these mn atoms , and another two mn atoms situated on the outside of the ring ( coordinated to only one w = ot ) , are shared between adjacent { p8w48 } rings . as has been observed previously , the four - fold symmetry of the parent cluster is distorted , and an additional 49th tungsten center is accommodated within the hinge region of the { p8w48 } rings , occupationally disordered across half of the clusters throughout the crystal . ball - and - stick representation of the { p8w48 } sbu in compound 1 from ( a ) top - down and ( b ) side - on . similarly , these alkali metal cations and water molecules are also situated inside the { p8w48 } ring cavities , and could potentially be exchanged by solid - state cation sorption experiments , as observed for similar structures . however , in contrast to the preparation of compound 1 , the synthesis of compound 2 utilized the li17(nh4)21h2[p8w48o184]85h2o starting material instead of k28li5h7[p8w48o184]92h2o . the influence of specific alkali metal cations on the assembly of { p8w48}-based frameworks has previously been discussed ; however , until the recent preparation of a potassium - deficient salt of { p8w48 } , this effect could not be directly investigated . ball - and - stick representation of the { p8w48 } sbu in compound 2 from ( a ) top - down and ( b ) side - on . dark green spheres , w ; red spheres , o ; orange spheres , mn ; dark blue spheres , p. compound 2 has eight mn centers situated on the outside of the { p8w48 } hinge regions , each coordinated to two pom w = ot sites ( these are all shared with adjacent rings , and so constitute only four mn atoms in the formula of the compound ) . the remaining coordinated mn centers in compound 2 reside in the center of the { p8w48 } cavity , disordered across the inside of each hinge site . additional mn content , identified by icp - oes but not observed crystallographically , is assumed not to be coordinated to { p8w48 } , but to reside in the pores of the framework structure as charge - balancing , and potentially exchangeable , [ mn(h2o)6 ] countercation . each ring in compound 2 retains the approximate d4h symmetry of the parent { p8w48 } structure , with no additional tungsten content in the hinge positions . this arrangement of { p8w48 } into parallel columns creates cylindrical channels of roughly 1 nm diameter , in which reside a mixture of the crystallographically unaccounted - for [ mn(h2o)6 ] cations , nh4 cations , li cations , and solvent water molecules . by recognizing the role played by nh4 cations in the synthesis of compound 2 , the potassium - free starting material , li17(nh4)21h2[p8w48o184]85h2o , was investigated for its effect on the synthesis of further { p8w48}-based compounds . similarly , the recent publication of the first ni - linked { p8w48 } framework indicated that ni could be a suitable tm to link { p8w48 } clusters and add further new compounds based on this system . ball - and - stick representation of the { p8w48 } sbu in compound 3 from ( a ) top - down and ( b ) side - on . dark green spheres , w ; red spheres , o ; light green spheres , mn ; dark blue spheres , p. compound 3 has a planar one - dimensional chain - type structure , and as with the two previously described compounds , each { p8w48 } cluster is oriented uniformly throughout the crystal . however , in contrast to the chains of compound 1 , each of the connected rings resides in the same plane . however , many of the compounds currently suffer from a lack of reproducibility and are yet to be fully characterized . however , the rapid expansion of this class of materials has highlighted the need for a greater understanding of the nature of tm coordination to { p8w48 } , and its subsequent extension into multidimensional frameworks . further , the establishment of a classification system to cover all one- , two- , and three - dimensional { p8w48}-based structures is needed to facilitate both the identification and discussion of new compounds . although the nanometer - sized cavity of { p8w48 } makes it an ideal building block for the modular construction of porous frameworks , its tm - mediated extension into multidimensional architectures has so far been plagued by unpredictability . this is primarily because there are several sites around the { p8w48 } ring to which tms may coordinate , causing rings which are connected through one or more common tm atoms to adopt a variety of different positions and orientations in relation to each other . crucially , the specific nanoscale arrangement of neighboring rings determines the long - range organization of { p8w48 } on the mesoscale , into coordinatively linked , one- , two- , or three - dimensional structures . in order to control the extension of the effectively 0-d { p8w48 } sbu into higher - dimensional structures , it is therefore crucial to gain a clearer insight into tm coordination to { p8w48 } by fully mapping the binding sites of the heteropolyanion , and using the wealth of information available from the tm - linked { p8w48 } structures which are currently known ( a complete list of the compounds considered in this study is given in the supporting information ) . in general , poms act as nucleophilic o - donor ligands for the coordination of first - row tms . more specifically , the { p8w48 } ring features a total of 64 reactive , coordinatively unsaturated terminal oxygen ( w = ot ) sites to which 3d tms may bind . despite this apparent complexity , however , in its idealized d4h configuration there are only four inequivalent groups of w = ot , each made up of 16 equiv sites around the ring , and labeled either a , b , c , or d according to their environment ( figure 5 ) . ( a ) { p8w48 } ring - shaped sbu node , with close - up of the smallest repeating [ p0.5w3o11.5 ] subunit , highlighting the three inequivalent w atoms and four inequivalent w = ot ( labeled a , b , c , and d ) . although tm binding is a prerequisite for the extension of { p8w48 } into a coordination polymer , it does not always result in ring connectivity . pendant metal coordination , in cases where tm cations are bound to only one { p8w48 } sbu , has been observed at each of the four binding sites around the ring ( figure 6 ) . in accordance with the labeling system for these sites , the binding modes of the four possible monodentate pendant positions can in turn be labeled a , b , c , or d according to the identity of the coordinated w = ot . four types of monodentate pendant coordination modes , labeled a , b , c , and d according to the identity of the coordinated terminal oxygen . in addition to the simple monodentate nonbridging modes of tm coordination to { p8w48 } , the two components may also be combined in bidentate fashion ( figure 7 ) . there are 10 potential bidentate modes of coordination based on the binary combination of recognized w = ot sites ; however , only aa and dd combinations have been observed for the first - row tms . labeled dd * , these binding sites are on the inner face of { p8w48 } , and are seemingly favored by bulkier cations , due to the typically larger spacing between the o - termini in comparison with the dd coordination mode . moreover , the three observed bidentate pendant modes of tm binding to { p8w48 } suggest that bidentate nonbridging coordination is forbidden for two different w = ot sites of the same { p8w48 } ring . transition metals which are bound to { p8w48 } in pendant modes undoubtedly have a significant influence on the general properties of the compound . we have also demonstrated that pendant sites in { p8w48}-based structures may be activated to form linkers , and in turn entirely new frameworks , which are only accessible through an intermediary , preorganized structure with nonbridging tms . although pendant sites must not be ignored in pomzite chemistry , the extension of { p8w48 } into multidimensional structures requires the coordination of tm centers which are common to two or more { p8w48 } sbus . as the seven common linker modes are crucial in determining the positional and orientational relationships between neighboring rings , they play a crucial structure - directing role in the extension of { p8w48 } into coordinatively linked frameworks , and are responsible for the structural diversity seen for the pomzite family of materials . the coordination number is also 2 if the rings are connected in only one dimension ; however , column structures may also be linked into two or three dimensions by edge - to - edge connectivity . for two - dimensional structures , which may be considered as rows of columns , the coordination number is typically 6 , while for three - dimensional structures rings are 8-connected and reside in a columnar matrix . one of the major benefits of disentangling the complex connectivity of { p8w48 } rings in pomzite structures by defining a limited number of linker modes , is that it allows simplified representations of the structures to be presented . this gives the reader a much greater insight into the three - dimensional arrangements of { p8w48}-based compounds than with conventional ball - and - stick models , and enables facile comparisons between pomzites to be made . additionally , if more than one compound has been identified with any given structure , it is the publication date of the first compound to be presented which determines its numbering here . for example , the first multidimensional { p8w48 } compound to be presented was the co - linked structure , k15li5[co10(h2o)34(p8w48o184)]54h2o , labeled compound 1 in a communication published in 2009 which presented two new compounds . compound 1 , presented herein , is a mn - linked analogue of pomzite-1(co ) , appropriately termed isostructural pomzites have the same tm substitution patterns and connectivity , although they may differ in the actual occupancy of each tm coordination site . indeed , we have observed small variations in heterometal content between crystals from different batches of a number of pom framework materials , although the batches are still considered to be of the same compound . the figures in parentheses are the tm occupancies of such sites per { p8w48 } ring . in pomzite-1 , the presence of an external tm coordinated in the b position sets it apart from the two other compounds for two reasons : ( 1 ) pendant functionality is important to the general properties of the framework and can be activated by single crystal to single crystal transformations , and ( 2 ) the steric effect of a tm positioned externally causes spatial displacement of adjacent chains in the solid state . in pomzite-4 and pomzite-9 , the same positions are occupied in both compounds , but the direction of the aab linkers is reversed . notably , the authors of the latter publication did not refer to the prior - published compound when presenting pomzite-9(mn ) , despite its close similarity to pomzite-4(co ) . three new compounds based on a minimal building block library of the cyclic polyoxometalate { p8w48 } and transition metal cations have been prepared . furthermore , a comprehensive { p8w48 } binding site analysis of the literature has been performed to gain a greater understanding of how the various structure - types are accessed . as a result , a previously disparate group of compounds has been united and proposed to be named pomzite materials have tremendous potential to bridge the gap in porous framework materials between mof tunability and zeolite functionality . previously , the structural complexity of these materials , and a lack of understanding regarding the connectivity within them , has held back their development .

over the past two decades , the emergence of metal organic frameworks ( mofs ) and covalent organic frameworks ( cofs ) has reinvigorated the field of crystalline microporous materials , previously dominated by zeolites . however , with widespread applications in areas including , but not restricted to , heterogeneous catalysis , the adsorption of pollutants , and resistant coatings , zeolites are still regarded as the benchmark for functional porous materials . although the porosity of zeolites and related materials has been exploited for centuries , real insights into the behavior of guest molecules in confined environments , and the role of the host in which they reside , have only recently become possible through advanced crystallographic techniques . composed primarily of the naturally abundant elements o , si , and al , zeolites have remarkable thermal stability upon evacuation , regular pore sizes , and high acidity . however , tuning zeolite functionality is generally limited to controlling the ratio of si : al in the framework , while extensive structure modulation of these otherwise simple inorganic materials relies on the introduction of organic molecules , either as structure - directing agents ( sdas ) or as functional organic platforms incorporated within the zeolite scaffold itself . mof materials , including hybrid zeolite analogues , are born of remarkably sophisticated design principles , and display a richly diverse range of properties . synthetic control in mof chemistry is largely attributable to the modular nature of mof preparation , with secondary building units ( sbus ) utilized as geometrically well - defined components to access many of the great variety of framework topologies currently realized . despite rapid progress made in this field and the widespread application of mofs , there remains scope to explore and extend the thermal and chemical stability range of the compounds due to the nature of the organic constituents . therefore , the development of porous transition metal oxide - based molecular materials could be interesting due to the promise of flexibility and tunable redox , catalytic , and thermal properties . as such , pom - frameworks promise to combine the thermal stability and general applicability of zeolites , combined with the synthetic control and tunable properties of mofs . polyoxometalates ( poms ) are a diverse class of early transition metal oxide clusters , typically comprised of multiple w , mo , or v centers connected through shared oxygen atoms , and commonly incorporating additional heteroelements such as si , p , or ge . although by their definition poms are discrete ( 0-d ) molecules , the introduction of transition metals ( tms ) and/or organic moieties to pom solutions may lead to connectivity between clusters , and their extension into coordinatively linked 1-d chains , 2-d sheets , or 3-d networks . by application of the topological node and linker designation given to the structural components of a mof , in multidimensional inorganic pom assemblies the pom units may be considered as inorganic nodes , which extend into infinite lattice arrangements through the coordination of tm linkers . however , in contrast to mofs , it is primarily the versatility of the node , and not the linker , which generates the diversity of structures found in tm - linked pom framework materials . furthermore , the recent trend for the incorporation of pom guests inside porous frameworks recognizes the acidic , electronic , and catalytic properties that these clusters may embellish on the overall framework structure . it is therefore clear that the direct incorporation of poms into porous framework scaffolds is a valid strategy for the modular synthesis of robust functional materials , without the need for an organic component . two main strategies are typically employed in the construction of modular frameworks with high porosity : ( 1 ) the selection of long , narrow linkers ( which may lead to the interpenetration of multiple frameworks ) , and ( 2 ) the pre - fabrication of a pore within one of the components . in particular , the crown - type heteropolyanion , [ p8w48o184 ] ( hereafter referred to as { p8w48 } ) is notable for several properties , including its high - negative charge and remarkable electrochemistry . such structures have already been prepared by introducing first - row tms to aqueous solutions of { p8w48 } ; however , the desired control in preparing { p8w48}-based porous frameworks is yet to be realized . detailed herein are the syntheses of three coordinatively linked framework structures , based on the minimal building block library of { p8w48 } nodes and tm linkers . in addition , the tm coordination sites of { p8w48 } are fully mapped , allowing a comprehensive analysis of ring connectivity within the known { p8w48}-based frameworks . despite the various similarities between pomzites and zeolites in terms of their composition and properties , the two compound families are accessed via contrasting routes of assembly . the modular nature of pomzite preparation affords a valuable synthetic handle to exert greater control over the eventual framework topologies , which could enable the precise tuning of these porous inorganic materials toward tailored applications . inductively coupled plasma optical emission spectroscopy ( icp - oes ) was carried out on a tja - iris - advantage spectrometer , with echelle optics and a cid detector used to observe in the 170900 nm wavelength range . where relevant , carbon , hydrogen , and nitrogen content were determined by the microanalysis services within the school of chemistry , university of glasgow , using an ea 1110 chns , ce-440 elemental analyzer . for fourier transform infrared ( ft - ir ) spectroscopy , the materials were prepared as kbr pellets , and ft - ir spectra were collected in transmission mode using a jasco ft / ir 4100 spectrometer . characteristic ftir bands : 1620 ( s ) , 1402 ( s ) , 1134 ( s , sh ) , 1084 ( s , sh ) , 1022 ( w ) , 932 ( s ) , 797 ( m ) , 673 ( br ) . characteristic ftir bands : 1635 ( br ) , 1399 ( sh ) , 1132 ( s , sh ) , 1083 ( s , sh ) , 1019 ( w ) , 934 ( s , br ) , 704 ( m ) , 663 ( br ) . two mn atoms are positioned in the internal cavity of the ring , occupying hinge sites on opposite sides of the cluster , in octahedral coordination environments of two pom terminal oxygen ligands ( w = ot ) , and four water molecules . as has been observed previously , the four - fold symmetry of the parent cluster is distorted , and an additional 49th tungsten center is accommodated within the hinge region of the { p8w48 } rings , occupationally disordered across half of the clusters throughout the crystal . ball - and - stick representation of the { p8w48 } sbu in compound 1 from ( a ) top - down and ( b ) side - on . similarly , these alkali metal cations and water molecules are also situated inside the { p8w48 } ring cavities , and could potentially be exchanged by solid - state cation sorption experiments , as observed for similar structures . however , in contrast to the preparation of compound 1 , the synthesis of compound 2 utilized the li17(nh4)21h2[p8w48o184]85h2o starting material instead of k28li5h7[p8w48o184]92h2o . the influence of specific alkali metal cations on the assembly of { p8w48}-based frameworks has previously been discussed ; however , until the recent preparation of a potassium - deficient salt of { p8w48 } , this effect could not be directly investigated . ball - and - stick representation of the { p8w48 } sbu in compound 2 from ( a ) top - down and ( b ) side - on . dark green spheres , w ; red spheres , o ; orange spheres , mn ; dark blue spheres , p. compound 2 has eight mn centers situated on the outside of the { p8w48 } hinge regions , each coordinated to two pom w = ot sites ( these are all shared with adjacent rings , and so constitute only four mn atoms in the formula of the compound ) . additional mn content , identified by icp - oes but not observed crystallographically , is assumed not to be coordinated to { p8w48 } , but to reside in the pores of the framework structure as charge - balancing , and potentially exchangeable , [ mn(h2o)6 ] countercation . each ring in compound 2 retains the approximate d4h symmetry of the parent { p8w48 } structure , with no additional tungsten content in the hinge positions . this arrangement of { p8w48 } into parallel columns creates cylindrical channels of roughly 1 nm diameter , in which reside a mixture of the crystallographically unaccounted - for [ mn(h2o)6 ] cations , nh4 cations , li cations , and solvent water molecules . by recognizing the role played by nh4 cations in the synthesis of compound 2 , the potassium - free starting material , li17(nh4)21h2[p8w48o184]85h2o , was investigated for its effect on the synthesis of further { p8w48}-based compounds . similarly , the recent publication of the first ni - linked { p8w48 } framework indicated that ni could be a suitable tm to link { p8w48 } clusters and add further new compounds based on this system . again similarly to compound 1 , the four - fold symmetry of the parent cluster is distorted , and an additional 49th tungsten center is accommodated within the hinge region of the rings . dark green spheres , w ; red spheres , o ; light green spheres , mn ; dark blue spheres , p. compound 3 has a planar one - dimensional chain - type structure , and as with the two previously described compounds , each { p8w48 } cluster is oriented uniformly throughout the crystal . vacancies in the structure of compound 3 are occupied by a combination of nh4 and li cations , solvent water molecules , and the remaining [ ni(h2o)6 ] cations which are not coordinated directly to the ring via dative bonds . among the strategies which have shown promise so far include the introduction of secondary cations to the reaction mixture , the results of which approach will be reported in due course . however , the rapid expansion of this class of materials has highlighted the need for a greater understanding of the nature of tm coordination to { p8w48 } , and its subsequent extension into multidimensional frameworks . further , the establishment of a classification system to cover all one- , two- , and three - dimensional { p8w48}-based structures is needed to facilitate both the identification and discussion of new compounds . although the nanometer - sized cavity of { p8w48 } makes it an ideal building block for the modular construction of porous frameworks , its tm - mediated extension into multidimensional architectures has so far been plagued by unpredictability . crucially , the specific nanoscale arrangement of neighboring rings determines the long - range organization of { p8w48 } on the mesoscale , into coordinatively linked , one- , two- , or three - dimensional structures . in order to control the extension of the effectively 0-d { p8w48 } sbu into higher - dimensional structures , it is therefore crucial to gain a clearer insight into tm coordination to { p8w48 } by fully mapping the binding sites of the heteropolyanion , and using the wealth of information available from the tm - linked { p8w48 } structures which are currently known ( a complete list of the compounds considered in this study is given in the supporting information ) . despite this apparent complexity , however , in its idealized d4h configuration there are only four inequivalent groups of w = ot , each made up of 16 equiv sites around the ring , and labeled either a , b , c , or d according to their environment ( figure 5 ) . ( a ) { p8w48 } ring - shaped sbu node , with close - up of the smallest repeating [ p0.5w3o11.5 ] subunit , highlighting the three inequivalent w atoms and four inequivalent w = ot ( labeled a , b , c , and d ) . in accordance with the labeling system for these sites , the binding modes of the four possible monodentate pendant positions can in turn be labeled a , b , c , or d according to the identity of the coordinated w = ot . in addition to the simple monodentate nonbridging modes of tm coordination to { p8w48 } , the two components may also be combined in bidentate fashion ( figure 7 ) . there are 10 potential bidentate modes of coordination based on the binary combination of recognized w = ot sites ; however , only aa and dd combinations have been observed for the first - row tms . labeled dd * , these binding sites are on the inner face of { p8w48 } , and are seemingly favored by bulkier cations , due to the typically larger spacing between the o - termini in comparison with the dd coordination mode . moreover , the three observed bidentate pendant modes of tm binding to { p8w48 } suggest that bidentate nonbridging coordination is forbidden for two different w = ot sites of the same { p8w48 } ring . we have also demonstrated that pendant sites in { p8w48}-based structures may be activated to form linkers , and in turn entirely new frameworks , which are only accessible through an intermediary , preorganized structure with nonbridging tms . although pendant sites must not be ignored in pomzite chemistry , the extension of { p8w48 } into multidimensional structures requires the coordination of tm centers which are common to two or more { p8w48 } sbus . as with the small number of observed bidentate pendant coordination modes , there are many potential combinations of pendant - type coordination modes which are not , or are only rarely , observed as linkers . as the seven common linker modes are crucial in determining the positional and orientational relationships between neighboring rings , they play a crucial structure - directing role in the extension of { p8w48 } into coordinatively linked frameworks , and are responsible for the structural diversity seen for the pomzite family of materials . four main heterotopic linker coordination modes observed in pomzites : a - type linkage , green ; b - type linkage , blue ; c - type linkage , yellow ; d - type linkage , red . the simplest structural type is the chain arrangement , seen for both compounds 1 and 3 , in which { p8w48 } rings are linked edge - to - edge in only one dimension . finally , the cube architecture is a three - dimensional construct of 8-coordinated rings in a stacked - box it seems highly likely that further structure types will be identified in future . one of the major benefits of disentangling the complex connectivity of { p8w48 } rings in pomzite structures by defining a limited number of linker modes , is that it allows simplified representations of the structures to be presented . this gives the reader a much greater insight into the three - dimensional arrangements of { p8w48}-based compounds than with conventional ball - and - stick models , and enables facile comparisons between pomzites to be made . for example , the first multidimensional { p8w48 } compound to be presented was the co - linked structure , k15li5[co10(h2o)34(p8w48o184)]54h2o , labeled compound 1 in a communication published in 2009 which presented two new compounds . compound 1 , presented herein , is a mn - linked analogue of pomzite-1(co ) , appropriately termed isostructural pomzites have the same tm substitution patterns and connectivity , although they may differ in the actual occupancy of each tm coordination site . indeed , we have observed small variations in heterometal content between crystals from different batches of a number of pom framework materials , although the batches are still considered to be of the same compound . for each pomzite the left - hand column details the pendant modes present and the right - hand column the linker modes ( as described in the main text ) . in pomzite-1 , the presence of an external tm coordinated in the b position sets it apart from the two other compounds for two reasons : ( 1 ) pendant functionality is important to the general properties of the framework and can be activated by single crystal to single crystal transformations , and ( 2 ) the steric effect of a tm positioned externally causes spatial displacement of adjacent chains in the solid state . three new compounds based on a minimal building block library of the cyclic polyoxometalate { p8w48 } and transition metal cations have been prepared . as a result , a previously disparate group of compounds has been united and proposed to be named pomzite materials have tremendous potential to bridge the gap in porous framework materials between mof tunability and zeolite functionality . in the future , the use of organic sdas , a well - known strategy in zeolite synthesis , will be employed to access novel pomzite archetypes .

root - end filling materials are in contact with periradicular tissues , so they should have good sealing ability and be biocompatible to promote healing . because many of these materials are not able to ensure a perfect seal , a microscopic space is likely to create at the root - end cavity / filling interface , through which micro organisms and their products can penetrate . thus , besides good sealing ability and biocompatibility , root - end filling materials should ideally have some antibacterial activity . more over , these materials should have low solubility to avoid that components leaching from the endodontic space might exercise undesirable biologic effects on the surrounding tissues . finally , radiopacity is a very important property for all root - end filling materials because the material has to be detected radiographically , and thus distinguished from surrounding anatomic structures . intermediate restorative material ( irm ) has addressed some drawbacks including moisture sensitivity , irritation to vital tissues and difficulty in handling . mta was developed by parirokh and torabinejad to address shortcomings of commonly used root - end filling materials . several studies have recognized mta as a bioactive material , that is , hard tissue conductive , hard tissue inductive , and biocompatible . the sealing ability of mta in root - end fillings was found to be superior to that of amalgam , irm , and super - ethoxy benzoic acid ( eba ) with both dye and bacterial leakage methods . however , its handling characteristics are less than ideal as a result of long setting time and difficulty in maintaining consistency of mixture . several new calcium silicate - based materials have recently been developed ; aiming to improve some mta drawbacks such as its difficult handling property and long setting time . biodentine is amongst these materials and is claimed to be used as a dentine restorative material in addition to endodontic indications similar to those of mta . the purpose of the present study is to evaluate and compare the biological ( cytotoxicity and antibacterial efficacy ) and chemical - physical ( solubility , ph , and radiopacity ) properties of four different root - end filling materials : biodentine ( septodont , saint - maur - des - fosses , france ) , mta - angelus ( angelus , londrina , pr , brazil ) , proroot mta ( dentsply , johnson city , tn , usa ) , and irm ( dentsply , johnson city , tn , usa ) . transwell insert methodology ( sigma - aldrich , st louis , mo , usa ) was performed . cytotoxicity was assessed with mdpc-23 cells grown in the lower chamber of a 24-mm diameter transwell plate with a 0.3 mm pore size polycarbonate membrane ( sigma - aldrich , st . the transwell membrane of the inner chamber , filled with the paper disks , were placed into the lower chamber of the 24-well culture plate containing at the bottom 5 10 cells / well and incubated at 37c in 5% co2 atmosphere for 24 , 48 , and 72 h , respectively . some wells were incubated with only tissue culture media ( negative control ) whereas others with a 10% dilution of 30% h2o2 ( positive control ) . at the end of incubation time the results were presented as percentage of cell viability referred at cells incubated in absence of materials set at 100% . the vitality test to alamar blue reagent acts as an indicator of cell health , determining the reducing power in order to measure quantitatively the proliferative capacity . the reagent was added in a ratio of 1:10 to the cell culture and then the cells were kept in the incubator for 3 - 4 h at 37c . the degree of fluorescence and the relative values of absorbance were then acquired by using a spectrophotometer at a wavelength of 595 nm . for a further control , the percentage of vitality of murine odontoblasts , at 72 h , was also assessed with the 3-(4,5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ( mtt ) assay ( sigma - aldrich , st louis , mo , usa ) . the mtt test is a standard colorimetric assay for measuring the activity of enzymes that reduce the mtt to formazan ( a salt blue ) in the mitochondria , giving the substance a blue / purple color . this reaction was assessed and measured by the spectrophotometric reading of the sample , at a wavelength of 570 nm by a microplate reader ( bio - rad laboratories , hercules , ca , usa ) . the streptococcal strains used in this study were provided from the culture collection of university of goteborg ( ccug ) : streptococcus mutans ( ccug 35176 ) , s. salivarius ( ccug 11878 ) , and s. sanguis ( ccug 17826 ) . the cultures were grown and maintained in a brain heart infusion ( bhi ; difco lab . , detroit , mi , usa ) . s. mutans culture medium was supplemented with 10% ( v / v ) heat - inactivated horse blood serum ( oxoid s.p.a . , the culture of all bacterial strains was statically incubated for 16 h at 37c under aerobic conditions . this overnight culture , used as source for the experiments , was reduced at a final density of 1 10 cells / ml as determined by comparing the optical density at 600 nm ( od600 ) of the sample with a standard curve relating od600 to cell number . sterile paper discs ( diameter : 6 mm , thickness : 1 mm ) ( watman international ltd . maidstone , uk ) were impregnated with 10 l of each root - endfilling material . then , bhi - agar plates were incubated with 1 10 cells / ml of an overnight culture of each streptococcal strain at 37c for 20 min . the excess of bacterial suspension was removed from the plates and incubated with the paper disks impregnated with the root - end filling materials at 37c for 24 h. the diameter of the halo formed around the paper disc ( inhibition zone ) was measured by the same operator in two perpendicular locations with a millimeter ruler with accuracy of 0.5 mm , after 24 and 48 h. the size of the inhibition zone was calculated as follows : size of inhibition zone = ( diameter of halo diameter of specimen ) . all the assays were conducted in triplicate and the results were recorded in terms of the average diameter of inhibition zone . stainless steel ring molds with an internal diameter of 20 0.1 mm and a height of 1.5 0.1 mm were used for sample preparation . all molds were weighed three times prior to use ( accuracy 0.0001 g ) on a mettler ae-163 balance ( mettler - toledo s.p.a . , novate milanese , mi , italy ) , which was used throughout the experiment . after filling the molds , a glass plate covered with a mylar strip was placed on top of the molds , exerting a light pressure in order to remove any excess . all samples were left to set for 24 h on a grating in a cabinet at 37c and 100% relative humidity . the samples in their molds were then exposed to air for 15 min , weighed three times , and the average reading was recorded to three decimal places . the specimens of each material were individually placed in tarred bottles , containing 5 ml of distilled water . the bottles were then transferred to an oven at 37c where they remained for 24 h. they were removed from the oven and rinsed with distilled water , which was then collected in the same bottle . bottles and residues were dried in an oven at 105c , cooled down in desiccators , and weighed . the differences found between this weight and the original bottle weight were divided by the initial dry weight of the specimens and multiplied by 100 . the solubility of the set root - end filling materials should not exceed 3% mass fraction ( iso 6876 clause 4.3.6 ) . the samples were stored at 37c , and ph measurement was performed after incubation of 3 and 24 h. the ph value was measured by a digital hi 2210 ph meter ( hanna instruments us , woonsocket , ri , usa ) , previously calibrated . biodentine , mta - angelus , proroot mta , and irm were mixed and placed into silicon molds measuring 1 mm in thickness and 4 mm in internal diameter . the specimens were covered with glass plates on each side to allow for the removal of excessive material . the specimens were kept in a chamber at 37c and 95% relative humidity for 24 h. ten arrays were set by collecting three samples for each material in order to scan them with prodigy dxa system ( ge healthcare , madison , wi , usa ) . all materials were scanned on a ge healthcare lunar prodigy and idxa in routine clinical manner per manufacturer recommendations . for the prodigy , encore software ( ge healthcare , madison , wi , usa ) version 9.2 was used for acquisition and 11.4 for analysis ; with idxa , encore software version 9.3 was used to acquire scans with version 11.0 used for analyses . the precision assessments were performed in routine clinical manner according to international society for clinical densitometry ( iscd ) recommendations . college station , tx : statacorp lp . ) . to assess the normality of the data obtained from the cytotoxicity assays , the antibacterial tests , the solubility evaluations , and from the radiographic densities ; the shapiro wilk test was applied ( p < 0.05 ) . the number of vital cells was analyzed with tukey 's test . whitney test ; the inhibition zones values after 48 h were used for the statistical analysis . whitney test . to determine whether time influenced the solubility of the pulp capping materials , an analysis of longitudinal data was performed using t - test for paired data ( p < 0.05 ) . t - test for paired data test was applied to determine whether significant differences existed in ph values after 3 and 24 h of incubation ( p < 0.05 ) . tukey 's test was then applied to evaluate the differences in ph values among the materials ( p < 0.05 ) . radiographic densities obtained from dual - energy x - ray absorptiometry ( dxa ) measurements were analyzed using tukey 's test . transwell insert methodology ( sigma - aldrich , st louis , mo , usa ) was performed . cytotoxicity was assessed with mdpc-23 cells grown in the lower chamber of a 24-mm diameter transwell plate with a 0.3 mm pore size polycarbonate membrane ( sigma - aldrich , st . the transwell membrane of the inner chamber , filled with the paper disks , were placed into the lower chamber of the 24-well culture plate containing at the bottom 5 10 cells / well and incubated at 37c in 5% co2 atmosphere for 24 , 48 , and 72 h , respectively . some wells were incubated with only tissue culture media ( negative control ) whereas others with a 10% dilution of 30% h2o2 ( positive control ) . at the end of incubation time the results were presented as percentage of cell viability referred at cells incubated in absence of materials set at 100% . the vitality test to alamar blue reagent acts as an indicator of cell health , determining the reducing power in order to measure quantitatively the proliferative capacity . the reagent was added in a ratio of 1:10 to the cell culture and then the cells were kept in the incubator for 3 - 4 h at 37c . the degree of fluorescence and the relative values of absorbance were then acquired by using a spectrophotometer at a wavelength of 595 nm . for a further control , the percentage of vitality of murine odontoblasts , at 72 h , was also assessed with the 3-(4,5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ( mtt ) assay ( sigma - aldrich , st louis , mo , usa ) . the mtt test is a standard colorimetric assay for measuring the activity of enzymes that reduce the mtt to formazan ( a salt blue ) in the mitochondria , giving the substance a blue / purple color . this reaction was assessed and measured by the spectrophotometric reading of the sample , at a wavelength of 570 nm by a microplate reader ( bio - rad laboratories , hercules , ca , usa ) . the streptococcal strains used in this study were provided from the culture collection of university of goteborg ( ccug ) : streptococcus mutans ( ccug 35176 ) , s. salivarius ( ccug 11878 ) , and s. sanguis ( ccug 17826 ) . the cultures were grown and maintained in a brain heart infusion ( bhi ; difco lab . , detroit , mi , usa ) . s. mutans culture medium was supplemented with 10% ( v / v ) heat - inactivated horse blood serum ( oxoid s.p.a . , rodano , mi , italy ) to improve its growth . the culture of all bacterial strains was statically incubated for 16 h at 37c under aerobic conditions . this overnight culture , used as source for the experiments , was reduced at a final density of 1 10 cells / ml as determined by comparing the optical density at 600 nm ( od600 ) of the sample with a standard curve relating od600 to cell number . sterile paper discs ( diameter : 6 mm , thickness : 1 mm ) ( watman international ltd . maidstone , uk ) were impregnated with 10 l of each root - endfilling material . then , bhi - agar plates were incubated with 1 10 cells / ml of an overnight culture of each streptococcal strain at 37c for 20 min . the excess of bacterial suspension was removed from the plates and incubated with the paper disks impregnated with the root - end filling materials at 37c for 24 h. the diameter of the halo formed around the paper disc ( inhibition zone ) was measured by the same operator in two perpendicular locations with a millimeter ruler with accuracy of 0.5 mm , after 24 and 48 h. the size of the inhibition zone was calculated as follows : size of inhibition zone = ( diameter of halo diameter of specimen ) . all the assays were conducted in triplicate and the results were recorded in terms of the average diameter of inhibition zone . stainless steel ring molds with an internal diameter of 20 0.1 mm and a height of 1.5 0.1 mm were used for sample preparation . all molds were weighed three times prior to use ( accuracy 0.0001 g ) on a mettler ae-163 balance ( mettler - toledo s.p.a . , novate milanese , mi , italy ) , which was used throughout the experiment . after filling the molds , a glass plate covered with a mylar strip was placed on top of the molds , exerting a light pressure in order to remove any excess . all samples were left to set for 24 h on a grating in a cabinet at 37c and 100% relative humidity . the samples in their molds were then exposed to air for 15 min , weighed three times , and the average reading was recorded to three decimal places . the specimens of each material were individually placed in tarred bottles , containing 5 ml of distilled water . the bottles were then transferred to an oven at 37c where they remained for 24 h. they were removed from the oven and rinsed with distilled water , which was then collected in the same bottle . bottles and residues were dried in an oven at 105c , cooled down in desiccators , and weighed . the differences found between this weight and the original bottle weight were divided by the initial dry weight of the specimens and multiplied by 100 . the solubility of the set root - end filling materials should not exceed 3% mass fraction ( iso 6876 clause 4.3.6 ) . the samples were stored at 37c , and ph measurement was performed after incubation of 3 and 24 h. the ph value was measured by a digital hi 2210 ph meter ( hanna instruments us , woonsocket , ri , usa ) , previously calibrated . biodentine , mta - angelus , proroot mta , and irm were mixed and placed into silicon molds measuring 1 mm in thickness and 4 mm in internal diameter . the specimens were covered with glass plates on each side to allow for the removal of excessive material . the specimens were kept in a chamber at 37c and 95% relative humidity for 24 h. ten arrays were set by collecting three samples for each material in order to scan them with prodigy dxa system ( ge healthcare , madison , wi , usa ) . all materials were scanned on a ge healthcare lunar prodigy and idxa in routine clinical manner per manufacturer recommendations . for the prodigy , encore software ( ge healthcare , madison , wi , usa ) version 9.2 was used for acquisition and 11.4 for analysis ; with idxa , encore software version 9.3 was used to acquire scans with version 11.0 used for analyses . the precision assessments were performed in routine clinical manner according to international society for clinical densitometry ( iscd ) recommendations . stata statistical software : release 12 . college station , tx : statacorp lp . ) . to assess the normality of the data obtained from the cytotoxicity assays , the antibacterial tests , the solubility evaluations , and from the radiographic densities ; the shapiro whitney test ; the inhibition zones values after 48 h were used for the statistical analysis . whitney test . to determine whether time influenced the solubility of the pulp capping materials , an analysis of longitudinal data was performed using t - test for paired data ( p < 0.05 ) . t - test for paired data test was applied to determine whether significant differences existed in ph values after 3 and 24 h of incubation ( p < 0.05 ) . tukey 's test was then applied to evaluate the differences in ph values among the materials ( p < 0.05 ) . radiographic densities obtained from dual - energy x - ray absorptiometry ( dxa ) measurements were analyzed using tukey 's test . descriptive statistics analysis for biocompatibility investigation is reported in table 1 . as regards alamar blue test [ table 1 ] , after 24 h there were no differences in the number of vital cells among proroot mta , mta - angelus , biodentine , and the negative control ( p > 0.05 ) . irm showed a significant reduction in the number of vital cells ( p < 0.05 ) . these results changed uniformly after 48 h and maintained no significant differences among the materials , except for irm . after 72 h all three materials varied from the negative control , in particular proroot mta and mta - angelus gained a similar and lower number of vital cells than the control , while biodentine showed no decrease , as confirmed by the analysis for paired data . when the mtt test [ table 1 ] was applied , no significant differences were recorded among proroot mta , mta - angelus , biodentine , and the negative control ( p > 0.05 ) , while irm maintained lower percentages of vitality ( p < 0.05 ) . mean standard deviation of the number of vital cells for each material tested with alamar blue at different times table 2 shows the mean diameter of the inhibition zones after 48 h and the standard deviation . except for biodentine when tested for s. mutans , the root - end filling materials caused zones of inhibition . however ; mta - angelus , proroot mta , and irm showed the highest values for the inhibition zones when tested for s. mutans ( p < 0.05 ) . no significant differences between mta - angelus , proroot mta , and irm ( p > 0.05 ) were found when materials were tested for s. salivarius . biodentine showed the largest inhibition zone when tested for s. sanguis ( p < 0.05 ) , while mta - angelus and proroot mta produced similar smaller inhibition zones ( p > 0.05 ) . mean diameter and standard deviation of inhibition zones ( mm ) formed by the root - endfilling materials after 48 h by the paper disc method the results of solubility test ( after 24 h and 2 months ) are listed in table 3 . all the materials fulfilled the requirements of the international standard 6876 , demonstrating a weight loss of less than 3% . there was no statistical significance in solubility among the materials tested after 24 h ( p < 0.05 ) . similar results were obtained after 2 months , except for irm for which solubility significantly raised ( p < 0.05 ) . for remnant materials the weight loss after 2 months was not significantly different from the weight loss after 24 h : all the materials tested provided a very alkaline ph after 3 h [ table 3 ] ; proroot mta showed the highest value among the materials tested . biodentine and irm provided similar ph values after 3 h ( p > 0.05 ) . for all materials tested , a nonsignificant reduction in ph value was recorded after 24 h ( p > 0.05 ) . for each root - end filling material descriptive statistics for radiographic densities and the results obtained with the analyses of multiple comparisons were calculated and reported in table 4 . proroot mta and mta - angelus showed the highest values of density when compared with the other materials ( p < 0.05 ) . mean percentage values of solubility and standard deviation ( sd ) for each material and for each immersion period radiographic densities : descriptive statistics for each material tested and multiple comparisons calculated with tukey test ( p = 0.05 ) when comparing the cytotoxicity of the root - end materials tested in this study , irm demonstrated the lower rates of vitality and a strong cytotoxic capability . irm has shown the lowest mean number of cells in the colorimetric assay performed with alamar blue , with assessments at 24 , 48 , and 72 h , and in the mtt assay at 72 h. these results are in agreement with previous studies . it has been demonstrated that cell response to irm was characterized by marked rounding of the cells and depletion of cell numbers , indicating that irm was not biocompatible . very different results were obtained by mta - based materials . at 72 h , with alamar blue test and mtt assay , parirokh and torabinejad found that proroot mta was less toxic than amalgam , super eba , and irm . biodentine , in measurements made at 24 , 48 , and 72 h ; reported percentage of vitality above the negative control . biodentine is new bioactive cement based on calcium silicate , whose high biocompatibility has been shown in recent studies . the agar diffusion test has been widely used to evaluate the antibacterial activity of dental materials . in our study , except for biodentine against s. mutans , all the materials demonstrate antibacterial activity , causing zones of inhibition against the three streptococcal strains tested small differences in the antibacterial effect on the different streptococcal strains were recorded . , in which proroot mta and irm proved to be strong inhibitors . while we have little data about the antibacterial capability of biodentine it has been shown that in aerobic conditions , mta could generate reactive oxygen species with antimicrobial activity . parirokh and torabinejad found that mta had no antibacterial effect against any of the strictly anaerobic bacteria . however , as showed by our results , it is possible that mta 's highly alkaline ph affords its antimicrobial activity even when in anaerobic condition . the test was repeated after 2 months , because schafer and zandbiglari stated that the 24-h period of the specification test is not sufficient . in our study all the materials tested showed minimal solubility , fulfilling the requirements of the iso 6876 and demonstrating a weight loss of less than 3% . these conclusions are in accordance with our previous study about solubility of proroot mta , irm , and super - eba . a very alkaline ph was recorded after 3 h ; with the highest value registered for proroot mta . no significant reduction in ph value was recorded after 24 h. proroot mta showed ph values greater than biodentine . this may be probably caused by the higher presence of metallic oxides in mta that could promote ion dissociation . finally , as concerned for radiopacity , in our study mta - based materials demonstrated higher values of density compared to irm and biodentine . radiopacity of biodentine has been recently evaluated . comparing the density of biodentine and irm , grech et al . , found higher values for irm ; while the relatively lower radiopacity of biodentine , compared to mta , has been recently shown in a study by tanalp et al . within the limitation of this in vitro study , the differences shown by the materials do not cover completely the ideal clinical requests for root - end filling . irm could not be considered as the ideal material due to the high cytotoxicity ; while mta - based cement and biodentine proved to be biocompatible . however , biodentine , comparing to mta , did not cause zone of inhibition for the entire microorganism tested and it showed lower values of radiopacity . therefore , mta - angelus and proroot mta can be considered as the best material for root - end filling among those tested due to the fact that they presented at once biocompatibility , antibacterial properties , radiopacity , and low solubility .

aim : the purpose of the study is to evaluate and compare the biological and chemical - physical properties of four different root - end filling materials.materials and methods : cytotoxicity towards murine odontoblasts cells ( mdpc-23 ) was evaluated using the transwell insert methodology by alamar blue test . streptococcus salivarius , s. sanguis , and s. mutans strains were selected to evaluate the antimicrobial activity by agar disc diffusion test . solubility was determined after 24 h and 2 months . ph values were measured after 3 and 24 h. to evaluate radiopacity , all materials were scanned on a ge healthcare lunar prodigy.results:excellent percentage of vitality were obtained by mineral trioxide aggregate ( mta)-based materials and biodentine . mta - angelus , proroot mta , and intermediate restorative material ( irm ) showed the highest values for the inhibition zones when tested for s. mutans , while biodentine showed the largest inhibition zone when tested for s. sanguis . all the materials fulfilled the requirements of the international standard 6876 , demonstrating low solubility with a weight loss of less than 3% . no significant reduction in ph value was demonstrated after 24 h. proroot mta and mta - angelus showed the highest values of radiographic density.conclusions:the differences showed by the root - end filling materials tested do not cover completely the ideal clinical requests .

INTRODUCTION MATERIALS AND METHODS Cytotoxicity assay Antibacterial test Solubility evaluations pH measurements Radiopacity DXA Measurements Statistical analysis RESULTS DISCUSSION CONCLUSIONS

root - end filling materials are in contact with periradicular tissues , so they should have good sealing ability and be biocompatible to promote healing . because many of these materials are not able to ensure a perfect seal , a microscopic space is likely to create at the root - end cavity / filling interface , through which micro organisms and their products can penetrate . thus , besides good sealing ability and biocompatibility , root - end filling materials should ideally have some antibacterial activity . finally , radiopacity is a very important property for all root - end filling materials because the material has to be detected radiographically , and thus distinguished from surrounding anatomic structures . intermediate restorative material ( irm ) has addressed some drawbacks including moisture sensitivity , irritation to vital tissues and difficulty in handling . mta was developed by parirokh and torabinejad to address shortcomings of commonly used root - end filling materials . the sealing ability of mta in root - end fillings was found to be superior to that of amalgam , irm , and super - ethoxy benzoic acid ( eba ) with both dye and bacterial leakage methods . however , its handling characteristics are less than ideal as a result of long setting time and difficulty in maintaining consistency of mixture . biodentine is amongst these materials and is claimed to be used as a dentine restorative material in addition to endodontic indications similar to those of mta . the purpose of the present study is to evaluate and compare the biological ( cytotoxicity and antibacterial efficacy ) and chemical - physical ( solubility , ph , and radiopacity ) properties of four different root - end filling materials : biodentine ( septodont , saint - maur - des - fosses , france ) , mta - angelus ( angelus , londrina , pr , brazil ) , proroot mta ( dentsply , johnson city , tn , usa ) , and irm ( dentsply , johnson city , tn , usa ) . transwell insert methodology ( sigma - aldrich , st louis , mo , usa ) was performed . the transwell membrane of the inner chamber , filled with the paper disks , were placed into the lower chamber of the 24-well culture plate containing at the bottom 5 10 cells / well and incubated at 37c in 5% co2 atmosphere for 24 , 48 , and 72 h , respectively . some wells were incubated with only tissue culture media ( negative control ) whereas others with a 10% dilution of 30% h2o2 ( positive control ) . at the end of incubation time the results were presented as percentage of cell viability referred at cells incubated in absence of materials set at 100% . the degree of fluorescence and the relative values of absorbance were then acquired by using a spectrophotometer at a wavelength of 595 nm . for a further control , the percentage of vitality of murine odontoblasts , at 72 h , was also assessed with the 3-(4,5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ( mtt ) assay ( sigma - aldrich , st louis , mo , usa ) . this reaction was assessed and measured by the spectrophotometric reading of the sample , at a wavelength of 570 nm by a microplate reader ( bio - rad laboratories , hercules , ca , usa ) . the streptococcal strains used in this study were provided from the culture collection of university of goteborg ( ccug ) : streptococcus mutans ( ccug 35176 ) , s. salivarius ( ccug 11878 ) , and s. sanguis ( ccug 17826 ) . this overnight culture , used as source for the experiments , was reduced at a final density of 1 10 cells / ml as determined by comparing the optical density at 600 nm ( od600 ) of the sample with a standard curve relating od600 to cell number . maidstone , uk ) were impregnated with 10 l of each root - endfilling material . the excess of bacterial suspension was removed from the plates and incubated with the paper disks impregnated with the root - end filling materials at 37c for 24 h. the diameter of the halo formed around the paper disc ( inhibition zone ) was measured by the same operator in two perpendicular locations with a millimeter ruler with accuracy of 0.5 mm , after 24 and 48 h. the size of the inhibition zone was calculated as follows : size of inhibition zone = ( diameter of halo diameter of specimen ) . all the assays were conducted in triplicate and the results were recorded in terms of the average diameter of inhibition zone . after filling the molds , a glass plate covered with a mylar strip was placed on top of the molds , exerting a light pressure in order to remove any excess . all samples were left to set for 24 h on a grating in a cabinet at 37c and 100% relative humidity . the samples in their molds were then exposed to air for 15 min , weighed three times , and the average reading was recorded to three decimal places . the bottles were then transferred to an oven at 37c where they remained for 24 h. they were removed from the oven and rinsed with distilled water , which was then collected in the same bottle . the differences found between this weight and the original bottle weight were divided by the initial dry weight of the specimens and multiplied by 100 . the solubility of the set root - end filling materials should not exceed 3% mass fraction ( iso 6876 clause 4.3.6 ) . the samples were stored at 37c , and ph measurement was performed after incubation of 3 and 24 h. the ph value was measured by a digital hi 2210 ph meter ( hanna instruments us , woonsocket , ri , usa ) , previously calibrated . biodentine , mta - angelus , proroot mta , and irm were mixed and placed into silicon molds measuring 1 mm in thickness and 4 mm in internal diameter . the specimens were kept in a chamber at 37c and 95% relative humidity for 24 h. ten arrays were set by collecting three samples for each material in order to scan them with prodigy dxa system ( ge healthcare , madison , wi , usa ) . all materials were scanned on a ge healthcare lunar prodigy and idxa in routine clinical manner per manufacturer recommendations . for the prodigy , encore software ( ge healthcare , madison , wi , usa ) version 9.2 was used for acquisition and 11.4 for analysis ; with idxa , encore software version 9.3 was used to acquire scans with version 11.0 used for analyses . to assess the normality of the data obtained from the cytotoxicity assays , the antibacterial tests , the solubility evaluations , and from the radiographic densities ; the shapiro wilk test was applied ( p < 0.05 ) . whitney test ; the inhibition zones values after 48 h were used for the statistical analysis . to determine whether time influenced the solubility of the pulp capping materials , an analysis of longitudinal data was performed using t - test for paired data ( p < 0.05 ) . t - test for paired data test was applied to determine whether significant differences existed in ph values after 3 and 24 h of incubation ( p < 0.05 ) . tukey 's test was then applied to evaluate the differences in ph values among the materials ( p < 0.05 ) . transwell insert methodology ( sigma - aldrich , st louis , mo , usa ) was performed . cytotoxicity was assessed with mdpc-23 cells grown in the lower chamber of a 24-mm diameter transwell plate with a 0.3 mm pore size polycarbonate membrane ( sigma - aldrich , st . the transwell membrane of the inner chamber , filled with the paper disks , were placed into the lower chamber of the 24-well culture plate containing at the bottom 5 10 cells / well and incubated at 37c in 5% co2 atmosphere for 24 , 48 , and 72 h , respectively . some wells were incubated with only tissue culture media ( negative control ) whereas others with a 10% dilution of 30% h2o2 ( positive control ) . the degree of fluorescence and the relative values of absorbance were then acquired by using a spectrophotometer at a wavelength of 595 nm . for a further control , the percentage of vitality of murine odontoblasts , at 72 h , was also assessed with the 3-(4,5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ( mtt ) assay ( sigma - aldrich , st louis , mo , usa ) . this reaction was assessed and measured by the spectrophotometric reading of the sample , at a wavelength of 570 nm by a microplate reader ( bio - rad laboratories , hercules , ca , usa ) . the streptococcal strains used in this study were provided from the culture collection of university of goteborg ( ccug ) : streptococcus mutans ( ccug 35176 ) , s. salivarius ( ccug 11878 ) , and s. sanguis ( ccug 17826 ) . this overnight culture , used as source for the experiments , was reduced at a final density of 1 10 cells / ml as determined by comparing the optical density at 600 nm ( od600 ) of the sample with a standard curve relating od600 to cell number . the excess of bacterial suspension was removed from the plates and incubated with the paper disks impregnated with the root - end filling materials at 37c for 24 h. the diameter of the halo formed around the paper disc ( inhibition zone ) was measured by the same operator in two perpendicular locations with a millimeter ruler with accuracy of 0.5 mm , after 24 and 48 h. the size of the inhibition zone was calculated as follows : size of inhibition zone = ( diameter of halo diameter of specimen ) . all the assays were conducted in triplicate and the results were recorded in terms of the average diameter of inhibition zone . after filling the molds , a glass plate covered with a mylar strip was placed on top of the molds , exerting a light pressure in order to remove any excess . all samples were left to set for 24 h on a grating in a cabinet at 37c and 100% relative humidity . the differences found between this weight and the original bottle weight were divided by the initial dry weight of the specimens and multiplied by 100 . the solubility of the set root - end filling materials should not exceed 3% mass fraction ( iso 6876 clause 4.3.6 ) . the samples were stored at 37c , and ph measurement was performed after incubation of 3 and 24 h. the ph value was measured by a digital hi 2210 ph meter ( hanna instruments us , woonsocket , ri , usa ) , previously calibrated . biodentine , mta - angelus , proroot mta , and irm were mixed and placed into silicon molds measuring 1 mm in thickness and 4 mm in internal diameter . the specimens were kept in a chamber at 37c and 95% relative humidity for 24 h. ten arrays were set by collecting three samples for each material in order to scan them with prodigy dxa system ( ge healthcare , madison , wi , usa ) . all materials were scanned on a ge healthcare lunar prodigy and idxa in routine clinical manner per manufacturer recommendations . for the prodigy , encore software ( ge healthcare , madison , wi , usa ) version 9.2 was used for acquisition and 11.4 for analysis ; with idxa , encore software version 9.3 was used to acquire scans with version 11.0 used for analyses . to assess the normality of the data obtained from the cytotoxicity assays , the antibacterial tests , the solubility evaluations , and from the radiographic densities ; the shapiro whitney test ; the inhibition zones values after 48 h were used for the statistical analysis . to determine whether time influenced the solubility of the pulp capping materials , an analysis of longitudinal data was performed using t - test for paired data ( p < 0.05 ) . t - test for paired data test was applied to determine whether significant differences existed in ph values after 3 and 24 h of incubation ( p < 0.05 ) . tukey 's test was then applied to evaluate the differences in ph values among the materials ( p < 0.05 ) . as regards alamar blue test [ table 1 ] , after 24 h there were no differences in the number of vital cells among proroot mta , mta - angelus , biodentine , and the negative control ( p > 0.05 ) . irm showed a significant reduction in the number of vital cells ( p < 0.05 ) . these results changed uniformly after 48 h and maintained no significant differences among the materials , except for irm . after 72 h all three materials varied from the negative control , in particular proroot mta and mta - angelus gained a similar and lower number of vital cells than the control , while biodentine showed no decrease , as confirmed by the analysis for paired data . when the mtt test [ table 1 ] was applied , no significant differences were recorded among proroot mta , mta - angelus , biodentine , and the negative control ( p > 0.05 ) , while irm maintained lower percentages of vitality ( p < 0.05 ) . mean standard deviation of the number of vital cells for each material tested with alamar blue at different times table 2 shows the mean diameter of the inhibition zones after 48 h and the standard deviation . except for biodentine when tested for s. mutans , the root - end filling materials caused zones of inhibition . however ; mta - angelus , proroot mta , and irm showed the highest values for the inhibition zones when tested for s. mutans ( p < 0.05 ) . no significant differences between mta - angelus , proroot mta , and irm ( p > 0.05 ) were found when materials were tested for s. salivarius . biodentine showed the largest inhibition zone when tested for s. sanguis ( p < 0.05 ) , while mta - angelus and proroot mta produced similar smaller inhibition zones ( p > 0.05 ) . mean diameter and standard deviation of inhibition zones ( mm ) formed by the root - endfilling materials after 48 h by the paper disc method the results of solubility test ( after 24 h and 2 months ) are listed in table 3 . all the materials fulfilled the requirements of the international standard 6876 , demonstrating a weight loss of less than 3% . there was no statistical significance in solubility among the materials tested after 24 h ( p < 0.05 ) . similar results were obtained after 2 months , except for irm for which solubility significantly raised ( p < 0.05 ) . for remnant materials the weight loss after 2 months was not significantly different from the weight loss after 24 h : all the materials tested provided a very alkaline ph after 3 h [ table 3 ] ; proroot mta showed the highest value among the materials tested . biodentine and irm provided similar ph values after 3 h ( p > 0.05 ) . for all materials tested , a nonsignificant reduction in ph value was recorded after 24 h ( p > 0.05 ) . for each root - end filling material descriptive statistics for radiographic densities and the results obtained with the analyses of multiple comparisons were calculated and reported in table 4 . proroot mta and mta - angelus showed the highest values of density when compared with the other materials ( p < 0.05 ) . mean percentage values of solubility and standard deviation ( sd ) for each material and for each immersion period radiographic densities : descriptive statistics for each material tested and multiple comparisons calculated with tukey test ( p = 0.05 ) when comparing the cytotoxicity of the root - end materials tested in this study , irm demonstrated the lower rates of vitality and a strong cytotoxic capability . irm has shown the lowest mean number of cells in the colorimetric assay performed with alamar blue , with assessments at 24 , 48 , and 72 h , and in the mtt assay at 72 h. these results are in agreement with previous studies . very different results were obtained by mta - based materials . at 72 h , with alamar blue test and mtt assay , parirokh and torabinejad found that proroot mta was less toxic than amalgam , super eba , and irm . biodentine , in measurements made at 24 , 48 , and 72 h ; reported percentage of vitality above the negative control . the agar diffusion test has been widely used to evaluate the antibacterial activity of dental materials . in our study , except for biodentine against s. mutans , all the materials demonstrate antibacterial activity , causing zones of inhibition against the three streptococcal strains tested small differences in the antibacterial effect on the different streptococcal strains were recorded . , in which proroot mta and irm proved to be strong inhibitors . however , as showed by our results , it is possible that mta 's highly alkaline ph affords its antimicrobial activity even when in anaerobic condition . the test was repeated after 2 months , because schafer and zandbiglari stated that the 24-h period of the specification test is not sufficient . in our study all the materials tested showed minimal solubility , fulfilling the requirements of the iso 6876 and demonstrating a weight loss of less than 3% . these conclusions are in accordance with our previous study about solubility of proroot mta , irm , and super - eba . a very alkaline ph was recorded after 3 h ; with the highest value registered for proroot mta . no significant reduction in ph value was recorded after 24 h. proroot mta showed ph values greater than biodentine . this may be probably caused by the higher presence of metallic oxides in mta that could promote ion dissociation . finally , as concerned for radiopacity , in our study mta - based materials demonstrated higher values of density compared to irm and biodentine . , found higher values for irm ; while the relatively lower radiopacity of biodentine , compared to mta , has been recently shown in a study by tanalp et al . within the limitation of this in vitro study , the differences shown by the materials do not cover completely the ideal clinical requests for root - end filling . irm could not be considered as the ideal material due to the high cytotoxicity ; while mta - based cement and biodentine proved to be biocompatible . however , biodentine , comparing to mta , did not cause zone of inhibition for the entire microorganism tested and it showed lower values of radiopacity . therefore , mta - angelus and proroot mta can be considered as the best material for root - end filling among those tested due to the fact that they presented at once biocompatibility , antibacterial properties , radiopacity , and low solubility .

because many of these materials are not able to ensure a perfect seal , a microscopic space is likely to create at the root - end cavity / filling interface , through which micro organisms and their products can penetrate . more over , these materials should have low solubility to avoid that components leaching from the endodontic space might exercise undesirable biologic effects on the surrounding tissues . finally , radiopacity is a very important property for all root - end filling materials because the material has to be detected radiographically , and thus distinguished from surrounding anatomic structures . intermediate restorative material ( irm ) has addressed some drawbacks including moisture sensitivity , irritation to vital tissues and difficulty in handling . mta was developed by parirokh and torabinejad to address shortcomings of commonly used root - end filling materials . several studies have recognized mta as a bioactive material , that is , hard tissue conductive , hard tissue inductive , and biocompatible . the sealing ability of mta in root - end fillings was found to be superior to that of amalgam , irm , and super - ethoxy benzoic acid ( eba ) with both dye and bacterial leakage methods . however , its handling characteristics are less than ideal as a result of long setting time and difficulty in maintaining consistency of mixture . several new calcium silicate - based materials have recently been developed ; aiming to improve some mta drawbacks such as its difficult handling property and long setting time . biodentine is amongst these materials and is claimed to be used as a dentine restorative material in addition to endodontic indications similar to those of mta . the purpose of the present study is to evaluate and compare the biological ( cytotoxicity and antibacterial efficacy ) and chemical - physical ( solubility , ph , and radiopacity ) properties of four different root - end filling materials : biodentine ( septodont , saint - maur - des - fosses , france ) , mta - angelus ( angelus , londrina , pr , brazil ) , proroot mta ( dentsply , johnson city , tn , usa ) , and irm ( dentsply , johnson city , tn , usa ) . cytotoxicity was assessed with mdpc-23 cells grown in the lower chamber of a 24-mm diameter transwell plate with a 0.3 mm pore size polycarbonate membrane ( sigma - aldrich , st . the transwell membrane of the inner chamber , filled with the paper disks , were placed into the lower chamber of the 24-well culture plate containing at the bottom 5 10 cells / well and incubated at 37c in 5% co2 atmosphere for 24 , 48 , and 72 h , respectively . at the end of incubation time the results were presented as percentage of cell viability referred at cells incubated in absence of materials set at 100% . the vitality test to alamar blue reagent acts as an indicator of cell health , determining the reducing power in order to measure quantitatively the proliferative capacity . the reagent was added in a ratio of 1:10 to the cell culture and then the cells were kept in the incubator for 3 - 4 h at 37c . for a further control , the percentage of vitality of murine odontoblasts , at 72 h , was also assessed with the 3-(4,5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ( mtt ) assay ( sigma - aldrich , st louis , mo , usa ) . the mtt test is a standard colorimetric assay for measuring the activity of enzymes that reduce the mtt to formazan ( a salt blue ) in the mitochondria , giving the substance a blue / purple color . this reaction was assessed and measured by the spectrophotometric reading of the sample , at a wavelength of 570 nm by a microplate reader ( bio - rad laboratories , hercules , ca , usa ) . the streptococcal strains used in this study were provided from the culture collection of university of goteborg ( ccug ) : streptococcus mutans ( ccug 35176 ) , s. salivarius ( ccug 11878 ) , and s. sanguis ( ccug 17826 ) . s. mutans culture medium was supplemented with 10% ( v / v ) heat - inactivated horse blood serum ( oxoid s.p.a . , the culture of all bacterial strains was statically incubated for 16 h at 37c under aerobic conditions . this overnight culture , used as source for the experiments , was reduced at a final density of 1 10 cells / ml as determined by comparing the optical density at 600 nm ( od600 ) of the sample with a standard curve relating od600 to cell number . the excess of bacterial suspension was removed from the plates and incubated with the paper disks impregnated with the root - end filling materials at 37c for 24 h. the diameter of the halo formed around the paper disc ( inhibition zone ) was measured by the same operator in two perpendicular locations with a millimeter ruler with accuracy of 0.5 mm , after 24 and 48 h. the size of the inhibition zone was calculated as follows : size of inhibition zone = ( diameter of halo diameter of specimen ) . all the assays were conducted in triplicate and the results were recorded in terms of the average diameter of inhibition zone . all molds were weighed three times prior to use ( accuracy 0.0001 g ) on a mettler ae-163 balance ( mettler - toledo s.p.a . , novate milanese , mi , italy ) , which was used throughout the experiment . after filling the molds , a glass plate covered with a mylar strip was placed on top of the molds , exerting a light pressure in order to remove any excess . the samples in their molds were then exposed to air for 15 min , weighed three times , and the average reading was recorded to three decimal places . the bottles were then transferred to an oven at 37c where they remained for 24 h. they were removed from the oven and rinsed with distilled water , which was then collected in the same bottle . the differences found between this weight and the original bottle weight were divided by the initial dry weight of the specimens and multiplied by 100 . the solubility of the set root - end filling materials should not exceed 3% mass fraction ( iso 6876 clause 4.3.6 ) . the samples were stored at 37c , and ph measurement was performed after incubation of 3 and 24 h. the ph value was measured by a digital hi 2210 ph meter ( hanna instruments us , woonsocket , ri , usa ) , previously calibrated . biodentine , mta - angelus , proroot mta , and irm were mixed and placed into silicon molds measuring 1 mm in thickness and 4 mm in internal diameter . the specimens were kept in a chamber at 37c and 95% relative humidity for 24 h. ten arrays were set by collecting three samples for each material in order to scan them with prodigy dxa system ( ge healthcare , madison , wi , usa ) . for the prodigy , encore software ( ge healthcare , madison , wi , usa ) version 9.2 was used for acquisition and 11.4 for analysis ; with idxa , encore software version 9.3 was used to acquire scans with version 11.0 used for analyses . to assess the normality of the data obtained from the cytotoxicity assays , the antibacterial tests , the solubility evaluations , and from the radiographic densities ; the shapiro wilk test was applied ( p < 0.05 ) . to determine whether time influenced the solubility of the pulp capping materials , an analysis of longitudinal data was performed using t - test for paired data ( p < 0.05 ) . t - test for paired data test was applied to determine whether significant differences existed in ph values after 3 and 24 h of incubation ( p < 0.05 ) . tukey 's test was then applied to evaluate the differences in ph values among the materials ( p < 0.05 ) . radiographic densities obtained from dual - energy x - ray absorptiometry ( dxa ) measurements were analyzed using tukey 's test . cytotoxicity was assessed with mdpc-23 cells grown in the lower chamber of a 24-mm diameter transwell plate with a 0.3 mm pore size polycarbonate membrane ( sigma - aldrich , st . the transwell membrane of the inner chamber , filled with the paper disks , were placed into the lower chamber of the 24-well culture plate containing at the bottom 5 10 cells / well and incubated at 37c in 5% co2 atmosphere for 24 , 48 , and 72 h , respectively . at the end of incubation time the results were presented as percentage of cell viability referred at cells incubated in absence of materials set at 100% . the vitality test to alamar blue reagent acts as an indicator of cell health , determining the reducing power in order to measure quantitatively the proliferative capacity . the reagent was added in a ratio of 1:10 to the cell culture and then the cells were kept in the incubator for 3 - 4 h at 37c . for a further control , the percentage of vitality of murine odontoblasts , at 72 h , was also assessed with the 3-(4,5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ( mtt ) assay ( sigma - aldrich , st louis , mo , usa ) . the mtt test is a standard colorimetric assay for measuring the activity of enzymes that reduce the mtt to formazan ( a salt blue ) in the mitochondria , giving the substance a blue / purple color . this reaction was assessed and measured by the spectrophotometric reading of the sample , at a wavelength of 570 nm by a microplate reader ( bio - rad laboratories , hercules , ca , usa ) . the streptococcal strains used in this study were provided from the culture collection of university of goteborg ( ccug ) : streptococcus mutans ( ccug 35176 ) , s. salivarius ( ccug 11878 ) , and s. sanguis ( ccug 17826 ) . s. mutans culture medium was supplemented with 10% ( v / v ) heat - inactivated horse blood serum ( oxoid s.p.a . this overnight culture , used as source for the experiments , was reduced at a final density of 1 10 cells / ml as determined by comparing the optical density at 600 nm ( od600 ) of the sample with a standard curve relating od600 to cell number . the excess of bacterial suspension was removed from the plates and incubated with the paper disks impregnated with the root - end filling materials at 37c for 24 h. the diameter of the halo formed around the paper disc ( inhibition zone ) was measured by the same operator in two perpendicular locations with a millimeter ruler with accuracy of 0.5 mm , after 24 and 48 h. the size of the inhibition zone was calculated as follows : size of inhibition zone = ( diameter of halo diameter of specimen ) . all the assays were conducted in triplicate and the results were recorded in terms of the average diameter of inhibition zone . all molds were weighed three times prior to use ( accuracy 0.0001 g ) on a mettler ae-163 balance ( mettler - toledo s.p.a . after filling the molds , a glass plate covered with a mylar strip was placed on top of the molds , exerting a light pressure in order to remove any excess . the samples in their molds were then exposed to air for 15 min , weighed three times , and the average reading was recorded to three decimal places . the differences found between this weight and the original bottle weight were divided by the initial dry weight of the specimens and multiplied by 100 . the solubility of the set root - end filling materials should not exceed 3% mass fraction ( iso 6876 clause 4.3.6 ) . the samples were stored at 37c , and ph measurement was performed after incubation of 3 and 24 h. the ph value was measured by a digital hi 2210 ph meter ( hanna instruments us , woonsocket , ri , usa ) , previously calibrated . biodentine , mta - angelus , proroot mta , and irm were mixed and placed into silicon molds measuring 1 mm in thickness and 4 mm in internal diameter . the specimens were kept in a chamber at 37c and 95% relative humidity for 24 h. ten arrays were set by collecting three samples for each material in order to scan them with prodigy dxa system ( ge healthcare , madison , wi , usa ) . for the prodigy , encore software ( ge healthcare , madison , wi , usa ) version 9.2 was used for acquisition and 11.4 for analysis ; with idxa , encore software version 9.3 was used to acquire scans with version 11.0 used for analyses . to assess the normality of the data obtained from the cytotoxicity assays , the antibacterial tests , the solubility evaluations , and from the radiographic densities ; the shapiro whitney test ; the inhibition zones values after 48 h were used for the statistical analysis . to determine whether time influenced the solubility of the pulp capping materials , an analysis of longitudinal data was performed using t - test for paired data ( p < 0.05 ) . t - test for paired data test was applied to determine whether significant differences existed in ph values after 3 and 24 h of incubation ( p < 0.05 ) . tukey 's test was then applied to evaluate the differences in ph values among the materials ( p < 0.05 ) . radiographic densities obtained from dual - energy x - ray absorptiometry ( dxa ) measurements were analyzed using tukey 's test . as regards alamar blue test [ table 1 ] , after 24 h there were no differences in the number of vital cells among proroot mta , mta - angelus , biodentine , and the negative control ( p > 0.05 ) . irm showed a significant reduction in the number of vital cells ( p < 0.05 ) . after 72 h all three materials varied from the negative control , in particular proroot mta and mta - angelus gained a similar and lower number of vital cells than the control , while biodentine showed no decrease , as confirmed by the analysis for paired data . when the mtt test [ table 1 ] was applied , no significant differences were recorded among proroot mta , mta - angelus , biodentine , and the negative control ( p > 0.05 ) , while irm maintained lower percentages of vitality ( p < 0.05 ) . mean standard deviation of the number of vital cells for each material tested with alamar blue at different times table 2 shows the mean diameter of the inhibition zones after 48 h and the standard deviation . except for biodentine when tested for s. mutans , the root - end filling materials caused zones of inhibition . however ; mta - angelus , proroot mta , and irm showed the highest values for the inhibition zones when tested for s. mutans ( p < 0.05 ) . no significant differences between mta - angelus , proroot mta , and irm ( p > 0.05 ) were found when materials were tested for s. salivarius . biodentine showed the largest inhibition zone when tested for s. sanguis ( p < 0.05 ) , while mta - angelus and proroot mta produced similar smaller inhibition zones ( p > 0.05 ) . mean diameter and standard deviation of inhibition zones ( mm ) formed by the root - endfilling materials after 48 h by the paper disc method the results of solubility test ( after 24 h and 2 months ) are listed in table 3 . all the materials fulfilled the requirements of the international standard 6876 , demonstrating a weight loss of less than 3% . there was no statistical significance in solubility among the materials tested after 24 h ( p < 0.05 ) . similar results were obtained after 2 months , except for irm for which solubility significantly raised ( p < 0.05 ) . for remnant materials the weight loss after 2 months was not significantly different from the weight loss after 24 h : all the materials tested provided a very alkaline ph after 3 h [ table 3 ] ; proroot mta showed the highest value among the materials tested . for each root - end filling material descriptive statistics for radiographic densities and the results obtained with the analyses of multiple comparisons were calculated and reported in table 4 . proroot mta and mta - angelus showed the highest values of density when compared with the other materials ( p < 0.05 ) . mean percentage values of solubility and standard deviation ( sd ) for each material and for each immersion period radiographic densities : descriptive statistics for each material tested and multiple comparisons calculated with tukey test ( p = 0.05 ) when comparing the cytotoxicity of the root - end materials tested in this study , irm demonstrated the lower rates of vitality and a strong cytotoxic capability . irm has shown the lowest mean number of cells in the colorimetric assay performed with alamar blue , with assessments at 24 , 48 , and 72 h , and in the mtt assay at 72 h. these results are in agreement with previous studies . it has been demonstrated that cell response to irm was characterized by marked rounding of the cells and depletion of cell numbers , indicating that irm was not biocompatible . in our study , except for biodentine against s. mutans , all the materials demonstrate antibacterial activity , causing zones of inhibition against the three streptococcal strains tested small differences in the antibacterial effect on the different streptococcal strains were recorded . while we have little data about the antibacterial capability of biodentine it has been shown that in aerobic conditions , mta could generate reactive oxygen species with antimicrobial activity . in our study all the materials tested showed minimal solubility , fulfilling the requirements of the iso 6876 and demonstrating a weight loss of less than 3% . finally , as concerned for radiopacity , in our study mta - based materials demonstrated higher values of density compared to irm and biodentine . , found higher values for irm ; while the relatively lower radiopacity of biodentine , compared to mta , has been recently shown in a study by tanalp et al . within the limitation of this in vitro study , the differences shown by the materials do not cover completely the ideal clinical requests for root - end filling . however , biodentine , comparing to mta , did not cause zone of inhibition for the entire microorganism tested and it showed lower values of radiopacity . therefore , mta - angelus and proroot mta can be considered as the best material for root - end filling among those tested due to the fact that they presented at once biocompatibility , antibacterial properties , radiopacity , and low solubility .

poxviruses are large , double - stranded dna viruses that infect a wide variety of hosts , ranging fromcaterpillars to humans . they are the largest animal virus , with a brick - shape , ranging in size from 200 to 400 nm long and 170 to 200 nm wide . due to their large size , poxviruses can be visualized with light microscopy , although the fine details of the virus remain obscure . poxvirus genomes encode the genes necessary for replication and immune modulation , with replication occurring in the host cell 's cytoplasm . the genome is large compared to most viruses , containing 130300 kb , depending on the virus type . poxviruses are classified into two categories : entomopox ( insect infecting ) and chordopox ( vertebrate infecting ) . the chordopox viruses are further divided into eight categories , including orthopox , which includes variola major and vaccinia virus , and avipox , which includes fowlpox virus . variola major , the virus that causes the contagious disease smallpox , has been considered one of the most destructive viruses in history , affecting 1/10th of all humankind and killing 300 million people in the 20th century alone . the disease was considered fully eradicated in 1980 . however , although variola major no longer exists in any known animal reservoirs and nature , stocks are still kept in the united states and russia . smallpox has a 2030% lethality rate in unvaccinated individuals , although analysis of historical data suggests that 77.6% of cases are still protected against severe disease 70 years after vaccination . vaccina virus , long considered the prototype of variola major , has been used as both a simulant and vaccine for variola major . they show remarkable sequence similarity ; however , vaccinia virus does not cause disease in healthy individuals , possibly due to the fact that variola major virus contains complement inhibitors 100 times more potent than those of vaccinia virus . however , vaccinia can still cause serious infection in humans and is therefore not an ideal simulant . the purpose of this paper is to investigate whether fowlpox , also a member of the chordopox family , can be used as a simulant for variola major . fowlpox virus naturally infects chicken and turkey ; however , it is not infectious to humans . fowlpox virus has a genome consisting of 260 genes . of the 260 genes , 65 are conserved homologues of chordopox genes . when compared by hexamer and glimer computer programs , these genes encode for transcription factors , rna - polymerase subunits , dna repair , protein modification , structural proteins , and immune evasion proteins . several genes encoding cellular function show homology with proteins of unknown function from yeast , bacteria , roundworm , plant , and human . another gene shows similar homology to a protein found in yeast , human , tomato and fruit fly . secondly , the fowlpox genome contains host range genes , limiting infection to members of the avian family . thirdly , the virus is so large due to the presence of novel cellular homologues . vaccinia strain wr ( vr-119 ) and fowlpox strain fpv - mdvgbh ( merck 's disease vaccine strain ) ( vr-2330 ) were obtained from the american type culture collection ( atcc , manassas , va ) . bhk-21 ( ccl-10 ) , bsc-40 cells ( crl-2761 ) and umnsah / df-1 cells ( chicken embryo fibroblast cells ) ( crl-12203 ) were also obtained from atcc . vaccinia virus was propagated in bhk-21 monolayer cultures in dulbecco 's modified eagle 's medium ( dmem ) containing 10% heat - inactivated fetal bovine serum ( fbs ) at 37c . infected cells were harvested and centrifuged at 3,210 g at 4c for 10 minutes . pellets were resuspended in dmem containing 10% fbs , freeze - thawed for three cycles , sonicated for four minutes on ice and then centrifuged at 3,210 g at 4c for 10 minutes . the supernatant was collected and titered in bsc-40 cells and served as the source of virus for all experiments described in this paper . bsc-40 cells were plated at a density of 5.0 10 cells per well in six well plates for vaccinia plaque assays . cells were allowed to reach confluence overnight at 37c . in dmem containing 10% fbs . following absorption , medium was removed and replaced with minimal essential medium ( mem ) containing 5% fbs and 1% seaplaque agarose . forty - eight hours after infection , plaques were visualized by staining with 0.01% crystal violet . the plaques were counted , and the results were reported in plaque forming units per milliliter ( pfu / ml ) . fowlpox virus was propagated in umnsah / df-1 monolayer cultures in dmem containing 10% fbs at 39c . infected cells were harvested and centrifuged at 3,210 g at 4c for 10 minutes . pellets were resuspended in dmem containing 10% fbs , freeze - thawed for three cycles , sonicated for four minutes on ice , and centrifuged at 3,210 g at 4c for 10 minutes . the supernatant was collected and titered in umnsah / df-1 cells and served as the source of virus for all experiments described in this paper . umnsah / df-1 cells were plated at a density of 1.0 10 cells per well in six well plates for the fowlpox plaque assay . cells were allowed to reach confluence overnight at 39c in dmem containing 10% fbs . following absorption , media was removed and replaced with 50% mem , 50% dmem , 2.5% fbs , and 1% seaplaque agarose . the following disinfectants were tested for their efficacy against vaccinia and fowlpox viruses : ethanol ( 70% ) , isopropyl alcohol ( 50% ) , sodium hypochlorite ( 0.5% ) , formaldehyde ( 30% ) , benzalkonium chloride ( 10% ) , a mixture of cetyltrimethylammonium chloride ( 6.67% ) and benzalkonium chloride ( 3.33% ) , and a mixture of iodine ( 1.75% ) and polyethyleneglycol nonylphenyl ether ( 10% ) . all disinfectants were prepared in distilled , deionized water and then filtered through a 0.25 m filter . the following concentrations of disinfectants were used to determine the inactivation kinetics of vaccinia and fowlpox viruses : ethanol ( 40% ) , sodium hypochlorite ( 0.05% and 0.025% ) , a mixture of cetyltrimethylammonium chloride ( 0.1% ) and benzalkonium chloride ( 0.05% ) , a mixture of iodine ( 0.1% ) and polyethyleneglycol nonylphenyl ether ( 5.7% ) , and a mixture of iodine ( 0.05% ) and polyethyleneglycol nonylphenyl ether ( 0.3% ) . all disinfectants were prepared in distilled , deionized water and then filtered through a 0.25 m filter . the plaque assays described above for vaccinia and fowlpox viruses were followed to determine the activity of the disinfectants , with minor changes . vaccinia and fowlpox viruses were diluted 1 : 10 in sterile water ( control ) or disinfectant . to test the infectivity of the treated viruses , the virus was treated with disinfectants for 1 , 3 , 5 , and 10 minutes . to determine the inactivation kinetics of the individual disinfectants , the viruses were treated for 15 , 30 , 45 , 60 , 90 , 120 , 150 , and 180 seconds . in order to determine the thermal stability of vaccinia and fowlpox viruses were diluted in 0.85% saline , ph 4.5 , phosphate buffered saline ( pbs ) , ph 7.4 , 10% skim milk , and heart infusion broth as described by hahon and kozikowski for their work with variola major . solutions were equilibrated to 40c , 45c , 50c , and 55c and then seeded with virus , resulting in a 1 : 100 dilution . samples were removed at 0 , 15 , 30 , 45 , and 60 minutes and cooled on wet ice . the plaque assays described above were then performed . aliquots of vaccinia and fowlpox viruses were stored at 80c and 4c for one week . after the one week storage period , the viruses were diluted 1 : 100 in sterile pbs heated to 56c as described by hahon and kozikowski . samples were incubated at 56c for 0 , 15 , 30 , 45 , and 60 minutes . at the indicated timepoint , samples were removed and cooled on wet ice . the plaque assays described above were then performed . to determine the order of the reaction , the following graphs were produced . to determine a first - order reaction , graphs of the natural log of the virus concentration versus time were produced . a linear line with a negative slope indicated a first - order reaction . to determine a second - order reaction , graphs of 1/virus concentration versus time were produced . the rate of reaction for the first - order reactions was determined using the following equation : vt / v0 = e , where vt is the concentration of virus at time , t , and v0 is the concentration of virus at the zero timepoint , and k is the reaction rate . the rate of reaction for the second - order reactions was determined using the following equation : ( 1/vt ) = ( 1/v0 ) + kt , where vt is the concentration of virus at time , t , v0 is the concentration of virus at the zero timepoint , and k is the reaction rate . the virus half - life ( t1/2 ) for viruses showing a first - order reaction rate was determined using the following equation : t1/2 = ln 2/k , where k is the rate constant determined using the equations described above . the t1/2 for viruses showing a second - order reaction rate was determined using the following equation : t1/2 = 1/(v0 k ) , where v0 is the concentration of virus at the zero timepoint . the rate constant and virus half - life for the thermal inactivation studies were calculated as described above . the calculations of hahon and kozikowski were used to determine h and s . as shown in figure 3 , h may be obtained by the arrhenius plot of the natural logarithm of the k values versus the reciprocal of the absolute temperature ( 1/t ) . a straight line with the slope h / r is obtained , where r is the universal gas constant , from which the h can be calculated from . the eyring equation is as follows : k = ( kt / h ) e(h / rt ) e(s / r ) , where k is boltzmann 's constant , t is the absolute temperature , h is plank 's constant , r is the universal gas constant , and k is the rate constant calculated for the reaction . chemical toxicity assays were performed by incubating bsc-40 and umnsah / df-1 cells with the highest dose of chemical for the viral absorption time ( one hour for bsc-40 and two hours for umnsah / df-1 ) . following incubation , toxicity effects were noted to ensure that cells were healthy enough to produce viral protein , allowing the infection process to occur . additionally , the determinations of toxicity effects were important to ensure that any cell death was due to viral infection and not the treatment chemical . the ethanol , isopropyl alcohol , and sodium hypochlorite had no observable effects on either cell line . the formaldehyde and the surface - active detergents ( benzalkonium chloride , cetyltrimethylammonium chloride , and a mixture of iodine and polyethyleneglycol nonylphenyl ether ) had some toxic effects on cells at the highest two dilutions , but cells that recovered and grew had normal morphology . all the chemicals tested disinfected both vaccinia virus and fowlpox virus ( tables 1 and 2 ) . . for each chemical tested , triplicate test plates of untreated virus ( positive control ) were also cultured on cells . in order for the results of the chemical decontamination timepoint to be considered valid , the average positive control titer was within 1-log unit of the known virus titer . these results were identical to the results reported by tanabe and hotta , using vaccinia and variola major . the four chemicals chosen for the inactivation kinetic studies were shown by tanabe and hotta to demonstrate high viracidal properties at minimum concentrations . as shown in figure 1 , 0.05% and 0.025% sodium hypochlorite inactivated the vaccinia virus within 15 seconds of treatment . within 30 and 45 seconds following treatment , 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride inactivated vaccinia virus , respectively . two concentrations of chemicals were not high enough to result in complete viral inactivation . following a three minute treatment with 40% ethanol , although treatment for three minutes with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether did not result in complete inactivation , viral titers dropped by 5-log pfu / ml units . the 40% ethanol inactivation kinetics in this study vary somewhat from the data reported by tanabe and hotta . purified variola major virus is inactivated by 40% ethanol within 1 minute , while a nonpurified culture is inactivated within three minutes . secondly , tanabe and hotta reported their data in percent reduction in plaque count . if the 40% ethanol inactivation kinetics described here were reported in percent reduction in plaque count , there would be a 95.4% reduction in plaques at the 60-second timepoint , which is the point at which tanabe and hotta describe a 100% reduction . fowlpox virus had an inactivation profile similar to that of vaccinia virus . like vaccinia virus , 0.05% and 0.025% sodium hypochlorite inactivated the fowlpox virus within 15 seconds of treatment , as shown in figure 2 . within 15 and 45 seconds following treatment , 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride inactivated fowlpox virus , respectively . the inactivation kinetic curves for 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride treated fowlpox are similar to the inactivation kinetic curves of vaccinia virus treated with those same chemicals . like vaccinia virus , treatment for 3 minutes with 40% ethanol and 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether did not result in complete inactivation of fowlpox virus following treatment of fowlpox virus with 40% ethanol for three minutes , the concentration of fowlpox virus dropped by approximately 2-log pfu / ml units . fowlpox virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether had a decreased titer of approximately 1-log pfu / ml unit following a three - minute treatment . inactivation rate constants were calculated for vaccinia and fowlpox viruses treated with ethanol ( 40% ) , sodium hypochlorite ( 0.05% and 0.025% ) , a mixture of cetyltrimethylammonium chloride ( 0.1% ) and benzalkonium chloride ( 0.05% ) , a mixture of iodine ( 0.1% ) and polyethyleneglycol nonylphenyl ether ( 5.7% ) , and a mixture of iodine ( 0.05% ) and polyethyleneglycol nonylphenyl ether ( 0.3% ) . the calculated rate constants for vaccinia and fowlpox viruses are shown in table 3 . overall , vaccinia virus exhibited greater inactivation rate constants than fowlpox virus treated with the same chemicals . for both viruses , the order of the inactivation reaction when treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether was a second - order reaction . vaccinia virus exhibited a first - order reaction , while fowlpox virus exhibited a second - order reaction . a first - order reaction was observed in both vaccinia and fowlpox virus treated with 0.05% and 0.025% sodium hypochlorite , a mixture of 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride , and 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether . vaccinia and fowlpox viruses treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.05% sodium hypochlorite , 0.1% sodium hypochlorite , and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride had a calculated half - life of less than 8.41 seconds ( table 3 ) . the difference in virus half - life between the two viruses treated with the above chemicals was less than two seconds . when treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.05% sodium hypochlorite , 0.1% sodium hypochlorite , and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride , fowlpox had a slightly greater half - life than vaccinia virus by 12 seconds . for two chemicals , fowlpox virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether had a half - life 2.4 10 times greater than the half - life of vaccinia virus treated with the same chemical . however , vaccinia virus treated with 40% ethanol had a virus half - life over 20 times greater than fowlpox virus treated with the same disinfectant . vaccinia virus was completely inactivated in 0.85% saline when heated to 55c . approximately two - log reductions of viral titer were observed in the vaccinia diluted in 0.85% saline and heated at 40c , 45c and 50c . according to the arrhenius plot , the h for vaccinia virus is 44,138 calories per mole in 0.85% saline ( see table 4 ) . unlike vaccinia virus , after one hour of heating at 55c , fowlpox virus had a 2-log decrease in plaques . after heating for one hour at 40c , 45c , and 50c , fowlpox virus titers did not decrease by even 1-log unit . the h for fowlpox virus in 0.85% saline was 53,481 calories per mol in 0.85% saline ( see table 4 ) . the entropy of activation ( s ) for vaccinia virus in 0.85% saline ranged from 67.61 cal/c at 40c to 65.85 cal/c at 55c ( see table 5 ) . the entropy of activation ( s ) for fowlpox virus in 0.85% saline ranged from 71.70 cal/c at 40c to 63.93 cal/c at 55c ( see table 6 ) . vaccinia and fowlpox viruses behaved oppositely in pbs , ph 7.4 as compared to their behavior in 0.85% saline , ph 4.5 . unlike the vaccinia virus treated in 0.85% saline that was completely inactivated at 55c , vaccinia virus in pbs , ph 7.4 is stable , decreasing by only 2-log units over the 60 minute incubation period . vaccinia virus heated for one hour at 40c , 45c , and 50c decreased by less than one - log unit . the calculated h for vaccinia virus in pbs , ph 7.4 , is 34,239 calories per mole ( see table 4 ) . unlike vaccinia virus , fowlpox virus was completely inactivated in pbs , ph 7.4 at 55c . however , at 40c , 45c , and 50c , fowlpox , like vaccinia , was stable , with titers decreasing by less than one - log unit over the hour long incubation period . the calculated h for fowlpox virus in pbs , ph 7.4 , is 35,868 calories per mole ( see table 4 ) . the entropy of activation ( s ) for vaccinia virus in pbs , ph 7.4 , ranged from 69.08 cal/c at 40c to 63.17 cal/c at 55c ( see table 5 ) . the entropy of activation ( s ) for fowlpox virus in pbs , ph 7.4 ranged from 68.34 cal/c at 40c to 62.45 cal/c at 55c ( see table 6 ) . vaccinia and fowlpox viruses behaved similarly when heated in a solution of 10% skim milk . after a one hour treatment at 55c in 10% skim milk , vaccinia titers decreased by 4-log units and fowlpox virus decreased by 2-log units . after a one hour treatment in 10% skim milk at 50c , vaccinia virus titers decreased by 2-log units , while fowlpox virus titers decreased by one - log unit . at the lower temperatures ( 40c and 45c ) titers only dropped by one - log unit for both vaccinia and fowlpox viruses the calculated h for vaccinia virus in 10% skim milk is 44,741 calories per mole ; while the calculated h fowlpox virus in 10% skim milk is 22,378 calories per mole ( see table 4 ) . the entropy of activation ( s ) for vaccinia virus in skim milk ranged from 70.04 cal/c at 40c to 62.82 cal/c at 55c ( see table 5 ) . the entropy of activation ( s ) for fowlpox virus in skim milk ranged from 67.74 cal/c at 40c to 65.12 cal/c at 55c ( see table 6 ) . vaccina and fowlpox viruses also behaved similarly in heart infusion broth , both in their inactivation curves , but also in the fact that both viruses showed decreased aggregation at lower temperatures in this test liquid . both viruses , especially noted in vaccinia as the time course progressed , plaque size dramatically decreased and titers actually increased between the 30- and 45- minute timepoints . at the 30- and 45-minute timepoints , both vaccinia and fowlpox plaques were approximately 10 times smaller than those observed during plaque assay optimization procedures , during which the virus was not treated with chemicals or heated . however , by the 60-minute timepoint , titers had decreased to below or similar to the starting titer and plaque size returned to the normal sizes observed during plaque assay optimization . neither vaccinia virus nor fowlpox virus was completely inactivated at 55c in brain heart infusion broth . vaccinia titers decreased by 4-log units after a one hour incubation in heart infusion broth , while fowlpox virus titers decreased by 2-log units after a one hour incubation . at both 45c and 50c for both vaccinia and fowlpox viruses , the calculated h for vaccinia virus in heart infusion broth is 95,204 calories per mole ; while the calculated h fowlpox virus in heart infusion broth is 65,229 calories per mole ( see table 4 ) . the entropy of activation ( s ) for vaccinia virus in heart infusion broth ranged from 63.12 cal/c at 40c to 64.86 cal/c at 55c ( see table 5 ) . the entropy of activation ( s ) for fowlpox virus in heart infusion broth ranged from 73.95 cal/c at 40c to 63.55 cal/c at 55c ( see table 6 ) . vaccinia and fowlpox viruses were stored for one week at either 4c or 80c , then diluted in pbs , ph 7.4 , and inactivated at 56c . vaccinia virus was more stable than fowlpox virus under both storage conditions ( see figure 3 ) . following 60 minutes of heating at 56c , vaccinia stored at 4c showed a 3-log decrease in plaque forming units . although titers decreased , viable virus still remained after the 60-minute incubation period . the reaction orders for both the virus stored at 4c and 80c were both first - order reactions . fowlpox varied significantly from vaccinia virus in its stability in pbs following different storage conditions ( see figure 4 ) . initially , fowlpox virus was heated at 56c for 60 minutes ; however , half - way through the incubation period , no viable fowlpox virus could be detected ( data not shown ) . the experiment was repeated and instead of incubating the virus for 60 minutes at 56c , the fowlpox was only incubated for 15 minutes at 56c . when incubated for 15 minutes , inactivation kinetics could be determined . when the virus stored at 4c was inactivated at 56c , viral titers only dropped approximately one - log at the 15-minute timepoint , however , by the 30 minute timepoint , no active virus could be detected . when the virus stored at 80c was inactivated at 56c , viral titers declined ~0.2-log unit within the first 15 minutes , however like the sample stored at 4c , after 30 minutes no active virus could be detected . like the vaccinia virus , the fowlpox inactivation curves were both first - order reactions . the viruses are so closely related that it has been hypothesized that vaccinia virus may have evolved from variola virus through continual passaging in cows and humans . thus , vaccinia virus data common between this work and that of tanabe and hotta and hahon and kozikowski allow for an approximate comparison of the decontamination and thermal stability of fowlpox and variola major . vaccinia virus is a well characterized virus that has been grown extensively in a variety of cell lines , from monkey and hamster kidney cells to chicken embryo fibroblasts . fowlpox , due to the presence of host limiting genes in the viral genome , does not infect a wide variety of species like vaccinia . based on literature searches , fowlpox virus is normally grown in primary chicken embryo fibroblast ( cef ) cells . due to the more difficult nature of maintaining primary cell lines , it has been demonstrated in this work that fowlpox can be grown in an immortalized cef line , the umnsah / df-1 cell line . although the concentration of fowlpox virus was on the order of 1 - 2 log units less than the concentration of vaccinia virus , the minimum concentration of fowlpox virus produced by these cells was approximately 7 10 pfu / ml . vaccinia virus titers were in the 2 10 pfu / ml range . the virus used in this work was cell - associated virus that was lysed out of the cells . the virus was then centrifuged to remove any cell debris , which is similar to the manner in which tanabe and hotta prepared the variola major used in their work . the purities of the vaccinia and fowlpox viruses are comparable , as the virus harvesting methods were very similar to the methods described by tanabe and hotta . data from this study suggest that fowlpox virus is a suitable decontamination simulant for variola major when using disinfectants at full strength ( as they would typically be used ) . fowlpox virus and vaccine virus are both inactivated within 1 minute when using the following disinfectants : 70% ethanol , 50% isopropyl alcohol , 0.5% sodium hypochlorite , 30% formaldehyde , 10% benzalkonium chloride , a mixture of 6.67% cetyltrimethylammonium chloride and 3.33% benzalkonium chloride , and a mixture of 1.75% iodine and 10% polyethyleneglycol nonylphenyl ether . viral inactivation with minimal concentrations of the same chemicals was also examined in the present study , for the purpose of determining the inactivation kinetics of both vaccinia and fowlpox . to first principles , the inactivation curves for each virus and chemical appear similar ; however mathematical analysis of the data reveals distinct differences for vaccinia and fowlpox viruses treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol . the reaction orders described in this study are both first- and second - order decay reactions . first - order reactions were observed with vaccinia and fowlpox viruses treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.025% and 0.05% sodium hypochlorite , and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . vaccinia virus treated with 40% ethanol also showed a first - order reaction , while a second - order reaction was observed when the virus was treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether . fowlpox virus exhibited second - order reactions when treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol . the first - order decay reaction is typically expected when observing viral kinetics . however , further analysis of the unique properties of poxviruses suggests possible reasons for the observed second - order reaction of fowlpox virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol and the vaccinia virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether . first , vertebrate poxviruses have the unique ability to nongenetically reactivate . in nongenetic reactivation , secondly , poxviruses ( like many other viruses ) tend to aggregate in solutions . the aggregated viruses that can not be neutralized by antibodies are called the persistent fraction or nonneutralizable fraction . studies have shown that vaccinia virus treated with nitrogen mustard bis ( -chloroethyl ) methylamine ( nitrogen mustard ) , a chemical known for virus inactivation , can survive treatment with this agent when the particles are aggregated . in fact , jolik showed that rabbitpox virus treated with nitrogen mustard is still able to act as an uncoating agent for other viruses that have been inactivated , but still genetically intact , which is the phenomenon described above for nongenetic reactivation . based on the data and published research , it could be concluded that fowlpox possibly undergoes greater aggregation than vaccinia virus in both 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol , resulting in nongenetic reactivation and therefore , a second - order reaction . thus , the second - order reaction might be due to the fact that fowlpox virus particles in the center of a viral aggregate might be protected from the disinfectant and have the ability to reactive the genetically intact , but inactivated fowlpox virus particles . kinetic analysis further elucidated the inactivation similarities and differences between vaccinia and fowlpox viruses . the rate constant , k , is calculated from the starting concentration of virus and the concentration of virus at time , t. the k values differed by no more than 0.7 1/s for the vaccinia and fowlpox viruses treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.025% sodium hypochlorite , 0.05% sodium hypochlorite , and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . however , the vaccinia and fowlpox viruses treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol show large differences in k values . these observed differences could be due to the fact that when treated with 40% ethanol , vaccinia undergoes a first - order reaction and fowlpox undergoes a second - order reaction . when treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether , the data suggests that fowlpox virus is more stable in that disinfectant . although the k value is an important variable for comparison , for the purpose of this work , the virus half - life ( t1/2 ) value is a better indicator of simulant suitability . the purpose of this work is to determine if fowlpox is a suitable decontamination simulant for variola minor , thus the amount of time the virus will survive is more important than the rate of the reaction . based on the data presented in table 1 , we can conclude that fowlpox is a suitable decontamination simulant for variola major when using the following disinfectants at concentrations used for inactivation kinetics : 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.025% sodium hypochlorite , 0.05% sodium hypochlorite and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . the similar virus half - life and reaction orders indicate that fowlpox and vaccinia virus undergo similar reactions at similar times . however , fowlpox is not a suitable decontamination simulant for variola major when using 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol . the differences in reaction order for the two viruses for 40% ethanol are important factors . additionally , vaccinia is two times more stable in that disinfectant than fowlpox . fowlpox virus is 20 times more stable than vaccinia virus in 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether . the studies presented in this paper suggest that fowlpox is a suitable decontamination simulant for variola major when using the following full strength disinfectants : 70% ethanol , 50% isopropyl alcohol , 0.5% sodium hypochlorite , 30% formaldehyde , 10% benzalkonium chloride , a mixture of 6.67% cetyltrimethylammonium chloride and 3.33% benzalkonium chloride and a mixture of 1.75% iodine and 10% polyethyleneglycol nonylphenyl ether . fowlpox virus is also a suitable inactivation kinetics simulant when treated with the following chemicals : 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.025% sodium hypochlorite , 0.05% sodium hypochlorite and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . fowlpox virus is not a suitable inactivation kinetics simulant for variola major when treated with the following chemicals : 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol . knowing the genetic and decontamination similarities of variola major and vaccinia from previous studies , fowlpox virus has been shown to be a suitable variola major simulant through identical decontamination results using vaccinia virus as the common link between the studies . like the decontamination studies , vaccinia virus served as the common link between variola virus and fowlpox virus for the thermal stability studies . hahon and kozikowski determined the h and s of variola major virus in various buffers at temperatures from 40c to 55c . previous studies with vaccinia virus demonstrated that vaccinia virus has a range of h values from 20,000 to 90,000 calories per mole . the calculated h values for the vaccinia virus and fowlpox virus described in this paper fall into the range described kaplan , with the exception of vaccinia virus heated in brain heart infusion broth . the difference in h values in this paper with vaccinia virus in brain heart infusion broth might be due to the decreased virus aggregation , which might have affected the inactivation and plaque counts . hahon and kozikowski did not mention decreased aggregation in the variola major virus heated in brain heart infusion broth ; however , their variola major sample was stored in brain heart infusion broth , while the viruses used in this work were stored in dmem containing 10% fbs . the h values calculated for vaccinia virus in 0.85% saline , ph 4.5 , pbs , ph 7.4 , and 10% skim milk were in the range described for both vaccinia and variola major virus . the h values calculated for fowlpox virus in 0.85% saline , ph 4.5 , pbs , ph 7.4 , 10% skim milk , and brain heart infusion broth were in the range described for both vaccinia ( kaplan ) and variola major virus . it is common to find values of s of the order of 10 to 100 calories per mol per degree for the denaturation of proteins . the s values described in this work for both vaccinia and fowlpox viruses fall well within the 10100 calories per mol per degree celsius range . these studies suggest that fowlpox can serve as a thermal stability simulant for variola major in 0.85% saline , ph 4.5 , pbs , ph 7.4 , 10% skim milk , and heart infusion broth at temperatures ranging from 40c to 55c . at temperatures above , 55c , fowlpox does not appear to be as stable as vaccinia virus . as the storage conditions studies demonstrate , even when stored at 80c , fowlpox quickly denatures when heated at 56c when compared to vaccinia virus .

variola major , the causative agent of smallpox , has been eradicated from nature . however , stocks still exist ; thus , there is a need for relevant decontamination studies , preferably with nonpathogenic simulants . previous studies have shown a similarity in response of vaccinia virus and variola major to various decontaminants and thermal inactivation . this study compared vaccinia and fowlpox viruses under similar conditions , using disinfectants and temperatures for which variola major data already existed . most disinfectants showed similar efficacy against vaccinia and fowlpox , suggesting the utility of fowlpox as a decontamination simulant . inactivation kinetics studies showed that fowlpox behaved similarly to variola major when treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.025% sodium hypochlorite , 0.05% sodium hypochlorite , and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride , but differed in its response to 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol . thermal inactivation studies demonstrated that fowlpox is a suitable thermal simulant for variola major between 40c and 55c .

1. Introduction 2. Materials and Methods 3. Results 4. Conclusion

the chordopox viruses are further divided into eight categories , including orthopox , which includes variola major and vaccinia virus , and avipox , which includes fowlpox virus . variola major , the virus that causes the contagious disease smallpox , has been considered one of the most destructive viruses in history , affecting 1/10th of all humankind and killing 300 million people in the 20th century alone . however , although variola major no longer exists in any known animal reservoirs and nature , stocks are still kept in the united states and russia . vaccina virus , long considered the prototype of variola major , has been used as both a simulant and vaccine for variola major . they show remarkable sequence similarity ; however , vaccinia virus does not cause disease in healthy individuals , possibly due to the fact that variola major virus contains complement inhibitors 100 times more potent than those of vaccinia virus . the purpose of this paper is to investigate whether fowlpox , also a member of the chordopox family , can be used as a simulant for variola major . the following disinfectants were tested for their efficacy against vaccinia and fowlpox viruses : ethanol ( 70% ) , isopropyl alcohol ( 50% ) , sodium hypochlorite ( 0.5% ) , formaldehyde ( 30% ) , benzalkonium chloride ( 10% ) , a mixture of cetyltrimethylammonium chloride ( 6.67% ) and benzalkonium chloride ( 3.33% ) , and a mixture of iodine ( 1.75% ) and polyethyleneglycol nonylphenyl ether ( 10% ) . the following concentrations of disinfectants were used to determine the inactivation kinetics of vaccinia and fowlpox viruses : ethanol ( 40% ) , sodium hypochlorite ( 0.05% and 0.025% ) , a mixture of cetyltrimethylammonium chloride ( 0.1% ) and benzalkonium chloride ( 0.05% ) , a mixture of iodine ( 0.1% ) and polyethyleneglycol nonylphenyl ether ( 5.7% ) , and a mixture of iodine ( 0.05% ) and polyethyleneglycol nonylphenyl ether ( 0.3% ) . the plaque assays described above for vaccinia and fowlpox viruses were followed to determine the activity of the disinfectants , with minor changes . vaccinia and fowlpox viruses were diluted 1 : 10 in sterile water ( control ) or disinfectant . to determine the inactivation kinetics of the individual disinfectants , the viruses were treated for 15 , 30 , 45 , 60 , 90 , 120 , 150 , and 180 seconds . in order to determine the thermal stability of vaccinia and fowlpox viruses were diluted in 0.85% saline , ph 4.5 , phosphate buffered saline ( pbs ) , ph 7.4 , 10% skim milk , and heart infusion broth as described by hahon and kozikowski for their work with variola major . aliquots of vaccinia and fowlpox viruses were stored at 80c and 4c for one week . the formaldehyde and the surface - active detergents ( benzalkonium chloride , cetyltrimethylammonium chloride , and a mixture of iodine and polyethyleneglycol nonylphenyl ether ) had some toxic effects on cells at the highest two dilutions , but cells that recovered and grew had normal morphology . these results were identical to the results reported by tanabe and hotta , using vaccinia and variola major . as shown in figure 1 , 0.05% and 0.025% sodium hypochlorite inactivated the vaccinia virus within 15 seconds of treatment . within 30 and 45 seconds following treatment , 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride inactivated vaccinia virus , respectively . following a three minute treatment with 40% ethanol , although treatment for three minutes with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether did not result in complete inactivation , viral titers dropped by 5-log pfu / ml units . the 40% ethanol inactivation kinetics in this study vary somewhat from the data reported by tanabe and hotta . if the 40% ethanol inactivation kinetics described here were reported in percent reduction in plaque count , there would be a 95.4% reduction in plaques at the 60-second timepoint , which is the point at which tanabe and hotta describe a 100% reduction . like vaccinia virus , 0.05% and 0.025% sodium hypochlorite inactivated the fowlpox virus within 15 seconds of treatment , as shown in figure 2 . within 15 and 45 seconds following treatment , 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride inactivated fowlpox virus , respectively . the inactivation kinetic curves for 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride treated fowlpox are similar to the inactivation kinetic curves of vaccinia virus treated with those same chemicals . like vaccinia virus , treatment for 3 minutes with 40% ethanol and 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether did not result in complete inactivation of fowlpox virus following treatment of fowlpox virus with 40% ethanol for three minutes , the concentration of fowlpox virus dropped by approximately 2-log pfu / ml units . fowlpox virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether had a decreased titer of approximately 1-log pfu / ml unit following a three - minute treatment . inactivation rate constants were calculated for vaccinia and fowlpox viruses treated with ethanol ( 40% ) , sodium hypochlorite ( 0.05% and 0.025% ) , a mixture of cetyltrimethylammonium chloride ( 0.1% ) and benzalkonium chloride ( 0.05% ) , a mixture of iodine ( 0.1% ) and polyethyleneglycol nonylphenyl ether ( 5.7% ) , and a mixture of iodine ( 0.05% ) and polyethyleneglycol nonylphenyl ether ( 0.3% ) . the calculated rate constants for vaccinia and fowlpox viruses are shown in table 3 . for both viruses , the order of the inactivation reaction when treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether was a second - order reaction . a first - order reaction was observed in both vaccinia and fowlpox virus treated with 0.05% and 0.025% sodium hypochlorite , a mixture of 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride , and 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether . vaccinia and fowlpox viruses treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.05% sodium hypochlorite , 0.1% sodium hypochlorite , and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride had a calculated half - life of less than 8.41 seconds ( table 3 ) . when treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.05% sodium hypochlorite , 0.1% sodium hypochlorite , and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride , fowlpox had a slightly greater half - life than vaccinia virus by 12 seconds . for two chemicals , fowlpox virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether had a half - life 2.4 10 times greater than the half - life of vaccinia virus treated with the same chemical . however , vaccinia virus treated with 40% ethanol had a virus half - life over 20 times greater than fowlpox virus treated with the same disinfectant . however , at 40c , 45c , and 50c , fowlpox , like vaccinia , was stable , with titers decreasing by less than one - log unit over the hour long incubation period . vaccinia and fowlpox viruses behaved similarly when heated in a solution of 10% skim milk . at the lower temperatures ( 40c and 45c ) titers only dropped by one - log unit for both vaccinia and fowlpox viruses the calculated h for vaccinia virus in 10% skim milk is 44,741 calories per mole ; while the calculated h fowlpox virus in 10% skim milk is 22,378 calories per mole ( see table 4 ) . vaccina and fowlpox viruses also behaved similarly in heart infusion broth , both in their inactivation curves , but also in the fact that both viruses showed decreased aggregation at lower temperatures in this test liquid . at the 30- and 45-minute timepoints , both vaccinia and fowlpox plaques were approximately 10 times smaller than those observed during plaque assay optimization procedures , during which the virus was not treated with chemicals or heated . at both 45c and 50c for both vaccinia and fowlpox viruses , the calculated h for vaccinia virus in heart infusion broth is 95,204 calories per mole ; while the calculated h fowlpox virus in heart infusion broth is 65,229 calories per mole ( see table 4 ) . vaccinia and fowlpox viruses were stored for one week at either 4c or 80c , then diluted in pbs , ph 7.4 , and inactivated at 56c . thus , vaccinia virus data common between this work and that of tanabe and hotta and hahon and kozikowski allow for an approximate comparison of the decontamination and thermal stability of fowlpox and variola major . vaccinia virus is a well characterized virus that has been grown extensively in a variety of cell lines , from monkey and hamster kidney cells to chicken embryo fibroblasts . due to the more difficult nature of maintaining primary cell lines , it has been demonstrated in this work that fowlpox can be grown in an immortalized cef line , the umnsah / df-1 cell line . although the concentration of fowlpox virus was on the order of 1 - 2 log units less than the concentration of vaccinia virus , the minimum concentration of fowlpox virus produced by these cells was approximately 7 10 pfu / ml . data from this study suggest that fowlpox virus is a suitable decontamination simulant for variola major when using disinfectants at full strength ( as they would typically be used ) . fowlpox virus and vaccine virus are both inactivated within 1 minute when using the following disinfectants : 70% ethanol , 50% isopropyl alcohol , 0.5% sodium hypochlorite , 30% formaldehyde , 10% benzalkonium chloride , a mixture of 6.67% cetyltrimethylammonium chloride and 3.33% benzalkonium chloride , and a mixture of 1.75% iodine and 10% polyethyleneglycol nonylphenyl ether . to first principles , the inactivation curves for each virus and chemical appear similar ; however mathematical analysis of the data reveals distinct differences for vaccinia and fowlpox viruses treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol . first - order reactions were observed with vaccinia and fowlpox viruses treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.025% and 0.05% sodium hypochlorite , and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . vaccinia virus treated with 40% ethanol also showed a first - order reaction , while a second - order reaction was observed when the virus was treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether . fowlpox virus exhibited second - order reactions when treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol . however , further analysis of the unique properties of poxviruses suggests possible reasons for the observed second - order reaction of fowlpox virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol and the vaccinia virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether . studies have shown that vaccinia virus treated with nitrogen mustard bis ( -chloroethyl ) methylamine ( nitrogen mustard ) , a chemical known for virus inactivation , can survive treatment with this agent when the particles are aggregated . in fact , jolik showed that rabbitpox virus treated with nitrogen mustard is still able to act as an uncoating agent for other viruses that have been inactivated , but still genetically intact , which is the phenomenon described above for nongenetic reactivation . based on the data and published research , it could be concluded that fowlpox possibly undergoes greater aggregation than vaccinia virus in both 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol , resulting in nongenetic reactivation and therefore , a second - order reaction . thus , the second - order reaction might be due to the fact that fowlpox virus particles in the center of a viral aggregate might be protected from the disinfectant and have the ability to reactive the genetically intact , but inactivated fowlpox virus particles . kinetic analysis further elucidated the inactivation similarities and differences between vaccinia and fowlpox viruses . the rate constant , k , is calculated from the starting concentration of virus and the concentration of virus at time , t. the k values differed by no more than 0.7 1/s for the vaccinia and fowlpox viruses treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.025% sodium hypochlorite , 0.05% sodium hypochlorite , and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . however , the vaccinia and fowlpox viruses treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol show large differences in k values . these observed differences could be due to the fact that when treated with 40% ethanol , vaccinia undergoes a first - order reaction and fowlpox undergoes a second - order reaction . when treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether , the data suggests that fowlpox virus is more stable in that disinfectant . the purpose of this work is to determine if fowlpox is a suitable decontamination simulant for variola minor , thus the amount of time the virus will survive is more important than the rate of the reaction . based on the data presented in table 1 , we can conclude that fowlpox is a suitable decontamination simulant for variola major when using the following disinfectants at concentrations used for inactivation kinetics : 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.025% sodium hypochlorite , 0.05% sodium hypochlorite and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . however , fowlpox is not a suitable decontamination simulant for variola major when using 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol . fowlpox virus is 20 times more stable than vaccinia virus in 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether . the studies presented in this paper suggest that fowlpox is a suitable decontamination simulant for variola major when using the following full strength disinfectants : 70% ethanol , 50% isopropyl alcohol , 0.5% sodium hypochlorite , 30% formaldehyde , 10% benzalkonium chloride , a mixture of 6.67% cetyltrimethylammonium chloride and 3.33% benzalkonium chloride and a mixture of 1.75% iodine and 10% polyethyleneglycol nonylphenyl ether . fowlpox virus is also a suitable inactivation kinetics simulant when treated with the following chemicals : 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.025% sodium hypochlorite , 0.05% sodium hypochlorite and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . fowlpox virus is not a suitable inactivation kinetics simulant for variola major when treated with the following chemicals : 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol . knowing the genetic and decontamination similarities of variola major and vaccinia from previous studies , fowlpox virus has been shown to be a suitable variola major simulant through identical decontamination results using vaccinia virus as the common link between the studies . like the decontamination studies , vaccinia virus served as the common link between variola virus and fowlpox virus for the thermal stability studies . the calculated h values for the vaccinia virus and fowlpox virus described in this paper fall into the range described kaplan , with the exception of vaccinia virus heated in brain heart infusion broth . the h values calculated for vaccinia virus in 0.85% saline , ph 4.5 , pbs , ph 7.4 , and 10% skim milk were in the range described for both vaccinia and variola major virus . the s values described in this work for both vaccinia and fowlpox viruses fall well within the 10100 calories per mol per degree celsius range . these studies suggest that fowlpox can serve as a thermal stability simulant for variola major in 0.85% saline , ph 4.5 , pbs , ph 7.4 , 10% skim milk , and heart infusion broth at temperatures ranging from 40c to 55c .

poxvirus genomes encode the genes necessary for replication and immune modulation , with replication occurring in the host cell 's cytoplasm . variola major , the virus that causes the contagious disease smallpox , has been considered one of the most destructive viruses in history , affecting 1/10th of all humankind and killing 300 million people in the 20th century alone . smallpox has a 2030% lethality rate in unvaccinated individuals , although analysis of historical data suggests that 77.6% of cases are still protected against severe disease 70 years after vaccination . vaccina virus , long considered the prototype of variola major , has been used as both a simulant and vaccine for variola major . they show remarkable sequence similarity ; however , vaccinia virus does not cause disease in healthy individuals , possibly due to the fact that variola major virus contains complement inhibitors 100 times more potent than those of vaccinia virus . the purpose of this paper is to investigate whether fowlpox , also a member of the chordopox family , can be used as a simulant for variola major . when compared by hexamer and glimer computer programs , these genes encode for transcription factors , rna - polymerase subunits , dna repair , protein modification , structural proteins , and immune evasion proteins . several genes encoding cellular function show homology with proteins of unknown function from yeast , bacteria , roundworm , plant , and human . secondly , the fowlpox genome contains host range genes , limiting infection to members of the avian family . vaccinia strain wr ( vr-119 ) and fowlpox strain fpv - mdvgbh ( merck 's disease vaccine strain ) ( vr-2330 ) were obtained from the american type culture collection ( atcc , manassas , va ) . bhk-21 ( ccl-10 ) , bsc-40 cells ( crl-2761 ) and umnsah / df-1 cells ( chicken embryo fibroblast cells ) ( crl-12203 ) were also obtained from atcc . the following disinfectants were tested for their efficacy against vaccinia and fowlpox viruses : ethanol ( 70% ) , isopropyl alcohol ( 50% ) , sodium hypochlorite ( 0.5% ) , formaldehyde ( 30% ) , benzalkonium chloride ( 10% ) , a mixture of cetyltrimethylammonium chloride ( 6.67% ) and benzalkonium chloride ( 3.33% ) , and a mixture of iodine ( 1.75% ) and polyethyleneglycol nonylphenyl ether ( 10% ) . the following concentrations of disinfectants were used to determine the inactivation kinetics of vaccinia and fowlpox viruses : ethanol ( 40% ) , sodium hypochlorite ( 0.05% and 0.025% ) , a mixture of cetyltrimethylammonium chloride ( 0.1% ) and benzalkonium chloride ( 0.05% ) , a mixture of iodine ( 0.1% ) and polyethyleneglycol nonylphenyl ether ( 5.7% ) , and a mixture of iodine ( 0.05% ) and polyethyleneglycol nonylphenyl ether ( 0.3% ) . the plaque assays described above for vaccinia and fowlpox viruses were followed to determine the activity of the disinfectants , with minor changes . to test the infectivity of the treated viruses , the virus was treated with disinfectants for 1 , 3 , 5 , and 10 minutes . to determine the inactivation kinetics of the individual disinfectants , the viruses were treated for 15 , 30 , 45 , 60 , 90 , 120 , 150 , and 180 seconds . in order to determine the thermal stability of vaccinia and fowlpox viruses were diluted in 0.85% saline , ph 4.5 , phosphate buffered saline ( pbs ) , ph 7.4 , 10% skim milk , and heart infusion broth as described by hahon and kozikowski for their work with variola major . to determine a first - order reaction , graphs of the natural log of the virus concentration versus time were produced . the rate of reaction for the first - order reactions was determined using the following equation : vt / v0 = e , where vt is the concentration of virus at time , t , and v0 is the concentration of virus at the zero timepoint , and k is the reaction rate . the rate of reaction for the second - order reactions was determined using the following equation : ( 1/vt ) = ( 1/v0 ) + kt , where vt is the concentration of virus at time , t , v0 is the concentration of virus at the zero timepoint , and k is the reaction rate . the virus half - life ( t1/2 ) for viruses showing a first - order reaction rate was determined using the following equation : t1/2 = ln 2/k , where k is the rate constant determined using the equations described above . as shown in figure 3 , h may be obtained by the arrhenius plot of the natural logarithm of the k values versus the reciprocal of the absolute temperature ( 1/t ) . the eyring equation is as follows : k = ( kt / h ) e(h / rt ) e(s / r ) , where k is boltzmann 's constant , t is the absolute temperature , h is plank 's constant , r is the universal gas constant , and k is the rate constant calculated for the reaction . chemical toxicity assays were performed by incubating bsc-40 and umnsah / df-1 cells with the highest dose of chemical for the viral absorption time ( one hour for bsc-40 and two hours for umnsah / df-1 ) . the formaldehyde and the surface - active detergents ( benzalkonium chloride , cetyltrimethylammonium chloride , and a mixture of iodine and polyethyleneglycol nonylphenyl ether ) had some toxic effects on cells at the highest two dilutions , but cells that recovered and grew had normal morphology . in order for the results of the chemical decontamination timepoint to be considered valid , the average positive control titer was within 1-log unit of the known virus titer . if the 40% ethanol inactivation kinetics described here were reported in percent reduction in plaque count , there would be a 95.4% reduction in plaques at the 60-second timepoint , which is the point at which tanabe and hotta describe a 100% reduction . like vaccinia virus , 0.05% and 0.025% sodium hypochlorite inactivated the fowlpox virus within 15 seconds of treatment , as shown in figure 2 . the inactivation kinetic curves for 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride treated fowlpox are similar to the inactivation kinetic curves of vaccinia virus treated with those same chemicals . like vaccinia virus , treatment for 3 minutes with 40% ethanol and 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether did not result in complete inactivation of fowlpox virus following treatment of fowlpox virus with 40% ethanol for three minutes , the concentration of fowlpox virus dropped by approximately 2-log pfu / ml units . inactivation rate constants were calculated for vaccinia and fowlpox viruses treated with ethanol ( 40% ) , sodium hypochlorite ( 0.05% and 0.025% ) , a mixture of cetyltrimethylammonium chloride ( 0.1% ) and benzalkonium chloride ( 0.05% ) , a mixture of iodine ( 0.1% ) and polyethyleneglycol nonylphenyl ether ( 5.7% ) , and a mixture of iodine ( 0.05% ) and polyethyleneglycol nonylphenyl ether ( 0.3% ) . for both viruses , the order of the inactivation reaction when treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether was a second - order reaction . a first - order reaction was observed in both vaccinia and fowlpox virus treated with 0.05% and 0.025% sodium hypochlorite , a mixture of 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride , and 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether . vaccinia and fowlpox viruses treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.05% sodium hypochlorite , 0.1% sodium hypochlorite , and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride had a calculated half - life of less than 8.41 seconds ( table 3 ) . when treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.05% sodium hypochlorite , 0.1% sodium hypochlorite , and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride , fowlpox had a slightly greater half - life than vaccinia virus by 12 seconds . for two chemicals , fowlpox virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether had a half - life 2.4 10 times greater than the half - life of vaccinia virus treated with the same chemical . unlike the vaccinia virus treated in 0.85% saline that was completely inactivated at 55c , vaccinia virus in pbs , ph 7.4 is stable , decreasing by only 2-log units over the 60 minute incubation period . the entropy of activation ( s ) for vaccinia virus in pbs , ph 7.4 , ranged from 69.08 cal/c at 40c to 63.17 cal/c at 55c ( see table 5 ) . at the lower temperatures ( 40c and 45c ) titers only dropped by one - log unit for both vaccinia and fowlpox viruses the calculated h for vaccinia virus in 10% skim milk is 44,741 calories per mole ; while the calculated h fowlpox virus in 10% skim milk is 22,378 calories per mole ( see table 4 ) . vaccina and fowlpox viruses also behaved similarly in heart infusion broth , both in their inactivation curves , but also in the fact that both viruses showed decreased aggregation at lower temperatures in this test liquid . at both 45c and 50c for both vaccinia and fowlpox viruses , the calculated h for vaccinia virus in heart infusion broth is 95,204 calories per mole ; while the calculated h fowlpox virus in heart infusion broth is 65,229 calories per mole ( see table 4 ) . the entropy of activation ( s ) for fowlpox virus in heart infusion broth ranged from 73.95 cal/c at 40c to 63.55 cal/c at 55c ( see table 6 ) . thus , vaccinia virus data common between this work and that of tanabe and hotta and hahon and kozikowski allow for an approximate comparison of the decontamination and thermal stability of fowlpox and variola major . fowlpox , due to the presence of host limiting genes in the viral genome , does not infect a wide variety of species like vaccinia . due to the more difficult nature of maintaining primary cell lines , it has been demonstrated in this work that fowlpox can be grown in an immortalized cef line , the umnsah / df-1 cell line . although the concentration of fowlpox virus was on the order of 1 - 2 log units less than the concentration of vaccinia virus , the minimum concentration of fowlpox virus produced by these cells was approximately 7 10 pfu / ml . the purities of the vaccinia and fowlpox viruses are comparable , as the virus harvesting methods were very similar to the methods described by tanabe and hotta . fowlpox virus and vaccine virus are both inactivated within 1 minute when using the following disinfectants : 70% ethanol , 50% isopropyl alcohol , 0.5% sodium hypochlorite , 30% formaldehyde , 10% benzalkonium chloride , a mixture of 6.67% cetyltrimethylammonium chloride and 3.33% benzalkonium chloride , and a mixture of 1.75% iodine and 10% polyethyleneglycol nonylphenyl ether . viral inactivation with minimal concentrations of the same chemicals was also examined in the present study , for the purpose of determining the inactivation kinetics of both vaccinia and fowlpox . to first principles , the inactivation curves for each virus and chemical appear similar ; however mathematical analysis of the data reveals distinct differences for vaccinia and fowlpox viruses treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol . first - order reactions were observed with vaccinia and fowlpox viruses treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.025% and 0.05% sodium hypochlorite , and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . vaccinia virus treated with 40% ethanol also showed a first - order reaction , while a second - order reaction was observed when the virus was treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether . however , further analysis of the unique properties of poxviruses suggests possible reasons for the observed second - order reaction of fowlpox virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol and the vaccinia virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether . based on the data and published research , it could be concluded that fowlpox possibly undergoes greater aggregation than vaccinia virus in both 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol , resulting in nongenetic reactivation and therefore , a second - order reaction . thus , the second - order reaction might be due to the fact that fowlpox virus particles in the center of a viral aggregate might be protected from the disinfectant and have the ability to reactive the genetically intact , but inactivated fowlpox virus particles . the rate constant , k , is calculated from the starting concentration of virus and the concentration of virus at time , t. the k values differed by no more than 0.7 1/s for the vaccinia and fowlpox viruses treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.025% sodium hypochlorite , 0.05% sodium hypochlorite , and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . however , the vaccinia and fowlpox viruses treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol show large differences in k values . although the k value is an important variable for comparison , for the purpose of this work , the virus half - life ( t1/2 ) value is a better indicator of simulant suitability . the purpose of this work is to determine if fowlpox is a suitable decontamination simulant for variola minor , thus the amount of time the virus will survive is more important than the rate of the reaction . based on the data presented in table 1 , we can conclude that fowlpox is a suitable decontamination simulant for variola major when using the following disinfectants at concentrations used for inactivation kinetics : 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.025% sodium hypochlorite , 0.05% sodium hypochlorite and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . the studies presented in this paper suggest that fowlpox is a suitable decontamination simulant for variola major when using the following full strength disinfectants : 70% ethanol , 50% isopropyl alcohol , 0.5% sodium hypochlorite , 30% formaldehyde , 10% benzalkonium chloride , a mixture of 6.67% cetyltrimethylammonium chloride and 3.33% benzalkonium chloride and a mixture of 1.75% iodine and 10% polyethyleneglycol nonylphenyl ether . fowlpox virus is also a suitable inactivation kinetics simulant when treated with the following chemicals : 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether , 0.025% sodium hypochlorite , 0.05% sodium hypochlorite and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride . fowlpox virus is not a suitable inactivation kinetics simulant for variola major when treated with the following chemicals : 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol . knowing the genetic and decontamination similarities of variola major and vaccinia from previous studies , fowlpox virus has been shown to be a suitable variola major simulant through identical decontamination results using vaccinia virus as the common link between the studies . the calculated h values for the vaccinia virus and fowlpox virus described in this paper fall into the range described kaplan , with the exception of vaccinia virus heated in brain heart infusion broth . the difference in h values in this paper with vaccinia virus in brain heart infusion broth might be due to the decreased virus aggregation , which might have affected the inactivation and plaque counts . hahon and kozikowski did not mention decreased aggregation in the variola major virus heated in brain heart infusion broth ; however , their variola major sample was stored in brain heart infusion broth , while the viruses used in this work were stored in dmem containing 10% fbs . the h values calculated for fowlpox virus in 0.85% saline , ph 4.5 , pbs , ph 7.4 , 10% skim milk , and brain heart infusion broth were in the range described for both vaccinia ( kaplan ) and variola major virus . these studies suggest that fowlpox can serve as a thermal stability simulant for variola major in 0.85% saline , ph 4.5 , pbs , ph 7.4 , 10% skim milk , and heart infusion broth at temperatures ranging from 40c to 55c .

phosphorylation of the smooth muscle 20 kda myosin light chain ( mlc ) regulates smooth muscle contraction or vascular tone ( 1 , 2 ) , and mlc phosphorylation is determined by the balance of the activities of mlc kinase and mlc phosphatase ( 3 ) . there are a number of important signaling pathways in smooth muscle that regulate vascular tone , and the vast majority influence the activity of mlc phosphatase ( 4 , 5 ) . the signaling pathways converging on mlc phosphatase either increase phosphatase activity to decrease mlc phosphorylation and force , which is referred to as ca desensitization , or decrease phosphatase activity to increase mlc phosphorylation and force , which is referred to as ca sensitization ( 4 ) . mlc phosphatase is a holoenzyme consisting of enzymatic , 20 kda and myosin targeting ( mypt1 ) subunits ( 5 ) . alternative mrna splicing of a central and a 3 ' exon produce a mypt1 central insert ( ci ) and a cooh - terminal leucine zipper ( lz ) , respectively ( 5 ) ; there are four mypt1 isoforms ( ci+lz+ , ci+lz- , ci - lz+ and ci - lz- ) , and mypt1 isoform expression is developmentally regulated , tissue specific ( 6,7,8 ) , and is modulated in disease ( 9,10,11,12,13 ) . the mypt1 lz domain is important for mediating ca desensitization during no mediated vasodilatation : during no / cgmp signaling , lz+ , but not lz- , mypt1 isoforms are phosphorylated by pkg ( 14 , 15 ) . therefore , lz+/lz- mypt1 isoform expression , in part , underlies the heterogenous response of the vasculature to no and no based vasodilators ( 14,15,16,17 ) . for g - protein coupled agonists , the stimulation of gq and g11 activates phospholipase c , and the subsequent increase in intracellular ca activates mlc kinase ( 4 ) . however , a number of agonists also activate rhoa / rho kinase through g12 and g13 ( 4 ) . rho kinase phosphorylates mypt1 at both thr696 and thr853 ( 18 ) , which decreases the activity of the mlc phosphatase ( 18,19,20,21,22 ) to produce ca sensitization ( 21 , 23 , 24 ) . however , similar to ca desensitization , there is variability in the response of the vasculature to agonists ; in smooth muscles , there is heterogeneity in the magnitude and sensitivity of ca sensitization ( 25 , 26 ) , and the mechanism to explain this variability is unknown . therefore , this study was designed to determine if mypt1 isoforms are differentially phosphorylated by rho kinase . to investigate this question , we determined the time course of mypt1 phosphorylation ( thr696 and thr853 ) following activation of rho kinase with gtps in hek293 t cells lines that express each of the mypt1 isoforms . for the present study , we used our human embryonic cell ( hek293 t ) lines that express the four avian mypt1 isoforms ( ci+lz+ , ci+lz- , ci - lz+ , ci - lz- ) . these hek293 t cell lines have the sv40 large t - antigen , which allows an episomal plasmid to be replicated , and the cloning and transfection techniques to generate these hek293 t cell lines have been previously described ( 15 ) . untransfected hek293 t cells expresses the four isoforms of human mypt1 ( 15 ) , and in the present experiments , the density of the ratio of ci+/ci- bands was 1.22x higher for the band containing the exogenous avian mypt1 isoform , which is consistent with an overexpression of the exogenous avian mypt1 isoform and our previous data ( 15 ) . the sequences of the avian and mammalian mypt1 are highly homologous and there are multiple regions of sequence identity ( 27 ) . rho kinase mediated mypt1 phosphorylation occurs at thr696 and thr853 of the mammalian sequence , which corresponds to thr695 and thr850 of the avian sequence . for consistency , we will refer to the numbering of the mammalian sequence throughout the manuscript . as previously described ( 15 ) , hek293 t cells lines were grown in p100 plates using 1x dulbecco 's modified eagle 's media ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) , 1% penicillin / streptomycin and 0.004% zeocin at 37 c with 5% co2 , and cells were split at 80100% confluence . to determine the time course of mypt1 phosphorylation , cells were grown to 7090% confluence , and then the hek293 t cells were starved for 24 h in low serum media ( 1% fbs , 1% penicillin / streptomycin and 0.004% zeocin ) at 37 c . as described in previous publications ( 28 ) , cells were placed in skinning solution ( 0.1% triton x-100 ) for 10 min and then the solution was changed to a low ca solution and the cells were treated at 37 c with 0.1 mm guanosine 5'-o-(3-thiotriphosphate ) tetralithium ( gtps ) . at 0 min , 1 min , 5 min and 30 min of gtps treatment , cells scraped off the plate , sonicated , spun and placed in fresh tubes , and stored at 80 c prior to immunoblotting . as we have described ( 15 ) , mypt1 phosphorylation was determined using phospho - specific antibodies and normalized to mypt1 expression . gels were run in pairs , one for mypt1 and the other for phospho - mypt1 . mypt1 was detected using a polyclonal anti - mypt1 antibody ( epitomics ) , while mypt1 phosphorylation at thr696 or thr853 was detected using phospho - specific antibodies to either phospho - thr696 or phospho - thr853 ( cell signaling ) . to determine relative thr696 and thr853 mypt1 phosphorylation , images were scanned ( epson perfection v750 pro ) and band density was determined using imagequant tl software ( ge healthcare ) . relative phosphorylation was computed as the density of the phosphorylated signal ( thr696 or thr853 ) divided by the density of the mypt1 protein band(s ) . the data for relative phosphorylation for both thr696 and thr853 were normalized . to normalize the data for each analysis , the density of the ratio of phospho - mypt1/mypt1 for the 30 min time point was averaged and all time points were subsequently normalized to this mean . the student 's t - test was used to determine the significance of the difference between the relative phosphorylation between the 0 and 30 min time points , and p<0.05 was considered significant . as previously described ( 15 ) , hek293 t cells lines were grown in p100 plates using 1x dulbecco 's modified eagle 's media ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) , 1% penicillin / streptomycin and 0.004% zeocin at 37 c with 5% co2 , and cells were split at 80100% confluence . to determine the time course of mypt1 phosphorylation , cells were grown to 7090% confluence , and then the hek293 t cells were starved for 24 h in low serum media ( 1% fbs , 1% penicillin / streptomycin and 0.004% zeocin ) at 37 c . as described in previous publications ( 28 ) , cells were placed in skinning solution ( 0.1% triton x-100 ) for 10 min and then the solution was changed to a low ca solution and the cells were treated at 37 c with 0.1 mm guanosine 5'-o-(3-thiotriphosphate ) tetralithium ( gtps ) . at 0 min , 1 min , 5 min and 30 min of gtps treatment , cells scraped off the plate , sonicated , spun and placed in fresh tubes , and stored at 80 c prior to immunoblotting . as we have described ( 15 ) , mypt1 phosphorylation was determined using phospho - specific antibodies and normalized to mypt1 expression . gels were run in pairs , one for mypt1 and the other for phospho - mypt1 . mypt1 was detected using a polyclonal anti - mypt1 antibody ( epitomics ) , while mypt1 phosphorylation at thr696 or thr853 was detected using phospho - specific antibodies to either phospho - thr696 or phospho - thr853 ( cell signaling ) . to determine relative thr696 and thr853 mypt1 phosphorylation , images were scanned ( epson perfection v750 pro ) and band density was determined using imagequant tl software ( ge healthcare ) . relative phosphorylation was computed as the density of the phosphorylated signal ( thr696 or thr853 ) divided by the density of the mypt1 protein band(s ) . the data for relative phosphorylation for both thr696 and thr853 were normalized . to normalize the data for each analysis , the density of the ratio of phospho - mypt1/mypt1 for the 30 min time point was averaged and all time points were subsequently normalized to this mean . the student 's t - test was used to determine the significance of the difference between the relative phosphorylation between the 0 and 30 min time points , and p<0.05 was considered significant . mypt1 phosphorylation at both thr853 and thr696 was detected prior to treatment with gtps . however , the signal representing phospho - thr853 was faint . for the hek293 t cells over expressing ci+ mypt1 isoforms ( ci+lz+mypt1 & ci+lz - mypt1 ) , gtps did not increase relative mypt1 phosphorylation at thr853 ( phospho - thr853/mypt1 ; table 1table 1 . relative thr853 mypt1 phosphorylationmypt1 isoform0 min30 minci+lz - mypt10.7 0.31.0 0.4ci+lz+mypt10.7 0.11.0 0.3ci - lz - mypt10.9 0.11.0 0.4ci - lz+mypt10.7 0.11.0 0.3following treatment with gtps , relative thr853 phosphorylation , computed as the density of both bands on the western blot in fig . 1 ( total phospho - thr853/total mypt1 ) did not change ( * , p<0.05 , n=46 ) . ( a ) western blots demonstrating time course of thr853 phosphorylation for ci+lz - mypt1 and ci+lz+mypt1 . ( b ) western blots demonstrating time course of thr853 phosphorylation for the ci - lz - mypt1 and ci - lz+mypt1 . relative total mypt1 thr853 phosphorylation ( total phospho - thr853/total mypt1 ) did not significantly change after gtps for any mypt1 isoform ( table 1 ) . ) . similarly , for the hek293 t cells expressing ci- mypt1 isoforms , there was no significant increase in relative thr853 mypt1 phosphorylation with gtps treatment ( table 1 , fig . following treatment with gtps , relative thr853 phosphorylation , computed as the density of both bands on the western blot in fig . 1 ( total phospho - thr853/total mypt1 ) did not change ( * , p<0.05 , n=46 ) . ( a ) western blots demonstrating time course of thr853 phosphorylation for ci+lz - mypt1 and ci+lz+mypt1 . ( b ) western blots demonstrating time course of thr853 phosphorylation for the ci - lz - mypt1 and ci - lz+mypt1 . relative total mypt1 thr853 phosphorylation ( total phospho - thr853/total mypt1 ) did not significantly change after gtps for any mypt1 isoform ( table 1 ) . in contrast to the results for thr853 mypt1 phosphorylation , the phospho - thr696 bands were readily apparent . ( a ) western blots demonstrating time course of thr696 phosphorylation for ci+lz - mypt1 and ci+lz+mypt1 . ( b ) western blots demonstrating time course of thr696 phosphorylation for the ci - lz - mypt1 and ci - lz+mypt1 . relative total mypt1 thr696 phosphorylation ( total phospho - thr696/total mypt1 ) increased after gtps for all mypt1 isoforms , except ci - lz+ mypt1 ( table 2 ) . , both bands ) . in the hek293 t cells expressing ci+ mypt1 isoforms ( ci+lz+mypt1 & ci+lz - mypt1 ) , there was a significant increase in total relative thr696 mypt1 phosphorylation following treatment with gtps ( table 2table 2 . relative thr696 mypt1 phosphorylationmypt1 isoform0 min30 minci+lz - mypt10.2 0.11.0 0.3*ci+lz+mypt10.6 0.21.0 0.3*ci - lz - mypt10.5 0.11.0 0.2*ci - lz+mypt10.8 0.21.0 0.2following treatment with gtps , relative thr696 phosphorylation , computed as the density of both bands on the western blot in fig . 2 ( total phospho - thr696/total mypt1 ) increased for every mypt1 isoform , except ci - lz+mypt1 ( * , p<0.05 , n=46 ) . note , the data in this table represent the results for both bands in fig . 2 , 3 is only for the single mypt1 band expressing the exogenous mypt1 isoform ( ci+ or ci- ) , which is indicated by the arrowhead . ) . however in the hek293 t cells expressing ci- mypt1 isoforms , the results were variable ; in hek293 t cells expressing ci - lz - mypt1 , gtps stimulation increased total relative mypt1 phosphorylation at thr696 , while in the hek293 t cells expressing ci - lz+mypt1 , total relative thr696 mypt1 phosphorylation did not change ( table 2 ) . ( a ) western blots demonstrating time course of thr696 phosphorylation for ci+lz - mypt1 and ci+lz+mypt1 . ( b ) western blots demonstrating time course of thr696 phosphorylation for the ci - lz - mypt1 and ci - lz+mypt1 . relative total mypt1 thr696 phosphorylation ( total phospho - thr696/total mypt1 ) increased after gtps for all mypt1 isoforms , except ci - lz+ mypt1 ( table 2 ) . following treatment with gtps , relative thr696 phosphorylation , computed as the density of both bands on the western blot in fig . 2 ( total phospho - thr696/total mypt1 ) increased for every mypt1 isoform , except ci - lz+mypt1 ( * , p<0.05 , n=46 ) . note , the data in this table represent the results for both bands in fig . 2 , while the time course of thr696 mypt1 phosphorylation in fig . 3 is only for the single mypt1 band expressing the exogenous mypt1 isoform ( ci+ or ci- ) , which is indicated by the arrowhead . to further define isoform specific mypt1 phosphorylation in response to gtps , we examined the time course of thr696 phosphorylation for only the single mypt1 band containing the exogenous mypt1 isoform ( indicated by the arrowhead in fig . 2 ) . for both lz- mypt1 isoforms ( ci+lz- & ci - lz- ) , gtps produced a significant increase in relative thr696 mypt1 phosphorylation ( fig . 3fig . the time course of relative thr696 mypt1 phosphorylation was computed for only the single band containing the exogenous mypt1 isoform ( ci+ or ci- ) , which is indicated by the arrowhead in fig 2 . ( a ) time course of relative thr696 phosphorylation for ci+lz - mypt1 ( , n=4 ) and ci+lz+mypt1 ( , n=4 ) . the increase in thr696 phosphorylation was significant for ci+lz- mypt1 ( p<0.05 ) , but not the ci+lz+mypt1 isoform . ( b ) time course of relative thr696 phosphorylation of ci - lz - mypt1 ( , n=6 ) and ci - lz+mypt1 ( , n=4 ) . there is a significant increase in thr696 phosphorylation for ci - lz- mypt1 ( p<0.05 ) and a significant decrease ( p<0.05 ) in thr696 phosphorylation for ci - lz+ mypt1 . the solid lines represent a single exponential fit of the time course of phosphorylation . ) . for the ci+lz+ mypt1 , 3a , p>0.05 ) , while for the ci - lz+ mypt1 isoform , relative thr696 phosphorylation decreased ( p<0.05 ) following gtps ( fig . the time course of relative thr696 mypt1 phosphorylation was computed for only the single band containing the exogenous mypt1 isoform ( ci+ or ci- ) , which is indicated by the arrowhead in fig 2 . ( a ) time course of relative thr696 phosphorylation for ci+lz - mypt1 ( , n=4 ) and ci+lz+mypt1 ( , n=4 ) . the increase in thr696 phosphorylation was significant for ci+lz- mypt1 ( p<0.05 ) , but not the ci+lz+mypt1 isoform . ( b ) time course of relative thr696 phosphorylation of ci - lz - mypt1 ( , n=6 ) and ci - lz+mypt1 ( , n=4 ) . there is a significant increase in thr696 phosphorylation for ci - lz- mypt1 ( p<0.05 ) and a significant decrease ( p<0.05 ) in thr696 phosphorylation for ci - lz+ mypt1 . the solid lines represent a single exponential fit of the time course of phosphorylation . in smooth muscle , in addition to mlc kinase ( 1 ) and rhoa / rho kinase ( 21 , 24 ) , there are multiple other signaling pathways that modulate force including pkc ( 29,30,31 ) , zip kinase ( 32 ) , integrin - linked kinase ( 33 ) and rac1 ( 34 ) , and further , the physiologically important signaling pathways that mediate ca sensitization are both agonist as well as tissue specific ( 4 , 35 ) . the rho kinase inhibitor fasudil reduces blood pressure in animal models of hypertension ( 36,37,38 ) and in humans , is effective in treating both cerebral vasospasm ( 39 , 40 ) and pulmonary hypertension ( 41 , 42 ) , which suggests that rho kinase mediated signaling is important in both health and disease . rho kinase has been demonstrated to phosphorylate both mypt1 as well as cpi-17 ( 43 , 44 ) , and similar to the other signaling pathways which influence mlc phosphatase activity , the importance of each of these proteins for ca sensitization is tissue specific ( 44 ) . in the present study , we examined whether gtps treatment results in isoform specific mypt1 phosphorylation . others have demonstrated rho kinase is expressed in hek293 t cells ( 45 , 46 ) and additionally , in hek293 t cells , gtps stimulates rho ( 47 , 48 ) . further , rho has been demonstrated to activate rho kinase , pkn and pip5 kinase ( 49 ) . therefore , the gtps stimulated increase in mypt1 phosphorylation could be mediated by rho kinase , pkn , pip5-kinase or another unknown kinase . however , only rho kinase and ilk have been demonstrated to phosphorylate mypt1 at thr696 and thr853 ( 50 ) , and ilk is not activated by rho kinase , pkn or pip5 kinase ( 51 ) . these data strongly suggest that following gtps treatment of hek293 t cells , mypt1 phosphorylation at thr696 and/or thr853 is mediated by a gtps induced activation of rho kinase . we have previously demonstrated that an overexpressed exogenous mypt1 isoform replaces the endogenous isoform in the mlc phosphatase holoenzyme ( 17 ) and alters the activity of mlc phosphatase ( 15 , 17 ) . similar to previous reports ( 15 , 17 ) , in the present experiments , the ratio of ci+/ci- changed consistent with overexpression of the exogenous mypt1 . these data suggest that mypt1 phosphorylation determined in these hek293 t cell lines reflects the phosphorylation of the exogenous mypt1 isoform ( 15 ) . however compared to our prior results , the expression of the exogenous avian mypt1 isoform is lower in the present study , which could result in an underestimation of the magnitude of the changes in mypt1 phosphorylation . in the present study , mypt1 phosphorylation at thr696 increased after treatment with gtps for the ci+lz - mypt1 and ci - lz - mypt1 isoforms ( fig . others have demonstrated that activation of pkg increases mypt1 phosphorylation at both ser695 and ser852 , which then inhibits rho kinase mediated mypt1 phosphorylation at thr696 and thr853 ( 52 , 53 ) . the lz domain is hypothesized to mediate the interaction of mypt1 and pkg ( 16 ) and is required for pkg mediated mypt1 phosphorylation ( 14 , 15 , 17 ) . these data could suggest that lz- mypt1 isoforms , but not lz+ mypt1 isoforms , are phosphorylated by rho kinase , which is consistent with our results . for the ci+lz+ mypt1 isoform , gtps treatment increased thr696 phosphorylation , but the increase did not reach statistical significance ( fig . 3 ) , which could suggest the ci domain modulates rho kinase phosphorylation . for the ci - lz+mypt1 isoform , it is unclear why relative thr696 phosphorylation decreased with gtps stimulation ( fig . one possibility is that rho kinase can not access thr696 due to differences in the three - dimensional structure of ci - lz+mypt1 and the other mypt1 isoforms , and thus for ci - lz+ mypt1 , thr696 is only subject to autodephosphorylation ( 54 ) . alternatively , this may be due to an interaction of ci - lz+mypt1 and another protein , such as m - rip ( 55 ) or prostate apoptosis response 4 ( 56 ) , which could alter the kinetics of rho kinase mediated mypt1 phosphorylation . in contrast to the signal for thr696 phosphorylation , the signal for phospho - thr853 mypt1 was low , and we could not detect a significant increase in relative thr853 mypt1 phosphorylation after gtps treatment ( table 1 , fig . these results contrast to the significant increase in mypt1 thr853 phosphorylation observed in smooth muscle ( 44 , 57 ) . the mechanism to explain the lack of thr853 phosphorylation in the hek293 t cells expressing avian mypt1 isoforms is beyond the scope of the present study . in our hek293 t cells , gtps could have activated a g - protein pathway that inactivated rho or even activated a kinase other than rho kinase . since gtps treatment increased phosphorylation at thr696 , a well documented mypt1 residue for rho kinase ( 4 ) , this possibility is unlikely . in isolated 500 aa mypt1 protein fragments , we have demonstrated that rho kinase does not phosphorylate thr853 ( 14 ) . however , in untransfected control hek293 t cells , we have previously demonstrated that the phosphorylation of the endogenous human ci+ mypt1 at both thr696 and thr853 ( 15 ) , which differs from the present results . these data could also suggest that for avian mypt1 , phospho - thr853 is poorly recognized by the anti - phosphothr853 antibody . however , there is a high degree of identity between the aa sequences of avian and mammalian mypt1 ( 27 ) ; 85% identity for the 60 aa flanking the thr850/thr853 phosphorylation site and we also detected a phospho - thr853 signal , albeit faint ( fig . similar to our results , there is also considerable variability in thr696 vs. thr853 mypt1 phosphorylation in mammalian smooth muscle ; mypt1 phosphorylation is both tissue and agonist dependent . studies have demonstrated that 1 ) a rho kinase mediated mypt1 phosphorylation at thr853 , but not thr696 , is responsible for the sustained phase of the force response in rat uterine smooth muscle ( 58 ) , 2 ) a rho kinase mediated increase in both thr696 and thr853 mypt1 phosphorylation occurs during serotonin induced vasoconstriction of cerebral arteries ( 59 ) , 3 ) a rho kinase mediated increase in mypt1 phosphorylation at thr853 , but not thr696 , contributes to the myogenic response of cerebral vessels ( 60 ) , 4 ) a rho kinase mediated increase in thr696 mypt1 phosphorylation occurs during ca sensitization of rat ileal smooth muscle ( 61 ) and 5 ) a rho kinase mediated increase in thr696 mypt1 phosphorylation is responsible for pgf2 induced ca sensitization in rabbit aorta ( 62 ) . additionally , during activation of mouse bladder smooth muscle , mypt1 phosphorylation at thr853 is mediated predominantly by rho kinase , while phosphorylation at thr696 did not change ( 63 ) . using mypt1 ala mutants , these investigators demonstrated that the increase in thr853 phosphorylation did not contribute to the force response , while the force maintenance was reduced in the ala696thr mypt1 mutant , suggesting that mypt1 phosphorylation at thr696 is important for the regulation of force maintenance ( 63 ) . our current results demonstrate that gtps treatment of hek293 t cells increased thr696 phosphorylation of ci+lz- and ci - lz- , but not ci+lz+ and ci - lz+ , mypt1 isoforms . these data suggest that following agonist activation , rho kinase will differentially phosphorylate mypt1 isoforms . we have previously demonstrated that mypt1 isoform expression determines the sensitivity of no mediated vasodilatation ( 14 , 15 , 17 ) ; our previous results show that smooth muscle tissues expressing lz- mypt1 isoforms ( ci+lz- , ci - lz- ) are not phosphorylated by pkg ( 14 , 15 ) . taken together , these results suggest that the lz mypt1 domain is an important determinant for the regulation of mlc phosphatase ; lz+ mypt1 isoforms are phosphorylated by pkg ( 14 , 15 ) , but poor substrates for rho kinase ( fig . 3 ) , while lz- mypt1 isoforms are phosphorylated by rho kinase ( fig . the ci domain appears to modulate rho kinase phosphorylation of lz+ mypt1 isoforms ; following gtps treatment mypt1 phosphorylation decreased for ci - lz+mypt1 , but did not change for ci+lz+mypt1 ( fig . 3 ) , which provides another mechanism to tune the vasculature 's response to vasoactive agents . these data demonstrate that alternative splicing to produce ci+/ci- and lz+/lz- mypt1 isoforms could contribute to the molecular mechanism producing the variable sensitivity of smooth muscle to signaling pathways for both ca sensitization ( rhoa / rho kinase ) and desensitization ( no / cgmp / pkg ) . changes in relative ci+/ci- mypt1 isoform expression have only been examined in an animal model of portal hypertension ( 10 ) , but relative lz+/lz- mypt1 isoform expression is well documented to decrease in a number of diseases including heart failure ( 9 , 11 , 64 , 65 ) and pulmonary hypertension ( 13 , 66 ) . therefore , modulation of mypt1 isoform expression may represent a mechanism to tune the vasculature 's response to both no / pkg and g - protein coupled agonists / rho kinase signaling , which will influence vascular tone and/or resistance in both health and disease .

agonist stimulation of smooth muscle is known to activate rhoa / rho kinase signaling , and rho kinase phosphorylates the myosin targeting subunit ( mypt1 ) of myosin light chain ( mlc ) phosphatase at thr696 and thr853 , which inhibits the activity of mlc phosphatase to produce a ca2 + independent increase in mlc phosphorylation and force ( ca2 + sensitization ) . alternative mrna splicing produces four mypt1 isoforms , which differ by the presence or absence of a central insert ( ci ) and leucine zipper ( lz ) . this study was designed to determine if rho kinase differentially phosphorylates mypt1 isoforms . in hek293 t cells expressing each of the four mypt1 isoforms , we could not detect a change in thr853 mypt1 phosphorylation following gtps treatment . however , there is differential phosphorylation of mypt1 isoforms at thr696 ; gtps treatment increases mypt1 phosphorylation for the ci+lz- and ci - lz- mypt1 isoforms , but not the ci+lz+ or ci - lz+ mypt1 isoforms . these data could suggest that in smooth muscle rho kinase differentially phosphorylates mypt1 isoforms .

Introduction Methods Cell culture Immunoblotting Results Discussion Conflict of Interest

phosphorylation of the smooth muscle 20 kda myosin light chain ( mlc ) regulates smooth muscle contraction or vascular tone ( 1 , 2 ) , and mlc phosphorylation is determined by the balance of the activities of mlc kinase and mlc phosphatase ( 3 ) . there are a number of important signaling pathways in smooth muscle that regulate vascular tone , and the vast majority influence the activity of mlc phosphatase ( 4 , 5 ) . the signaling pathways converging on mlc phosphatase either increase phosphatase activity to decrease mlc phosphorylation and force , which is referred to as ca desensitization , or decrease phosphatase activity to increase mlc phosphorylation and force , which is referred to as ca sensitization ( 4 ) . mlc phosphatase is a holoenzyme consisting of enzymatic , 20 kda and myosin targeting ( mypt1 ) subunits ( 5 ) . alternative mrna splicing of a central and a 3 ' exon produce a mypt1 central insert ( ci ) and a cooh - terminal leucine zipper ( lz ) , respectively ( 5 ) ; there are four mypt1 isoforms ( ci+lz+ , ci+lz- , ci - lz+ and ci - lz- ) , and mypt1 isoform expression is developmentally regulated , tissue specific ( 6,7,8 ) , and is modulated in disease ( 9,10,11,12,13 ) . the mypt1 lz domain is important for mediating ca desensitization during no mediated vasodilatation : during no / cgmp signaling , lz+ , but not lz- , mypt1 isoforms are phosphorylated by pkg ( 14 , 15 ) . however , a number of agonists also activate rhoa / rho kinase through g12 and g13 ( 4 ) . rho kinase phosphorylates mypt1 at both thr696 and thr853 ( 18 ) , which decreases the activity of the mlc phosphatase ( 18,19,20,21,22 ) to produce ca sensitization ( 21 , 23 , 24 ) . however , similar to ca desensitization , there is variability in the response of the vasculature to agonists ; in smooth muscles , there is heterogeneity in the magnitude and sensitivity of ca sensitization ( 25 , 26 ) , and the mechanism to explain this variability is unknown . therefore , this study was designed to determine if mypt1 isoforms are differentially phosphorylated by rho kinase . to investigate this question , we determined the time course of mypt1 phosphorylation ( thr696 and thr853 ) following activation of rho kinase with gtps in hek293 t cells lines that express each of the mypt1 isoforms . for the present study , we used our human embryonic cell ( hek293 t ) lines that express the four avian mypt1 isoforms ( ci+lz+ , ci+lz- , ci - lz+ , ci - lz- ) . these hek293 t cell lines have the sv40 large t - antigen , which allows an episomal plasmid to be replicated , and the cloning and transfection techniques to generate these hek293 t cell lines have been previously described ( 15 ) . untransfected hek293 t cells expresses the four isoforms of human mypt1 ( 15 ) , and in the present experiments , the density of the ratio of ci+/ci- bands was 1.22x higher for the band containing the exogenous avian mypt1 isoform , which is consistent with an overexpression of the exogenous avian mypt1 isoform and our previous data ( 15 ) . rho kinase mediated mypt1 phosphorylation occurs at thr696 and thr853 of the mammalian sequence , which corresponds to thr695 and thr850 of the avian sequence . to determine the time course of mypt1 phosphorylation , cells were grown to 7090% confluence , and then the hek293 t cells were starved for 24 h in low serum media ( 1% fbs , 1% penicillin / streptomycin and 0.004% zeocin ) at 37 c . to determine relative thr696 and thr853 mypt1 phosphorylation , images were scanned ( epson perfection v750 pro ) and band density was determined using imagequant tl software ( ge healthcare ) . to determine the time course of mypt1 phosphorylation , cells were grown to 7090% confluence , and then the hek293 t cells were starved for 24 h in low serum media ( 1% fbs , 1% penicillin / streptomycin and 0.004% zeocin ) at 37 c . to determine relative thr696 and thr853 mypt1 phosphorylation , images were scanned ( epson perfection v750 pro ) and band density was determined using imagequant tl software ( ge healthcare ) . the data for relative phosphorylation for both thr696 and thr853 were normalized . for the hek293 t cells over expressing ci+ mypt1 isoforms ( ci+lz+mypt1 & ci+lz - mypt1 ) , gtps did not increase relative mypt1 phosphorylation at thr853 ( phospho - thr853/mypt1 ; table 1table 1 . ( b ) western blots demonstrating time course of thr853 phosphorylation for the ci - lz - mypt1 and ci - lz+mypt1 . similarly , for the hek293 t cells expressing ci- mypt1 isoforms , there was no significant increase in relative thr853 mypt1 phosphorylation with gtps treatment ( table 1 , fig . ( b ) western blots demonstrating time course of thr853 phosphorylation for the ci - lz - mypt1 and ci - lz+mypt1 . ( b ) western blots demonstrating time course of thr696 phosphorylation for the ci - lz - mypt1 and ci - lz+mypt1 . relative total mypt1 thr696 phosphorylation ( total phospho - thr696/total mypt1 ) increased after gtps for all mypt1 isoforms , except ci - lz+ mypt1 ( table 2 ) . in the hek293 t cells expressing ci+ mypt1 isoforms ( ci+lz+mypt1 & ci+lz - mypt1 ) , there was a significant increase in total relative thr696 mypt1 phosphorylation following treatment with gtps ( table 2table 2 . however in the hek293 t cells expressing ci- mypt1 isoforms , the results were variable ; in hek293 t cells expressing ci - lz - mypt1 , gtps stimulation increased total relative mypt1 phosphorylation at thr696 , while in the hek293 t cells expressing ci - lz+mypt1 , total relative thr696 mypt1 phosphorylation did not change ( table 2 ) . ( b ) western blots demonstrating time course of thr696 phosphorylation for the ci - lz - mypt1 and ci - lz+mypt1 . relative total mypt1 thr696 phosphorylation ( total phospho - thr696/total mypt1 ) increased after gtps for all mypt1 isoforms , except ci - lz+ mypt1 ( table 2 ) . to further define isoform specific mypt1 phosphorylation in response to gtps , we examined the time course of thr696 phosphorylation for only the single mypt1 band containing the exogenous mypt1 isoform ( indicated by the arrowhead in fig . for both lz- mypt1 isoforms ( ci+lz- & ci - lz- ) , gtps produced a significant increase in relative thr696 mypt1 phosphorylation ( fig . the increase in thr696 phosphorylation was significant for ci+lz- mypt1 ( p<0.05 ) , but not the ci+lz+mypt1 isoform . ( b ) time course of relative thr696 phosphorylation of ci - lz - mypt1 ( , n=6 ) and ci - lz+mypt1 ( , n=4 ) . there is a significant increase in thr696 phosphorylation for ci - lz- mypt1 ( p<0.05 ) and a significant decrease ( p<0.05 ) in thr696 phosphorylation for ci - lz+ mypt1 . for the ci+lz+ mypt1 , 3a , p>0.05 ) , while for the ci - lz+ mypt1 isoform , relative thr696 phosphorylation decreased ( p<0.05 ) following gtps ( fig . the increase in thr696 phosphorylation was significant for ci+lz- mypt1 ( p<0.05 ) , but not the ci+lz+mypt1 isoform . ( b ) time course of relative thr696 phosphorylation of ci - lz - mypt1 ( , n=6 ) and ci - lz+mypt1 ( , n=4 ) . there is a significant increase in thr696 phosphorylation for ci - lz- mypt1 ( p<0.05 ) and a significant decrease ( p<0.05 ) in thr696 phosphorylation for ci - lz+ mypt1 . in smooth muscle , in addition to mlc kinase ( 1 ) and rhoa / rho kinase ( 21 , 24 ) , there are multiple other signaling pathways that modulate force including pkc ( 29,30,31 ) , zip kinase ( 32 ) , integrin - linked kinase ( 33 ) and rac1 ( 34 ) , and further , the physiologically important signaling pathways that mediate ca sensitization are both agonist as well as tissue specific ( 4 , 35 ) . rho kinase has been demonstrated to phosphorylate both mypt1 as well as cpi-17 ( 43 , 44 ) , and similar to the other signaling pathways which influence mlc phosphatase activity , the importance of each of these proteins for ca sensitization is tissue specific ( 44 ) . others have demonstrated rho kinase is expressed in hek293 t cells ( 45 , 46 ) and additionally , in hek293 t cells , gtps stimulates rho ( 47 , 48 ) . therefore , the gtps stimulated increase in mypt1 phosphorylation could be mediated by rho kinase , pkn , pip5-kinase or another unknown kinase . however , only rho kinase and ilk have been demonstrated to phosphorylate mypt1 at thr696 and thr853 ( 50 ) , and ilk is not activated by rho kinase , pkn or pip5 kinase ( 51 ) . these data strongly suggest that following gtps treatment of hek293 t cells , mypt1 phosphorylation at thr696 and/or thr853 is mediated by a gtps induced activation of rho kinase . we have previously demonstrated that an overexpressed exogenous mypt1 isoform replaces the endogenous isoform in the mlc phosphatase holoenzyme ( 17 ) and alters the activity of mlc phosphatase ( 15 , 17 ) . these data suggest that mypt1 phosphorylation determined in these hek293 t cell lines reflects the phosphorylation of the exogenous mypt1 isoform ( 15 ) . in the present study , mypt1 phosphorylation at thr696 increased after treatment with gtps for the ci+lz - mypt1 and ci - lz - mypt1 isoforms ( fig . others have demonstrated that activation of pkg increases mypt1 phosphorylation at both ser695 and ser852 , which then inhibits rho kinase mediated mypt1 phosphorylation at thr696 and thr853 ( 52 , 53 ) . these data could suggest that lz- mypt1 isoforms , but not lz+ mypt1 isoforms , are phosphorylated by rho kinase , which is consistent with our results . for the ci+lz+ mypt1 isoform , gtps treatment increased thr696 phosphorylation , but the increase did not reach statistical significance ( fig . one possibility is that rho kinase can not access thr696 due to differences in the three - dimensional structure of ci - lz+mypt1 and the other mypt1 isoforms , and thus for ci - lz+ mypt1 , thr696 is only subject to autodephosphorylation ( 54 ) . alternatively , this may be due to an interaction of ci - lz+mypt1 and another protein , such as m - rip ( 55 ) or prostate apoptosis response 4 ( 56 ) , which could alter the kinetics of rho kinase mediated mypt1 phosphorylation . in contrast to the signal for thr696 phosphorylation , the signal for phospho - thr853 mypt1 was low , and we could not detect a significant increase in relative thr853 mypt1 phosphorylation after gtps treatment ( table 1 , fig . the mechanism to explain the lack of thr853 phosphorylation in the hek293 t cells expressing avian mypt1 isoforms is beyond the scope of the present study . however , in untransfected control hek293 t cells , we have previously demonstrated that the phosphorylation of the endogenous human ci+ mypt1 at both thr696 and thr853 ( 15 ) , which differs from the present results . these data could also suggest that for avian mypt1 , phospho - thr853 is poorly recognized by the anti - phosphothr853 antibody . however , there is a high degree of identity between the aa sequences of avian and mammalian mypt1 ( 27 ) ; 85% identity for the 60 aa flanking the thr850/thr853 phosphorylation site and we also detected a phospho - thr853 signal , albeit faint ( fig . similar to our results , there is also considerable variability in thr696 vs. thr853 mypt1 phosphorylation in mammalian smooth muscle ; mypt1 phosphorylation is both tissue and agonist dependent . studies have demonstrated that 1 ) a rho kinase mediated mypt1 phosphorylation at thr853 , but not thr696 , is responsible for the sustained phase of the force response in rat uterine smooth muscle ( 58 ) , 2 ) a rho kinase mediated increase in both thr696 and thr853 mypt1 phosphorylation occurs during serotonin induced vasoconstriction of cerebral arteries ( 59 ) , 3 ) a rho kinase mediated increase in mypt1 phosphorylation at thr853 , but not thr696 , contributes to the myogenic response of cerebral vessels ( 60 ) , 4 ) a rho kinase mediated increase in thr696 mypt1 phosphorylation occurs during ca sensitization of rat ileal smooth muscle ( 61 ) and 5 ) a rho kinase mediated increase in thr696 mypt1 phosphorylation is responsible for pgf2 induced ca sensitization in rabbit aorta ( 62 ) . additionally , during activation of mouse bladder smooth muscle , mypt1 phosphorylation at thr853 is mediated predominantly by rho kinase , while phosphorylation at thr696 did not change ( 63 ) . using mypt1 ala mutants , these investigators demonstrated that the increase in thr853 phosphorylation did not contribute to the force response , while the force maintenance was reduced in the ala696thr mypt1 mutant , suggesting that mypt1 phosphorylation at thr696 is important for the regulation of force maintenance ( 63 ) . our current results demonstrate that gtps treatment of hek293 t cells increased thr696 phosphorylation of ci+lz- and ci - lz- , but not ci+lz+ and ci - lz+ , mypt1 isoforms . these data suggest that following agonist activation , rho kinase will differentially phosphorylate mypt1 isoforms . we have previously demonstrated that mypt1 isoform expression determines the sensitivity of no mediated vasodilatation ( 14 , 15 , 17 ) ; our previous results show that smooth muscle tissues expressing lz- mypt1 isoforms ( ci+lz- , ci - lz- ) are not phosphorylated by pkg ( 14 , 15 ) . taken together , these results suggest that the lz mypt1 domain is an important determinant for the regulation of mlc phosphatase ; lz+ mypt1 isoforms are phosphorylated by pkg ( 14 , 15 ) , but poor substrates for rho kinase ( fig . the ci domain appears to modulate rho kinase phosphorylation of lz+ mypt1 isoforms ; following gtps treatment mypt1 phosphorylation decreased for ci - lz+mypt1 , but did not change for ci+lz+mypt1 ( fig . these data demonstrate that alternative splicing to produce ci+/ci- and lz+/lz- mypt1 isoforms could contribute to the molecular mechanism producing the variable sensitivity of smooth muscle to signaling pathways for both ca sensitization ( rhoa / rho kinase ) and desensitization ( no / cgmp / pkg ) . therefore , modulation of mypt1 isoform expression may represent a mechanism to tune the vasculature 's response to both no / pkg and g - protein coupled agonists / rho kinase signaling , which will influence vascular tone and/or resistance in both health and disease .

phosphorylation of the smooth muscle 20 kda myosin light chain ( mlc ) regulates smooth muscle contraction or vascular tone ( 1 , 2 ) , and mlc phosphorylation is determined by the balance of the activities of mlc kinase and mlc phosphatase ( 3 ) . there are a number of important signaling pathways in smooth muscle that regulate vascular tone , and the vast majority influence the activity of mlc phosphatase ( 4 , 5 ) . the signaling pathways converging on mlc phosphatase either increase phosphatase activity to decrease mlc phosphorylation and force , which is referred to as ca desensitization , or decrease phosphatase activity to increase mlc phosphorylation and force , which is referred to as ca sensitization ( 4 ) . alternative mrna splicing of a central and a 3 ' exon produce a mypt1 central insert ( ci ) and a cooh - terminal leucine zipper ( lz ) , respectively ( 5 ) ; there are four mypt1 isoforms ( ci+lz+ , ci+lz- , ci - lz+ and ci - lz- ) , and mypt1 isoform expression is developmentally regulated , tissue specific ( 6,7,8 ) , and is modulated in disease ( 9,10,11,12,13 ) . therefore , lz+/lz- mypt1 isoform expression , in part , underlies the heterogenous response of the vasculature to no and no based vasodilators ( 14,15,16,17 ) . for g - protein coupled agonists , the stimulation of gq and g11 activates phospholipase c , and the subsequent increase in intracellular ca activates mlc kinase ( 4 ) . however , similar to ca desensitization , there is variability in the response of the vasculature to agonists ; in smooth muscles , there is heterogeneity in the magnitude and sensitivity of ca sensitization ( 25 , 26 ) , and the mechanism to explain this variability is unknown . to investigate this question , we determined the time course of mypt1 phosphorylation ( thr696 and thr853 ) following activation of rho kinase with gtps in hek293 t cells lines that express each of the mypt1 isoforms . for the present study , we used our human embryonic cell ( hek293 t ) lines that express the four avian mypt1 isoforms ( ci+lz+ , ci+lz- , ci - lz+ , ci - lz- ) . these hek293 t cell lines have the sv40 large t - antigen , which allows an episomal plasmid to be replicated , and the cloning and transfection techniques to generate these hek293 t cell lines have been previously described ( 15 ) . untransfected hek293 t cells expresses the four isoforms of human mypt1 ( 15 ) , and in the present experiments , the density of the ratio of ci+/ci- bands was 1.22x higher for the band containing the exogenous avian mypt1 isoform , which is consistent with an overexpression of the exogenous avian mypt1 isoform and our previous data ( 15 ) . the sequences of the avian and mammalian mypt1 are highly homologous and there are multiple regions of sequence identity ( 27 ) . as previously described ( 15 ) , hek293 t cells lines were grown in p100 plates using 1x dulbecco 's modified eagle 's media ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) , 1% penicillin / streptomycin and 0.004% zeocin at 37 c with 5% co2 , and cells were split at 80100% confluence . to determine the time course of mypt1 phosphorylation , cells were grown to 7090% confluence , and then the hek293 t cells were starved for 24 h in low serum media ( 1% fbs , 1% penicillin / streptomycin and 0.004% zeocin ) at 37 c . as described in previous publications ( 28 ) , cells were placed in skinning solution ( 0.1% triton x-100 ) for 10 min and then the solution was changed to a low ca solution and the cells were treated at 37 c with 0.1 mm guanosine 5'-o-(3-thiotriphosphate ) tetralithium ( gtps ) . to normalize the data for each analysis , the density of the ratio of phospho - mypt1/mypt1 for the 30 min time point was averaged and all time points were subsequently normalized to this mean . the student 's t - test was used to determine the significance of the difference between the relative phosphorylation between the 0 and 30 min time points , and p<0.05 was considered significant . as previously described ( 15 ) , hek293 t cells lines were grown in p100 plates using 1x dulbecco 's modified eagle 's media ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) , 1% penicillin / streptomycin and 0.004% zeocin at 37 c with 5% co2 , and cells were split at 80100% confluence . to determine the time course of mypt1 phosphorylation , cells were grown to 7090% confluence , and then the hek293 t cells were starved for 24 h in low serum media ( 1% fbs , 1% penicillin / streptomycin and 0.004% zeocin ) at 37 c . as described in previous publications ( 28 ) , cells were placed in skinning solution ( 0.1% triton x-100 ) for 10 min and then the solution was changed to a low ca solution and the cells were treated at 37 c with 0.1 mm guanosine 5'-o-(3-thiotriphosphate ) tetralithium ( gtps ) . to normalize the data for each analysis , the density of the ratio of phospho - mypt1/mypt1 for the 30 min time point was averaged and all time points were subsequently normalized to this mean . the student 's t - test was used to determine the significance of the difference between the relative phosphorylation between the 0 and 30 min time points , and p<0.05 was considered significant . relative thr853 mypt1 phosphorylationmypt1 isoform0 min30 minci+lz - mypt10.7 0.31.0 0.4ci+lz+mypt10.7 0.11.0 0.3ci - lz - mypt10.9 0.11.0 0.4ci - lz+mypt10.7 0.11.0 0.3following treatment with gtps , relative thr853 phosphorylation , computed as the density of both bands on the western blot in fig . similarly , for the hek293 t cells expressing ci- mypt1 isoforms , there was no significant increase in relative thr853 mypt1 phosphorylation with gtps treatment ( table 1 , fig . following treatment with gtps , relative thr853 phosphorylation , computed as the density of both bands on the western blot in fig . in the hek293 t cells expressing ci+ mypt1 isoforms ( ci+lz+mypt1 & ci+lz - mypt1 ) , there was a significant increase in total relative thr696 mypt1 phosphorylation following treatment with gtps ( table 2table 2 . relative thr696 mypt1 phosphorylationmypt1 isoform0 min30 minci+lz - mypt10.2 0.11.0 0.3*ci+lz+mypt10.6 0.21.0 0.3*ci - lz - mypt10.5 0.11.0 0.2*ci - lz+mypt10.8 0.21.0 0.2following treatment with gtps , relative thr696 phosphorylation , computed as the density of both bands on the western blot in fig . however in the hek293 t cells expressing ci- mypt1 isoforms , the results were variable ; in hek293 t cells expressing ci - lz - mypt1 , gtps stimulation increased total relative mypt1 phosphorylation at thr696 , while in the hek293 t cells expressing ci - lz+mypt1 , total relative thr696 mypt1 phosphorylation did not change ( table 2 ) . following treatment with gtps , relative thr696 phosphorylation , computed as the density of both bands on the western blot in fig . note , the data in this table represent the results for both bands in fig . to further define isoform specific mypt1 phosphorylation in response to gtps , we examined the time course of thr696 phosphorylation for only the single mypt1 band containing the exogenous mypt1 isoform ( indicated by the arrowhead in fig . the time course of relative thr696 mypt1 phosphorylation was computed for only the single band containing the exogenous mypt1 isoform ( ci+ or ci- ) , which is indicated by the arrowhead in fig 2 . ( b ) time course of relative thr696 phosphorylation of ci - lz - mypt1 ( , n=6 ) and ci - lz+mypt1 ( , n=4 ) . there is a significant increase in thr696 phosphorylation for ci - lz- mypt1 ( p<0.05 ) and a significant decrease ( p<0.05 ) in thr696 phosphorylation for ci - lz+ mypt1 . for the ci+lz+ mypt1 , 3a , p>0.05 ) , while for the ci - lz+ mypt1 isoform , relative thr696 phosphorylation decreased ( p<0.05 ) following gtps ( fig . the time course of relative thr696 mypt1 phosphorylation was computed for only the single band containing the exogenous mypt1 isoform ( ci+ or ci- ) , which is indicated by the arrowhead in fig 2 . there is a significant increase in thr696 phosphorylation for ci - lz- mypt1 ( p<0.05 ) and a significant decrease ( p<0.05 ) in thr696 phosphorylation for ci - lz+ mypt1 . in smooth muscle , in addition to mlc kinase ( 1 ) and rhoa / rho kinase ( 21 , 24 ) , there are multiple other signaling pathways that modulate force including pkc ( 29,30,31 ) , zip kinase ( 32 ) , integrin - linked kinase ( 33 ) and rac1 ( 34 ) , and further , the physiologically important signaling pathways that mediate ca sensitization are both agonist as well as tissue specific ( 4 , 35 ) . the rho kinase inhibitor fasudil reduces blood pressure in animal models of hypertension ( 36,37,38 ) and in humans , is effective in treating both cerebral vasospasm ( 39 , 40 ) and pulmonary hypertension ( 41 , 42 ) , which suggests that rho kinase mediated signaling is important in both health and disease . rho kinase has been demonstrated to phosphorylate both mypt1 as well as cpi-17 ( 43 , 44 ) , and similar to the other signaling pathways which influence mlc phosphatase activity , the importance of each of these proteins for ca sensitization is tissue specific ( 44 ) . in the present study , we examined whether gtps treatment results in isoform specific mypt1 phosphorylation . therefore , the gtps stimulated increase in mypt1 phosphorylation could be mediated by rho kinase , pkn , pip5-kinase or another unknown kinase . these data strongly suggest that following gtps treatment of hek293 t cells , mypt1 phosphorylation at thr696 and/or thr853 is mediated by a gtps induced activation of rho kinase . similar to previous reports ( 15 , 17 ) , in the present experiments , the ratio of ci+/ci- changed consistent with overexpression of the exogenous mypt1 . however compared to our prior results , the expression of the exogenous avian mypt1 isoform is lower in the present study , which could result in an underestimation of the magnitude of the changes in mypt1 phosphorylation . in the present study , mypt1 phosphorylation at thr696 increased after treatment with gtps for the ci+lz - mypt1 and ci - lz - mypt1 isoforms ( fig . the lz domain is hypothesized to mediate the interaction of mypt1 and pkg ( 16 ) and is required for pkg mediated mypt1 phosphorylation ( 14 , 15 , 17 ) . one possibility is that rho kinase can not access thr696 due to differences in the three - dimensional structure of ci - lz+mypt1 and the other mypt1 isoforms , and thus for ci - lz+ mypt1 , thr696 is only subject to autodephosphorylation ( 54 ) . alternatively , this may be due to an interaction of ci - lz+mypt1 and another protein , such as m - rip ( 55 ) or prostate apoptosis response 4 ( 56 ) , which could alter the kinetics of rho kinase mediated mypt1 phosphorylation . in contrast to the signal for thr696 phosphorylation , the signal for phospho - thr853 mypt1 was low , and we could not detect a significant increase in relative thr853 mypt1 phosphorylation after gtps treatment ( table 1 , fig . however , in untransfected control hek293 t cells , we have previously demonstrated that the phosphorylation of the endogenous human ci+ mypt1 at both thr696 and thr853 ( 15 ) , which differs from the present results . however , there is a high degree of identity between the aa sequences of avian and mammalian mypt1 ( 27 ) ; 85% identity for the 60 aa flanking the thr850/thr853 phosphorylation site and we also detected a phospho - thr853 signal , albeit faint ( fig . studies have demonstrated that 1 ) a rho kinase mediated mypt1 phosphorylation at thr853 , but not thr696 , is responsible for the sustained phase of the force response in rat uterine smooth muscle ( 58 ) , 2 ) a rho kinase mediated increase in both thr696 and thr853 mypt1 phosphorylation occurs during serotonin induced vasoconstriction of cerebral arteries ( 59 ) , 3 ) a rho kinase mediated increase in mypt1 phosphorylation at thr853 , but not thr696 , contributes to the myogenic response of cerebral vessels ( 60 ) , 4 ) a rho kinase mediated increase in thr696 mypt1 phosphorylation occurs during ca sensitization of rat ileal smooth muscle ( 61 ) and 5 ) a rho kinase mediated increase in thr696 mypt1 phosphorylation is responsible for pgf2 induced ca sensitization in rabbit aorta ( 62 ) . using mypt1 ala mutants , these investigators demonstrated that the increase in thr853 phosphorylation did not contribute to the force response , while the force maintenance was reduced in the ala696thr mypt1 mutant , suggesting that mypt1 phosphorylation at thr696 is important for the regulation of force maintenance ( 63 ) . our current results demonstrate that gtps treatment of hek293 t cells increased thr696 phosphorylation of ci+lz- and ci - lz- , but not ci+lz+ and ci - lz+ , mypt1 isoforms . we have previously demonstrated that mypt1 isoform expression determines the sensitivity of no mediated vasodilatation ( 14 , 15 , 17 ) ; our previous results show that smooth muscle tissues expressing lz- mypt1 isoforms ( ci+lz- , ci - lz- ) are not phosphorylated by pkg ( 14 , 15 ) . taken together , these results suggest that the lz mypt1 domain is an important determinant for the regulation of mlc phosphatase ; lz+ mypt1 isoforms are phosphorylated by pkg ( 14 , 15 ) , but poor substrates for rho kinase ( fig . the ci domain appears to modulate rho kinase phosphorylation of lz+ mypt1 isoforms ; following gtps treatment mypt1 phosphorylation decreased for ci - lz+mypt1 , but did not change for ci+lz+mypt1 ( fig . these data demonstrate that alternative splicing to produce ci+/ci- and lz+/lz- mypt1 isoforms could contribute to the molecular mechanism producing the variable sensitivity of smooth muscle to signaling pathways for both ca sensitization ( rhoa / rho kinase ) and desensitization ( no / cgmp / pkg ) . changes in relative ci+/ci- mypt1 isoform expression have only been examined in an animal model of portal hypertension ( 10 ) , but relative lz+/lz- mypt1 isoform expression is well documented to decrease in a number of diseases including heart failure ( 9 , 11 , 64 , 65 ) and pulmonary hypertension ( 13 , 66 ) . therefore , modulation of mypt1 isoform expression may represent a mechanism to tune the vasculature 's response to both no / pkg and g - protein coupled agonists / rho kinase signaling , which will influence vascular tone and/or resistance in both health and disease .

the chemokine receptor cxcr3 is a class a seven - transmembrane - domain or g protein - coupled receptor ( gpcr ) that is involved primarily in chemotaxis of certain immune cells , inhibition of angiogenesis , and th1 cell polarization [ 13 ] . cxcr3 is expressed by various effector t lymphocytes , including cd4 t helper 1 ( th1 ) cells , cd8 cytotoxic t lymphocytes ( ctl ) , and cd4 and cd8 memory t cells , as well as monocytes , m1 macrophages , natural killer ( nk ) cells , subsets of b - cells , mast cells , endothelial cells , and vascular smooth muscle cells [ 14 ] . cxcr3 couples to gi protein [ 5 , 6 ] and although not extensively studied , it has been shown to activate a number of signaling pathways that are generally associated with gpcrs such as increases in intracellular calcium and activation of map kinases and pi3k / akt signaling [ 4 , 7 , 8 ] . the principal agonists of cxcr3 are cxcl9 ( mig ) , cxcl10 ( ip-10 ) , and cxcl11 ( i - tac ) . the human equivalent of the murine form of cxcr3 is cxcr3a and unless noted otherwise cxcr3 is used in this review to include both murine and human isoforms . cxcr3b , which couples to gs , is the receptor isoform expressed in microvascular endothelial cells and is linked to inhibition of angiogenesis and induction of apoptosis [ 2 , 3 ] . besides cxcl9 , cxcl10 , and cxcl11 , cxcr3b and cxcr3 are activated by cxcl4 and cxcl4l1 , chemokines that are released by platelets and have been implicated in atherogenesis and acute coronary syndrome [ 3 , 9 , 10 ] . cxcr3-alt is a truncated form of cxcr3 that is selectively activated by cxcl11 [ 24 ] . cxcr3 is associated with the pathophysiology of th1-type diseases , including infections of various etiologies and autoimmune disorders [ 1 , 3 ] . although cxcr3 is activated by cxcl9 , cxcl10 , and cxcl11 , the outcome is different with growing evidence that cxcl9 and cxcl10 are essentially proinflammatory , while cxcl11 has anti - inflammatory actions [ 11 , 12 ] . over the last decade , numerous studies have documented elevated circulating levels of cxcl10 in wide - ranging infectious and autoimmune diseases , autoimmune encephalomyelitis , crohn 's disease , tuberculosis , thyroid autoimmune diseases , and type 1 diabetes , as well as several cancers [ 1318 ] . recent evidence from us and others has revealed the importance of the cxcl10/cxcr3 axis in cardiovascular diseases . as discussed elsewhere , cxcl9 and cxcl10 in addition , the homing signature for memory t cells to the heart from mediastinal lymph nodes is c - metccr4cxcr3 . while c - met triggering supports cardiotropic t cell recirculation , cxcr3 and ccr4 engagement via tissue - released cxcl10 and ccl4 , respectively , sustains recruitment in heart inflammation , we present an overview of the role of cxcl9 and cxcl10 in infectious and noninfectious diseases of the heart and its implications for immunotherapy . recently , zohar et al . showed that cxcl9 and cxcl10 drive effector th1/th17 cell polarization via stat1 , stat4 , and stat5 activation , thereby promoting inflammation . in contrast , cxcl11 , which exhibits relatively higher binding affinity for cxcr3 , drives development of foxp3 ( forkhead box p3)-negative il-10 t regulatory 1 ( tr1 ) cells and il-4 th2 cells via stat3 and stat6 activation and was demonstrated to dampen inflammation . the opposite actions of the cxcr3 agonists are likely the consequence of the biased signaling that is a fixture of gpcrs , which can activate both g protein - dependent and protein - independent signaling cascades , the latter occurring via -arrestin 2 recruitment [ 1 , 11 , 12 ] . biased allosteric agonists of cxcr3 that selectively activate -arrestin or g protein - dependent signaling are in development and may have utility in immunotherapy . coronary artery disease ( cad ) , which progresses to coronary heart disease , is a leading cause of death in the usa and globally [ 23 , 24 ] . cad is caused by atherosclerosis within the arteries of the heart , a chronic inflammatory condition associated with waxy plaque buildup . cxcr3 expressing monocytes / macrophages , th1 cells , nk cells , and ctl cells play a critical role in atheromatous plaque progression and eventual disruption . ruptured or ulcerated plaques cause formation of a thrombus that precipitates an acute coronary syndrome , such as unstable angina or a heart attack . endothelial dysfunction , increased vascular permeability , increased expression of adhesion molecules on endothelial cells for leukocytes , and increased plasma levels of low density lipoprotein ( ldl ) are initiating factors in atherosclerosis . ldl , which accumulates in the intima , undergoes oxidation by macrophages and endothelial cells , as well as by vsmc that migrate into the intima from the media and proliferate . in response to the oxidized ldl and plasma ldl , endothelial cells secrete proinflammatory cytokines and chemokines ( mcp-1/ccl2 , fractalkine , and cxcr3 ligands ) that attract monocytes , which differentiate into dendritic cells or macrophages that accumulate oxidized ldl to become foam cells . t cells are recruited into the intima , and dendritic and nk cells help induce the cd4 th1 phenotype , which is the most abundant t cell population in human atherosclerotic plaques . th1 cells , as do nk cells , produce ifn- , which contributes to th1 polarization , activates proinflammatory m1 macrophages , and induces apoptosis . the atheromatous plaque that builds up in the artery wall is made up of an accumulation of lipids , fibrous connective tissue , macrophages , and cellular debris that arises from the cytolytic actions of oxidized ldl , nk cells , ifn- , and ctl cells on macrophages , foam cells , vsmc , and endothelial cells . a fibrous coat of extracellular matrix proteins produced by vsmc stabilizes the plaque , but proinflammatory m1 macrophages secrete metalloproteinases in response to ifn- that degrade the fibrous cap and enhance its vulnerability to rupture . cxcl10 is reported to be expressed by endothelial cells , smooth muscle cells , and macrophages during the formation of atherosclerotic lesions in both preclinical and clinical studies [ 28 , 29 ] . suppression of cxcl10 bioactivity in apo - e deficient mice resulted in a more stable plaque phenotype with less macrophage activation , along with more smooth muscle cells and collagen abundance . the mechanistic role of cxcl10 in the pathogenesis of atherosclerotic plaque growth and destabilization is not yet resolved . of note , unstable plaques have increased levels of th1 , nk , and ctl cells and decreased levels of anti - inflammatory regulatory t ( treg ) cells . recent studies show that the relative levels of treg cells are reduced and their functionality is impaired in patients with cad [ 32 , 33 ] . knockout of cxcl10 in the apolipoprotein e - deficient mouse model of atherosclerosis was associated with increased treg cell numbers and activity , along with a reduction in lesion formation . circulating levels of cxcl10 notably , cxcl10 was also reported to be produced by the endothelium of mouse coronary blood vessels infused with angiotensin ii , human coronary artery endothelial cells treated with tnf- , and rat cardiac microvascular endothelial cells subjected to hypoxia / ischemia . patients with acute myocardial infarction ( ami ) showed significantly higher serum levels of cxcl10 than control subjects and patients with stable angina pectoris . although serum cxcl10 levels were negatively correlated with infarct size , these results in terms of pathogenic implications and determining cause versus effect relationships have limitations . first , during ami there is a massive systemic inflammatory insult in which cxcl10 levels are expected to be high . it would be interesting to test blood concentration of cxcl10 within the first 3 hours after angina onset during ami when systemic activation is not yet started secondly , the pathogenic mechanisms of plaque rupture may involve factors acting locally without necessarily showing a high systemic blood concentration . it would be interesting to analyze cxcl10 in samples of blood obtained by thrombus - aspiration during coronary artery percutaneous intervention ( pci ) in patients with unstable coronary artery disease . in patients with first - time st - segment elevation ami , high circulating levels of ccl4 , cxcl16 , cxcl8 , and cxcl10 within the first week after pci were found to be positively correlated with the degree of myocardial damage . kawasaki disease is an autoimmune disease that manifests as a systemic vasculitis with a predilection for coronary arteries . the disease occurs in children under 5 years of age and a preexisting viral infection may have a role in its development . during the acute phase of kawasaki disease the immune system is highly activated and includes both th1 and th2 subsets . recently , ko et al . reported that cxcl10 is a good biomarker / predictor of kawasaki disease and , furthermore , that cxcr3 is activated in the t cells of patients with acute kawasaki disease . in addition , several studies report that numbers and functionality of treg cells are reduced in kawasaki patients [ 4548 ] . myocarditis or inflammation of the myocardium is a heterogeneous group of disorders initiated by various pathogens , including worms , bacteria , protozoa , rickettsia , and most commonly viruses . autoimmunity after viral myocarditis is thought to cause dilated cardiomyopathy , which is characterized by ventricular dilation and contractile dysfunction [ 50 , 51 ] . cxcl10 is elevated in the heart following viral and nonviral infection and has the characteristics of a biomarker in rodent models of myocarditis [ 52 , 53 ] . reported findings showing that cxcl10 contributes to the pathogenesis of viral myocarditis . in their study , myocarditis was induced with coxsackievirus b3 ( cvb3 ) , the primary cause of viral myocarditis , in mice that overexpressed a cxcl10 mutant protein without functional activity in order to antagonize endogenous cxcl10 . these mice exhibited ameliorated disease progression , including reduced cardiac thickening ( due to inflammatory edema ) , inflammation , and cell death , as well as improved survival when compared to wild - type mice . the authors concluded that cxcl10 plays a crucial role in recruitment of th1 cells to the heart , leading to the increase in detrimental proinflammatory th1 cytokines . these findings have implications for interferon treatment , which is beneficial for some forms of viral myocarditis . following cvb3 infection , the rise in ifn- stimulates cxcl10 expression in cardiac myocytes and other cardiac cells . yuan et al . reported that cxcl10 inhibits cvb3 replication at early stage of infection , consequently protecting cardiac myocytes from damage and improving heart function . this antiviral activity of cxcl10 entails the regulation of natural killer ( nk ) cell infiltration into the myocardium and associated ifn- expression . however , the transient antiviral effect of cxcl10 was shown to be insufficient for viral clearance and in preventing death during acute inflammation stages in their mouse model . other chemokines or cytokines were proposed to play an important role in clearance of viruses . no simple explanation seems to explain the disparate findings of yuan et al . and yue et al . on whether myocardial cxcl10 is harmful or beneficial in the context of acute cvb3 myocarditis , although timing and dosage levels of cxcl10 and effective viral clearance versus th1 recruitment are likely contributing factors . evidence indicates the major contribution of autoimmunity to the etiology of myocarditis and thus adoptive transfer of tregs and/or stimulating their differentiation are promising therapeutic approaches [ 49 , 50 ] . both th1 and th17 cells drive myocarditis , with th17 cells playing an important part in the development of dilated cardiomyopathy . chagas disease is a tropical disease that results from infection with the protozoan parasite trypanosoma cruzi and affects ~10 million individuals worldwide but is most prevalent in latin america [ 58 , 59 ] . in some , 2030% , of infected individuals , chronic infection leads to a potentially fatal cardiomyopathy known as chagas heart disease , generally 1020 years after the initial infection [ 60 , 61 ] . chagas heart disease is characterized by marked inflammation and fibrosis of the heart , along with cardiac edema , myofibrillar destruction , chamber dilation , and loss of contractile function . the etiology of chagas heart disease is not fully understood and likely multifactorial , with a contribution of an autoimmune response due in part to molecular mimicry between antigenic determinants of t. cruzi and human ( cardiac ) antigens [ 62 , 63 ] . with heart failure in chagas disease , myocardial levels of cd8 and cd4 t cells are increased , with a predominance of cd8 t cells [ 61 , 64 , 65 ] . in addition , the myocardium exhibits a strong th1 cytokine profile with increased expression of ifn- and il-18 genes that correlate with ventricular dilation [ 64 , 65 ] . circulating levels of ifn- are elevated during chronic chagas disease and were reported to be inversely correlated to left ventricular ejection fraction ( lvef ) . in contrast , chagas - related heart failure is associated with reduced myocardial levels of treg cells [ 61 , 64 ] , and circulating treg activity was reported to be reduced in moderate or severe cardiomyopathy with activity directly correlated to lvef . were detected in patients with chronic chagas disease , and lv mrna expression levels of cxcl10 were found to be elevated in patients with chagas cardiomyopathy . recently , evidence was provided that polymorphisms in the cxcl9 and cxcl10 genes controlled the expression of chemokines in the myocardium and the degree of myocarditis in chagas cardiomyopathy . behet 's disease is an autoimmune or autoinflammatory disorder common in the middle east , asia , and japan . the basis for the pathogenesis of behet 's disease is not known , although a number of factors have been proposed to have a role , including viral , bacterial , environmental , genetic , and immune factors . behet 's is caused by small - vessel systemic vasculitis that very often affects the heart in diverse ways , including endomyocardial fibrosis , intracardiac thrombus , endocarditis , pericarditis , myocarditis , coronary arteritis , myocardial infarction , and valvular disease . the cardiomyopathy may be ischemic , nonischemic , or inflammatory in nature and may manifest as asymptomatic systolic or diastolic dysfunction or overt systolic or diastolic heart failure . recently , monocytes of behet 's patients were found to have dysfunctional posttranscriptional regulation of cxcl10 mrna that resulted in overexpression of cxcl10 protein with ifn- stimulation . in general , the role of cxcl10 in immune - mediated and autoimmune myocarditis is little studied ; however , based on studies of infective myocarditis , a critical role for cxcl10 is likely . after cad , hypertension is the most common risk factor for heart failure and accounts for ~25% of heart failure cases . in the elderly , as many as 68% of heart failure cases are linked to hypertension and community - based studies indicate that hypertension contributes to heart failure in 60% of patients . hypertension causes a number of adverse remodeling events at the cellular and tissue level of the heart , including cardiac myocyte hypertrophy and gene reprograming , activation of cardiac fibroblasts , interstitial and perivascular fibrosis , and capillary refraction [ 7375 ] . these alterations ultimately cause marked changes in the overall geometry of the heart that may progress to heart failure and the inability of the heart to adequately meet the oxygen and energy demands of the body . heart failure ( hf ) may manifest clinically with either preserved or reduced lvef , which are designated hfpef ( so - called diastolic heart failure ) and hfref ( systolic heart failure ) , respectively . hypertension results in concentric lv hypertrophy , which may progress to ventricular dilation and eventual hfref because of poorly understood means that may include ischemic injury [ 76 , 77 ] . related to this , volume overload due to fluid retention and impaired kidney function may cause a dilated pattern of eccentric lv hypertrophy with hypertension that leads to hfref . generally , cardiac remodeling with hypertension reflects a combination of both concentric and eccentric patterns of remodeling . concentric hypertrophy is also a characteristic of hfpef , which typically has hypertension as the major comorbidity . in addition , microvascular dysfunction concomitant to hypertension is thought to be a contributing factor for hfpef . the relative importance of cxcl10 in concentric versus eccentric lv hypertrophy , as well as their progression to heart failure , is not known . numerous preclinical and clinical studies have implicated marked activation of neurohormonal drive to the heart in the pathoetiology of lv hypertrophy and its progression to heart failure . neurohormonal drive , namely , activation of the sympathetic and renin - angiotensin - aldosterone systems , is generally thought to directly cause adverse remodeling of the heart . at the same time , there is evidence for indirect actions of neurohormonal stimulation on cardiac remodeling via activation of innate immunity and inflammation , especially the induction of heart - derived proinflammatory cytokines . in chronic heart failure , an increased th1/th2 ratio is seen , but whether increased th1 cell levels contribute to heart failure progression or simply are a consequence of heart failure is unresolved . exciting new findings have now implicated adaptive immunity and t cells more directly as causal agents in hypertensive lv hypertrophy and resultant heart failure by poorly understood means . these preclinical studies employed the mouse model of transverse aortic constriction- ( tac- ) induced heart failure to mimic the impact of high blood pressure on the heart . laroumanie et al . reported increased recruitment of activated cd4 and cd8 t cells and elevated levels of several chemokines for t cells and monocytes , including cxcl10 , in ventricular tissues from mice with tac - induced heart failure . tac - induced ventricular dilation and fibrosis was prevented and contractile dysfunction was attenuated in mice deficient in mature b and t lymphocytes due to knockout of rag2 , although cardiac hypertrophy was still observed . t cell replenishment in rag2 knockout mice restored the tac - induced heart failure phenotype . in addition , elimination of cd4 t cells ( mhcii knockout ) but not cd8 t cells ( cd8 knockout ) prevented tac - induced cardiac fibrosis and failure , suggesting a critical involvement of t helper cells . this conclusion was further supported by the observation that mice with transgenic t cell receptor specific for ovalbumin did not develop heart failure and fibrosis with tac . altogether these findings suggest that activation of cd4 t cells in hypertension causes interstitial and perivascular fibrosis that leads to functional and morphological changes in the heart conducive to the development of heart failure . however , it should be noted that an earlier study reported that coronary vessels of rag1 knockout mice exhibited more intimal hyperplasia and perivascular fibrosis compared to wild - type mice following tac . more recently , nevers et al . also investigated the role of t cells in cardiac remodeling in response to tac - induced pressure overload . they observed that the development of systolic dysfunction was associated with the kinetics of t cell infiltration into the left ventricle and evidence was provided that most of the infiltrating t cells were ifn- secreting th1 cells . lv systolic and diastolic function were preserved with tac in t cell deficient mice ( t cell receptor ( tcr ) knockout ) , and lv hypertrophy , fibrosis , and inflammation were markedly attenuated . in addition , t cell depletion with an anti - cd3 antibody prevented heart failure in wild - type mice . unresolved at present is the identity of the antigen(s ) responsible for t cell activation in lv hypertrophy and heart failure , and the potential contribution played by the loss of regulatory mechanisms that normally protect the heart from t cells . in contrast to the involvement of adaptive immunity in tac , ma et al . provided evidence that cd8 t cells play a critical role in perivascular and interstitial fibrosis in the angiotensin ii infusion model of hypertensive cardiac remodeling through the recruitment and activation of macrophages . they found that cd8 t cells are recruited to the heart and activated by ifn- secreting myocardial cells ; recruited macrophages in turn are activated by cd8 t cells in contact - dependent , but tcr - independent means . a possible contribution of cd4 t cells to the actions of cd8 t cells will need to be explored . circulating levels of cxcl10 are elevated in patients with untreated essential hypertension . in a small cohort , we observed that the cxcr3 chemokines , including cxcl10 , were present in elevated concentrations in the plasma of patients with symptomatic diastolic lv dysfunction indicative of hfpef or early stage hfref . the magnitude of their increase was independent of the extent of hypertension and the cxcr3 agonists enhanced diagnostic accuracy over and beyond nt - pro bnp . more recently , we reported that circulating cxcl10 , mip-1 , and cd40 ligand were the best indicators for differentiating healthy and heart failure subjects . we found that serum cxcl10 levels were increased in patients with symptomatic heart failure as indexed by nyha classification ii through iv and were positively correlated with serum levels of th1 proinflammatory cytokines . the findings of these two studies are consistent with the idea that inflammation is involved in the pathogenesis of heart failure with cxcl10 playing a central role . numerous preclinical studies and recent genome - wide association studies ( gwas ) support a role for both cytotoxic ( cd8 ) t cells and th ( cd4 ) lymphocytes in human hypertension [ 92 , 93 ] . however , accumulating evidence from experimental studies indicates that increasing treg cell levels in hypertension is an effective strategy to preserve cardiac function , attenuate cardiac hypertrophy and fibrosis , and prevent heart failure progression , independent of any blood pressure lowering effects [ 9496 ] . reduced circulating levels of treg cells in heart failure patients have been reported in several studies [ 9799 ] . the role of cxcl10 in other forms of nonischemic heart failure with reduced ejection fraction , such as restrictive cardiomyopathy , ion channelopathies , and diabetic cardiomyopathy , awaits investigation . recently , di luigi et al . reported that the phosphodiesterase type 5 inhibitor sildenafil decreased elevated circulating cxcl10 levels in subjects with diabetic cardiomyopathy , suggesting that sildenafil could be used pharmacologically to mitigate cxcl10-associated inflammation in diabetic cardiomyopathy . recent findings support a role for cxcl10 in right ventricular ( rv ) remodeling as well . . found that several chemokines , most notably cxcl10 , are upregulated in the pressure - overloaded right ventricle and play a role in myocardial extracellular matrix remodeling in an animal model of pulmonary stenosis . cxcl10 is implicated also in rv dysfunction and inflammation following experimental pulmonary embolism in rats . the chemokine receptor cxcr3 and its agonist cxcl10 are potential drug targets to treat various cardiovascular diseases . potential immunotherapies for cardiac inflammation are as follows : treg stimulation il-2/anti - il-2 complex treatment to enhance treg number and activity targeted cytokine - infused nanoparticles to stabilize and expand tregs in vivointravenous immunoglobulin ( ivig ) therapy to boost treg activity vitamin d to modulate formation and activity of tregs atorvastatin to enhance treg number and activity fty720 to increase treg levels and activityadoptive treg cell transfer il-2/anti - il-2 complex treatment to enhance treg number and activity targeted cytokine - infused nanoparticles to stabilize and expand tregs in vivo intravenous immunoglobulin ( ivig ) therapy to boost treg activity vitamin d to modulate formation and activity of tregs atorvastatin to enhance treg number and activity fty720 to increase treg levels and activity adoptive treg cell transfer immunosuppression immunoadsorption to remove circulating antibodies and boost treg activityphosphodiesterase type 5 inhibitor to decrease cxcl10 formation cxcl11 to stimulate biased gpcr anti - inflammatory signaling ppar- agonists to block cxcl9 , cxcl10 , and cxcl11 formation immunoadsorption to remove circulating antibodies and boost treg activity phosphodiesterase type 5 inhibitor to decrease cxcl10 formation cxcl11 to stimulate biased gpcr anti - inflammatory signaling ppar- agonists to block cxcl9 , cxcl10 , and cxcl11 formation levels of cxcl10 are generally elevated with chronic cardiac inflammation , which is associated with enhanced th1 polarization and infiltration into the myocardium . cxcr3 plays a key role in recruiting various leukocytes to the heart , including monocytes , effector lymphocytes , and ctl cells . peroxisome proliferator - activated receptor- ( ppar- ) agonists may be a potential pharmacological treatment to block cxcl9 , cxcl10 , and cxcl11 formation in patients , as ppar- agonists show a strong inhibitory effect on their expression and production in vitro . pioglitazone , which lacks the adverse cardiovascular effects of older thiazolidinediones and may be cardiovascular protective , looks promising in this regard , although pioglitazone is contraindicated in heart failure patients likely due to fluid retention [ 104107 ] . another potential therapeutic approach is the phosphodiesterase type 5 inhibitor sildenafil , which was recently reported to decrease cxcl10 gene expression and protein secretion in human cardiac myocytes and decrease circulating cxcl10 in subjects with diabetic cardiomyopathy . there is substantial evidence that sildenafil has protective effects against adverse remodeling of the heart . although cxcl9 , cxcl10 , and cxcl11 all bind to cxcr3 , there is evidence that these agonists activate opposing responses due to biased signaling that is a fixture of g protein - coupled receptors [ 1 , 11 , 12 ] . whereas cxcl9/cxcl10/cxcr3 interactions drive effector th1 polarization , cxcl11/cxcr3 binding seems to induce an immunotolerant state characterized by t lymphocyte polarization into regulatory tr1 lymphocytes that produce anti - inflammatory il-10 [ 11 , 12 ] . biased agonists have been developed that exert cxcl11-like actions at cxcr3 but have not as yet been assessed in experimental models of cardiovascular diseases . cxcl11 has a short half - life in vivo . to address this shortcoming , zohar et al . generated a stabilized form of cxcl11 by creating a fusion protein in which cxcl11 was linked to igg1 . when administered during ongoing autoimmune encephalomyelitis , the fusion protein suppressed the disease by increasing the number of il-10-secreting tr1-like cells ( direct effect ) and reducing th1 polarization . targeting cxcr3 might be more effective in treating chronic heart inflammation in combination with approaches to enhance treg numbers or activity , which are generally reduced in cardiovascular diseases ( table 1 ) . regulatory t cells are immunosuppressive and anti - inflammatory , and a growing number of experimental studies have shown their beneficial effects on the heart in various experimental models of coronary artery disease [ 109 , 110 ] , kawasaki disease , myocarditis and dilated cardiomyopathies [ 112117 ] , chagas heart disease [ 118 , 119 ] , hypertensive lv hypertrophy [ 95 , 96 ] , and nonischemic heart failure [ 94 , 109 , 120 ] . although not discussed here , boosting treg numbers or activity in the heart is reported to be beneficial as well in experimental models of infarction - driven remodeling [ 121124 ] . increasing levels of il-10-secreting treg / tr1 cells may be particularly advantageous , as il-10 has anti - inflammatory and protective actions on the vasculature and heart [ 125132 ] . intravenous immunoglobulin ( ivig ) therapy has been shown to be effective in treating acute kawasaki disease in 8090% of kawasaki patients with rapid resolution of clinical symptoms and reduced risk of coronary disease . although the exact basis for the effectiveness of ivig therapy is not clear , ivig therapy is thought to modulate the inflammatory process and recent evidence indicates that ivig therapy acts in part by stimulating an immature myeloid population of dendritic cells that secretes il-10 and favors expansion of fc - specific natural treg cells . ivig may have promise for treating viral myocarditis and might be effective in treating chagas , as well as chronic heart failure , including both ischemic and idiopathic dilated cardiomyopathies . adoptive transfer of tregs and/or stimulating their differentiation are promising therapeutic approaches to target cardiac inflammation [ 49 , 50 ] , although the concerns that must be overcome to make adaptive transfer routine therapy in humans are considerable . however , the safety and efficacy of treg immunotherapy in humans is supported by preliminary clinical trials for treating graft versus host disease [ 137 , 138 ] . immunoadsorption , which is a promising approach for treating myocarditis and dilated cardiomyopathy , may have beneficial effects by not merely removing circulating antibodies , but increasing treg activity [ 49 , 139141 ] . the prodrug fty720 ( fingolimod ) is phosphorylated in vivo and has been shown to trap nave and memory t cells in the thymus and secondary lymphoid organs by downregulating sphingosine 1-phosphate receptor 1 ( s1p1 ) . activation of s1p1 is linked to inhibition of treg cell differentiation ; however , fty720 and phosphorylated fty720 increase treg levels and activity in vivo and in vitro [ 143146 ] . the inhibitory effects of atorvastatin on inflammation in acute coronary syndrome ( acs ) may be due to its beneficial effects on natural tregs . in patients with acs , il-2 plays a critical role in treg cell activity , growth , and survival , but there may be insufficient il-2 to sustain their in vivo potentiation . emerging evidence indicates that enhancing treg cell numbers and activity with low - dose il-2 treatment is effective in treating autoimmune and inflammatory diseases [ 12 , 148151 ] , although there may be intrinsic risk as il-2 is also a key growth factor for effector cd4 t cells and nk cells . an il-2/anti - il-2 immune complex is reported to preferentially expand treg cells with little or no effect on other cells . delivery of the complex to mice before tac was recently reported to attenuate lv hypertrophy , inflammation , and contractile dysfunction , while increasing lv levels of treg cells . recently reported success in using nanoparticles loaded with the treg inducers il-2 and tgf- and targeted to cd4 t cells with conjugated antibodies . these nanoparticles were demonstrated to induce cd4 tregs in vitro , even in the presence of proinflammatory cytokines , and expand their number in mice in vivo . vitamin d status has been linked to chronic heart failure and vitamin d supplementation improves lv structure and function in heart failure patients . recent evidence indicates a modulatory role of the vitamin d system in the formation and activity of regulatory t cells . vitamin d deficiency was associated with reduced numbers and impaired function of nave cd45ra regulatory t cell in chronic heart failure patients . in addition , the vitamin d receptor agonist bxl-01 - 0029 was shown to inhibit ifn- and tnf--induced cxcl10 secretion by fetal human cardiac myocytes and reduce cxcl10 protein secretion and gene expression by cd4 t cells . whether a vitamin d receptor agonist or supplementation increases treg levels and reduces cxcl10 levels in heart failure patients will need to be assessed . the last decade has witnessed an impressive advance in our understanding of the role of the immune system in the pathophysiology of cardiovascular diseases . beyond an expected role in allograft disease and the development and progression of atherosclerosis , an abundant body of evidence supports a significant contribution of the immune system to many other cardiovascular settings , ranging from the development and maintenance of hypertension to cardiac and vessel remodeling ( whether constrictive or expansive ) in response to hemodynamic stress , in both health and disease . the challenge now is to translate this knowledge for the benefit of patients suffering from cardiovascular diseases . targeting the cxcl10/cxcr3 axis and cardiac inflammation may open new pharmacological venues for treating heart failure or coronary artery disease to supplement current drugs that target the sympathetic or renin - angiotensin systems , or platelets . this will require selectively targeting the most critical immune pathways in order to optimize interference with pathological processes , while preserving protective and homeostatic immune functions . antigen - specific modulation of the immune system , for example , through systemic delivery of nanoparticles coated with disease - relevant peptides bound to major histocompatibility complex class ii ( pmhcii ) to expand endogenous antigen - specific tregs , is an optimal strategy in settings where an antigen - specific adaptive immune response has been involved in disease development or progression . however , several cardiovascular diseases will probably resist the identification of a specific pathogenic antigen and will require a broader , although targeted , regulation of the immune response , for example , through administration of low - dose il-2 to promote endogenous tregs . with regard to cxcr3 pathway , besides the therapeutic possibilities listed above , we believe that the identification and development of selective evasins or evasin - like peptides that differentially bind and neutralize cxcl9 or cxcl10 versus cxcl11 may provide an interesting therapeutic strategy to limit pathogenic while preserving regulatory cxcr3 functions .

accumulating evidence reveals involvement of t lymphocytes and adaptive immunity in the chronic inflammation associated with infectious and noninfectious diseases of the heart , including coronary artery disease , kawasaki disease , myocarditis , dilated cardiomyopathies , chagas , hypertensive left ventricular ( lv ) hypertrophy , and nonischemic heart failure . chemokine cxcl10 is elevated in cardiovascular diseases , along with increased cardiac infiltration of proinflammatory th1 and cytotoxic t cells . cxcl10 is a chemoattractant for these t cells and polarizing factor for the proinflammatory phenotype . thus , targeting the cxcl10 receptor cxcr3 is a promising therapeutic approach to treating cardiac inflammation . due to biased signaling cxcr3 also couples to anti - inflammatory signaling and immunosuppressive regulatory t cell formation when activated by cxcl11 . numbers and functionality of regulatory t cells are reduced in patients with cardiac inflammation , supporting the utility of biased agonists or biologicals to simultaneously block the pro - inflammatory and activate the anti - inflammatory actions of cxcr3 . other immunotherapy strategies to boost regulatory t cell actions include intravenous immunoglobulin ( ivig ) therapy , adoptive transfer , immunoadsorption , and low - dose interleukin-2/interleukin-2 antibody complexes . pharmacological approaches include sphingosine 1-phosphate receptor 1 agonists and vitamin d supplementation . a combined strategy of switching cxcr3 signaling from pro- to anti - inflammatory and improving treg functionality is predicted to synergistically lessen adverse cardiac remodeling .

1. Introduction 2. CXCR3 Biased Signaling 3. Coronary Artery Disease (Ischemic Heart Disease) 4. Kawasaki Disease 5. Myocarditis and Chagas Heart Disease 6. LV Hypertrophy and Nonischemic Heart Failure 7. Implications for Immunotherapy 8. Conclusions and Future Perspectives

the chemokine receptor cxcr3 is a class a seven - transmembrane - domain or g protein - coupled receptor ( gpcr ) that is involved primarily in chemotaxis of certain immune cells , inhibition of angiogenesis , and th1 cell polarization [ 13 ] . cxcr3 is expressed by various effector t lymphocytes , including cd4 t helper 1 ( th1 ) cells , cd8 cytotoxic t lymphocytes ( ctl ) , and cd4 and cd8 memory t cells , as well as monocytes , m1 macrophages , natural killer ( nk ) cells , subsets of b - cells , mast cells , endothelial cells , and vascular smooth muscle cells [ 14 ] . the principal agonists of cxcr3 are cxcl9 ( mig ) , cxcl10 ( ip-10 ) , and cxcl11 ( i - tac ) . the human equivalent of the murine form of cxcr3 is cxcr3a and unless noted otherwise cxcr3 is used in this review to include both murine and human isoforms . besides cxcl9 , cxcl10 , and cxcl11 , cxcr3b and cxcr3 are activated by cxcl4 and cxcl4l1 , chemokines that are released by platelets and have been implicated in atherogenesis and acute coronary syndrome [ 3 , 9 , 10 ] . cxcr3-alt is a truncated form of cxcr3 that is selectively activated by cxcl11 [ 24 ] . cxcr3 is associated with the pathophysiology of th1-type diseases , including infections of various etiologies and autoimmune disorders [ 1 , 3 ] . although cxcr3 is activated by cxcl9 , cxcl10 , and cxcl11 , the outcome is different with growing evidence that cxcl9 and cxcl10 are essentially proinflammatory , while cxcl11 has anti - inflammatory actions [ 11 , 12 ] . over the last decade , numerous studies have documented elevated circulating levels of cxcl10 in wide - ranging infectious and autoimmune diseases , autoimmune encephalomyelitis , crohn 's disease , tuberculosis , thyroid autoimmune diseases , and type 1 diabetes , as well as several cancers [ 1318 ] . recent evidence from us and others has revealed the importance of the cxcl10/cxcr3 axis in cardiovascular diseases . as discussed elsewhere , cxcl9 and cxcl10 in addition , the homing signature for memory t cells to the heart from mediastinal lymph nodes is c - metccr4cxcr3 . while c - met triggering supports cardiotropic t cell recirculation , cxcr3 and ccr4 engagement via tissue - released cxcl10 and ccl4 , respectively , sustains recruitment in heart inflammation , we present an overview of the role of cxcl9 and cxcl10 in infectious and noninfectious diseases of the heart and its implications for immunotherapy . the opposite actions of the cxcr3 agonists are likely the consequence of the biased signaling that is a fixture of gpcrs , which can activate both g protein - dependent and protein - independent signaling cascades , the latter occurring via -arrestin 2 recruitment [ 1 , 11 , 12 ] . coronary artery disease ( cad ) , which progresses to coronary heart disease , is a leading cause of death in the usa and globally [ 23 , 24 ] . cad is caused by atherosclerosis within the arteries of the heart , a chronic inflammatory condition associated with waxy plaque buildup . t cells are recruited into the intima , and dendritic and nk cells help induce the cd4 th1 phenotype , which is the most abundant t cell population in human atherosclerotic plaques . the atheromatous plaque that builds up in the artery wall is made up of an accumulation of lipids , fibrous connective tissue , macrophages , and cellular debris that arises from the cytolytic actions of oxidized ldl , nk cells , ifn- , and ctl cells on macrophages , foam cells , vsmc , and endothelial cells . cxcl10 is reported to be expressed by endothelial cells , smooth muscle cells , and macrophages during the formation of atherosclerotic lesions in both preclinical and clinical studies [ 28 , 29 ] . suppression of cxcl10 bioactivity in apo - e deficient mice resulted in a more stable plaque phenotype with less macrophage activation , along with more smooth muscle cells and collagen abundance . of note , unstable plaques have increased levels of th1 , nk , and ctl cells and decreased levels of anti - inflammatory regulatory t ( treg ) cells . recent studies show that the relative levels of treg cells are reduced and their functionality is impaired in patients with cad [ 32 , 33 ] . knockout of cxcl10 in the apolipoprotein e - deficient mouse model of atherosclerosis was associated with increased treg cell numbers and activity , along with a reduction in lesion formation . it would be interesting to analyze cxcl10 in samples of blood obtained by thrombus - aspiration during coronary artery percutaneous intervention ( pci ) in patients with unstable coronary artery disease . in patients with first - time st - segment elevation ami , high circulating levels of ccl4 , cxcl16 , cxcl8 , and cxcl10 within the first week after pci were found to be positively correlated with the degree of myocardial damage . reported that cxcl10 is a good biomarker / predictor of kawasaki disease and , furthermore , that cxcr3 is activated in the t cells of patients with acute kawasaki disease . in addition , several studies report that numbers and functionality of treg cells are reduced in kawasaki patients [ 4548 ] . myocarditis or inflammation of the myocardium is a heterogeneous group of disorders initiated by various pathogens , including worms , bacteria , protozoa , rickettsia , and most commonly viruses . cxcl10 is elevated in the heart following viral and nonviral infection and has the characteristics of a biomarker in rodent models of myocarditis [ 52 , 53 ] . these mice exhibited ameliorated disease progression , including reduced cardiac thickening ( due to inflammatory edema ) , inflammation , and cell death , as well as improved survival when compared to wild - type mice . the authors concluded that cxcl10 plays a crucial role in recruitment of th1 cells to the heart , leading to the increase in detrimental proinflammatory th1 cytokines . on whether myocardial cxcl10 is harmful or beneficial in the context of acute cvb3 myocarditis , although timing and dosage levels of cxcl10 and effective viral clearance versus th1 recruitment are likely contributing factors . both th1 and th17 cells drive myocarditis , with th17 cells playing an important part in the development of dilated cardiomyopathy . chagas heart disease is characterized by marked inflammation and fibrosis of the heart , along with cardiac edema , myofibrillar destruction , chamber dilation , and loss of contractile function . with heart failure in chagas disease , myocardial levels of cd8 and cd4 t cells are increased , with a predominance of cd8 t cells [ 61 , 64 , 65 ] . in contrast , chagas - related heart failure is associated with reduced myocardial levels of treg cells [ 61 , 64 ] , and circulating treg activity was reported to be reduced in moderate or severe cardiomyopathy with activity directly correlated to lvef . were detected in patients with chronic chagas disease , and lv mrna expression levels of cxcl10 were found to be elevated in patients with chagas cardiomyopathy . behet 's disease is an autoimmune or autoinflammatory disorder common in the middle east , asia , and japan . the basis for the pathogenesis of behet 's disease is not known , although a number of factors have been proposed to have a role , including viral , bacterial , environmental , genetic , and immune factors . behet 's is caused by small - vessel systemic vasculitis that very often affects the heart in diverse ways , including endomyocardial fibrosis , intracardiac thrombus , endocarditis , pericarditis , myocarditis , coronary arteritis , myocardial infarction , and valvular disease . hypertension causes a number of adverse remodeling events at the cellular and tissue level of the heart , including cardiac myocyte hypertrophy and gene reprograming , activation of cardiac fibroblasts , interstitial and perivascular fibrosis , and capillary refraction [ 7375 ] . these alterations ultimately cause marked changes in the overall geometry of the heart that may progress to heart failure and the inability of the heart to adequately meet the oxygen and energy demands of the body . numerous preclinical and clinical studies have implicated marked activation of neurohormonal drive to the heart in the pathoetiology of lv hypertrophy and its progression to heart failure . neurohormonal drive , namely , activation of the sympathetic and renin - angiotensin - aldosterone systems , is generally thought to directly cause adverse remodeling of the heart . at the same time , there is evidence for indirect actions of neurohormonal stimulation on cardiac remodeling via activation of innate immunity and inflammation , especially the induction of heart - derived proinflammatory cytokines . exciting new findings have now implicated adaptive immunity and t cells more directly as causal agents in hypertensive lv hypertrophy and resultant heart failure by poorly understood means . reported increased recruitment of activated cd4 and cd8 t cells and elevated levels of several chemokines for t cells and monocytes , including cxcl10 , in ventricular tissues from mice with tac - induced heart failure . t cell replenishment in rag2 knockout mice restored the tac - induced heart failure phenotype . in addition , elimination of cd4 t cells ( mhcii knockout ) but not cd8 t cells ( cd8 knockout ) prevented tac - induced cardiac fibrosis and failure , suggesting a critical involvement of t helper cells . altogether these findings suggest that activation of cd4 t cells in hypertension causes interstitial and perivascular fibrosis that leads to functional and morphological changes in the heart conducive to the development of heart failure . also investigated the role of t cells in cardiac remodeling in response to tac - induced pressure overload . they observed that the development of systolic dysfunction was associated with the kinetics of t cell infiltration into the left ventricle and evidence was provided that most of the infiltrating t cells were ifn- secreting th1 cells . lv systolic and diastolic function were preserved with tac in t cell deficient mice ( t cell receptor ( tcr ) knockout ) , and lv hypertrophy , fibrosis , and inflammation were markedly attenuated . in addition , t cell depletion with an anti - cd3 antibody prevented heart failure in wild - type mice . unresolved at present is the identity of the antigen(s ) responsible for t cell activation in lv hypertrophy and heart failure , and the potential contribution played by the loss of regulatory mechanisms that normally protect the heart from t cells . in contrast to the involvement of adaptive immunity in tac , ma et al . provided evidence that cd8 t cells play a critical role in perivascular and interstitial fibrosis in the angiotensin ii infusion model of hypertensive cardiac remodeling through the recruitment and activation of macrophages . they found that cd8 t cells are recruited to the heart and activated by ifn- secreting myocardial cells ; recruited macrophages in turn are activated by cd8 t cells in contact - dependent , but tcr - independent means . circulating levels of cxcl10 are elevated in patients with untreated essential hypertension . in a small cohort , we observed that the cxcr3 chemokines , including cxcl10 , were present in elevated concentrations in the plasma of patients with symptomatic diastolic lv dysfunction indicative of hfpef or early stage hfref . more recently , we reported that circulating cxcl10 , mip-1 , and cd40 ligand were the best indicators for differentiating healthy and heart failure subjects . we found that serum cxcl10 levels were increased in patients with symptomatic heart failure as indexed by nyha classification ii through iv and were positively correlated with serum levels of th1 proinflammatory cytokines . the findings of these two studies are consistent with the idea that inflammation is involved in the pathogenesis of heart failure with cxcl10 playing a central role . however , accumulating evidence from experimental studies indicates that increasing treg cell levels in hypertension is an effective strategy to preserve cardiac function , attenuate cardiac hypertrophy and fibrosis , and prevent heart failure progression , independent of any blood pressure lowering effects [ 9496 ] . the role of cxcl10 in other forms of nonischemic heart failure with reduced ejection fraction , such as restrictive cardiomyopathy , ion channelopathies , and diabetic cardiomyopathy , awaits investigation . potential immunotherapies for cardiac inflammation are as follows : treg stimulation il-2/anti - il-2 complex treatment to enhance treg number and activity targeted cytokine - infused nanoparticles to stabilize and expand tregs in vivointravenous immunoglobulin ( ivig ) therapy to boost treg activity vitamin d to modulate formation and activity of tregs atorvastatin to enhance treg number and activity fty720 to increase treg levels and activityadoptive treg cell transfer il-2/anti - il-2 complex treatment to enhance treg number and activity targeted cytokine - infused nanoparticles to stabilize and expand tregs in vivo intravenous immunoglobulin ( ivig ) therapy to boost treg activity vitamin d to modulate formation and activity of tregs atorvastatin to enhance treg number and activity fty720 to increase treg levels and activity adoptive treg cell transfer immunosuppression immunoadsorption to remove circulating antibodies and boost treg activityphosphodiesterase type 5 inhibitor to decrease cxcl10 formation cxcl11 to stimulate biased gpcr anti - inflammatory signaling ppar- agonists to block cxcl9 , cxcl10 , and cxcl11 formation immunoadsorption to remove circulating antibodies and boost treg activity phosphodiesterase type 5 inhibitor to decrease cxcl10 formation cxcl11 to stimulate biased gpcr anti - inflammatory signaling ppar- agonists to block cxcl9 , cxcl10 , and cxcl11 formation levels of cxcl10 are generally elevated with chronic cardiac inflammation , which is associated with enhanced th1 polarization and infiltration into the myocardium . cxcr3 plays a key role in recruiting various leukocytes to the heart , including monocytes , effector lymphocytes , and ctl cells . peroxisome proliferator - activated receptor- ( ppar- ) agonists may be a potential pharmacological treatment to block cxcl9 , cxcl10 , and cxcl11 formation in patients , as ppar- agonists show a strong inhibitory effect on their expression and production in vitro . although cxcl9 , cxcl10 , and cxcl11 all bind to cxcr3 , there is evidence that these agonists activate opposing responses due to biased signaling that is a fixture of g protein - coupled receptors [ 1 , 11 , 12 ] . targeting cxcr3 might be more effective in treating chronic heart inflammation in combination with approaches to enhance treg numbers or activity , which are generally reduced in cardiovascular diseases ( table 1 ) . regulatory t cells are immunosuppressive and anti - inflammatory , and a growing number of experimental studies have shown their beneficial effects on the heart in various experimental models of coronary artery disease [ 109 , 110 ] , kawasaki disease , myocarditis and dilated cardiomyopathies [ 112117 ] , chagas heart disease [ 118 , 119 ] , hypertensive lv hypertrophy [ 95 , 96 ] , and nonischemic heart failure [ 94 , 109 , 120 ] . increasing levels of il-10-secreting treg / tr1 cells may be particularly advantageous , as il-10 has anti - inflammatory and protective actions on the vasculature and heart [ 125132 ] . intravenous immunoglobulin ( ivig ) therapy has been shown to be effective in treating acute kawasaki disease in 8090% of kawasaki patients with rapid resolution of clinical symptoms and reduced risk of coronary disease . ivig may have promise for treating viral myocarditis and might be effective in treating chagas , as well as chronic heart failure , including both ischemic and idiopathic dilated cardiomyopathies . adoptive transfer of tregs and/or stimulating their differentiation are promising therapeutic approaches to target cardiac inflammation [ 49 , 50 ] , although the concerns that must be overcome to make adaptive transfer routine therapy in humans are considerable . immunoadsorption , which is a promising approach for treating myocarditis and dilated cardiomyopathy , may have beneficial effects by not merely removing circulating antibodies , but increasing treg activity [ 49 , 139141 ] . the prodrug fty720 ( fingolimod ) is phosphorylated in vivo and has been shown to trap nave and memory t cells in the thymus and secondary lymphoid organs by downregulating sphingosine 1-phosphate receptor 1 ( s1p1 ) . in patients with acs , il-2 plays a critical role in treg cell activity , growth , and survival , but there may be insufficient il-2 to sustain their in vivo potentiation . emerging evidence indicates that enhancing treg cell numbers and activity with low - dose il-2 treatment is effective in treating autoimmune and inflammatory diseases [ 12 , 148151 ] , although there may be intrinsic risk as il-2 is also a key growth factor for effector cd4 t cells and nk cells . delivery of the complex to mice before tac was recently reported to attenuate lv hypertrophy , inflammation , and contractile dysfunction , while increasing lv levels of treg cells . these nanoparticles were demonstrated to induce cd4 tregs in vitro , even in the presence of proinflammatory cytokines , and expand their number in mice in vivo . vitamin d status has been linked to chronic heart failure and vitamin d supplementation improves lv structure and function in heart failure patients . recent evidence indicates a modulatory role of the vitamin d system in the formation and activity of regulatory t cells . vitamin d deficiency was associated with reduced numbers and impaired function of nave cd45ra regulatory t cell in chronic heart failure patients . the last decade has witnessed an impressive advance in our understanding of the role of the immune system in the pathophysiology of cardiovascular diseases . the challenge now is to translate this knowledge for the benefit of patients suffering from cardiovascular diseases . targeting the cxcl10/cxcr3 axis and cardiac inflammation may open new pharmacological venues for treating heart failure or coronary artery disease to supplement current drugs that target the sympathetic or renin - angiotensin systems , or platelets . however , several cardiovascular diseases will probably resist the identification of a specific pathogenic antigen and will require a broader , although targeted , regulation of the immune response , for example , through administration of low - dose il-2 to promote endogenous tregs .

the chemokine receptor cxcr3 is a class a seven - transmembrane - domain or g protein - coupled receptor ( gpcr ) that is involved primarily in chemotaxis of certain immune cells , inhibition of angiogenesis , and th1 cell polarization [ 13 ] . cxcr3 is expressed by various effector t lymphocytes , including cd4 t helper 1 ( th1 ) cells , cd8 cytotoxic t lymphocytes ( ctl ) , and cd4 and cd8 memory t cells , as well as monocytes , m1 macrophages , natural killer ( nk ) cells , subsets of b - cells , mast cells , endothelial cells , and vascular smooth muscle cells [ 14 ] . cxcr3 couples to gi protein [ 5 , 6 ] and although not extensively studied , it has been shown to activate a number of signaling pathways that are generally associated with gpcrs such as increases in intracellular calcium and activation of map kinases and pi3k / akt signaling [ 4 , 7 , 8 ] . cxcr3b , which couples to gs , is the receptor isoform expressed in microvascular endothelial cells and is linked to inhibition of angiogenesis and induction of apoptosis [ 2 , 3 ] . besides cxcl9 , cxcl10 , and cxcl11 , cxcr3b and cxcr3 are activated by cxcl4 and cxcl4l1 , chemokines that are released by platelets and have been implicated in atherogenesis and acute coronary syndrome [ 3 , 9 , 10 ] . although cxcr3 is activated by cxcl9 , cxcl10 , and cxcl11 , the outcome is different with growing evidence that cxcl9 and cxcl10 are essentially proinflammatory , while cxcl11 has anti - inflammatory actions [ 11 , 12 ] . over the last decade , numerous studies have documented elevated circulating levels of cxcl10 in wide - ranging infectious and autoimmune diseases , autoimmune encephalomyelitis , crohn 's disease , tuberculosis , thyroid autoimmune diseases , and type 1 diabetes , as well as several cancers [ 1318 ] . as discussed elsewhere , cxcl9 and cxcl10 in addition , the homing signature for memory t cells to the heart from mediastinal lymph nodes is c - metccr4cxcr3 . while c - met triggering supports cardiotropic t cell recirculation , cxcr3 and ccr4 engagement via tissue - released cxcl10 and ccl4 , respectively , sustains recruitment in heart inflammation , we present an overview of the role of cxcl9 and cxcl10 in infectious and noninfectious diseases of the heart and its implications for immunotherapy . in contrast , cxcl11 , which exhibits relatively higher binding affinity for cxcr3 , drives development of foxp3 ( forkhead box p3)-negative il-10 t regulatory 1 ( tr1 ) cells and il-4 th2 cells via stat3 and stat6 activation and was demonstrated to dampen inflammation . the opposite actions of the cxcr3 agonists are likely the consequence of the biased signaling that is a fixture of gpcrs , which can activate both g protein - dependent and protein - independent signaling cascades , the latter occurring via -arrestin 2 recruitment [ 1 , 11 , 12 ] . endothelial dysfunction , increased vascular permeability , increased expression of adhesion molecules on endothelial cells for leukocytes , and increased plasma levels of low density lipoprotein ( ldl ) are initiating factors in atherosclerosis . ldl , which accumulates in the intima , undergoes oxidation by macrophages and endothelial cells , as well as by vsmc that migrate into the intima from the media and proliferate . in response to the oxidized ldl and plasma ldl , endothelial cells secrete proinflammatory cytokines and chemokines ( mcp-1/ccl2 , fractalkine , and cxcr3 ligands ) that attract monocytes , which differentiate into dendritic cells or macrophages that accumulate oxidized ldl to become foam cells . t cells are recruited into the intima , and dendritic and nk cells help induce the cd4 th1 phenotype , which is the most abundant t cell population in human atherosclerotic plaques . the atheromatous plaque that builds up in the artery wall is made up of an accumulation of lipids , fibrous connective tissue , macrophages , and cellular debris that arises from the cytolytic actions of oxidized ldl , nk cells , ifn- , and ctl cells on macrophages , foam cells , vsmc , and endothelial cells . cxcl10 is reported to be expressed by endothelial cells , smooth muscle cells , and macrophages during the formation of atherosclerotic lesions in both preclinical and clinical studies [ 28 , 29 ] . of note , unstable plaques have increased levels of th1 , nk , and ctl cells and decreased levels of anti - inflammatory regulatory t ( treg ) cells . knockout of cxcl10 in the apolipoprotein e - deficient mouse model of atherosclerosis was associated with increased treg cell numbers and activity , along with a reduction in lesion formation . circulating levels of cxcl10 notably , cxcl10 was also reported to be produced by the endothelium of mouse coronary blood vessels infused with angiotensin ii , human coronary artery endothelial cells treated with tnf- , and rat cardiac microvascular endothelial cells subjected to hypoxia / ischemia . patients with acute myocardial infarction ( ami ) showed significantly higher serum levels of cxcl10 than control subjects and patients with stable angina pectoris . it would be interesting to test blood concentration of cxcl10 within the first 3 hours after angina onset during ami when systemic activation is not yet started secondly , the pathogenic mechanisms of plaque rupture may involve factors acting locally without necessarily showing a high systemic blood concentration . in patients with first - time st - segment elevation ami , high circulating levels of ccl4 , cxcl16 , cxcl8 , and cxcl10 within the first week after pci were found to be positively correlated with the degree of myocardial damage . reported that cxcl10 is a good biomarker / predictor of kawasaki disease and , furthermore , that cxcr3 is activated in the t cells of patients with acute kawasaki disease . in their study , myocarditis was induced with coxsackievirus b3 ( cvb3 ) , the primary cause of viral myocarditis , in mice that overexpressed a cxcl10 mutant protein without functional activity in order to antagonize endogenous cxcl10 . on whether myocardial cxcl10 is harmful or beneficial in the context of acute cvb3 myocarditis , although timing and dosage levels of cxcl10 and effective viral clearance versus th1 recruitment are likely contributing factors . evidence indicates the major contribution of autoimmunity to the etiology of myocarditis and thus adoptive transfer of tregs and/or stimulating their differentiation are promising therapeutic approaches [ 49 , 50 ] . the etiology of chagas heart disease is not fully understood and likely multifactorial , with a contribution of an autoimmune response due in part to molecular mimicry between antigenic determinants of t. cruzi and human ( cardiac ) antigens [ 62 , 63 ] . with heart failure in chagas disease , myocardial levels of cd8 and cd4 t cells are increased , with a predominance of cd8 t cells [ 61 , 64 , 65 ] . in contrast , chagas - related heart failure is associated with reduced myocardial levels of treg cells [ 61 , 64 ] , and circulating treg activity was reported to be reduced in moderate or severe cardiomyopathy with activity directly correlated to lvef . were detected in patients with chronic chagas disease , and lv mrna expression levels of cxcl10 were found to be elevated in patients with chagas cardiomyopathy . recently , evidence was provided that polymorphisms in the cxcl9 and cxcl10 genes controlled the expression of chemokines in the myocardium and the degree of myocarditis in chagas cardiomyopathy . the basis for the pathogenesis of behet 's disease is not known , although a number of factors have been proposed to have a role , including viral , bacterial , environmental , genetic , and immune factors . behet 's is caused by small - vessel systemic vasculitis that very often affects the heart in diverse ways , including endomyocardial fibrosis , intracardiac thrombus , endocarditis , pericarditis , myocarditis , coronary arteritis , myocardial infarction , and valvular disease . recently , monocytes of behet 's patients were found to have dysfunctional posttranscriptional regulation of cxcl10 mrna that resulted in overexpression of cxcl10 protein with ifn- stimulation . in general , the role of cxcl10 in immune - mediated and autoimmune myocarditis is little studied ; however , based on studies of infective myocarditis , a critical role for cxcl10 is likely . in the elderly , as many as 68% of heart failure cases are linked to hypertension and community - based studies indicate that hypertension contributes to heart failure in 60% of patients . hypertension causes a number of adverse remodeling events at the cellular and tissue level of the heart , including cardiac myocyte hypertrophy and gene reprograming , activation of cardiac fibroblasts , interstitial and perivascular fibrosis , and capillary refraction [ 7375 ] . these alterations ultimately cause marked changes in the overall geometry of the heart that may progress to heart failure and the inability of the heart to adequately meet the oxygen and energy demands of the body . numerous preclinical and clinical studies have implicated marked activation of neurohormonal drive to the heart in the pathoetiology of lv hypertrophy and its progression to heart failure . at the same time , there is evidence for indirect actions of neurohormonal stimulation on cardiac remodeling via activation of innate immunity and inflammation , especially the induction of heart - derived proinflammatory cytokines . tac - induced ventricular dilation and fibrosis was prevented and contractile dysfunction was attenuated in mice deficient in mature b and t lymphocytes due to knockout of rag2 , although cardiac hypertrophy was still observed . altogether these findings suggest that activation of cd4 t cells in hypertension causes interstitial and perivascular fibrosis that leads to functional and morphological changes in the heart conducive to the development of heart failure . they observed that the development of systolic dysfunction was associated with the kinetics of t cell infiltration into the left ventricle and evidence was provided that most of the infiltrating t cells were ifn- secreting th1 cells . lv systolic and diastolic function were preserved with tac in t cell deficient mice ( t cell receptor ( tcr ) knockout ) , and lv hypertrophy , fibrosis , and inflammation were markedly attenuated . unresolved at present is the identity of the antigen(s ) responsible for t cell activation in lv hypertrophy and heart failure , and the potential contribution played by the loss of regulatory mechanisms that normally protect the heart from t cells . provided evidence that cd8 t cells play a critical role in perivascular and interstitial fibrosis in the angiotensin ii infusion model of hypertensive cardiac remodeling through the recruitment and activation of macrophages . in a small cohort , we observed that the cxcr3 chemokines , including cxcl10 , were present in elevated concentrations in the plasma of patients with symptomatic diastolic lv dysfunction indicative of hfpef or early stage hfref . we found that serum cxcl10 levels were increased in patients with symptomatic heart failure as indexed by nyha classification ii through iv and were positively correlated with serum levels of th1 proinflammatory cytokines . numerous preclinical studies and recent genome - wide association studies ( gwas ) support a role for both cytotoxic ( cd8 ) t cells and th ( cd4 ) lymphocytes in human hypertension [ 92 , 93 ] . however , accumulating evidence from experimental studies indicates that increasing treg cell levels in hypertension is an effective strategy to preserve cardiac function , attenuate cardiac hypertrophy and fibrosis , and prevent heart failure progression , independent of any blood pressure lowering effects [ 9496 ] . the role of cxcl10 in other forms of nonischemic heart failure with reduced ejection fraction , such as restrictive cardiomyopathy , ion channelopathies , and diabetic cardiomyopathy , awaits investigation . reported that the phosphodiesterase type 5 inhibitor sildenafil decreased elevated circulating cxcl10 levels in subjects with diabetic cardiomyopathy , suggesting that sildenafil could be used pharmacologically to mitigate cxcl10-associated inflammation in diabetic cardiomyopathy . found that several chemokines , most notably cxcl10 , are upregulated in the pressure - overloaded right ventricle and play a role in myocardial extracellular matrix remodeling in an animal model of pulmonary stenosis . potential immunotherapies for cardiac inflammation are as follows : treg stimulation il-2/anti - il-2 complex treatment to enhance treg number and activity targeted cytokine - infused nanoparticles to stabilize and expand tregs in vivointravenous immunoglobulin ( ivig ) therapy to boost treg activity vitamin d to modulate formation and activity of tregs atorvastatin to enhance treg number and activity fty720 to increase treg levels and activityadoptive treg cell transfer il-2/anti - il-2 complex treatment to enhance treg number and activity targeted cytokine - infused nanoparticles to stabilize and expand tregs in vivo intravenous immunoglobulin ( ivig ) therapy to boost treg activity vitamin d to modulate formation and activity of tregs atorvastatin to enhance treg number and activity fty720 to increase treg levels and activity adoptive treg cell transfer immunosuppression immunoadsorption to remove circulating antibodies and boost treg activityphosphodiesterase type 5 inhibitor to decrease cxcl10 formation cxcl11 to stimulate biased gpcr anti - inflammatory signaling ppar- agonists to block cxcl9 , cxcl10 , and cxcl11 formation immunoadsorption to remove circulating antibodies and boost treg activity phosphodiesterase type 5 inhibitor to decrease cxcl10 formation cxcl11 to stimulate biased gpcr anti - inflammatory signaling ppar- agonists to block cxcl9 , cxcl10 , and cxcl11 formation levels of cxcl10 are generally elevated with chronic cardiac inflammation , which is associated with enhanced th1 polarization and infiltration into the myocardium . peroxisome proliferator - activated receptor- ( ppar- ) agonists may be a potential pharmacological treatment to block cxcl9 , cxcl10 , and cxcl11 formation in patients , as ppar- agonists show a strong inhibitory effect on their expression and production in vitro . pioglitazone , which lacks the adverse cardiovascular effects of older thiazolidinediones and may be cardiovascular protective , looks promising in this regard , although pioglitazone is contraindicated in heart failure patients likely due to fluid retention [ 104107 ] . another potential therapeutic approach is the phosphodiesterase type 5 inhibitor sildenafil , which was recently reported to decrease cxcl10 gene expression and protein secretion in human cardiac myocytes and decrease circulating cxcl10 in subjects with diabetic cardiomyopathy . when administered during ongoing autoimmune encephalomyelitis , the fusion protein suppressed the disease by increasing the number of il-10-secreting tr1-like cells ( direct effect ) and reducing th1 polarization . regulatory t cells are immunosuppressive and anti - inflammatory , and a growing number of experimental studies have shown their beneficial effects on the heart in various experimental models of coronary artery disease [ 109 , 110 ] , kawasaki disease , myocarditis and dilated cardiomyopathies [ 112117 ] , chagas heart disease [ 118 , 119 ] , hypertensive lv hypertrophy [ 95 , 96 ] , and nonischemic heart failure [ 94 , 109 , 120 ] . although not discussed here , boosting treg numbers or activity in the heart is reported to be beneficial as well in experimental models of infarction - driven remodeling [ 121124 ] . although the exact basis for the effectiveness of ivig therapy is not clear , ivig therapy is thought to modulate the inflammatory process and recent evidence indicates that ivig therapy acts in part by stimulating an immature myeloid population of dendritic cells that secretes il-10 and favors expansion of fc - specific natural treg cells . the prodrug fty720 ( fingolimod ) is phosphorylated in vivo and has been shown to trap nave and memory t cells in the thymus and secondary lymphoid organs by downregulating sphingosine 1-phosphate receptor 1 ( s1p1 ) . in patients with acs , il-2 plays a critical role in treg cell activity , growth , and survival , but there may be insufficient il-2 to sustain their in vivo potentiation . emerging evidence indicates that enhancing treg cell numbers and activity with low - dose il-2 treatment is effective in treating autoimmune and inflammatory diseases [ 12 , 148151 ] , although there may be intrinsic risk as il-2 is also a key growth factor for effector cd4 t cells and nk cells . in addition , the vitamin d receptor agonist bxl-01 - 0029 was shown to inhibit ifn- and tnf--induced cxcl10 secretion by fetal human cardiac myocytes and reduce cxcl10 protein secretion and gene expression by cd4 t cells . beyond an expected role in allograft disease and the development and progression of atherosclerosis , an abundant body of evidence supports a significant contribution of the immune system to many other cardiovascular settings , ranging from the development and maintenance of hypertension to cardiac and vessel remodeling ( whether constrictive or expansive ) in response to hemodynamic stress , in both health and disease . antigen - specific modulation of the immune system , for example , through systemic delivery of nanoparticles coated with disease - relevant peptides bound to major histocompatibility complex class ii ( pmhcii ) to expand endogenous antigen - specific tregs , is an optimal strategy in settings where an antigen - specific adaptive immune response has been involved in disease development or progression . however , several cardiovascular diseases will probably resist the identification of a specific pathogenic antigen and will require a broader , although targeted , regulation of the immune response , for example , through administration of low - dose il-2 to promote endogenous tregs . with regard to cxcr3 pathway , besides the therapeutic possibilities listed above , we believe that the identification and development of selective evasins or evasin - like peptides that differentially bind and neutralize cxcl9 or cxcl10 versus cxcl11 may provide an interesting therapeutic strategy to limit pathogenic while preserving regulatory cxcr3 functions .

the columnar organization of neocortex at the minicolumnar ( 2050 m ) and macrocolumnar ( 300600 m ) scales has long been known ( see mountcastle , 1997 ; horton and adams , 2005 for reviews ) . minicolumn - scale organization has been demonstrated on several anatomical bases ( lorente de no , 1938 ; defelipe et al . , 1990 ; peters and sethares , 1996 ) . there has been substantial debate as to whether this highly regular minicolumn - scale structure has some accompanying generic dynamics or functionality . i.e. , one that would apply equally well to all cortical areas and species has been determined . one basis upon which a functionality for the minicolumn has been suggested is possession of highly similar receptive field characteristics , or tuning , by the cells comprising the minicolumn , e.g. , v1 orientation columns ( hubel and wiesel , 1962 , 1968 ) and minicolumn - sized regions innervating cutaneous zones ( favorov and diamond , 1990 ) . the reasoning here appears to be that because a group of cells all have very similar tuning to a particular feature , , e.g. , an edge at a particular orientation , they form a unit whose function is to recognize . however , in searching for a possible generic minicolumn function , we need not limit ourselves to considering only recognition functions . furthermore , possession of highly similar tuning can not be a basis for a generic minicolumn functionality since in many cortical areas , the cells encountered along vertical penetrations can have quite variable tuning ( cf . on closer analysis , this is true in orientation column data as well ( hetherington and swindale , 1999 ; ringach et al . , 2002 ) . if there is a generic minicolumn functionality , then it must be compatible with varying degrees of tuning correlation amongst the minicolumn 's cells . i propose that the minicolumn does have a generic functionality ( given shortly ) , but one that only becomes clear when seen in the context of the function of the higher - level , subsuming unit , namely , the macrocolumn , which has been demonstrated anatomically ( goldman and nauta , 1977 ; lbke et al . , 2003 ; egger et al . , 2008 ) and functionally ( mountcastle , 1957 ; woolsey and van der loos , 1970 ; hubel and wiesel , 1974 ; albright et al . , 1984 ) . i propose that the function of a macrocolumn ( e.g. , hypercolumn , segregate , barrel ) is to store sparse distributed representations of its overall input patterns , and to act as a recognizer of those patterns . by overall input pattern , i mean the macrocolumn 's overall input at a given moment , including not only its bottom - up ( bu ) inputs from thalamus or lower cortical areas , but also its top - down ( td ) inputs from higher cortical areas and its horizontal ( h ) inputs , which i propose carry temporal ( sequential ) context information ( recurrently ) from the representations previously active in the same and nearby macrocolumns . thus , an overall input pattern can equally well be termed , a context - dependent input . thus , it is in fact the macrocolumn whose generic functionality is appropriately characterized as recognition ; i.e. , recognition of a class determined by the history of context - dependent inputs to which it has been exposed . a distributed representation of an item of information is one in which multiple units collectively represent that item , and crucially , such that each of those units generally participates in the representations of other items as well . distributed representations are also referred to as cell assemblies , population codes , or ensembles . in this paper , representation and a sparse distributed code ( sdc ) is one in which only a small fraction of the pool of available representing units is part of any particular code ( palm , 1982 ; lynch et al . , 1986 ; kanerva , 1988 ) . if the macrocolumn stores sdcs , then there must be some mechanism that enforces sparseness and this , i propose , is the generic function of the minicolumn . specifically , i propose that small , physically localized pools of l2/3 pyramidals , e.g. , 20 such cells , act as winner - take - all ( wta ) competitive modules ( cms ) . this implies that macrocolumnar codes should consist of about 6080 active l2/3 cells , one per minicolumn : for simplicity , assume 70 minicolumns per macrocolumn hereafter . defined in this way , the minicolumn has a more flexible relation to the ontogenetic column , the apical dendrite bundle , the dbc horsetail , etc . for example , a given minicolumn might include l2/3 pyramidals from more than one apical dendrite bundle , consistent with the findings of yoshimura and callaway ( 2005 ) of fine - scale networks of preferentially interconnected l2/3 pyramidals . there is increasing evidence for the use of sdc in the cortex and other brain structures ; e.g. , auditory cortex ( hromdka et al . , 2008 ) , visual areas ( young and yamane , 1992 ; vinje and gallant , 2000 ; waydo et al . , 2006 ; quian quiroga et al . , 2008 ) , zebra finch neopallium ( hahnloser et al . , 2002 ) , olfactory structures ( jortner et al . , 2007 ; linster and cleland , 2009 ; poo and isaacson , 2009 ) , and hippocampus ( leutgeb et al . , 2007 ) . particularly supportive of the proposed hypothesis is the reid lab 's calcium imaging data of rat v1 during stimulation by drifting square - wave gratings ( ohki et al . , their movie ( http://reid.med.harvard.edu/movies.html ) shows sparse collections of l2/3 cells extending over an approximately macrocolumn - sized region synchronously turning on and off in response to particular grating orientations . ( two frames from the movie ) show distinct sets of cells , i.e. , codes , responding to bars moving left and right , respectively , and emphasize that individual units may participate in multiple codes ( red - circled cells ) . in terms of the proposed model , the active neurons would be the winners in their respective minicolumns , as suggested in figures 1c , d . figure s1 in supplementary material provides another view of how the proposed model maps onto cortex . calcium ( tangential ) images of l2/3 of rat visual cortex showing sparse sets of cells activating in unison ( see movie link in text ) in response to leftward ( a ) and rightward ( b ) drifting gratings . from ohki ( c , d ) sketch of proposed sparse distributed coding concept , which could plausibly give rise to data like ( a ) and ( b ) . : to make the sketches look more like the calcium images , black is used for inactive units and white for active : this is reversed in subsequent figures . if the macrocolumn does indeed function as a sdc field in the way suggested here , then we must answer two key questions regarding its dynamics . how is any particular set of winners , one in each of the 70 minicolumns , initially chosen in response to an input pattern and bound into a holistic code ? that is , how are macrocolumnar codes learned ? how is a previously learned code reactivated in response to future presentations of the input pattern that it was initially chosen to represent ? that is , how are stored macrocolumnar codes retrieved ( reactivated ) ? in the next section , i describe an algorithm , referred to as the code selection algorithm ( csa ) , which answers both questions . a key property of the csa is that it causes similar inputs to be assigned to similar , i.e. , more highly intersecting , codes . this property , which will be referred to as sisc ( similar inputs map to similar codes ) , is very important in terms of computational efficiency ( see discussion ) and is possessed by most distributed coding schemes . however , the csa achieves it in a novel , probabilistic fashion , which can be summarized as follows : determine the familiarity of a macrocolumn 's input . to a first approximation , an input 's familiarity is its maximum similarity to any input previously stored in the macrocolumn . set the amount of randomness ( noise ) in the process of selecting winners in the wta modules in inverse proportion to the input 's familiarity . the algorithm 's rationale is described in detail in the next section , but broadly , the idea is as follows . when an input , 1 , is familiar , we want to reactivate the code , 1 , to which 1 was previously assigned . the cells comprising 1 will have had their synapses ( from the cells comprising 1 ) increased during learning . thus , if 1 is presented again , the cells of 1 will have the highest synaptic input summations in their respective wta modules . in this case , winners should be chosen on the deterministic basis of these summations : no noise should be present in the winner selection process . on the other hand , increasingly novel inputs should be assigned to increasingly distinct codes , i.e. , codes having progressively smaller intersection with existing codes . this can be achieved by increasing the noisiness of the winner selection process in each wta module , which can be achieved by suppressing the influence of the deterministic synaptic inputs ( which reflect prior learning ) relative to baseline random ( spontaneous ) activity . by adjusting parameters that control the global ( i.e. , across the whole macrocolumn ) noise level , we can modulate the expected intersection between the set of cells which have the maximal input summations in their respective wta modules and the set of winners that are actually chosen , thus implementing sisc . many experimental and theoretical studies implicate neuromodulators , notably norepinephrine ( ne ) and acetylcholine ( ach ) , in functionality similar to the above , which can be described generally as modulating signal - to - noise ratio ( snr ) . doya ( 2002 ) proposed that ne levels control the amount of noise in a process of choosing output actions . however , doya 's model assumes a localist representation of the choices , which precludes possession of the sisc property ( see discussion ) . in addition , increased ach has been shown to selectively increase the strength of afferent relative to intrinsic inputs in piriform cortex ( hasselmo and bower , 1992 ) and other brain structures ( see hasselmo , 2006 for review ) . these ach findings have been summarized as showing that increased ach adjusts network dynamics to favor encoding new memories compared to retrieving old memories , which fits well with the proposed csa functionality . following the model description , i offer a speculative mapping of my proposed model onto neural circuitry and discuss evidence for novelty - contingent noise modulation by both ne and ach . however , the specifics of this mechanism are a subject of ongoing research and likely will involve interactions between neuromodulatory systems ( cf . any discussion of columnar function of course centrally concerns cortical circuitry , and more specifically , the putative canonical cortical microcircuit ( rockland and pandya , 1979 ; douglas et al . , 1989 ; douglas and martin , 1991 ) . we have made huge progress in understanding many of the components of cortical microcircuitry a tiny sample of which includes ( defelipe et al . , 1990 ; , 2004 ; feldmeyer et al . , 2006 ; fukuda et al . , 2006 ; , 2008 ; hirata and castro - alamancos , 2008 ; berger et al . , 2009 ; murayama et al . , 2009 ; symes and wennekers , 2009 ; briggs , 2010 ; and see thomson et al . , 2002 ; bannister , 2005 ; silberberg et al . , 2005 for reviews ) nevertheless , we remain far from any sort of comprehensive and consensual understanding of how cortical columnar circuitry manipulates , i.e. , stores and retrieves , specific items of information . in the main , only very broad conclusions regarding information processing are asserted in the experimental literature , e.g. , horizontal connections between fast - spiking l4 interneurons and pyramidals are involved in formation of l4 assemblies and sharpening of tuning ( sun et al . , 2006 ) ; that both the populations of l4 pyramidals and of l5a pyramidals have transcolumnar connectivity patterns allowing them to act as integrators of information coming in from multiple vibrissae in parallel , or in close sequence ( schubert et al . , 2007 ) ; that the receptive fields of barrel - related l2/3 pyramids are dynamic and thus may reflect learning to recognize spatiotemporal patterns of vibrissae deflections ( brecht et al . , 2003 ) ; that wta competition occurs in the supra- and infragranular layers ( douglas and martin , 2004 ) ; and that local ( 100 m ) l2/3-to - l2/3 connections might serve to synchronize firing of l2/3 cell assemblies ( lbke and feldmeyer , 2007 ) ; etc . i believe the hypothetical model described herein to be a significant contribution because it goes beyond broad conclusions and offers a mechanistic explanation of how specific informational items are learned and retrieved and in so doing , proposes a generic function for the minicolumn , i.e. , that it functions as a wta module in support of manipulating sdcs at the next higher , i.e. , macrocolumnar , scale . in particular , it was stated in the introduction that sparse macrocolumnar codes are chosen in response to a macrocolumn 's overall input , which includes its bu , h , and td inputs . however , illustrations of the model in operation in that general case become rather complex , particularly since the h ( and td ) weights carry temporal information , which necessitates showing the model at multiple successive time steps while processing spatiotemporal patterns . more importantly , the core elements of the hypothesis which are : ( a ) that the macrocolumn stores sdcs consisting of one winning l2/3 cell per minicolumn ; and ( b ) that the sisc property is achieved by modulating the amount of randomness ( noise ) present in the winner selection process in inverse proportion to input familiarity can be clearly and more simply described for the bu - only case ( i.e. , where inputs are purely spatial patterns ) . therefore , the model description in this paper will be limited to the bu - only case . however , the generalized model ( with bu , h , and td inputs ) and the accompanying generalized version of the csa are given in figures s2 and table s1 in supplementary material . functional architecture . the input field ( f1 ) consists of binary feature detectors : a particular input consisting of five active features is shown . it consists of winner - take - all competitive modules ( cms ) proposed as analogous to minicolumns . bottom - up connectivity is all - to - all : gray lines signify initially 0 weights . the familiarity ( g ) of the input is proposed to be computed via a subcortical , neuromodulator - based mechanism , which then modulates the f2 unit activation function parameters ( e.g. , sigmoid height ) contingent on g ( purple arrows ) . the input field , f1 , is comprised of 12 binary units and can be considered analogous to a specific thalamic nucleus , though topographical organization is not assumed . the coding field , f2 , consists of q = 4 wta cms , each containing k = 3 binary units . complete ( all - to - all ) bu connectivity from f1 to f2 is assumed for simplicity . these bu weights ( synapses ) are binary , initially 0 , and are permanently set to a weight of 1 the first time the pre- and postsynaptic units are co - active ( i.e. , hebbian learning ) . the model 's core algorithm , the csa , determines which cells are chosen to represent an input , during both learning and retrieval . a single iteration of the algorithm involves two rounds of competition in the cms of f2 . the first round is a hard wta competition ( represented by boxes labeled max in figure 2 ) . the purpose of the first round is to compute a global familiarity measure , g , of the input pattern . g then drives a global modulation of the f2 unit activation function ( figure 2 : purple arrows ) in preparation for the second competitive round , which is a soft wta competition , the intent of which is that : as g goes to 1 ( indicating a completely familiar input ) , the probability that the unit with the highest input summation in a cm wins approaches 1 , and as g goes to 0 ( indicating a completely novel input ) , all units in a cm become equally likely to win ( regardless of their input summations ) . ( 1)u(i)=j nw(j , i ) where n is the current input ( f1 ) pattern . because unit activations are binary , we can simply sum the weights , w(j , i ) , which are also binary . normalize u(i ) to [ 0 .. 1 ] , yielding v(i ) . v(i ) is a local measure of support , or likelihood , that f2 unit i should be activated . it reflects how well unit i 's receptive field ( rf ) , specified by its afferent weight vector , matches the current input vector . ( round 1 competition ) the maximum v(i ) , v^x , is found in each of the q cms . ( 3)v^x = maxi cx{v(i ) } where x indexes the cms and i indexes the units in a cm , cx . average the q v^xvalues , yielding g , a global measure of the familiarity of the current input . ( 4)gx=1qv^x / q the expansivity , , of the probabilistic activation function ( which is implemented via steps 68 ) is set as an increasing nonlinear function of g ( eq . 5 , expressed as a table ) . the idea is to increase the range of relative win likelihoods , (i ) ( defined in step 6 ) over any given cm 's units as g goes to 1 . this in turn , serves to nonlinearly exaggerate the difference in the final win probabilities ( eq . the specific parameters of any instance of the g - to- mapping will determine the specifics of the relation between input similarity and code similarity , i.e. , the expected code intersection as a function of input similarity . 5 were chosen to yield the -distributions in the examples of figures 3 and 4 . circled numbers refer to algorithm steps in text . here , i show the case of the first input , 1 , presented to the model . the algorithm detects that 1 is completely unfamiliar , i.e. , g is computed to be 0 , and sets the f2 activation function to a constant function ( red line ) , which makes the choice of winner in each cm completely random , and thus , the overall f2 code , 1 , also completely random . green f1 ( f2 ) units denote units not in common with 1 ( 1 ) . circled numbers refer to algorithm steps in text . here , i show the case of the first input , 1 , presented to the model . the algorithm detects that 1 is completely unfamiliar , i.e. , g is computed to be 0 , and sets the f2 activation function to a constant function ( red line ) , which makes the choice of winner in each cm completely random , and thus , the overall f2 code , 1 , also completely random . the csa and the sisc property . green f1 ( f2 ) units denote units not in common with 1 ( 1 ) . the v values of all units in all cms are then passed through the sigmoidal activation function ( eq . 6 ) whose shape / scale reflects g. again , particular parameter values affect the relation of input similarity to code similarity ( and therefore , storage capacity ) : values of = 28 and = 5 produce the v - to- mappings in figure 4 . as noted above , within each cm , the output variable , (i ) , can be viewed as a relative likelihood that unit i should be chosen winner . ( 6)(i)=1+e ( v(i ) + )+1 when g = 1 ( perfectly familiar ) , is maximized ( in eq . 7 ) probabilities of winning for units with the maximum v value in their respective cms . in contrast , when g = 0 ( completely novel ) , = 0 , which collapses the sigmoid to the constant function , = 1 , thus making all units in a cm equally likely to win . this causes the expected intersection of the code being chosen in the current instance with any previously assigned code to be at chance level . in general , this modulation of the sigmoid activation function tends toward code completion in proportion to the familiarity of the input and code separation in proportion to its novelty . transform relative likelihood distribution ( ) in each cm to true probability distribution ( ) . ( 7)(i)=(i)k cm(k ) ( round 2 competition ) choose an f2 code by drawing a winner from the -distribution ( soft max ) in each cm . thus , choosing an f2 code is actually performed as q separate draws . when g = 0 , these draws are statistically independent , as in figures 3 and 4d . as we consider increasingly familiar inputs , i.e. , for g approaching 1 ( and , assuming the model is still operating in a regime where crosstalk is sufficiently low ) , the draws become increasingly correlated ( dependent ) , as can be seen in going from figure 4c to 4b to 4a . figure 3 graphically illustrates the operation of the csa in the case of the model being presented with its first input , 1 . the gray arrows indicate that the bu signals propagating from the active f1 units will be traversing connections with zero synaptic strength . this leads to unnormalized ( u ) and normalized ( v ) input summations of 0 for all 12 f2 units ( steps 1,2 ) . in step 3 , the max v , v^ , in each cm is found ( ties broken at random ) . in step 4 , g is computed as the average of the v^ values : in this case all the v^ are 0 , so g = 0 . in step 5 , the value , g = 0 , maps to = 0 , which causes the activation function of the f2 units to collapse to the constant function , = 1 . in step 6 , each f2 unit applies this activation function to its v value , yielding the uniform relative likelihood distribution in each cm . in step 7 , the relative likelihood function in each cm is normalized to a true probability ( ) distribution , which in this case , is again uniform . finally , in step 8 , a winner is drawn in each cm , resulting in a random f2 code , e.g. , 1 . figure 4 demonstrates that the csa realizes the sisc property by considering four possibilities ( a d ) for the second input presented to the model of figure 3 . these four inputs , 25 , range from being identical to 1 ( completely familiar ) to having zero overlap with 1 ( completely unfamiliar ) . to save space , the panels of figure 4 use an abbreviated version of the format of figure 3 . most noticeably , the intermediate variable , ( relative likelihood ) , is not shown . black bu connections are ones that were increased to one when 1 was learned ( figure 3 ) . working from figure 4a to 4d , the inputs have progressively lower similarity ( intersection ) with 1 : f1 units not in common with 1 are shown in green . as g drops , the sigmoid expansivity drops ( note the changing scale ) . thus , the -distributions become progressively flatter , which in turn results in f2 codes , 25 , having progressively smaller intersection with 1 . figure 4a shows the case of presenting a completely familiar input again , and is thus a recognition test trial , demonstrating retrieval . this leads , via csa steps 3 and 4 , to g = 1 , which yields , via steps 5 and 6 , the expansive nonlinear v - to- mapping shown ( red sigmoid ) . this nonlinearity is applied to every f2 unit , yielding the highly peaked -distributions shown . finally , one unit is drawn in each cm . the probability of drawing the correct unit in any single cm is approximately 98% . of course , what 's crucial in this case , i.e. , when the input is completely familiar ( g = 1 ) , is that the entire correct f2 code is reactivated . in this case , that probability is ( 0.98 ) 92% . thus , the familiarity , g , which depends on the entire f2 layer and is thus global information , influences the local activation functions so as to produce the desired overall result , in this case , reactivation of the code ( memory trace ) , 1 , of the familiar input pattern , 1 . the explanations of the remaining panels follow that of figures 3 and 4a . in going from figure 4b to 4d , one can readily see decreasing intersection with 1 , decreasing u and v values , decreasing g , decreasing sigmoid expansivity , progressively flatter -distributions , and ultimately , decreasing intersection with 1 . given a desired probability , r , of correctly reactivating an entire code ( i.e. , of choosing the correct unit in each cm ) , when g = 1 , given values for q and k , we could compute the needed value of . however , the macrocolumn model is a content - addressable associative memory and in actual usage , multiple sparse codes will be stored in superposition . any thorough analysis of the model 's expected retrieval error must include the effect of overlap between the stored codes ( i.e. , cross - talk ) : this influences the shapes of the -distributions and thus , the expected retrieval accuracy for any given number of stored codes . such an analysis will be conducted empirically and reported in a separate paper . before leaving figure 4 first , while the -distributions become flatter as g decreases , the units comprising the code of the most similar previously learned input ( here , 1 ) remain most likely to win in their respective cms . if we simply deterministically chose the unit with maximum v(i ) in each cm , we would have chosen the same f2 code , 1 , in response to all four inputs , 25 . thus , the computation of a quantity , g , which depends on all the cms is essential to achieving the sisc property . it constitutes a channel through which information transfers between all f2 units throughout the whole macrocolumn . as noted earlier , the full model also assumes direct h connections between f2 units , analogous to the horizontal matrix of l2/3 ( see figure s2 in supplementary material ) . these also mediate communication , but of the prior code active in the macrocolumn , not of the simultaneous state of all f2 units throughout the macrocolumn . second , learning is single - trial and involves only one iteration of the csa . this is largely facilitated by the fact that when a given input - code association , jj , is learned , each of j 's f2 units simultaneously has its afferent weight from all of j 's f1 units increased . the effect of these simultaneous correlated potentiations allows a rapid , even single - trial , formation of an association , even if the individual synaptic potentiations are small , consistent with the recent characterization of thalamocortical learning described in bruno and sakmann ( 2006 ) . third , figure 4a shows that recognizing an exact instance of a previous input also requires only one iteration of the csa . although this example does not directly show it , this holds for recognition of non - exact matches as well . evidence for this will be presented in a separate work . that both learning and recognition require only a single csa iteration is especially significant since , as can readily be seen , none of the csa steps involves iterations over stored codes : thus , the time it takes for the csa to either store a new input or retrieve the closest matching stored input remains constant as the number of stored codes increases . this does not imply that an infinite number of codes can be stored : of course , the model has finite storage capacity . this capacity will be characterized in future research , but should be similar to other sparse associative memories ( willshaw et al . , 1969 ; palm , 1982 ; moll and miikkulainen , 1995 ; knoblauch et al . , 2010 ) . there remain huge gaps in our knowledge of the intrinsic physiological , synaptic , morphological , and connectional properties of all classes of cortical cell and of functional relationships between cortical and sub - cortical structures . nevertheless , figure 5 shows a possible neural realization of the model 's wta cm , i.e. , minicolumn , and dynamics ( csa ) . i have attempted to respect known constraints but the realization is admittedly speculative and ongoing modifications will undoubtedly be required . figures 5a e show the critical events transpiring in a single minicolumn at five points in time during the csa 's computational cycle . note that due to space limitations figure 5 can not depict the true extents of the various axonal and dendritic systems of the cells involved . ( a e ) depict critical events transpiring in a single minicolumn at five points in time during the csa 's computational cycle , which i propose corresponds to one gamma cycle : approximate timings relative to start of csa cycle are shown across top . the units labeled c ( b ) represent the local chandelier ( basket cell ) populations , respectively ; see text for detailed explanation . figure 5a shows the initial csa steps 1 and 2 when the l2/3 pyramidals ( here only two cells , but in reality , 20 ) integrate their inputs . while the csa explanation in the prior section included only the bu inputs , all three input classes are included here : bu inputs ( labeled 2 ) are mediated via l4 ( rockland and pandya , 1979 ) td inputs ( 1 ) are mediated via l2/3 apical tufts ( rockland and drash , 1996 ) h inputs ( 3 ) are mediated via the horizontal matrix of l2/3 ( gilbert and wiesel , 1989 ; feldmeyer et al . , 2006 ) all three input vectors arrive in parallel and collectively give rise to postsynaptic potentials ( psps ) in the l2/3 pyramidals . the three ( normalized ) inputs are combined multiplicatively ; see the generalized version of the csa ( table s1 in supplementary material ) . c ) are firing at this time , preventing the l2/3 pyramids from firing . in figure 5b , the chandeliers shut off ( grayed out ) and the l2/3 pyramid with the highest psp ( cell 2 ) is assumed to be the first to spike ( csa step 3 ) . this winning cell , and more specifically , its psp value ( v in eq . 2 ) , represents this minicolumn 's local judgment of how similar the macrocolumn 's closest - matching stored input is to the current overall ( i.e. , bu , h , and td ) input . the output of cell 2 is then communicated to some locus where it is averaged with the round 1 winners from the other 70 minicolumns of the macrocolumn , yielding g ( csa step 4 ) . as noted in the introduction , the functionality related to g seems most consistent with the phenomenology of neuromodulatory systems , especially ach and ne . note that the communication of cell 2 s psp value is hypothesized to be mediated via l5 , one of whose pyramidal cells is seen integrating here ( light green ) ; this is based loosely on evidence that l5 cells , specifically l5b pyramidals , almost exclusively target pontine areas ( with collaterals to thalamus ) ( deschnes et al . , 1994 ; krieger et al . , 2007 ) . l2/3 pyramidals are the primary source of bu signals to higher cortical areas ( rockland and pandya , 1979 ; and see thomson et al . , 2002 ; brecht et al . , 2003 ; schubert et al . , 2003 ; bannister , 2005 ; helmstaedter et al . , 2007 ; lbke and feldmeyer , 2007 ; petersen , 2007 ; egger et al . , 2008 ; , 2009 for wider background on cortical columnar circuitry relevant to the current proposal ) . in addition , as stated earlier , the horizontal l2/3-to - l2/3 connections are proposed to communicate this macrocolumn 's final hypothesis regarding its total input pattern in the current csa cycle recurrently back to the same ( and surrounding ) macrocolumns on the next csa cycle . hence , it is crucial that since that final hypothesis is not present until the second round of competition completes ( figure 5e ) , the output pathways carrying those signals must be closed ( red xs on paths 4 and 5 ) . though not depicted here , one possible mechanism for selectively preventing horizontal signaling in l2/3 is activation of the double bouquet cells ( defelipe et al . , 1990 , 2006 ; peters and sethares , 1997 ) . axons , being interdigitated , nearly one - to - one with minicolumns would allow them to make contact with a substantial portion of the horizontally ( and obliquely ) oriented dendritic and axonal processes , in l2/3 , and thus prevent passage of horizontal signals . in figure 5c , the l5 pyramidal mediating the winning l2/3 cell 's psp value has reached threshold and sends that output to the sub - cortical averaging locus ( path 6 ) . in addition , the winning cell itself has activated the local basket cell network ( electrically coupled , cf . b , which rapidly deactivates and re - polarizes ( resets ) the l2/3 pyramidals ( grayed out ) . this reset need not be back to a completely even baseline : some remnant of the relative psp distribution prior to basket cell activation might remain , biasing the second round of competition . in figure 5d , the result of the subcortical computation of g is returned to the macrocolumn ( path 7 ) in the form of neuromodulator release ( purple cloud surrounding the l2/3 pyramidals ) . cloud actually extends across a broad , i.e. , macrocolumnar ( or wider ) , expanse of l2/3 , not just within a single minicolumn as this figure may suggest . the chandeliers are again firing to prevent any l2/3 from firing as the second round of integration occurs . the basket cells are inactive , allowing this integration to take place . in figure 5e , in general , the pyramidal cell winning on this second round of competition may differ from the first round winner . in particular , the probability that the second round winner is the same as the first round winner increases with g. the set of l2/3 winners , one per minicolumn , across the whole macrocolumn represents that macrocolumn 's final decision ( hypothesis ) as to identity of its current overall ( td , h , and bu ) input . thus , the output of the winning l2/3 cell in each minicolumn is now communicated to : lower cortical regions via l5 and its backprojections to the lower regions l1 ( labeled 8 ) ( rockland and drash , 1996 ) . l2/3 pyramids in the same and neighboring macrocolumns via the local horizontal l2/3 matrix ( 5 ) ( gilbert and wiesel , 1989 ; feldmeyer et al . , 2006 ) , thus delivering temporal context information ( recurrently ) to the local region to be integrated on the next csa cycle . the l4 layer of higher cortical regions ( 4 ) ( rockland and pandya , 1979 ) . note that the output of the winning l2/3 cell should be prevented from recurring to the local basket network at this time so as to allow the integration period to occur at the beginning of the next computational cycle ; hence , the red x on the link to basket cell . i reiterate that the above possible cortical realization of the proposed sdc model is highly speculative . it clearly lacks numerous details , especially regarding processing in the intermediate processing stages , e.g. , l4 , and output processing involving l5 ( and l6 ) . nevertheless , it is a starting point and can be falsified , especially as experimental methods mature . we still have decidedly little in the way of hard constraints on the time courses of activation of the many cell types involved in cortical ( and hippocampal ) circuits , though progress is being made ( klausberger et al . , 2003 , 2004 ; silberberg et al . , 2005 ; silberberg and markram , 2007 ; klausberger and somogyi , 2008 ; otsuka and kawaguchi , 2009 ; woodruff et al . , 2009 ) . moreover , the proposed theory 's key assumption that the l2/3 pyramidals are the core repository of information in cortex and that the codes laid down in l2/3 are the context - dependent memories of our experiences , is subject to challenge . specifically , the anatomy of the l5 thick tufted cells suggests that they too have access to bu ( via l4 ) , td ( via their apical tufts in l1 ) , and h ( via an extensive intra - l5 horizontal network , schubert et al . , 2007 ) inputs , and therefore that l5 might store the most detailed and context - dependent codes in cortex , a view supported by findings such as ( de kock et al . , 2007 ) . in the end , for the purpose of this hypothesis and theory paper , i believe the architecture and algorithm ( csa ) to be more important than the specifics of any particular neural realization . in this section , i consider evidence relating to six model predictions : a}signals generated in the macrocolumn [ i.e. , the v^x ( eq . strictly interpreted , figure 2 suggests that the model can only be true of cortical areas that have direct projections to the activation function modulator ( afm ) , hypothesized to be instantiated in midbrain neuromodulator source areas , e.g. , basal forebrain ( bf , source of ach ) and locus coeruleus ( lc , source of ne ) . direct cortical afferents to bf arise mainly from prepyriform , anterior insula , orbitofrontal , entorhinal and medial temporal areas ( mesulam and mufson , 1984 ) . direct cortical afferents to lc arise from dorsal prefrontal cortex ( arnsten and goldman - rakic , 1984 ) , medial prefrontal cortex ( jodo et al . , 1998 ) . while direct projections are limited , a much larger fraction of cortex may be able to influence the hypothesized afm via multi - synaptic pathways involving thalamus and other subcortical structures , especially via pathways interconnecting bf , lc , and other neuromodulator source areas . for example , bf cholinergic neurons are excited by lc ( jones et al . , 2004 ) , which allows dorsal and medial prefrontal areas indirect influence on bf . similarly , lc receives input from the raphe nuclei ( reviewed in samuels and szabadi , 2008 ) which would allow further extension of the sphere of cortical influence upon the afm . however , it is clearly possible that my macrocolumnar model applies only to the smaller set of cortical areas suggested above . after all , there would be some advantage to deferring the decision as to the overall familiarity / novelty of the current input ( moment ) to the very highest cortical levels , which are in position to make the most informed decisions . in this case , once g is computed , it is then broadcast pan - cortically , i.e. , to all levels of the hierarchy , allowing the local choice of code to proceed accordingly , i.e. , with a level of randomness appropriate to g. figure s2 in supplementary material illustrates this view . 5 ) , which correlates with the detection of familiarity , and/or inversely with the detection of novelty . such correlations have been shown for both ach ( miranda et al . , 2000 , 2003 ) and ne ( sara et al . , 1994 ; vankov et al . , lc projects to all of cortex ( foote and morrison , 1987 ; berridge and waterhouse , 2003 ; samuels and szabadi , 2008 ) . bf projects to almost all cortical areas ( reviewed in briand et al . , 2007 ) . the amount of randomness to be added to the winner selection process is a global parameter , which applies non - specifically to all the minicolumns . this is consistent with volume transmission believed to be used by neuromodulatory systems ( descarries et al . , 1997 ; see sarter et al . d}the signal determines ( eqs 68 ) the amount of noise ( randomness ) in the selection ( activation ) of cortical ( i.e. , macrocolumnar ) codes . controlling the noisiness of a process of choosing a winner from a competing group of neurons can be achieved by some combination of two actions : ( i ) increasing the spike probability of cells with high input summations relative to those with low summations and ( ii ) lowering the spike probability of low - input cells relative to high - input cells . both of these actions can be achieved by uniformly ( i.e. , to all competing cells in a wta module ) modulating intrinsic cell properties such as excitability . numerous studies have demonstrated both excitatory and suppressive effects on target cell responses ( both principal neurons and interneurons ) for both ach ( kawasaki and avoli , 1996 ; shalinsky et al . , 2002 ; cobb and davies , 2005 ; tateno et al . , 2005 ; isakova and mednikova , 2007 ; lawrence , 2008 ; eggermann and feldmeyer , 2009 ) and ne ( foote et al . , 1975 ; kawaguchi and shindou , 1998 ; harley and helen , 2007 ; moxon et al . , 2007 ) . it is not my intention here to argue for a precise realization of this mechanism . as suggested in many reviews ( berridge and waterhouse , 2003 ; lucas - meunier et al . , 2003 ; sara , 2009 ) , the landscape of this research is very complex and we are far from a comprehensive understanding of the how the various neuromodulatory systems affect high - level cognitive processing either alone or in concert ( briand et al . , 2007 ) . nevertheless , the large and varied body of evidence at least admits the possibility that one or more of these neuromodulators could implement the familiarity - contingent afm mechanism ( csa steps 58 ; see doya , 2002 , p. 502 e}decreased , i.e. , increased noise , leads to greater code separation ( decreased intersection ) . recently , goard and dan ( 2009 ) showed that increased bf stimulation decreased the correlation amongst a population of rat v1 cells . this decreased correlation essentially shows increased separation between population codes , which in the model proposed here , would manifest as decreased intersection between sparse codes . f}disabling of the brain 's ability to produce high noise , i.e. , causing to be permanently high , should reduce the ability to learn new inputs , while sparing or having much less effect on recognition of known items . looking at figure 4 , if the afm was stuck in the highly expansive sigmoid condition ( low noise ) , all four inputs , 25 , would have high probability of mapping to the same code , 1 . however , in general , inputs that were mapped to unique codes prior to such a disabling event will reliably activate those codes on future presentations . in accord with this , browning et al . ( 2010 ) found that severely diminishing cholinergic inputs to inferotemporal cortex severely reduced macaques performance on a visual episodic memory task , while having little effect on a dnms task . ( 2005 ) found a similar effect : cholinergic deafferentation of entorhinal cortex reduced performance on dnms tasks involving novel odors but not familiar odors . the model 's core algorithm , the csa , determines which cells are chosen to represent an input , during both learning and retrieval . a single iteration of the algorithm involves two rounds of competition in the cms of f2 . the first round is a hard wta competition ( represented by boxes labeled max in figure 2 ) . the purpose of the first round is to compute a global familiarity measure , g , of the input pattern . g then drives a global modulation of the f2 unit activation function ( figure 2 : purple arrows ) in preparation for the second competitive round , which is a soft wta competition , the intent of which is that : as g goes to 1 ( indicating a completely familiar input ) , the probability that the unit with the highest input summation in a cm wins approaches 1 , and as g goes to 0 ( indicating a completely novel input ) , all units in a cm become equally likely to win ( regardless of their input summations ) . ( 1)u(i)=j nw(j , i ) where n is the current input ( f1 ) pattern . because unit activations are binary , we can simply sum the weights , w(j , i ) , which are also binary . normalize u(i ) to [ 0 .. 1 ] , yielding v(i ) . v(i ) is a local measure of support , or likelihood , that f2 unit i should be activated . it reflects how well unit i 's receptive field ( rf ) , specified by its afferent weight vector , matches the current input vector . ( round 1 competition ) the maximum v(i ) , v^x , is found in each of the q cms . ( 3)v^x = maxi cx{v(i ) } where x indexes the cms and i indexes the units in a cm , cx . average the q v^xvalues , yielding g , a global measure of the familiarity of the current input . ( 4)gx=1qv^x / q the expansivity , , of the probabilistic activation function ( which is implemented via steps 68 ) is set as an increasing nonlinear function of g ( eq . the idea is to increase the range of relative win likelihoods , (i ) ( defined in step 6 ) over any given cm 's units as g goes to 1 . this in turn , serves to nonlinearly exaggerate the difference in the final win probabilities ( eq . the specific parameters of any instance of the g - to- mapping will determine the specifics of the relation between input similarity and code similarity , i.e. , the expected code intersection as a function of input similarity . 5 were chosen to yield the -distributions in the examples of figures 3 and 4 . circled numbers refer to algorithm steps in text . here , i show the case of the first input , 1 , presented to the model . the algorithm detects that 1 is completely unfamiliar , i.e. , g is computed to be 0 , and sets the f2 activation function to a constant function ( red line ) , which makes the choice of winner in each cm completely random , and thus , the overall f2 code , 1 , also completely random . green f1 ( f2 ) units denote units not in common with 1 ( 1 ) . , i show the case of the first input , 1 , presented to the model . the algorithm detects that 1 is completely unfamiliar , i.e. , g is computed to be 0 , and sets the f2 activation function to a constant function ( red line ) , which makes the choice of winner in each cm completely random , and thus , the overall f2 code , 1 , also completely random . the csa and the sisc property . green f1 ( f2 ) units denote units not in common with 1 ( 1 ) . the v values of all units in all cms are then passed through the sigmoidal activation function ( eq . 6 ) whose shape / scale reflects g. again , particular parameter values affect the relation of input similarity to code similarity ( and therefore , storage capacity ) : values of = 28 and = 5 produce the v - to- mappings in figure 4 . as noted above , within each cm , the output variable , (i ) , can be viewed as a relative likelihood that unit i should be chosen winner . ( 6)(i)=1+e ( v(i ) + )+1 when g = 1 ( perfectly familiar ) , is maximized ( in eq . 7 ) probabilities of winning for units with the maximum v value in their respective cms . in contrast , when g = 0 ( completely novel ) , = 0 , which collapses the sigmoid to the constant function , = 1 , thus making all units in a cm equally likely to win . this causes the expected intersection of the code being chosen in the current instance with any previously assigned code to be at chance level . in general , this modulation of the sigmoid activation function tends toward code completion in proportion to the familiarity of the input and code separation in proportion to its novelty . transform relative likelihood distribution ( ) in each cm to true probability distribution ( ) . ( 7)(i)=(i)k cm(k ) ( round 2 competition ) choose an f2 code by drawing a winner from the -distribution ( soft max ) in each cm . thus , choosing an f2 code is actually performed as q separate draws . when g = 0 , these draws are statistically independent , as in figures 3 and 4d . as we consider increasingly familiar inputs , i.e. , for g approaching 1 ( and , assuming the model is still operating in a regime where crosstalk is sufficiently low ) , the draws become increasingly correlated ( dependent ) , as can be seen in going from figure 4c to 4b to 4a . figure 3 graphically illustrates the operation of the csa in the case of the model being presented with its first input , 1 . the gray arrows indicate that the bu signals propagating from the active f1 units will be traversing connections with zero synaptic strength . this leads to unnormalized ( u ) and normalized ( v ) input summations of 0 for all 12 f2 units ( steps 1,2 ) . in step 3 , the max v , v^ , in each cm is found ( ties broken at random ) . in step 4 , g is computed as the average of the v^ values : in this case all the v^ are 0 , so g = 0 . in step 5 , the value , g = 0 , maps to = 0 , which causes the activation function of the f2 units to collapse to the constant function , = 1 . in step 6 , each f2 unit applies this activation function to its v value , yielding the uniform relative likelihood distribution in each cm . in step 7 , the relative likelihood function in each cm is normalized to a true probability ( ) distribution , which in this case , is again uniform . finally , in step 8 , a winner is drawn in each cm , resulting in a random f2 code , e.g. , 1 . figure 4 demonstrates that the csa realizes the sisc property by considering four possibilities ( a d ) for the second input presented to the model of figure 3 . these four inputs , 25 , range from being identical to 1 ( completely familiar ) to having zero overlap with 1 ( completely unfamiliar ) . to save space , the panels of figure 4 use an abbreviated version of the format of figure 3 . most noticeably , the intermediate variable , ( relative likelihood ) , is not shown . black bu connections are ones that were increased to one when 1 was learned ( figure 3 ) . working from figure 4a to 4d , the inputs have progressively lower similarity ( intersection ) with 1 : f1 units not in common with 1 are shown in green . as g drops , the sigmoid expansivity drops ( note the changing scale ) . thus , the -distributions become progressively flatter , which in turn results in f2 codes , 25 , having progressively smaller intersection with 1 . f2 units not in common with 1 also shown in green . figure 4a shows the case of presenting a completely familiar input again , and is thus a recognition test trial , demonstrating retrieval . this leads , via csa steps 3 and 4 , to g = 1 , which yields , via steps 5 and 6 , the expansive nonlinear v - to- mapping shown ( red sigmoid ) . this nonlinearity is applied to every f2 unit , yielding the highly peaked -distributions shown . the probability of drawing the correct unit in any single cm is approximately 98% . of course , what 's crucial in this case , i.e. , when the input is completely familiar ( g = 1 ) , is that the entire correct f2 code is reactivated . in this case , thus , the familiarity , g , which depends on the entire f2 layer and is thus global information , influences the local activation functions so as to produce the desired overall result , in this case , reactivation of the code ( memory trace ) , 1 , of the familiar input pattern , 1 . the explanations of the remaining panels follow that of figures 3 and 4a . in going from figure 4b to 4d , one can readily see decreasing intersection with 1 , decreasing u and v values , decreasing g , decreasing sigmoid expansivity , progressively flatter -distributions , and ultimately , decreasing intersection with 1 . given a desired probability , r , of correctly reactivating an entire code ( i.e. , of choosing the correct unit in each cm ) , when g = 1 , given values for q and k , we could compute the needed value of . however , the macrocolumn model is a content - addressable associative memory and in actual usage , multiple sparse codes will be stored in superposition . any thorough analysis of the model 's expected retrieval error must include the effect of overlap between the stored codes ( i.e. , cross - talk ) : this influences the shapes of the -distributions and thus , the expected retrieval accuracy for any given number of stored codes . such an analysis will be conducted empirically and reported in a separate paper . before leaving figure 4 first , while the -distributions become flatter as g decreases , the units comprising the code of the most similar previously learned input ( here , 1 ) remain most likely to win in their respective cms . if we simply deterministically chose the unit with maximum v(i ) in each cm , we would have chosen the same f2 code , 1 , in response to all four inputs , 25 . thus , the computation of a quantity , g , which depends on all the cms is essential to achieving the sisc property . it constitutes a channel through which information transfers between all f2 units throughout the whole macrocolumn . as noted earlier , the full model also assumes direct h connections between f2 units , analogous to the horizontal matrix of l2/3 ( see figure s2 in supplementary material ) . these also mediate communication , but of the prior code active in the macrocolumn , not of the simultaneous state of all f2 units throughout the macrocolumn . second , learning is single - trial and involves only one iteration of the csa . this is largely facilitated by the fact that when a given input - code association , jj , is learned , each of j 's f2 units simultaneously has its afferent weight from all of j 's f1 units increased . the effect of these simultaneous correlated potentiations allows a rapid , even single - trial , formation of an association , even if the individual synaptic potentiations are small , consistent with the recent characterization of thalamocortical learning described in bruno and sakmann ( 2006 ) . third , figure 4a shows that recognizing an exact instance of a previous input also requires only one iteration of the csa . although this example does not directly show it , this holds for recognition of non - exact matches as well . evidence for this will be presented in a separate work . that both learning and recognition require only a single csa iteration is especially significant since , as can readily be seen , none of the csa steps involves iterations over stored codes : thus , the time it takes for the csa to either store a new input or retrieve the closest matching stored input remains constant as the number of stored codes increases . this does not imply that an infinite number of codes can be stored : of course , the model has finite storage capacity . this capacity will be characterized in future research , but should be similar to other sparse associative memories ( willshaw et al . , 1969 ; palm , 1982 ; moll and miikkulainen , 1995 ; knoblauch et al . , 2010 ) . there remain huge gaps in our knowledge of the intrinsic physiological , synaptic , morphological , and connectional properties of all classes of cortical cell and of functional relationships between cortical and sub - cortical structures . nevertheless , figure 5 shows a possible neural realization of the model 's wta cm , i.e. , minicolumn , and dynamics ( csa ) . i have attempted to respect known constraints but the realization is admittedly speculative and ongoing modifications will undoubtedly be required . figures 5a e show the critical events transpiring in a single minicolumn at five points in time during the csa 's computational cycle . note that due to space limitations figure 5 can not depict the true extents of the various axonal and dendritic systems of the cells involved . ( a e ) depict critical events transpiring in a single minicolumn at five points in time during the csa 's computational cycle , which i propose corresponds to one gamma cycle : approximate timings relative to start of csa cycle are shown across top . the units labeled c ( b ) represent the local chandelier ( basket cell ) populations , respectively ; see text for detailed explanation . figure 5a shows the initial csa steps 1 and 2 when the l2/3 pyramidals ( here only two cells , but in reality , 20 ) integrate their inputs . while the csa explanation in the prior section included only the bu inputs , all three input classes are included here : bu inputs ( labeled 2 ) are mediated via l4 ( rockland and pandya , 1979 ) td inputs ( 1 ) are mediated via l2/3 apical tufts ( rockland and drash , 1996 ) h inputs ( 3 ) are mediated via the horizontal matrix of l2/3 ( gilbert and wiesel , 1989 ; feldmeyer et al . , 2006 ) all three input vectors arrive in parallel and collectively give rise to postsynaptic potentials ( psps ) in the l2/3 pyramidals . the three ( normalized ) inputs are combined multiplicatively ; see the generalized version of the csa ( table s1 in supplementary material ) . c ) are firing at this time , preventing the l2/3 pyramids from firing . in figure 5b , the chandeliers shut off ( grayed out ) and the l2/3 pyramid with the highest psp ( cell 2 ) is assumed to be the first to spike ( csa step 3 ) . this winning cell , and more specifically , its psp value ( v in eq . 2 ) , represents this minicolumn 's local judgment of how similar the macrocolumn 's closest - matching stored input is to the current overall ( i.e. , bu , h , and td ) input . the output of cell 2 is then communicated to some locus where it is averaged with the round 1 winners from the other 70 minicolumns of the macrocolumn , yielding g ( csa step 4 ) . as noted in the introduction , the functionality related to g seems most consistent with the phenomenology of neuromodulatory systems , especially ach and ne . note that the communication of cell 2 s psp value is hypothesized to be mediated via l5 , one of whose pyramidal cells is seen integrating here ( light green ) ; this is based loosely on evidence that l5 cells , specifically l5b pyramidals , almost exclusively target pontine areas ( with collaterals to thalamus ) ( deschnes et al . l2/3 pyramidals are the primary source of bu signals to higher cortical areas ( rockland and pandya , 1979 ; and see thomson et al . , 2002 ; brecht et al . , 2003 ; schubert et al . , 2003 ; bannister , 2005 ; helmstaedter et al . , 2007 ; lbke and feldmeyer , 2007 ; petersen , 2007 ; egger et al . , 2008 ; , 2009 for wider background on cortical columnar circuitry relevant to the current proposal ) . in addition , as stated earlier , the horizontal l2/3-to - l2/3 connections are proposed to communicate this macrocolumn 's final hypothesis regarding its total input pattern in the current csa cycle recurrently back to the same ( and surrounding ) macrocolumns on the next csa cycle . hence , it is crucial that since that final hypothesis is not present until the second round of competition completes ( figure 5e ) , the output pathways carrying those signals must be closed ( red xs on paths 4 and 5 ) . though not depicted here , one possible mechanism for selectively preventing horizontal signaling in l2/3 is activation of the double bouquet cells ( defelipe et al . , 1990 , 2006 ; peters and sethares , 1997 ) . axons , being interdigitated , nearly one - to - one with minicolumns would allow them to make contact with a substantial portion of the horizontally ( and obliquely ) oriented dendritic and axonal processes , in l2/3 , and thus prevent passage of horizontal signals . in figure 5c , the l5 pyramidal mediating the winning l2/3 cell 's psp value has reached threshold and sends that output to the sub - cortical averaging locus ( path 6 ) . in addition , the winning cell itself has activated the local basket cell network ( electrically coupled , cf . b , which rapidly deactivates and re - polarizes ( resets ) the l2/3 pyramidals ( grayed out ) . this reset need not be back to a completely even baseline : some remnant of the relative psp distribution prior to basket cell activation might remain , biasing the second round of competition . in figure 5d , the result of the subcortical computation of g is returned to the macrocolumn ( path 7 ) in the form of neuromodulator release ( purple cloud surrounding the l2/3 pyramidals ) . cloud actually extends across a broad , i.e. , macrocolumnar ( or wider ) , expanse of l2/3 , not just within a single minicolumn as this figure may suggest . the chandeliers are again firing to prevent any l2/3 from firing as the second round of integration occurs . the basket cells are inactive , allowing this integration to take place . in figure 5e , the chandeliers again deactivate . , the pyramidal cell winning on this second round of competition may differ from the first round winner . in particular , the probability that the second round winner is the same as the first round winner increases with g. the set of l2/3 winners , one per minicolumn , across the whole macrocolumn represents that macrocolumn 's final decision ( hypothesis ) as to identity of its current overall ( td , h , and bu ) input . thus , the output of the winning l2/3 cell in each minicolumn is now communicated to : lower cortical regions via l5 and its backprojections to the lower regions l1 ( labeled 8 ) ( rockland and drash , 1996 ) . l2/3 pyramids in the same and neighboring macrocolumns via the local horizontal l2/3 matrix ( 5 ) ( gilbert and wiesel , 1989 ; feldmeyer et al . , 2006 ) , thus delivering temporal context information ( recurrently ) to the local region to be integrated on the next csa cycle . the l4 layer of higher cortical regions ( 4 ) ( rockland and pandya , 1979 ) . note that the output of the winning l2/3 cell should be prevented from recurring to the local basket network at this time so as to allow the integration period to occur at the beginning of the next computational cycle ; hence , the red x on the link to basket cell . i reiterate that the above possible cortical realization of the proposed sdc model is highly speculative . it clearly lacks numerous details , especially regarding processing in the intermediate processing stages , e.g. , l4 , and output processing involving l5 ( and l6 ) . nevertheless , it is a starting point and can be falsified , especially as experimental methods mature . for example , the many timing relationships in the circuit can be tested . we still have decidedly little in the way of hard constraints on the time courses of activation of the many cell types involved in cortical ( and hippocampal ) circuits , though progress is being made ( klausberger et al . silberberg and markram , 2007 ; klausberger and somogyi , 2008 ; otsuka and kawaguchi , 2009 ; woodruff et al . , 2009 ) . moreover , the proposed theory 's key assumption that the l2/3 pyramidals are the core repository of information in cortex and that the codes laid down in l2/3 are the context - dependent memories of our experiences , is subject to challenge . specifically , the anatomy of the l5 thick tufted cells suggests that they too have access to bu ( via l4 ) , td ( via their apical tufts in l1 ) , and h ( via an extensive intra - l5 horizontal network , schubert et al . , 2007 ) inputs , and therefore that l5 might store the most detailed and context - dependent codes in cortex , a view supported by findings such as ( de kock et al . , 2007 ) . in the end , for the purpose of this hypothesis and theory paper , i believe the architecture and algorithm ( csa ) to be more important than the specifics of any particular neural realization . in this section , i consider evidence relating to six model predictions : a}signals generated in the macrocolumn [ i.e. , the v^x ( eq . strictly interpreted , figure 2 suggests that the model can only be true of cortical areas that have direct projections to the activation function modulator ( afm ) , hypothesized to be instantiated in midbrain neuromodulator source areas , e.g. , basal forebrain ( bf , source of ach ) and locus coeruleus ( lc , source of ne ) . direct cortical afferents to bf arise mainly from prepyriform , anterior insula , orbitofrontal , entorhinal and medial temporal areas ( mesulam and mufson , 1984 ) . direct cortical afferents to lc arise from dorsal prefrontal cortex ( arnsten and goldman - rakic , 1984 ) , medial prefrontal cortex ( jodo et al . , 1998 ) . while direct projections are limited , a much larger fraction of cortex may be able to influence the hypothesized afm via multi - synaptic pathways involving thalamus and other subcortical structures , especially via pathways interconnecting bf , lc , and other neuromodulator source areas . , 2004 ) , which allows dorsal and medial prefrontal areas indirect influence on bf . similarly , lc receives input from the raphe nuclei ( reviewed in samuels and szabadi , 2008 ) which would allow further extension of the sphere of cortical influence upon the afm . however , it is clearly possible that my macrocolumnar model applies only to the smaller set of cortical areas suggested above . after all , there would be some advantage to deferring the decision as to the overall familiarity / novelty of the current input ( moment ) to the very highest cortical levels , which are in position to make the most informed decisions . in this case , once g is computed , it is then broadcast pan - cortically , i.e. , to all levels of the hierarchy , allowing the local choice of code to proceed accordingly , i.e. , with a level of randomness appropriate to g. figure s2 in supplementary material illustrates this view . 5 ) , which correlates with the detection of familiarity , and/or inversely with the detection of novelty . such correlations have been shown for both ach ( miranda et al . , 2000 , 2003 ) and ne ( sara et al . , 1994 ; vankov et al . , lc projects to all of cortex ( foote and morrison , 1987 ; berridge and waterhouse , 2003 ; samuels and szabadi , 2008 ) . bf projects to almost all cortical areas ( reviewed in briand et al . , 2007 ) . the amount of randomness to be added to the winner selection process is a global parameter , which applies non - specifically to all the minicolumns . this is consistent with volume transmission believed to be used by neuromodulatory systems ( descarries et al . , 1997 ; see sarter et al . , 2009 for discussion of the complexities of the evidence regarding volume transmission ) . d}the signal determines ( eqs 68 ) the amount of noise ( randomness ) in the selection ( activation ) of cortical ( i.e. , macrocolumnar ) codes . controlling the noisiness of a process of choosing a winner from a competing group of neurons can be achieved by some combination of two actions : ( i ) increasing the spike probability of cells with high input summations relative to those with low summations and ( ii ) lowering the spike probability of low - input cells relative to high - input cells . both of these actions can be achieved by uniformly ( i.e. , to all competing cells in a wta module ) modulating intrinsic cell properties such as excitability . numerous studies have demonstrated both excitatory and suppressive effects on target cell responses ( both principal neurons and interneurons ) for both ach ( kawasaki and avoli , 1996 ; shalinsky et al . , 2002 ; cobb and davies , 2005 ; tateno et al . , 2005 ; isakova and mednikova , 2007 ; lawrence , 2008 ; eggermann and feldmeyer , 2009 ) and ne ( foote et al . , 1975 ; kawaguchi and shindou , 1998 ; harley and helen , 2007 ; moxon et al . it is not my intention here to argue for a precise realization of this mechanism . as suggested in many reviews ( berridge and waterhouse , 2003 ; lucas - meunier et al . , 2003 ; sara , 2009 ) , the landscape of this research is very complex and we are far from a comprehensive understanding of the how the various neuromodulatory systems affect high - level cognitive processing either alone or in concert ( briand et al . , 2007 ) . nevertheless , the large and varied body of evidence at least admits the possibility that one or more of these neuromodulators could implement the familiarity - contingent afm mechanism ( csa steps 58 ; see doya , 2002 , p. 502 e}decreased , i.e. , increased noise , leads to greater code separation ( decreased intersection ) . recently , goard and dan ( 2009 ) showed that increased bf stimulation decreased the correlation amongst a population of rat v1 cells . this decreased correlation essentially shows increased separation between population codes , which in the model proposed here , would manifest as decreased intersection between sparse codes . f}disabling of the brain 's ability to produce high noise , i.e. , causing to be permanently high , should reduce the ability to learn new inputs , while sparing or having much less effect on recognition of known items . looking at figure 4 , if the afm was stuck in the highly expansive sigmoid condition ( low noise ) , all four inputs , 25 , would have high probability of mapping to the same code , 1 . this would prevent the model from being able to distinguish them in future presentations . however , in general , inputs that were mapped to unique codes prior to such a disabling event will reliably activate those codes on future presentations . in accord with this , browning et al . ( 2010 ) found that severely diminishing cholinergic inputs to inferotemporal cortex severely reduced macaques performance on a visual episodic memory task , while having little effect on a dnms task . ( 2005 ) found a similar effect : cholinergic deafferentation of entorhinal cortex reduced performance on dnms tasks involving novel odors but not familiar odors . in this section , i consider evidence relating to six model predictions : a}signals generated in the macrocolumn [ i.e. , the v^x ( eq . strictly interpreted , figure 2 suggests that the model can only be true of cortical areas that have direct projections to the activation function modulator ( afm ) , hypothesized to be instantiated in midbrain neuromodulator source areas , e.g. , basal forebrain ( bf , source of ach ) and locus coeruleus ( lc , source of ne ) . relatively few cortical areas project directly to bf or lc . direct cortical afferents to bf arise mainly from prepyriform , anterior insula , orbitofrontal , entorhinal and medial temporal areas ( mesulam and mufson , 1984 ) . direct cortical afferents to lc arise from dorsal prefrontal cortex ( arnsten and goldman - rakic , 1984 ) , medial prefrontal cortex ( jodo et al . , 1998 ) . while direct projections are limited , a much larger fraction of cortex may be able to influence the hypothesized afm via multi - synaptic pathways involving thalamus and other subcortical structures , especially via pathways interconnecting bf , lc , and other neuromodulator source areas . for example , bf cholinergic neurons are excited by lc ( jones et al . , 2004 ) , which allows dorsal and medial prefrontal areas indirect influence on bf . similarly , lc receives input from the raphe nuclei ( reviewed in samuels and szabadi , 2008 ) which would allow further extension of the sphere of cortical influence upon the afm . however , it is clearly possible that my macrocolumnar model applies only to the smaller set of cortical areas suggested above . after all , there would be some advantage to deferring the decision as to the overall familiarity / novelty of the current input ( moment ) to the very highest cortical levels , which are in position to make the most informed decisions . in this case , once g is computed , it is then broadcast pan - cortically , i.e. , to all levels of the hierarchy , allowing the local choice of code to proceed accordingly , i.e. , with a level of randomness appropriate to g. figure s2 in supplementary material illustrates this view . 5 ) , which correlates with the detection of familiarity , and/or inversely with the detection of novelty . such correlations have been shown for both ach ( miranda et al . , 2000 , 2003 ) and ne ( sara et al . , 1994 ; vankov et al . , lc projects to all of cortex ( foote and morrison , 1987 ; berridge and waterhouse , 2003 ; samuels and szabadi , 2008 ) . bf projects to almost all cortical areas ( reviewed in briand et al . , 2007 ) . the amount of randomness to be added to the winner selection process is a global parameter , which applies non - specifically to all the minicolumns . this is consistent with volume transmission believed to be used by neuromodulatory systems ( descarries et al . , 1997 ; see sarter et al . , 2009 for discussion of the complexities of the evidence regarding volume transmission ) . d}the signal determines ( eqs 68 ) the amount of noise ( randomness ) in the selection ( activation ) of cortical ( i.e. , macrocolumnar ) codes . controlling the noisiness of a process of choosing a winner from a competing group of neurons can be achieved by some combination of two actions : ( i ) increasing the spike probability of cells with high input summations relative to those with low summations and ( ii ) lowering the spike probability of low - input cells relative to high - input cells . both of these actions can be achieved by uniformly ( i.e. , to all competing cells in a wta module ) modulating intrinsic cell properties such as excitability . numerous studies have demonstrated both excitatory and suppressive effects on target cell responses ( both principal neurons and interneurons ) for both ach ( kawasaki and avoli , 1996 ; shalinsky et al . , 2002 ; cobb and davies , 2005 ; tateno et al . , 2005 ; isakova and mednikova , 2007 ; lawrence , 2008 ; eggermann and feldmeyer , 2009 ) and ne ( foote et al . , 1975 ; kawaguchi and shindou , 1998 ; harley and helen , 2007 ; moxon et al . , 2007 ) . it is not my intention here to argue for a precise realization of this mechanism . as suggested in many reviews ( berridge and waterhouse , 2003 ; lucas - meunier et al . , 2003 ; sara , 2009 ) , the landscape of this research is very complex and we are far from a comprehensive understanding of the how the various neuromodulatory systems affect high - level cognitive processing either alone or in concert ( briand et al . , 2007 ) . nevertheless , the large and varied body of evidence at least admits the possibility that one or more of these neuromodulators could implement the familiarity - contingent afm mechanism ( csa steps 58 ; see doya , 2002 , p. 502 e}decreased , i.e. , increased noise , leads to greater code separation ( decreased intersection ) . recently , goard and dan ( 2009 ) showed that increased bf stimulation decreased the correlation amongst a population of rat v1 cells . this decreased correlation essentially shows increased separation between population codes , which in the model proposed here , would manifest as decreased intersection between sparse codes . f}disabling of the brain 's ability to produce high noise , i.e. , causing to be permanently high , should reduce the ability to learn new inputs , while sparing or having much less effect on recognition of known items . looking at figure 4 , if the afm was stuck in the highly expansive sigmoid condition ( low noise ) , all four inputs , 25 , would have high probability of mapping to the same code , 1 . this would prevent the model from being able to distinguish them in future presentations . however , in general , inputs that were mapped to unique codes prior to such a disabling event will reliably activate those codes on future presentations . ( 2010 ) found that severely diminishing cholinergic inputs to inferotemporal cortex severely reduced macaques performance on a visual episodic memory task , while having little effect on a dnms task . ( 2005 ) found a similar effect : cholinergic deafferentation of entorhinal cortex reduced performance on dnms tasks involving novel odors but not familiar odors . i have described a theoretical model of cortical function that explains the functional relationship between the minicolumn and macrocolumn . specifically : a}the macrocolumn ( in any of its forms ) is proposed to store information in the form of sdcs , and b}the minicolumn ( specifically , its l2/3 pool of pyramidals ) is proposed to operate as a wta cm , the purpose of which is to enforce the sparseness of the macrocolumnar code . two key motivations for this model are the computational advantages of sdc and the increasingly strong evidence for sdc in the brain , cited in the section introduction . one important advantage of sdc over a localist code is that the number of unique items that can be stored can be far larger than the number of representing units . for example , the 12 f2 units of the model in figure 2 allow 3 = 81 unique codes , though in realistic systems , e.g. , with less than complete connectivity leading to and from a coding field like f2 , the number of those codes that can safely ( i.e. , while maintaining retrieval error rates below some acceptable criterion ) be assigned will be substantially lower than 81 . nevertheless , if the number of input items that will need to be distinguished is not known a priori , sdc is more flexible . a second computational advantage of sdc is that , if used in conjunction with an appropriate storage / retrieval algorithm it possesses the sisc property . i demonstrated , with the small but statistically reasonable example of figures 3 and 4 , that the csa yields the sisc property . the sisc property strongly differentiates sdc from localist models : it is not even defined for a localist model since every code is formally disjoint with every other code . hence , there is no structural way to represent degrees of similarity in a localist code . if there is no way to represent a measure , e.g. , similarity , structurally , then whenever that measure is required e.g. , when the closest matching stored item in a database ( i.e. , macrocolumn ) to an input must be returned it must be computed , which takes time and energy . in contrast , when items codes are stored in physically overlapped fashion such that the degree of code overlap represents item similarity , as is the case for the proposed model , the most closely matching stored item will be returned immediately , i.e. , without requiring any serial search through the stored items . figure s4 in supplementary material shows test retrievals of the four unique codes stored in the model of figures 3 and 4 , demonstrating possession of this immediate access property for this small example . i consider the representation and the csa to be the most important contributions of this paper because of the computational advantages just described . however , i believe the suggestion that the minicolumn 's generic function is to act as a wta cm is also important . saying only that a group of l2/3 units forms a wta cm places no a priori constraints on what their tuning functions or receptive fields should look like . this is what gives that functionality a chance of being truly generic , i.e. , of applying across all areas and species , regardless of the observed tuning profiles of closely neighboring units . indeed , a recent calcium imaging study of mouse auditory cortex by rothschild et al . ( 2010 ) shows highly heterogeneous small - scale ( even immediately adjacent cells ) tuning even though the large - scale tuning is tonotopic . experimental methods are only now just reaching the point where this hypothesis might be directly testable , e.g. , modifying calcium imaging methods to have millisecond temporal granularity ; see ohki and reid ( 2007 ) . in a sense , the main point of this paper is that a generic minicolumnar function becomes apparent as soon as we postulate that what the cortex , i.e. , a macrocolumn , generally does is store and retrieve ( access ) sdcs of specific context - dependent inputs . as noted in the section introduction , the experimental literature contains little in the way of proposals linking the formation and retrieval of specific sdcs ( i.e. , of specific input items , especially of temporal context - dependent items ) to the cortical microcircuitry . my proposed model goes beyond broad conclusions and offers a mechanistic explanation of how specific informational items are learned and retrieved and in so doing , proposes a generic function for the minicolumn , i.e. , that it functions as a wta module in support of manipulating sdcs at the next higher , i.e. , macrocolumnar , scale . there have been several recent models linking formation / retrieval of specific items to cortical circuitry and which describe specific roles for the minicolumn ( kupper et al . , 2007 ; george and hawkins , 2009 ; litvak and ullman , 2009 ; schrader et al . , however , all of these models use localist representations and therefore would not possess the advantages of sdc described above . the cortext model ( kupper et al . , 2007 ; schrader et al . , 2009 ) assumes that each minicolumn in a hypercolumn represents one particular input feature . on each computational cycle , a wta process runs within each hypercolumn , such that exactly one minicolumn wins , which would be strongly at odds with the calcium image data ( ohki et al . , 2005 ) . a second problem is that the assumption that whole minicolumns compete with each other implies that any given hypercolumn ( at any level of the cortical hierarchy ) can represent only 70 unique features ( equivalence classes ) , which seems severely restrictive , especially for hypercolumns at higher cortical levels , e.g. , it . the litvak and ullman ( 2009 ) model also postulates that the l2/3 pool of neurons in a minicolumn implements a max function . however , their model proposes that each single minicolumn ( specifically , its l2/3 pool ) is partitioned into several disjoint groups ( cliques ) of cells , each representing a different item . since any particular cell can participate in only one clique , this constitutes a localist code . george and hawkins ( 2009 ) also assume that minicolumns represent informational items in a localist fashion . note however that both george and hawkins ( 2009 ) and litvak and ullman ( 2009 ) explicitly mention moving to a more general combinatorial code , a.k.a . a core principle of the proposed model is this notion of controlling the amount of noise in the process of choosing ( activating ) a macrocolumnar code as an inverse function of input similarity . doya ( 2002 ) uses the same principle , referred to as boltzmann selection , to modulate the amount of noise in the process of choosing amongst output action actions . doya specifically hypothesizes that ne controls the noise whereas i can assert only that it is some neuromodulator - based mechanism . in doya 's model , as ne levels increase , the action with the greatest expected reward is chosen with probability approaching 1 . this is suggested as corresponding to the exploitation end of the exploitation exploration continuum . as ne levels drop to 0 , all actions become equally probable , i.e. , exploration , which is appropriate if no single action has a particular high expected reward , which generally correlates with the condition of novelty , i.e. , of being in a novel environment wherein it is harder to anticipate the outcome of known actions . the analogy to high expected reward in my model is a highly familiar input ( g 1 ) in which case we want the stored code for that familiar input to be reactivated with probability approaching 1 ; the condition where no action has a high expected reward is analogous to low familiarity , i.e. , where no stored input is very similar to the current input , in which case we want to lay down a new memory trace for the novel input . despite the similarities , doya 's model also assumes a localist representation of the choices and , like the other models just mentioned , can not realize the advantages of sdc . i have identified several avenues of active and future research at various points in the text and as noted in the previous section , the prospective neural realization is highly speculative and very incomplete . several additional questions / issues for future research are : is the current proposal that the l2/3 cells engage in two rounds of competition in each computational ( putatively , gamma ) cycle plausible ? for simplicity , i have described the model in the simplest case of having only one internal coding field ( f2 ) and processing only purely spatial input patterns . however the core model was originally developed for the spatiotemporal pattern ( sequence ) case ( rinkus , 1996 ) and was generalized some time ago to have an arbitrarily deep hierarchy of coding fields ( rinkus and lisman , 2005 ) . see figure s2 in supplementary material . how do these generalized versions of the model map to neural structures ? is there evidence that chandeliers become active twice as frequently as baskets , as the proposed realization predicts ? is there evidence for the converse ? although not elaborated herein , the proposed mini-/macrocolumn model is easily generalized to allow substantial overlap between minicolumns ( see figure s5 in supplementary material ) and multiple winners in a minicolumn on each computational cycle . we know of the fast , phasic , time scales of operation for ne ( rajkowski et al . , 2004 ) and da ( schultz , 1998 ) and of slightly slower but still phasic mode for ach ( gulledge and kawaguchi , 2007 ) , but these have been proposed as signaling other measures besides pure novelty ( redgrave et al . , 1999 ; bouret and sara , 2005 ; dayan and yu , 2006 ) . how might all these signals conspire to implement a pure novelty signal ? the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

no generic function for the minicolumn i.e. , one that would apply equally well to all cortical areas and species has yet been proposed . i propose that the minicolumn does have a generic functionality , which only becomes clear when seen in the context of the function of the higher - level , subsuming unit , the macrocolumn . i propose that : ( a ) a macrocolumn 's function is to store sparse distributed representations of its inputs and to be a recognizer of those inputs ; and ( b ) the generic function of the minicolumn is to enforce macrocolumnar code sparseness . the minicolumn , defined here as a physically localized pool of 20 l2/3 pyramidals , does this by acting as a winner - take - all ( wta ) competitive module , implying that macrocolumnar codes consist of 70 active l2/3 cells , assuming 70 minicolumns per macrocolumn . i describe an algorithm for activating these codes during both learning and retrievals , which causes more similar inputs to map to more highly intersecting codes , a property which yields ultra - fast ( immediate , first - shot ) storage and retrieval . the algorithm achieves this by adding an amount of randomness ( noise ) into the code selection process , which is inversely proportional to an input 's familiarity . i propose a possible mapping of the algorithm onto cortical circuitry , and adduce evidence for a neuromodulatory implementation of this familiarity - contingent noise mechanism . the model is distinguished from other recent columnar cortical circuit models in proposing a generic minicolumnar function in which a group of cells within the minicolumn , the l2/3 pyramidals , compete ( wta ) to be part of the sparse distributed macrocolumnar code .

Introduction Results: Model Description Model dynamics: the code selection algorithm Prospective mapping to cortical circuitry Support for neuromodulator-based implementation of familiarity-contingent noise Discussion Supplementary Material Conflict of Interest Statement

, one that would apply equally well to all cortical areas and species has been determined . the reasoning here appears to be that because a group of cells all have very similar tuning to a particular feature , , e.g. furthermore , possession of highly similar tuning can not be a basis for a generic minicolumn functionality since in many cortical areas , the cells encountered along vertical penetrations can have quite variable tuning ( cf . i propose that the minicolumn does have a generic functionality ( given shortly ) , but one that only becomes clear when seen in the context of the function of the higher - level , subsuming unit , namely , the macrocolumn , which has been demonstrated anatomically ( goldman and nauta , 1977 ; lbke et al . i propose that the function of a macrocolumn ( e.g. , hypercolumn , segregate , barrel ) is to store sparse distributed representations of its overall input patterns , and to act as a recognizer of those patterns . by overall input pattern , i mean the macrocolumn 's overall input at a given moment , including not only its bottom - up ( bu ) inputs from thalamus or lower cortical areas , but also its top - down ( td ) inputs from higher cortical areas and its horizontal ( h ) inputs , which i propose carry temporal ( sequential ) context information ( recurrently ) from the representations previously active in the same and nearby macrocolumns . a distributed representation of an item of information is one in which multiple units collectively represent that item , and crucially , such that each of those units generally participates in the representations of other items as well . in this paper , representation and a sparse distributed code ( sdc ) is one in which only a small fraction of the pool of available representing units is part of any particular code ( palm , 1982 ; lynch et al . if the macrocolumn stores sdcs , then there must be some mechanism that enforces sparseness and this , i propose , is the generic function of the minicolumn . specifically , i propose that small , physically localized pools of l2/3 pyramidals , e.g. , 20 such cells , act as winner - take - all ( wta ) competitive modules ( cms ) . this implies that macrocolumnar codes should consist of about 6080 active l2/3 cells , one per minicolumn : for simplicity , assume 70 minicolumns per macrocolumn hereafter . calcium ( tangential ) images of l2/3 of rat visual cortex showing sparse sets of cells activating in unison ( see movie link in text ) in response to leftward ( a ) and rightward ( b ) drifting gratings . from ohki ( c , d ) sketch of proposed sparse distributed coding concept , which could plausibly give rise to data like ( a ) and ( b ) . how is any particular set of winners , one in each of the 70 minicolumns , initially chosen in response to an input pattern and bound into a holistic code ? in the next section , i describe an algorithm , referred to as the code selection algorithm ( csa ) , which answers both questions . a key property of the csa is that it causes similar inputs to be assigned to similar , i.e. however , the csa achieves it in a novel , probabilistic fashion , which can be summarized as follows : determine the familiarity of a macrocolumn 's input . to a first approximation , an input 's familiarity is its maximum similarity to any input previously stored in the macrocolumn . set the amount of randomness ( noise ) in the process of selecting winners in the wta modules in inverse proportion to the input 's familiarity . this can be achieved by increasing the noisiness of the winner selection process in each wta module , which can be achieved by suppressing the influence of the deterministic synaptic inputs ( which reflect prior learning ) relative to baseline random ( spontaneous ) activity . following the model description , i offer a speculative mapping of my proposed model onto neural circuitry and discuss evidence for novelty - contingent noise modulation by both ne and ach . any discussion of columnar function of course centrally concerns cortical circuitry , and more specifically , the putative canonical cortical microcircuit ( rockland and pandya , 1979 ; douglas et al . i believe the hypothetical model described herein to be a significant contribution because it goes beyond broad conclusions and offers a mechanistic explanation of how specific informational items are learned and retrieved and in so doing , proposes a generic function for the minicolumn , i.e. in particular , it was stated in the introduction that sparse macrocolumnar codes are chosen in response to a macrocolumn 's overall input , which includes its bu , h , and td inputs . more importantly , the core elements of the hypothesis which are : ( a ) that the macrocolumn stores sdcs consisting of one winning l2/3 cell per minicolumn ; and ( b ) that the sisc property is achieved by modulating the amount of randomness ( noise ) present in the winner selection process in inverse proportion to input familiarity can be clearly and more simply described for the bu - only case ( i.e. it consists of winner - take - all competitive modules ( cms ) proposed as analogous to minicolumns . the model 's core algorithm , the csa , determines which cells are chosen to represent an input , during both learning and retrieval . g then drives a global modulation of the f2 unit activation function ( figure 2 : purple arrows ) in preparation for the second competitive round , which is a soft wta competition , the intent of which is that : as g goes to 1 ( indicating a completely familiar input ) , the probability that the unit with the highest input summation in a cm wins approaches 1 , and as g goes to 0 ( indicating a completely novel input ) , all units in a cm become equally likely to win ( regardless of their input summations ) . , g is computed to be 0 , and sets the f2 activation function to a constant function ( red line ) , which makes the choice of winner in each cm completely random , and thus , the overall f2 code , 1 , also completely random . , g is computed to be 0 , and sets the f2 activation function to a constant function ( red line ) , which makes the choice of winner in each cm completely random , and thus , the overall f2 code , 1 , also completely random . this causes the expected intersection of the code being chosen in the current instance with any previously assigned code to be at chance level . , for g approaching 1 ( and , assuming the model is still operating in a regime where crosstalk is sufficiently low ) , the draws become increasingly correlated ( dependent ) , as can be seen in going from figure 4c to 4b to 4a . in step 5 , the value , g = 0 , maps to = 0 , which causes the activation function of the f2 units to collapse to the constant function , = 1 . figure 4 demonstrates that the csa realizes the sisc property by considering four possibilities ( a d ) for the second input presented to the model of figure 3 . thus , the familiarity , g , which depends on the entire f2 layer and is thus global information , influences the local activation functions so as to produce the desired overall result , in this case , reactivation of the code ( memory trace ) , 1 , of the familiar input pattern , 1 . that both learning and recognition require only a single csa iteration is especially significant since , as can readily be seen , none of the csa steps involves iterations over stored codes : thus , the time it takes for the csa to either store a new input or retrieve the closest matching stored input remains constant as the number of stored codes increases . note that the communication of cell 2 s psp value is hypothesized to be mediated via l5 , one of whose pyramidal cells is seen integrating here ( light green ) ; this is based loosely on evidence that l5 cells , specifically l5b pyramidals , almost exclusively target pontine areas ( with collaterals to thalamus ) ( deschnes et al . b , which rapidly deactivates and re - polarizes ( resets ) the l2/3 pyramidals ( grayed out ) . in figure 5d , the result of the subcortical computation of g is returned to the macrocolumn ( path 7 ) in the form of neuromodulator release ( purple cloud surrounding the l2/3 pyramidals ) . in particular , the probability that the second round winner is the same as the first round winner increases with g. the set of l2/3 winners , one per minicolumn , across the whole macrocolumn represents that macrocolumn 's final decision ( hypothesis ) as to identity of its current overall ( td , h , and bu ) input . moreover , the proposed theory 's key assumption that the l2/3 pyramidals are the core repository of information in cortex and that the codes laid down in l2/3 are the context - dependent memories of our experiences , is subject to challenge . in the end , for the purpose of this hypothesis and theory paper , i believe the architecture and algorithm ( csa ) to be more important than the specifics of any particular neural realization . strictly interpreted , figure 2 suggests that the model can only be true of cortical areas that have direct projections to the activation function modulator ( afm ) , hypothesized to be instantiated in midbrain neuromodulator source areas , e.g. the amount of randomness to be added to the winner selection process is a global parameter , which applies non - specifically to all the minicolumns . d}the signal determines ( eqs 68 ) the amount of noise ( randomness ) in the selection ( activation ) of cortical ( i.e. controlling the noisiness of a process of choosing a winner from a competing group of neurons can be achieved by some combination of two actions : ( i ) increasing the spike probability of cells with high input summations relative to those with low summations and ( ii ) lowering the spike probability of low - input cells relative to high - input cells . , 2003 ; sara , 2009 ) , the landscape of this research is very complex and we are far from a comprehensive understanding of the how the various neuromodulatory systems affect high - level cognitive processing either alone or in concert ( briand et al . nevertheless , the large and varied body of evidence at least admits the possibility that one or more of these neuromodulators could implement the familiarity - contingent afm mechanism ( csa steps 58 ; see doya , 2002 , p. 502 e}decreased , i.e. this decreased correlation essentially shows increased separation between population codes , which in the model proposed here , would manifest as decreased intersection between sparse codes . the model 's core algorithm , the csa , determines which cells are chosen to represent an input , during both learning and retrieval . g then drives a global modulation of the f2 unit activation function ( figure 2 : purple arrows ) in preparation for the second competitive round , which is a soft wta competition , the intent of which is that : as g goes to 1 ( indicating a completely familiar input ) , the probability that the unit with the highest input summation in a cm wins approaches 1 , and as g goes to 0 ( indicating a completely novel input ) , all units in a cm become equally likely to win ( regardless of their input summations ) . , g is computed to be 0 , and sets the f2 activation function to a constant function ( red line ) , which makes the choice of winner in each cm completely random , and thus , the overall f2 code , 1 , also completely random . , g is computed to be 0 , and sets the f2 activation function to a constant function ( red line ) , which makes the choice of winner in each cm completely random , and thus , the overall f2 code , 1 , also completely random . this causes the expected intersection of the code being chosen in the current instance with any previously assigned code to be at chance level . , for g approaching 1 ( and , assuming the model is still operating in a regime where crosstalk is sufficiently low ) , the draws become increasingly correlated ( dependent ) , as can be seen in going from figure 4c to 4b to 4a . in step 5 , the value , g = 0 , maps to = 0 , which causes the activation function of the f2 units to collapse to the constant function , = 1 . figure 4 demonstrates that the csa realizes the sisc property by considering four possibilities ( a d ) for the second input presented to the model of figure 3 . in this case , thus , the familiarity , g , which depends on the entire f2 layer and is thus global information , influences the local activation functions so as to produce the desired overall result , in this case , reactivation of the code ( memory trace ) , 1 , of the familiar input pattern , 1 . that both learning and recognition require only a single csa iteration is especially significant since , as can readily be seen , none of the csa steps involves iterations over stored codes : thus , the time it takes for the csa to either store a new input or retrieve the closest matching stored input remains constant as the number of stored codes increases . nevertheless , figure 5 shows a possible neural realization of the model 's wta cm , i.e. 2 ) , represents this minicolumn 's local judgment of how similar the macrocolumn 's closest - matching stored input is to the current overall ( i.e. note that the communication of cell 2 s psp value is hypothesized to be mediated via l5 , one of whose pyramidal cells is seen integrating here ( light green ) ; this is based loosely on evidence that l5 cells , specifically l5b pyramidals , almost exclusively target pontine areas ( with collaterals to thalamus ) ( deschnes et al . b , which rapidly deactivates and re - polarizes ( resets ) the l2/3 pyramidals ( grayed out ) . in figure 5d , the result of the subcortical computation of g is returned to the macrocolumn ( path 7 ) in the form of neuromodulator release ( purple cloud surrounding the l2/3 pyramidals ) . in particular , the probability that the second round winner is the same as the first round winner increases with g. the set of l2/3 winners , one per minicolumn , across the whole macrocolumn represents that macrocolumn 's final decision ( hypothesis ) as to identity of its current overall ( td , h , and bu ) input . moreover , the proposed theory 's key assumption that the l2/3 pyramidals are the core repository of information in cortex and that the codes laid down in l2/3 are the context - dependent memories of our experiences , is subject to challenge . in the end , for the purpose of this hypothesis and theory paper , i believe the architecture and algorithm ( csa ) to be more important than the specifics of any particular neural realization . strictly interpreted , figure 2 suggests that the model can only be true of cortical areas that have direct projections to the activation function modulator ( afm ) , hypothesized to be instantiated in midbrain neuromodulator source areas , e.g. the amount of randomness to be added to the winner selection process is a global parameter , which applies non - specifically to all the minicolumns . d}the signal determines ( eqs 68 ) the amount of noise ( randomness ) in the selection ( activation ) of cortical ( i.e. controlling the noisiness of a process of choosing a winner from a competing group of neurons can be achieved by some combination of two actions : ( i ) increasing the spike probability of cells with high input summations relative to those with low summations and ( ii ) lowering the spike probability of low - input cells relative to high - input cells . , 2003 ; sara , 2009 ) , the landscape of this research is very complex and we are far from a comprehensive understanding of the how the various neuromodulatory systems affect high - level cognitive processing either alone or in concert ( briand et al . nevertheless , the large and varied body of evidence at least admits the possibility that one or more of these neuromodulators could implement the familiarity - contingent afm mechanism ( csa steps 58 ; see doya , 2002 , p. 502 e}decreased , i.e. this decreased correlation essentially shows increased separation between population codes , which in the model proposed here , would manifest as decreased intersection between sparse codes . the amount of randomness to be added to the winner selection process is a global parameter , which applies non - specifically to all the minicolumns . d}the signal determines ( eqs 68 ) the amount of noise ( randomness ) in the selection ( activation ) of cortical ( i.e. controlling the noisiness of a process of choosing a winner from a competing group of neurons can be achieved by some combination of two actions : ( i ) increasing the spike probability of cells with high input summations relative to those with low summations and ( ii ) lowering the spike probability of low - input cells relative to high - input cells . nevertheless , the large and varied body of evidence at least admits the possibility that one or more of these neuromodulators could implement the familiarity - contingent afm mechanism ( csa steps 58 ; see doya , 2002 , p. 502 e}decreased , i.e. this decreased correlation essentially shows increased separation between population codes , which in the model proposed here , would manifest as decreased intersection between sparse codes . specifically : a}the macrocolumn ( in any of its forms ) is proposed to store information in the form of sdcs , and b}the minicolumn ( specifically , its l2/3 pool of pyramidals ) is proposed to operate as a wta cm , the purpose of which is to enforce the sparseness of the macrocolumnar code . for example , the 12 f2 units of the model in figure 2 allow 3 = 81 unique codes , though in realistic systems , e.g. , macrocolumn ) to an input must be returned it must be computed , which takes time and energy . in contrast , when items codes are stored in physically overlapped fashion such that the degree of code overlap represents item similarity , as is the case for the proposed model , the most closely matching stored item will be returned immediately , i.e. figure s4 in supplementary material shows test retrievals of the four unique codes stored in the model of figures 3 and 4 , demonstrating possession of this immediate access property for this small example . however , i believe the suggestion that the minicolumn 's generic function is to act as a wta cm is also important . in a sense , the main point of this paper is that a generic minicolumnar function becomes apparent as soon as we postulate that what the cortex , i.e. my proposed model goes beyond broad conclusions and offers a mechanistic explanation of how specific informational items are learned and retrieved and in so doing , proposes a generic function for the minicolumn , i.e. a core principle of the proposed model is this notion of controlling the amount of noise in the process of choosing ( activating ) a macrocolumnar code as an inverse function of input similarity . the analogy to high expected reward in my model is a highly familiar input ( g 1 ) in which case we want the stored code for that familiar input to be reactivated with probability approaching 1 ; the condition where no action has a high expected reward is analogous to low familiarity , i.e.

i propose that the minicolumn does have a generic functionality ( given shortly ) , but one that only becomes clear when seen in the context of the function of the higher - level , subsuming unit , namely , the macrocolumn , which has been demonstrated anatomically ( goldman and nauta , 1977 ; lbke et al . by overall input pattern , i mean the macrocolumn 's overall input at a given moment , including not only its bottom - up ( bu ) inputs from thalamus or lower cortical areas , but also its top - down ( td ) inputs from higher cortical areas and its horizontal ( h ) inputs , which i propose carry temporal ( sequential ) context information ( recurrently ) from the representations previously active in the same and nearby macrocolumns . in this paper , representation and a sparse distributed code ( sdc ) is one in which only a small fraction of the pool of available representing units is part of any particular code ( palm , 1982 ; lynch et al . in terms of the proposed model , the active neurons would be the winners in their respective minicolumns , as suggested in figures 1c , d . calcium ( tangential ) images of l2/3 of rat visual cortex showing sparse sets of cells activating in unison ( see movie link in text ) in response to leftward ( a ) and rightward ( b ) drifting gratings . however , the csa achieves it in a novel , probabilistic fashion , which can be summarized as follows : determine the familiarity of a macrocolumn 's input . this can be achieved by increasing the noisiness of the winner selection process in each wta module , which can be achieved by suppressing the influence of the deterministic synaptic inputs ( which reflect prior learning ) relative to baseline random ( spontaneous ) activity . , across the whole macrocolumn ) noise level , we can modulate the expected intersection between the set of cells which have the maximal input summations in their respective wta modules and the set of winners that are actually chosen , thus implementing sisc . many experimental and theoretical studies implicate neuromodulators , notably norepinephrine ( ne ) and acetylcholine ( ach ) , in functionality similar to the above , which can be described generally as modulating signal - to - noise ratio ( snr ) . in addition , increased ach has been shown to selectively increase the strength of afferent relative to intrinsic inputs in piriform cortex ( hasselmo and bower , 1992 ) and other brain structures ( see hasselmo , 2006 for review ) . following the model description , i offer a speculative mapping of my proposed model onto neural circuitry and discuss evidence for novelty - contingent noise modulation by both ne and ach . , 2006 ) ; that both the populations of l4 pyramidals and of l5a pyramidals have transcolumnar connectivity patterns allowing them to act as integrators of information coming in from multiple vibrissae in parallel , or in close sequence ( schubert et al . , 2003 ) ; that wta competition occurs in the supra- and infragranular layers ( douglas and martin , 2004 ) ; and that local ( 100 m ) l2/3-to - l2/3 connections might serve to synchronize firing of l2/3 cell assemblies ( lbke and feldmeyer , 2007 ) ; etc . i believe the hypothetical model described herein to be a significant contribution because it goes beyond broad conclusions and offers a mechanistic explanation of how specific informational items are learned and retrieved and in so doing , proposes a generic function for the minicolumn , i.e. however , illustrations of the model in operation in that general case become rather complex , particularly since the h ( and td ) weights carry temporal information , which necessitates showing the model at multiple successive time steps while processing spatiotemporal patterns . more importantly , the core elements of the hypothesis which are : ( a ) that the macrocolumn stores sdcs consisting of one winning l2/3 cell per minicolumn ; and ( b ) that the sisc property is achieved by modulating the amount of randomness ( noise ) present in the winner selection process in inverse proportion to input familiarity can be clearly and more simply described for the bu - only case ( i.e. however , the generalized model ( with bu , h , and td inputs ) and the accompanying generalized version of the csa are given in figures s2 and table s1 in supplementary material . the familiarity ( g ) of the input is proposed to be computed via a subcortical , neuromodulator - based mechanism , which then modulates the f2 unit activation function parameters ( e.g. g then drives a global modulation of the f2 unit activation function ( figure 2 : purple arrows ) in preparation for the second competitive round , which is a soft wta competition , the intent of which is that : as g goes to 1 ( indicating a completely familiar input ) , the probability that the unit with the highest input summation in a cm wins approaches 1 , and as g goes to 0 ( indicating a completely novel input ) , all units in a cm become equally likely to win ( regardless of their input summations ) . , for g approaching 1 ( and , assuming the model is still operating in a regime where crosstalk is sufficiently low ) , the draws become increasingly correlated ( dependent ) , as can be seen in going from figure 4c to 4b to 4a . thus , the familiarity , g , which depends on the entire f2 layer and is thus global information , influences the local activation functions so as to produce the desired overall result , in this case , reactivation of the code ( memory trace ) , 1 , of the familiar input pattern , 1 . before leaving figure 4 first , while the -distributions become flatter as g decreases , the units comprising the code of the most similar previously learned input ( here , 1 ) remain most likely to win in their respective cms . the effect of these simultaneous correlated potentiations allows a rapid , even single - trial , formation of an association , even if the individual synaptic potentiations are small , consistent with the recent characterization of thalamocortical learning described in bruno and sakmann ( 2006 ) . that both learning and recognition require only a single csa iteration is especially significant since , as can readily be seen , none of the csa steps involves iterations over stored codes : thus , the time it takes for the csa to either store a new input or retrieve the closest matching stored input remains constant as the number of stored codes increases . there remain huge gaps in our knowledge of the intrinsic physiological , synaptic , morphological , and connectional properties of all classes of cortical cell and of functional relationships between cortical and sub - cortical structures . while the csa explanation in the prior section included only the bu inputs , all three input classes are included here : bu inputs ( labeled 2 ) are mediated via l4 ( rockland and pandya , 1979 ) td inputs ( 1 ) are mediated via l2/3 apical tufts ( rockland and drash , 1996 ) h inputs ( 3 ) are mediated via the horizontal matrix of l2/3 ( gilbert and wiesel , 1989 ; feldmeyer et al . note that the communication of cell 2 s psp value is hypothesized to be mediated via l5 , one of whose pyramidal cells is seen integrating here ( light green ) ; this is based loosely on evidence that l5 cells , specifically l5b pyramidals , almost exclusively target pontine areas ( with collaterals to thalamus ) ( deschnes et al . in addition , as stated earlier , the horizontal l2/3-to - l2/3 connections are proposed to communicate this macrocolumn 's final hypothesis regarding its total input pattern in the current csa cycle recurrently back to the same ( and surrounding ) macrocolumns on the next csa cycle . in figure 5d , the result of the subcortical computation of g is returned to the macrocolumn ( path 7 ) in the form of neuromodulator release ( purple cloud surrounding the l2/3 pyramidals ) . in particular , the probability that the second round winner is the same as the first round winner increases with g. the set of l2/3 winners , one per minicolumn , across the whole macrocolumn represents that macrocolumn 's final decision ( hypothesis ) as to identity of its current overall ( td , h , and bu ) input . thus , the output of the winning l2/3 cell in each minicolumn is now communicated to : lower cortical regions via l5 and its backprojections to the lower regions l1 ( labeled 8 ) ( rockland and drash , 1996 ) . note that the output of the winning l2/3 cell should be prevented from recurring to the local basket network at this time so as to allow the integration period to occur at the beginning of the next computational cycle ; hence , the red x on the link to basket cell . we still have decidedly little in the way of hard constraints on the time courses of activation of the many cell types involved in cortical ( and hippocampal ) circuits , though progress is being made ( klausberger et al . moreover , the proposed theory 's key assumption that the l2/3 pyramidals are the core repository of information in cortex and that the codes laid down in l2/3 are the context - dependent memories of our experiences , is subject to challenge . specifically , the anatomy of the l5 thick tufted cells suggests that they too have access to bu ( via l4 ) , td ( via their apical tufts in l1 ) , and h ( via an extensive intra - l5 horizontal network , schubert et al . in the end , for the purpose of this hypothesis and theory paper , i believe the architecture and algorithm ( csa ) to be more important than the specifics of any particular neural realization . strictly interpreted , figure 2 suggests that the model can only be true of cortical areas that have direct projections to the activation function modulator ( afm ) , hypothesized to be instantiated in midbrain neuromodulator source areas , e.g. while direct projections are limited , a much larger fraction of cortex may be able to influence the hypothesized afm via multi - synaptic pathways involving thalamus and other subcortical structures , especially via pathways interconnecting bf , lc , and other neuromodulator source areas . controlling the noisiness of a process of choosing a winner from a competing group of neurons can be achieved by some combination of two actions : ( i ) increasing the spike probability of cells with high input summations relative to those with low summations and ( ii ) lowering the spike probability of low - input cells relative to high - input cells . nevertheless , the large and varied body of evidence at least admits the possibility that one or more of these neuromodulators could implement the familiarity - contingent afm mechanism ( csa steps 58 ; see doya , 2002 , p. 502 e}decreased , i.e. g then drives a global modulation of the f2 unit activation function ( figure 2 : purple arrows ) in preparation for the second competitive round , which is a soft wta competition , the intent of which is that : as g goes to 1 ( indicating a completely familiar input ) , the probability that the unit with the highest input summation in a cm wins approaches 1 , and as g goes to 0 ( indicating a completely novel input ) , all units in a cm become equally likely to win ( regardless of their input summations ) . in this case , thus , the familiarity , g , which depends on the entire f2 layer and is thus global information , influences the local activation functions so as to produce the desired overall result , in this case , reactivation of the code ( memory trace ) , 1 , of the familiar input pattern , 1 . the effect of these simultaneous correlated potentiations allows a rapid , even single - trial , formation of an association , even if the individual synaptic potentiations are small , consistent with the recent characterization of thalamocortical learning described in bruno and sakmann ( 2006 ) . that both learning and recognition require only a single csa iteration is especially significant since , as can readily be seen , none of the csa steps involves iterations over stored codes : thus , the time it takes for the csa to either store a new input or retrieve the closest matching stored input remains constant as the number of stored codes increases . while the csa explanation in the prior section included only the bu inputs , all three input classes are included here : bu inputs ( labeled 2 ) are mediated via l4 ( rockland and pandya , 1979 ) td inputs ( 1 ) are mediated via l2/3 apical tufts ( rockland and drash , 1996 ) h inputs ( 3 ) are mediated via the horizontal matrix of l2/3 ( gilbert and wiesel , 1989 ; feldmeyer et al . note that the communication of cell 2 s psp value is hypothesized to be mediated via l5 , one of whose pyramidal cells is seen integrating here ( light green ) ; this is based loosely on evidence that l5 cells , specifically l5b pyramidals , almost exclusively target pontine areas ( with collaterals to thalamus ) ( deschnes et al . in addition , as stated earlier , the horizontal l2/3-to - l2/3 connections are proposed to communicate this macrocolumn 's final hypothesis regarding its total input pattern in the current csa cycle recurrently back to the same ( and surrounding ) macrocolumns on the next csa cycle . in figure 5d , the result of the subcortical computation of g is returned to the macrocolumn ( path 7 ) in the form of neuromodulator release ( purple cloud surrounding the l2/3 pyramidals ) . in particular , the probability that the second round winner is the same as the first round winner increases with g. the set of l2/3 winners , one per minicolumn , across the whole macrocolumn represents that macrocolumn 's final decision ( hypothesis ) as to identity of its current overall ( td , h , and bu ) input . thus , the output of the winning l2/3 cell in each minicolumn is now communicated to : lower cortical regions via l5 and its backprojections to the lower regions l1 ( labeled 8 ) ( rockland and drash , 1996 ) . note that the output of the winning l2/3 cell should be prevented from recurring to the local basket network at this time so as to allow the integration period to occur at the beginning of the next computational cycle ; hence , the red x on the link to basket cell . we still have decidedly little in the way of hard constraints on the time courses of activation of the many cell types involved in cortical ( and hippocampal ) circuits , though progress is being made ( klausberger et al . moreover , the proposed theory 's key assumption that the l2/3 pyramidals are the core repository of information in cortex and that the codes laid down in l2/3 are the context - dependent memories of our experiences , is subject to challenge . specifically , the anatomy of the l5 thick tufted cells suggests that they too have access to bu ( via l4 ) , td ( via their apical tufts in l1 ) , and h ( via an extensive intra - l5 horizontal network , schubert et al . in the end , for the purpose of this hypothesis and theory paper , i believe the architecture and algorithm ( csa ) to be more important than the specifics of any particular neural realization . strictly interpreted , figure 2 suggests that the model can only be true of cortical areas that have direct projections to the activation function modulator ( afm ) , hypothesized to be instantiated in midbrain neuromodulator source areas , e.g. while direct projections are limited , a much larger fraction of cortex may be able to influence the hypothesized afm via multi - synaptic pathways involving thalamus and other subcortical structures , especially via pathways interconnecting bf , lc , and other neuromodulator source areas . after all , there would be some advantage to deferring the decision as to the overall familiarity / novelty of the current input ( moment ) to the very highest cortical levels , which are in position to make the most informed decisions . controlling the noisiness of a process of choosing a winner from a competing group of neurons can be achieved by some combination of two actions : ( i ) increasing the spike probability of cells with high input summations relative to those with low summations and ( ii ) lowering the spike probability of low - input cells relative to high - input cells . , 2003 ; sara , 2009 ) , the landscape of this research is very complex and we are far from a comprehensive understanding of the how the various neuromodulatory systems affect high - level cognitive processing either alone or in concert ( briand et al . nevertheless , the large and varied body of evidence at least admits the possibility that one or more of these neuromodulators could implement the familiarity - contingent afm mechanism ( csa steps 58 ; see doya , 2002 , p. 502 e}decreased , i.e. strictly interpreted , figure 2 suggests that the model can only be true of cortical areas that have direct projections to the activation function modulator ( afm ) , hypothesized to be instantiated in midbrain neuromodulator source areas , e.g. while direct projections are limited , a much larger fraction of cortex may be able to influence the hypothesized afm via multi - synaptic pathways involving thalamus and other subcortical structures , especially via pathways interconnecting bf , lc , and other neuromodulator source areas . controlling the noisiness of a process of choosing a winner from a competing group of neurons can be achieved by some combination of two actions : ( i ) increasing the spike probability of cells with high input summations relative to those with low summations and ( ii ) lowering the spike probability of low - input cells relative to high - input cells . , 2003 ; sara , 2009 ) , the landscape of this research is very complex and we are far from a comprehensive understanding of the how the various neuromodulatory systems affect high - level cognitive processing either alone or in concert ( briand et al . nevertheless , the large and varied body of evidence at least admits the possibility that one or more of these neuromodulators could implement the familiarity - contingent afm mechanism ( csa steps 58 ; see doya , 2002 , p. 502 e}decreased , i.e. specifically : a}the macrocolumn ( in any of its forms ) is proposed to store information in the form of sdcs , and b}the minicolumn ( specifically , its l2/3 pool of pyramidals ) is proposed to operate as a wta cm , the purpose of which is to enforce the sparseness of the macrocolumnar code . a core principle of the proposed model is this notion of controlling the amount of noise in the process of choosing ( activating ) a macrocolumnar code as an inverse function of input similarity . the analogy to high expected reward in my model is a highly familiar input ( g 1 ) in which case we want the stored code for that familiar input to be reactivated with probability approaching 1 ; the condition where no action has a high expected reward is analogous to low familiarity , i.e. however the core model was originally developed for the spatiotemporal pattern ( sequence ) case ( rinkus , 1996 ) and was generalized some time ago to have an arbitrarily deep hierarchy of coding fields ( rinkus and lisman , 2005 ) .

femtosecond laser - assisted cataract surgery ( flacs ) is superior to conventional phacoemulsification in terms of precise wound construction , capsulotomy , intraocular lens ( iol ) centration , and decreased effective phacoemulsification time.14 flacs is comparable to traditional cataract surgery in terms of intraoperative complications , although further research is needed to evaluate its long - term safety aspects.57 it may be especially advantageous in white cataracts , where achieving an adequately sized continuous curvilinear capsulorhexis is the major surgical challenge . the increased incidence of capsulorhexis - related complications in white cataracts is often associated with posterior capsular rent , vitreous loss , and nucleus drop.8 various maneuvers have been used to facilitate capsulorhexis , such as use of high - molecular - weight viscoelastic,9 trypan blue dye to stain the anterior capsule,10 two - stage capsulorhexis,11 frequent aspiration of liquefied cortical matter,12 use of vannas scissors,12 and endoilluminator.9 however , limited literature exists on the application of femtosecond lasers in white cataracts.13 comparative evaluation of flacs and conventional phacoemulsification has been done for varying grades of nuclear sclerosis ; however , there are no studies that have compared the outcomes of flacs with conventional phacoemulsification in cases with white cataract.5,7 we herein report our results of comparison of femtosecond laser capsulotomies with conventional capsulorhexis in cases of white cataract . in this prospective comparative study , 80 eyes of 80 patients with white cataract were enrolled at dr rp centre for ophthalmic sciences , all india institute of medical sciences , new delhi , india . ethical clearance was obtained from the all india institute of medical sciences institutional review board . the white cataracts were classified into type i , ii , and iii according to the classification provided by brazitikos et al.8 type i intumescent , white cataracts are characterized by the presence of liquefied cortex , type ii white cataracts have a voluminous nucleus with little amount of solid white cortex , and type iii white cataracts have anterior capsule fibrosis . type iii white cataracts with calcific and fibrotic capsules were excluded from the study.8 eyes with preexisting glaucoma , retinal pathology , pseudoexfoliation syndrome , or pupillary dilatation < 6 mm were also excluded . the patients were divided into two groups , each consisting of 40 eyes ( 40 patients ) . group i ( n=40 ) underwent flacs and group ii ( n=40 ) underwent conventional phacoemulsification performed by an experienced phacoemulsification surgeon ( jst ) . the patients with white cataract scheduled to undergo flacs or conventional phacoemulsification ( based on patient s choice and affordability of the procedure ) were compared . the preoperative detailed examination included uncorrected distance visual acuity ( udva ) , anterior segment evaluation , slit - lamp biomicroscopy , applanation tonometry , biometry ( tonoref iii , nidek co. , ltd . and ocuscan rxp , alcon laboratories , inc . ) , and ultrasonography b - scan ( master - vu ophthalmic ultrasound , sonomed escalon ) . the secondary outcome measures were intraoperative phacoemulsification parameters , intraoperative complications , and postoperative visual acuity . in group i , the preoperative planning of the corneal incisions , capsulotomy , and nucleotomy was done on lensx version 2.23 femtosecond laser system ( alcon lensx , inc . , a 4.9 mm capsulotomy was planned , with a 2.2 mm temporal clear corneal incision and two 1.1 mm side ports at 90 and 240. the chop pattern of nucleotomy was selected , with three chops of length 6 mm each . the anterior segment structures were imaged with intraoperative optical coherence tomography and the planned treatment was verified . the femtosecond laser parameters were the same as those used in our routine cases ( nonwhite cataract cases ) of flacs . the energy was kept at 6 j for corneal incisions , 8 j for capsulotomy , and 12 j for nucleotomy . the spot and layer separation was 5 m for the side port incisions , 6 m for 2.2 mm corneal incision , and 14 m for the nucleotomy . for capsulotomy , the spot separation was 5 m , the layer separation was 4 m , and the anterior and posterior delta value was 350 m each . after femtosecond laser application , the corneal incisions were opened with the help of flap lifter . sodium hyaluronate 1% was injected to maintain the anterior chamber and trypan blue 0.06% ( visionblue , dorc international , the netherlands ) was injected to stain the anterior capsule . the capsulotomies were lifted and removed with the microcapsulorhexis forceps , and microadhesions , if any , were released ( figure 1 , video s1 ) . gentle hydrodissection was performed , followed by phacoemulsification , irrigation aspiration ( ia ) , and implantation of single - piece hydrophobic acrylic iol . a hybrid technique of ia using a combination of coaxial and bimanual ia hand pieces was frequently employed to aspirate the subincisional cortical matter in cases with difficulty during removal of cortical matter.14 the corneal wounds were hydrated at the end of the procedure . in group ii , a 2.2 keratome was used to construct a temporal corneal incision and two side ports were constructed using a 20 g microvitreoretinal blade . capsulorhexis was performed with a 26-gauge needle cystotome ; microcapsulorhexis forceps was used if required . in cases with intumescence , an initial smaller capsulorhexis was followed by the aspiration of liquefied cortex , thus decreasing the raised intralenticular pressure . the aim was to provide ~0.5 mm iol coverage all around as the optic of the iol was 6 mm . after iol implantation , the capsulorhexis was further enlarged if required to achieve an adequate size . intraoperatively , the total surgical time , cumulative dissipated energy , total ultrasound time , and total aspiration time were noted . intraoperative complications such as anterior capsular tears / extension , posterior capsular tears , vitreous loss , need for anterior vitrectomy , and inability to implant iol were noted . postoperatively , clinical photographs were captured with the help of slit - lamp - mounted camera on postoperative day 30 and after 6 months , and the capsulorhexis size , shape , continuity , and iol optic coverage were evaluated . image - processing software ( imagej 1.46r , national institutes of health , md , usa ) was used to measure the diameter of the capsulorhexis and calculate the circularity index . the diameter of the implanted iol was used as a scale to calibrate the image . the circularity index was given by the formula 4 ( area / perimeter2 ) and a circularity index of 1 implied a perfect circle.3 the iol centration and coverage was assessed on the basis of coverage of iol optic in all four quadrants by the capsulotomy / capsulorhexis margin . continuity of the capsulorhexis refers to the absence of radial extension / tear of the capsulorhexis edge . udva and corrected distance visual acuity ( cdva ) were assessed on postoperative day 30 and at 6 months . statistical analysis was done using statistical package for the social sciences ( spss 11.0 ; spss inc . , chicago , il , usa ) . normally distributed continuous variables were expressed as mean standard deviation and were compared using independent samples t - test . nominal data were compared using chi - square test or fisher s exact test as appropriate . a p - value less than 0.05 was considered significant . in group i , the preoperative planning of the corneal incisions , capsulotomy , and nucleotomy was done on lensx version 2.23 femtosecond laser system ( alcon lensx , inc . , a 4.9 mm capsulotomy was planned , with a 2.2 mm temporal clear corneal incision and two 1.1 mm side ports at 90 and 240. the chop pattern of nucleotomy was selected , with three chops of length 6 mm each . the anterior segment structures were imaged with intraoperative optical coherence tomography and the planned treatment was verified . the femtosecond laser parameters were the same as those used in our routine cases ( nonwhite cataract cases ) of flacs . the energy was kept at 6 j for corneal incisions , 8 j for capsulotomy , and 12 j for nucleotomy . the spot and layer separation was 5 m for the side port incisions , 6 m for 2.2 mm corneal incision , and 14 m for the nucleotomy . for capsulotomy , the spot separation was 5 m , the layer separation was 4 m , and the anterior and posterior delta value was 350 m each . after femtosecond laser application , the corneal incisions were opened with the help of flap lifter . sodium hyaluronate 1% was injected to maintain the anterior chamber and trypan blue 0.06% ( visionblue , dorc international , the netherlands ) was injected to stain the anterior capsule . the capsulotomies were lifted and removed with the microcapsulorhexis forceps , and microadhesions , if any , were released ( figure 1 , video s1 ) . gentle hydrodissection was performed , followed by phacoemulsification , irrigation aspiration ( ia ) , and implantation of single - piece hydrophobic acrylic iol . a hybrid technique of ia using a combination of coaxial and bimanual ia hand pieces was frequently employed to aspirate the subincisional cortical matter in cases with difficulty during removal of cortical matter.14 the corneal wounds were hydrated at the end of the procedure . in group ii , a 2.2 keratome was used to construct a temporal corneal incision and two side ports were constructed using a 20 g microvitreoretinal blade . capsulorhexis was performed with a 26-gauge needle cystotome ; microcapsulorhexis forceps was used if required . in cases with intumescence , an initial smaller capsulorhexis was followed by the aspiration of liquefied cortex , thus decreasing the raised intralenticular pressure . the aim was to provide ~0.5 mm iol coverage all around as the optic of the iol was 6 mm . after iol implantation , the capsulorhexis was further enlarged if required to achieve an adequate size . intraoperatively , the total surgical time , cumulative dissipated energy , total ultrasound time , and total aspiration time were noted . intraoperative complications such as anterior capsular tears / extension , posterior capsular tears , vitreous loss , need for anterior vitrectomy , and inability to implant iol were noted . postoperatively , clinical photographs were captured with the help of slit - lamp - mounted camera on postoperative day 30 and after 6 months , and the capsulorhexis size , shape , continuity , and iol optic coverage were evaluated . image - processing software ( imagej 1.46r , national institutes of health , md , usa ) was used to measure the diameter of the capsulorhexis and calculate the circularity index . the diameter of the implanted iol was used as a scale to calibrate the image . the circularity index was given by the formula 4 ( area / perimeter2 ) and a circularity index of 1 implied a perfect circle.3 the iol centration and coverage was assessed on the basis of coverage of iol optic in all four quadrants by the capsulotomy / capsulorhexis margin . continuity of the capsulorhexis refers to the absence of radial extension / tear of the capsulorhexis edge . udva and corrected distance visual acuity ( cdva ) were assessed on postoperative day 30 and at 6 months . statistical analysis was done using statistical package for the social sciences ( spss 11.0 ; spss inc . , chicago , il , usa ) . normally distributed continuous variables were expressed as mean standard deviation and were compared using independent samples t - test . nominal data were compared using chi - square test or fisher s exact test as appropriate . a p - value less than 0.05 was considered significant . the mean age of the patients was 62.99.1 years in group i and 64.88.3 years in group ii ( p=0.34 ) . group i consisted of 20 males and 20 females ; group ii had 17 males and 23 females . a continuous capsulotomy / capsulorhexis was achieved in 97.5% ( 39/40 ) of eyes , each in groups i and ii . type i ( free floating ) , type ii ( microadhesions ; ie , an area of complete cut mixed with minute areas of adhesions ) , type iii ( incomplete treatment pattern no visible femtosecond laser cut in the anterior capsule ) , and type iv ( irregular complete pattern ) . in group i , free - floating capsulotomies ( type i ) were achieved in 52.5% ( 21/40 ) of eyes . however , 47.5% ( 19/40 ) of eyes needed manual completion of the capsulotomy , of these 37.5% ( 15/40 ) had microadhesions ( type ii capsulotomy ) and 10% ( 4/40 ) had incomplete area of capsulotomy in 12 clock hours ( type iii capsulotomy ) . in group the mean diameter of the capsulotomy / capsulorhexis was 4.90.1 mm in group i and 5.30.4 mm in group ii ( p<0.001 , difference 0.39 ; 95% confidence interval [ ci ] : 0.27 , 0.52 ) . the mean circularity index was 0.9960.003 in group i and 0.9100.047 in group ii ( p - value < 0.001 , difference : 0.09 [ 95% ci : 0.07 , 0.10 ] ) . inadequate coverage of iol by the capsulotomy / capsulorhexis edge was noted in 7.5% ( 3/40 ) cases in group i and 12.5% ( 5/40 ) cases in group ii ( p=0.712 ) . at 6 months , the mean diameter of the capsulotomy / capsulorhexis was 4.90.1 mm in group i and 5.30.4 mm in group ii ( p<0.001 , difference : 0.37 [ 95% ci : 0.25 , 0.50 ] ) . the mean circularity index was 0.9910.023 in group i and 0.9100.047 in group ii ( p - value < 0.001 , difference : 0.08 [ 95% ci : 0.06 , 0.10 ] ) . the cumulative dissipated energy , total ultrasound time , total aspiration time , and total surgical time were comparable between the two groups ( table 2 ) . there was no case of posterior capsular rent , vitreous loss , nucleus drop , or inability to implant iol in group i or ii . the mean logarithm of the minimum angle of resolution ( logmar ) udva was 0.1280.122 in group i and 0.1150.127 in group ii ( p=0.655 ) at 1 month postoperatively . the mean logmar cdva was 0.0100.044 in group i and 0.0150.053 in group ii ( p=0.649 ) at 1 month postoperatively . at 6 months , mean logmar udva was 0.130.12 in group i and 0.100.12 in group ii ( p=0.315 ) . at 6 months , mean logmar cdva was 0.0050.031 in group i and 0.0100.044 in group ii ( p=0.562 ) . the white cataracts in each group were divided into two subgroups based on the release of white milky fluid on initiation of the capsulotomy / capsulorhexis . the assessment of milky fluid egress was qualitative , and no attempt was made to quantify the amount of fluid by the surgeon . in group i , 17 cases had release of white milky fluid ( subgroup a ) and 23 cases did not ( subgroup b ) . in group ii , 19 cases were in the fluid group ( subgroup a ) and 21 cases were in the no - fluid group ( subgroup b ) . the capsulotomy / capsulorhexis characteristics in the fluid cases were compared with the no - fluid cases in each group . continuous capsulotomies were achieved in 94.1% ( 16/17 ) cases with fluid and 100% ( 23/23 ) cases with no - fluid ( p=0.42 ) . in the cases with fluid , free - floating ( type i capsulotomy ) capsulotomies were achieved in 23.5% ( 4/17 ) cases . manual completion of the capsulotomy was needed in 76.5% ( 13/17 ) cases , of these 52.9% ( 9/17 ) had microadhesions ( type ii capsulotomy ) and 23.5% ( 4/17 ) had incomplete area of capsulotomy in 12 clock hours ( type iii capsulotomy ) . in cases with no - fluid , approximately 26.1% ( 6/23 ) of eyes needed manual completion of the capsulotomy and release of the microadhesions ( type ii capsulotomy ) . the incidence of residual adhesions and incomplete capsulotomies was significantly more in cases with release of white milky fluid ( p=0.003 ) . anterior capsular extension with radial tear was observed in one eye in fluid release group only ( p=0.42 ) . the mean diameter of the capsulotomy was 4.90.1 mm in fluid and no - fluid group ( p=0.9 ) . the mean circularity index was 0.9960.003 in the fluid group and 0.9970.003 in the no - fluid group ( p=0.1 ) . inadequate coverage of iol by the capsulotomy edge was noted in 17.6% ( 3/17 ) cases in fluid group only ( p=0.09 ) . a continuous capsulorhexis was achieved in 100% ( 19/19 ) cases with fluid and 95.2% ( 20/21 ) cases with no - fluid ( p=1.0 ) . a multistep capsulorhexis was performed in 84.2% ( 16/19 ) cases with fluid and 57.1% ( 12/21 ) cases with no - fluid . there was one case of anterior capsular extension with radial tear in no - fluid group only ( p=1.00 ) . the mean diameter of the capsulorhexis was 5.30.3 mm in the fluid group and 5.40.4 mm in the no - fluid group ( p=0.7 , difference : 0.05 [ 95% ci : 0.3 , 0.2 ] ) . the mean circularity index was 0.880.04 in the fluid group and 0.940.04 in the no - fluid group ( p - value < 0.001 , difference : 0.05 [ 95% ci : 0.02 , 0.08 ] ) . inadequate coverage of iol by the capsulorhexis edge was noted in 15.8% ( 3/19 ) cases with fluid and 9.5% ( 2/21 ) cases with no - fluid ( p=0.65 ) . continuous capsulotomies were achieved in 94.1% ( 16/17 ) cases with fluid and 100% ( 23/23 ) cases with no - fluid ( p=0.42 ) . in the cases with fluid , free - floating ( type i capsulotomy ) capsulotomies were achieved in 23.5% ( 4/17 ) cases . manual completion of the capsulotomy was needed in 76.5% ( 13/17 ) cases , of these 52.9% ( 9/17 ) had microadhesions ( type ii capsulotomy ) and 23.5% ( 4/17 ) had incomplete area of capsulotomy in 12 clock hours ( type iii capsulotomy ) . in cases with no - fluid , approximately 26.1% ( 6/23 ) of eyes needed manual completion of the capsulotomy and release of the microadhesions ( type ii capsulotomy ) . the incidence of residual adhesions and incomplete capsulotomies was significantly more in cases with release of white milky fluid ( p=0.003 ) . anterior capsular extension with radial tear was observed in one eye in fluid release group only ( p=0.42 ) . the mean diameter of the capsulotomy was 4.90.1 mm in fluid and no - fluid group ( p=0.9 ) . the mean circularity index was 0.9960.003 in the fluid group and 0.9970.003 in the no - fluid group ( p=0.1 ) . inadequate coverage of iol by the capsulotomy edge was noted in 17.6% ( 3/17 ) cases in fluid group only ( p=0.09 ) . a continuous capsulorhexis was achieved in 100% ( 19/19 ) cases with fluid and 95.2% ( 20/21 ) cases with no - fluid ( p=1.0 ) . a multistep capsulorhexis was performed in 84.2% ( 16/19 ) cases with fluid and 57.1% ( 12/21 ) cases with no - fluid . there was one case of anterior capsular extension with radial tear in no - fluid group only ( p=1.00 ) . the mean diameter of the capsulorhexis was 5.30.3 mm in the fluid group and 5.40.4 mm in the no - fluid group ( p=0.7 , difference : 0.05 [ 95% ci : 0.3 , 0.2 ] ) . the mean circularity index was 0.880.04 in the fluid group and 0.940.04 in the no - fluid group ( p - value < 0.001 , difference : 0.05 [ 95% ci : 0.02 , 0.08 ] ) . inadequate coverage of iol by the capsulorhexis edge was noted in 15.8% ( 3/19 ) cases with fluid and 9.5% ( 2/21 ) cases with no - fluid ( p=0.65 ) . the main challenge in phacoemulsification in cases of white cataracts is achieving a continuous curvilinear capsulorhexis . the absence of a red reflex , raised intralenticular pressure , and a fragile anterior capsule complicate the management of white cataracts , and an incomplete capsulorhexis has been reported in 3.85%28.3% cases of white cataracts.912,16 an ideal capsulorhexis is a well - centered circular opening with smooth margins and a slightly smaller diameter than the iol providing 360 iol coverage . inadequate coverage of iol may lead to posterior capsular opacification and change in the effective lens position,17 whereas an excessively small capsulorhexis may cause anterior capsular contraction.18 in our study , reproducible circular capsulotomies were achieved with the help of femtosecond laser , providing adequate iol coverage in 92.5% ( 37/40 ) of cases . the femtosecond laser - assisted capsulotomies may be of four types based on the pattern of completion.15 in our study , type i free - floating capsulotomies were achieved in 52.5% ( 21/40 ) cases . type ii capsulotomy with microadhesions was observed in 37.5% ( 15/40 ) cases and 10% ( 4/40 ) had type iii capsulotomy with incomplete treatment pattern in 12 clock hours . a significantly higher incidence of residual adhesions ( type ii and type iii capsulotomies ) was observed in the subgroup with release of white milky fluid after femtosecond laser delivery ( p<0.003 ) . the reason for microadhesions / incomplete capsulotomies was the explosive egress of white milky fluid on initiation of the capsulotomy , which obscures the visual field and hampers the subsequent delivery of the laser at the same plane . the release of the intralenticular pressure after initiation of the capsulotomy causes a slight change in the position of the anterior capsular plane and hence may result in the occurrence of incomplete capsulotomy . during the lifting of the capsulotomy flap with the microcapsulorhexis forceps , the areas obscured by the white milky fluid during femtosecond laser application should be carefully assessed , as they are likely to have microadhesions . it is imperative to use trypan blue dye to stain the anterior capsule in all cases to delineate the area of microadhesions or incomplete treatment pattern . in each of these cases , the free edge of the capsulotomy was grasped with the help of microcapsulorhexis forceps and a circumferential motion tracing the capsulotomy margins was performed , which was akin to the maneuver that one does while performing a manual capsulorhexis ( figure 1 , video s1 ) . this crucial step ensures the effective release of the underlying residual adhesions in femtosecond laser - assisted capsulotomies . despite the presence of microadhesions and incomplete treatment patterns , the continuity and circularity of the capsulotomies was achieved in 97.5% ( 39/40 ) of the eyes . the incidence of incomplete capsulotomies and microadhesions is more in white hypermature cataracts as compared to immature senile cataracts . in a case series of 1,300 cases of senile cataract undergoing flacs , 96% capsulotomies were free floating or had small areas of nonperforations.19 in our study , one case of anterior capsular extension with radial tear was noted during flacs . the radial extension occurred while polishing the anterior capsule after iol implantation ( figure 3 ) . this case was characterized by raised intralenticular pressure as evidenced by a gush of white milky fluid after the initial capsulotomy opening and had a type ii capsulotomy with microadhesions . in cases with microadhesions , the capsular edge is fragile and careful anterior capsular polishing is needed to prevent extension and radial tears . in our experience , a raised intralenticular pressure with release of white milky fluid is the most significant factor affecting the femtosecond laser application . the milky fluid results in microadhesions and incomplete sectors of capsulotomy , which may increase the chances of tear extension during subsequent steps of phacoemulsification . this is in contrast to the manual capsulorhexis where the release of fluid is associated with a subsequent decrease in the intralenticular pressure and more safety in completion of capsulorhexis . a two - stage femtosecond laser capsulotomy has also been described in six cases of intumescent white cataract ; however , remnant tissue bridges were present in one - third of the mini - capsulotomies.20 in cases undergoing conventional phacoemulsification , a multistep capsulorhexis was performed in 70% cases to achieve an adequately sized capsulorhexis . the circularity of the capsulorhexis was compromised , more so in cases with release of milky fluid . microadhesions if present can be easily released during capsulotomy removal with forceps , without jeopardizing the size , circularity , and continuity of the capsulotomy . a previous study has demonstrated the safety and efficacy of femtosecond laser - assisted capsulotomy in 25 cases of white cataracts , with radial anterior tears in two eyes , an adherent tongue - like capsule adhesion in nine eyes , and an incomplete capsulotomy in three eyes.13 the total aspiration time and total surgical duration was longer in flacs , but the difference between the two groups was not statistically significant . a hybrid technique of ia using a combination of coaxial and bimanual ia hand pieces was frequently employed to aspirate the subincisional cortical matter in cases undergoing flacs , which may explain the longer aspiration times.14 to conclude , femtosecond laser creates single - step , circular , adequately sized capsulotomies and eliminates the difficulty associated with capsulorhexis in white cataracts . the release of white milky fluid during femtosecond laser delivery is the most important factor affecting the creation of a free - floating capsulotomy , and microadhesions and incomplete capsulotomies may be present in up to 47.5% cases of flacs . there is no significant difference in terms of the intraoperative complications and the final visual outcome between flacs and conventional phacoemulsification . to the best of our knowledge , this is the first prospective study comparing conventional phacoemulsification with flacs in white cataracts in terms of intraoperative parameters and postoperative outcomes . further randomized control trials with a larger sample size should be undertaken to further evaluate the long - term superiority of femtosecond laser over conventional phacoemulsification .

purposeto compare femtosecond laser - assisted capsulotomy with conventional manual capsulorhexis in cases of white cataract.patients and methodsthe prospective comparative study enrolled 80 eyes ( 80 patients ) with white cataract that underwent either femtosecond laser - assisted cataract surgery ( group i , n=40 ) or conventional manual phacoemulsification ( group ii , n=40 ) at a tertiary care ophthalmic institution . the groups were divided based on the patient s choice and affordability of the procedure . capsulotomy / capsulorhexis was evaluated in terms of size , circularity index ( 4 [ area / perimeter2 ] ) , intraocular lens coverage , and continuity . each group was further subdivided based on the release of white milky fluid on initiation of the capsulotomy / capsulorhexis , and the fluid cases were compared with the no - fluid cases . the primary outcome measure was capsulotomy / capsulorhexis characteristics in the two groups . the secondary outcome measures were intraoperative phacoemulsification parameters , intraoperative complications , and postoperative visual acuity.resultsthe size of the capsulotomy / capsulorhexis was 4.90.1 mm in group i and 5.30.4 mm in group ii ( p<0.001 ) . mean circularity index was 0.9960.003 and 0.9090.047 in groups i and ii , respectively ( p<0.001 ) . in group i , free - floating circular capsulotomies were obtained in 52.5% ( 21/40 ) eyes ; 37.5% ( 15/40 ) eyes had microadhesions ; and 10% ( 4/40 ) eyes had incomplete capsulotomy in 12 clock hours . the incidence of residual adhesions was more in cases with release of white milky fluid ( p=0.003 ) . in group ii , a multistep capsulorhexis was performed in 70% ( 28/40 ) of the eyes . there was no difference in terms of visual outcomes and intraoperative complications.conclusionfemtosecond laser - assisted cataract surgery has the advantage of creating a circular and optimally sized capsulotomy in cases of white cataract . the release of white milky fluid during femtosecond laser delivery is the most important factor affecting the creation of a free - floating capsulotomy .

Introduction Patients and methods Surgical technique Statistical analysis Results Capsulotomy characteristics in cases with fluid and no-fluid in Group I Capsulorhexis characteristics in cases with fluid and no-fluid in Group II Discussion Conclusion None

femtosecond laser - assisted cataract surgery ( flacs ) is superior to conventional phacoemulsification in terms of precise wound construction , capsulotomy , intraocular lens ( iol ) centration , and decreased effective phacoemulsification time.14 flacs is comparable to traditional cataract surgery in terms of intraoperative complications , although further research is needed to evaluate its long - term safety aspects.57 it may be especially advantageous in white cataracts , where achieving an adequately sized continuous curvilinear capsulorhexis is the major surgical challenge . the increased incidence of capsulorhexis - related complications in white cataracts is often associated with posterior capsular rent , vitreous loss , and nucleus drop.8 various maneuvers have been used to facilitate capsulorhexis , such as use of high - molecular - weight viscoelastic,9 trypan blue dye to stain the anterior capsule,10 two - stage capsulorhexis,11 frequent aspiration of liquefied cortical matter,12 use of vannas scissors,12 and endoilluminator.9 however , limited literature exists on the application of femtosecond lasers in white cataracts.13 comparative evaluation of flacs and conventional phacoemulsification has been done for varying grades of nuclear sclerosis ; however , there are no studies that have compared the outcomes of flacs with conventional phacoemulsification in cases with white cataract.5,7 we herein report our results of comparison of femtosecond laser capsulotomies with conventional capsulorhexis in cases of white cataract . in this prospective comparative study , 80 eyes of 80 patients with white cataract were enrolled at dr rp centre for ophthalmic sciences , all india institute of medical sciences , new delhi , india . the patients were divided into two groups , each consisting of 40 eyes ( 40 patients ) . group i ( n=40 ) underwent flacs and group ii ( n=40 ) underwent conventional phacoemulsification performed by an experienced phacoemulsification surgeon ( jst ) . the patients with white cataract scheduled to undergo flacs or conventional phacoemulsification ( based on patient s choice and affordability of the procedure ) were compared . the secondary outcome measures were intraoperative phacoemulsification parameters , intraoperative complications , and postoperative visual acuity . in group i , the preoperative planning of the corneal incisions , capsulotomy , and nucleotomy was done on lensx version 2.23 femtosecond laser system ( alcon lensx , inc . a hybrid technique of ia using a combination of coaxial and bimanual ia hand pieces was frequently employed to aspirate the subincisional cortical matter in cases with difficulty during removal of cortical matter.14 the corneal wounds were hydrated at the end of the procedure . in group ii , a 2.2 keratome was used to construct a temporal corneal incision and two side ports were constructed using a 20 g microvitreoretinal blade . postoperatively , clinical photographs were captured with the help of slit - lamp - mounted camera on postoperative day 30 and after 6 months , and the capsulorhexis size , shape , continuity , and iol optic coverage were evaluated . the circularity index was given by the formula 4 ( area / perimeter2 ) and a circularity index of 1 implied a perfect circle.3 the iol centration and coverage was assessed on the basis of coverage of iol optic in all four quadrants by the capsulotomy / capsulorhexis margin . in group i , the preoperative planning of the corneal incisions , capsulotomy , and nucleotomy was done on lensx version 2.23 femtosecond laser system ( alcon lensx , inc . gentle hydrodissection was performed , followed by phacoemulsification , irrigation aspiration ( ia ) , and implantation of single - piece hydrophobic acrylic iol . a hybrid technique of ia using a combination of coaxial and bimanual ia hand pieces was frequently employed to aspirate the subincisional cortical matter in cases with difficulty during removal of cortical matter.14 the corneal wounds were hydrated at the end of the procedure . in group ii , a 2.2 keratome was used to construct a temporal corneal incision and two side ports were constructed using a 20 g microvitreoretinal blade . postoperatively , clinical photographs were captured with the help of slit - lamp - mounted camera on postoperative day 30 and after 6 months , and the capsulorhexis size , shape , continuity , and iol optic coverage were evaluated . the circularity index was given by the formula 4 ( area / perimeter2 ) and a circularity index of 1 implied a perfect circle.3 the iol centration and coverage was assessed on the basis of coverage of iol optic in all four quadrants by the capsulotomy / capsulorhexis margin . the mean age of the patients was 62.99.1 years in group i and 64.88.3 years in group ii ( p=0.34 ) . a continuous capsulotomy / capsulorhexis was achieved in 97.5% ( 39/40 ) of eyes , each in groups i and ii . type i ( free floating ) , type ii ( microadhesions ; ie , an area of complete cut mixed with minute areas of adhesions ) , type iii ( incomplete treatment pattern no visible femtosecond laser cut in the anterior capsule ) , and type iv ( irregular complete pattern ) . in group i , free - floating capsulotomies ( type i ) were achieved in 52.5% ( 21/40 ) of eyes . however , 47.5% ( 19/40 ) of eyes needed manual completion of the capsulotomy , of these 37.5% ( 15/40 ) had microadhesions ( type ii capsulotomy ) and 10% ( 4/40 ) had incomplete area of capsulotomy in 12 clock hours ( type iii capsulotomy ) . in group the mean diameter of the capsulotomy / capsulorhexis was 4.90.1 mm in group i and 5.30.4 mm in group ii ( p<0.001 , difference 0.39 ; 95% confidence interval [ ci ] : 0.27 , 0.52 ) . the mean circularity index was 0.9960.003 in group i and 0.9100.047 in group ii ( p - value < 0.001 , difference : 0.09 [ 95% ci : 0.07 , 0.10 ] ) . inadequate coverage of iol by the capsulotomy / capsulorhexis edge was noted in 7.5% ( 3/40 ) cases in group i and 12.5% ( 5/40 ) cases in group ii ( p=0.712 ) . at 6 months , the mean diameter of the capsulotomy / capsulorhexis was 4.90.1 mm in group i and 5.30.4 mm in group ii ( p<0.001 , difference : 0.37 [ 95% ci : 0.25 , 0.50 ] ) . the mean circularity index was 0.9910.023 in group i and 0.9100.047 in group ii ( p - value < 0.001 , difference : 0.08 [ 95% ci : 0.06 , 0.10 ] ) . there was no case of posterior capsular rent , vitreous loss , nucleus drop , or inability to implant iol in group i or ii . the mean logarithm of the minimum angle of resolution ( logmar ) udva was 0.1280.122 in group i and 0.1150.127 in group ii ( p=0.655 ) at 1 month postoperatively . the mean logmar cdva was 0.0100.044 in group i and 0.0150.053 in group ii ( p=0.649 ) at 1 month postoperatively . at 6 months , mean logmar udva was 0.130.12 in group i and 0.100.12 in group ii ( p=0.315 ) . at 6 months , mean logmar cdva was 0.0050.031 in group i and 0.0100.044 in group ii ( p=0.562 ) . the white cataracts in each group were divided into two subgroups based on the release of white milky fluid on initiation of the capsulotomy / capsulorhexis . in group i , 17 cases had release of white milky fluid ( subgroup a ) and 23 cases did not ( subgroup b ) . in group ii , 19 cases were in the fluid group ( subgroup a ) and 21 cases were in the no - fluid group ( subgroup b ) . the capsulotomy / capsulorhexis characteristics in the fluid cases were compared with the no - fluid cases in each group . continuous capsulotomies were achieved in 94.1% ( 16/17 ) cases with fluid and 100% ( 23/23 ) cases with no - fluid ( p=0.42 ) . in the cases with fluid , free - floating ( type i capsulotomy ) capsulotomies were achieved in 23.5% ( 4/17 ) cases . manual completion of the capsulotomy was needed in 76.5% ( 13/17 ) cases , of these 52.9% ( 9/17 ) had microadhesions ( type ii capsulotomy ) and 23.5% ( 4/17 ) had incomplete area of capsulotomy in 12 clock hours ( type iii capsulotomy ) . in cases with no - fluid , approximately 26.1% ( 6/23 ) of eyes needed manual completion of the capsulotomy and release of the microadhesions ( type ii capsulotomy ) . the incidence of residual adhesions and incomplete capsulotomies was significantly more in cases with release of white milky fluid ( p=0.003 ) . the mean diameter of the capsulotomy was 4.90.1 mm in fluid and no - fluid group ( p=0.9 ) . the mean circularity index was 0.9960.003 in the fluid group and 0.9970.003 in the no - fluid group ( p=0.1 ) . a continuous capsulorhexis was achieved in 100% ( 19/19 ) cases with fluid and 95.2% ( 20/21 ) cases with no - fluid ( p=1.0 ) . a multistep capsulorhexis was performed in 84.2% ( 16/19 ) cases with fluid and 57.1% ( 12/21 ) cases with no - fluid . the mean diameter of the capsulorhexis was 5.30.3 mm in the fluid group and 5.40.4 mm in the no - fluid group ( p=0.7 , difference : 0.05 [ 95% ci : 0.3 , 0.2 ] ) . the mean circularity index was 0.880.04 in the fluid group and 0.940.04 in the no - fluid group ( p - value < 0.001 , difference : 0.05 [ 95% ci : 0.02 , 0.08 ] ) . inadequate coverage of iol by the capsulorhexis edge was noted in 15.8% ( 3/19 ) cases with fluid and 9.5% ( 2/21 ) cases with no - fluid ( p=0.65 ) . continuous capsulotomies were achieved in 94.1% ( 16/17 ) cases with fluid and 100% ( 23/23 ) cases with no - fluid ( p=0.42 ) . in the cases with fluid , free - floating ( type i capsulotomy ) capsulotomies were achieved in 23.5% ( 4/17 ) cases . manual completion of the capsulotomy was needed in 76.5% ( 13/17 ) cases , of these 52.9% ( 9/17 ) had microadhesions ( type ii capsulotomy ) and 23.5% ( 4/17 ) had incomplete area of capsulotomy in 12 clock hours ( type iii capsulotomy ) . in cases with no - fluid , approximately 26.1% ( 6/23 ) of eyes needed manual completion of the capsulotomy and release of the microadhesions ( type ii capsulotomy ) . the incidence of residual adhesions and incomplete capsulotomies was significantly more in cases with release of white milky fluid ( p=0.003 ) . the mean diameter of the capsulotomy was 4.90.1 mm in fluid and no - fluid group ( p=0.9 ) . the mean circularity index was 0.9960.003 in the fluid group and 0.9970.003 in the no - fluid group ( p=0.1 ) . a continuous capsulorhexis was achieved in 100% ( 19/19 ) cases with fluid and 95.2% ( 20/21 ) cases with no - fluid ( p=1.0 ) . a multistep capsulorhexis was performed in 84.2% ( 16/19 ) cases with fluid and 57.1% ( 12/21 ) cases with no - fluid . the mean diameter of the capsulorhexis was 5.30.3 mm in the fluid group and 5.40.4 mm in the no - fluid group ( p=0.7 , difference : 0.05 [ 95% ci : 0.3 , 0.2 ] ) . the mean circularity index was 0.880.04 in the fluid group and 0.940.04 in the no - fluid group ( p - value < 0.001 , difference : 0.05 [ 95% ci : 0.02 , 0.08 ] ) . inadequate coverage of iol by the capsulorhexis edge was noted in 15.8% ( 3/19 ) cases with fluid and 9.5% ( 2/21 ) cases with no - fluid ( p=0.65 ) . the absence of a red reflex , raised intralenticular pressure , and a fragile anterior capsule complicate the management of white cataracts , and an incomplete capsulorhexis has been reported in 3.85%28.3% cases of white cataracts.912,16 an ideal capsulorhexis is a well - centered circular opening with smooth margins and a slightly smaller diameter than the iol providing 360 iol coverage . inadequate coverage of iol may lead to posterior capsular opacification and change in the effective lens position,17 whereas an excessively small capsulorhexis may cause anterior capsular contraction.18 in our study , reproducible circular capsulotomies were achieved with the help of femtosecond laser , providing adequate iol coverage in 92.5% ( 37/40 ) of cases . the femtosecond laser - assisted capsulotomies may be of four types based on the pattern of completion.15 in our study , type i free - floating capsulotomies were achieved in 52.5% ( 21/40 ) cases . type ii capsulotomy with microadhesions was observed in 37.5% ( 15/40 ) cases and 10% ( 4/40 ) had type iii capsulotomy with incomplete treatment pattern in 12 clock hours . a significantly higher incidence of residual adhesions ( type ii and type iii capsulotomies ) was observed in the subgroup with release of white milky fluid after femtosecond laser delivery ( p<0.003 ) . the reason for microadhesions / incomplete capsulotomies was the explosive egress of white milky fluid on initiation of the capsulotomy , which obscures the visual field and hampers the subsequent delivery of the laser at the same plane . the release of the intralenticular pressure after initiation of the capsulotomy causes a slight change in the position of the anterior capsular plane and hence may result in the occurrence of incomplete capsulotomy . during the lifting of the capsulotomy flap with the microcapsulorhexis forceps , the areas obscured by the white milky fluid during femtosecond laser application should be carefully assessed , as they are likely to have microadhesions . in each of these cases , the free edge of the capsulotomy was grasped with the help of microcapsulorhexis forceps and a circumferential motion tracing the capsulotomy margins was performed , which was akin to the maneuver that one does while performing a manual capsulorhexis ( figure 1 , video s1 ) . this crucial step ensures the effective release of the underlying residual adhesions in femtosecond laser - assisted capsulotomies . despite the presence of microadhesions and incomplete treatment patterns , the continuity and circularity of the capsulotomies was achieved in 97.5% ( 39/40 ) of the eyes . this case was characterized by raised intralenticular pressure as evidenced by a gush of white milky fluid after the initial capsulotomy opening and had a type ii capsulotomy with microadhesions . in our experience , a raised intralenticular pressure with release of white milky fluid is the most significant factor affecting the femtosecond laser application . this is in contrast to the manual capsulorhexis where the release of fluid is associated with a subsequent decrease in the intralenticular pressure and more safety in completion of capsulorhexis . a two - stage femtosecond laser capsulotomy has also been described in six cases of intumescent white cataract ; however , remnant tissue bridges were present in one - third of the mini - capsulotomies.20 in cases undergoing conventional phacoemulsification , a multistep capsulorhexis was performed in 70% cases to achieve an adequately sized capsulorhexis . the circularity of the capsulorhexis was compromised , more so in cases with release of milky fluid . microadhesions if present can be easily released during capsulotomy removal with forceps , without jeopardizing the size , circularity , and continuity of the capsulotomy . a previous study has demonstrated the safety and efficacy of femtosecond laser - assisted capsulotomy in 25 cases of white cataracts , with radial anterior tears in two eyes , an adherent tongue - like capsule adhesion in nine eyes , and an incomplete capsulotomy in three eyes.13 the total aspiration time and total surgical duration was longer in flacs , but the difference between the two groups was not statistically significant . the release of white milky fluid during femtosecond laser delivery is the most important factor affecting the creation of a free - floating capsulotomy , and microadhesions and incomplete capsulotomies may be present in up to 47.5% cases of flacs . there is no significant difference in terms of the intraoperative complications and the final visual outcome between flacs and conventional phacoemulsification . to the best of our knowledge , this is the first prospective study comparing conventional phacoemulsification with flacs in white cataracts in terms of intraoperative parameters and postoperative outcomes .

femtosecond laser - assisted cataract surgery ( flacs ) is superior to conventional phacoemulsification in terms of precise wound construction , capsulotomy , intraocular lens ( iol ) centration , and decreased effective phacoemulsification time.14 flacs is comparable to traditional cataract surgery in terms of intraoperative complications , although further research is needed to evaluate its long - term safety aspects.57 it may be especially advantageous in white cataracts , where achieving an adequately sized continuous curvilinear capsulorhexis is the major surgical challenge . the increased incidence of capsulorhexis - related complications in white cataracts is often associated with posterior capsular rent , vitreous loss , and nucleus drop.8 various maneuvers have been used to facilitate capsulorhexis , such as use of high - molecular - weight viscoelastic,9 trypan blue dye to stain the anterior capsule,10 two - stage capsulorhexis,11 frequent aspiration of liquefied cortical matter,12 use of vannas scissors,12 and endoilluminator.9 however , limited literature exists on the application of femtosecond lasers in white cataracts.13 comparative evaluation of flacs and conventional phacoemulsification has been done for varying grades of nuclear sclerosis ; however , there are no studies that have compared the outcomes of flacs with conventional phacoemulsification in cases with white cataract.5,7 we herein report our results of comparison of femtosecond laser capsulotomies with conventional capsulorhexis in cases of white cataract . in this prospective comparative study , 80 eyes of 80 patients with white cataract were enrolled at dr rp centre for ophthalmic sciences , all india institute of medical sciences , new delhi , india . the white cataracts were classified into type i , ii , and iii according to the classification provided by brazitikos et al.8 type i intumescent , white cataracts are characterized by the presence of liquefied cortex , type ii white cataracts have a voluminous nucleus with little amount of solid white cortex , and type iii white cataracts have anterior capsule fibrosis . type iii white cataracts with calcific and fibrotic capsules were excluded from the study.8 eyes with preexisting glaucoma , retinal pathology , pseudoexfoliation syndrome , or pupillary dilatation < 6 mm were also excluded . group i ( n=40 ) underwent flacs and group ii ( n=40 ) underwent conventional phacoemulsification performed by an experienced phacoemulsification surgeon ( jst ) . the preoperative detailed examination included uncorrected distance visual acuity ( udva ) , anterior segment evaluation , slit - lamp biomicroscopy , applanation tonometry , biometry ( tonoref iii , nidek co. , ltd . in group i , the preoperative planning of the corneal incisions , capsulotomy , and nucleotomy was done on lensx version 2.23 femtosecond laser system ( alcon lensx , inc . , a 4.9 mm capsulotomy was planned , with a 2.2 mm temporal clear corneal incision and two 1.1 mm side ports at 90 and 240. the chop pattern of nucleotomy was selected , with three chops of length 6 mm each . the spot and layer separation was 5 m for the side port incisions , 6 m for 2.2 mm corneal incision , and 14 m for the nucleotomy . a hybrid technique of ia using a combination of coaxial and bimanual ia hand pieces was frequently employed to aspirate the subincisional cortical matter in cases with difficulty during removal of cortical matter.14 the corneal wounds were hydrated at the end of the procedure . in cases with intumescence , an initial smaller capsulorhexis was followed by the aspiration of liquefied cortex , thus decreasing the raised intralenticular pressure . intraoperatively , the total surgical time , cumulative dissipated energy , total ultrasound time , and total aspiration time were noted . intraoperative complications such as anterior capsular tears / extension , posterior capsular tears , vitreous loss , need for anterior vitrectomy , and inability to implant iol were noted . postoperatively , clinical photographs were captured with the help of slit - lamp - mounted camera on postoperative day 30 and after 6 months , and the capsulorhexis size , shape , continuity , and iol optic coverage were evaluated . image - processing software ( imagej 1.46r , national institutes of health , md , usa ) was used to measure the diameter of the capsulorhexis and calculate the circularity index . the circularity index was given by the formula 4 ( area / perimeter2 ) and a circularity index of 1 implied a perfect circle.3 the iol centration and coverage was assessed on the basis of coverage of iol optic in all four quadrants by the capsulotomy / capsulorhexis margin . continuity of the capsulorhexis refers to the absence of radial extension / tear of the capsulorhexis edge . in group i , the preoperative planning of the corneal incisions , capsulotomy , and nucleotomy was done on lensx version 2.23 femtosecond laser system ( alcon lensx , inc . , a 4.9 mm capsulotomy was planned , with a 2.2 mm temporal clear corneal incision and two 1.1 mm side ports at 90 and 240. the chop pattern of nucleotomy was selected , with three chops of length 6 mm each . the femtosecond laser parameters were the same as those used in our routine cases ( nonwhite cataract cases ) of flacs . the spot and layer separation was 5 m for the side port incisions , 6 m for 2.2 mm corneal incision , and 14 m for the nucleotomy . for capsulotomy , the spot separation was 5 m , the layer separation was 4 m , and the anterior and posterior delta value was 350 m each . a hybrid technique of ia using a combination of coaxial and bimanual ia hand pieces was frequently employed to aspirate the subincisional cortical matter in cases with difficulty during removal of cortical matter.14 the corneal wounds were hydrated at the end of the procedure . in group ii , a 2.2 keratome was used to construct a temporal corneal incision and two side ports were constructed using a 20 g microvitreoretinal blade . the aim was to provide ~0.5 mm iol coverage all around as the optic of the iol was 6 mm . intraoperatively , the total surgical time , cumulative dissipated energy , total ultrasound time , and total aspiration time were noted . intraoperative complications such as anterior capsular tears / extension , posterior capsular tears , vitreous loss , need for anterior vitrectomy , and inability to implant iol were noted . postoperatively , clinical photographs were captured with the help of slit - lamp - mounted camera on postoperative day 30 and after 6 months , and the capsulorhexis size , shape , continuity , and iol optic coverage were evaluated . image - processing software ( imagej 1.46r , national institutes of health , md , usa ) was used to measure the diameter of the capsulorhexis and calculate the circularity index . the circularity index was given by the formula 4 ( area / perimeter2 ) and a circularity index of 1 implied a perfect circle.3 the iol centration and coverage was assessed on the basis of coverage of iol optic in all four quadrants by the capsulotomy / capsulorhexis margin . continuity of the capsulorhexis refers to the absence of radial extension / tear of the capsulorhexis edge . the mean age of the patients was 62.99.1 years in group i and 64.88.3 years in group ii ( p=0.34 ) . type i ( free floating ) , type ii ( microadhesions ; ie , an area of complete cut mixed with minute areas of adhesions ) , type iii ( incomplete treatment pattern no visible femtosecond laser cut in the anterior capsule ) , and type iv ( irregular complete pattern ) . in group i , free - floating capsulotomies ( type i ) were achieved in 52.5% ( 21/40 ) of eyes . however , 47.5% ( 19/40 ) of eyes needed manual completion of the capsulotomy , of these 37.5% ( 15/40 ) had microadhesions ( type ii capsulotomy ) and 10% ( 4/40 ) had incomplete area of capsulotomy in 12 clock hours ( type iii capsulotomy ) . in group the mean diameter of the capsulotomy / capsulorhexis was 4.90.1 mm in group i and 5.30.4 mm in group ii ( p<0.001 , difference 0.39 ; 95% confidence interval [ ci ] : 0.27 , 0.52 ) . the mean circularity index was 0.9960.003 in group i and 0.9100.047 in group ii ( p - value < 0.001 , difference : 0.09 [ 95% ci : 0.07 , 0.10 ] ) . inadequate coverage of iol by the capsulotomy / capsulorhexis edge was noted in 7.5% ( 3/40 ) cases in group i and 12.5% ( 5/40 ) cases in group ii ( p=0.712 ) . at 6 months , the mean diameter of the capsulotomy / capsulorhexis was 4.90.1 mm in group i and 5.30.4 mm in group ii ( p<0.001 , difference : 0.37 [ 95% ci : 0.25 , 0.50 ] ) . the mean circularity index was 0.9910.023 in group i and 0.9100.047 in group ii ( p - value < 0.001 , difference : 0.08 [ 95% ci : 0.06 , 0.10 ] ) . the cumulative dissipated energy , total ultrasound time , total aspiration time , and total surgical time were comparable between the two groups ( table 2 ) . the mean logarithm of the minimum angle of resolution ( logmar ) udva was 0.1280.122 in group i and 0.1150.127 in group ii ( p=0.655 ) at 1 month postoperatively . the mean logmar cdva was 0.0100.044 in group i and 0.0150.053 in group ii ( p=0.649 ) at 1 month postoperatively . at 6 months , mean logmar udva was 0.130.12 in group i and 0.100.12 in group ii ( p=0.315 ) . at 6 months , mean logmar cdva was 0.0050.031 in group i and 0.0100.044 in group ii ( p=0.562 ) . in group ii , 19 cases were in the fluid group ( subgroup a ) and 21 cases were in the no - fluid group ( subgroup b ) . manual completion of the capsulotomy was needed in 76.5% ( 13/17 ) cases , of these 52.9% ( 9/17 ) had microadhesions ( type ii capsulotomy ) and 23.5% ( 4/17 ) had incomplete area of capsulotomy in 12 clock hours ( type iii capsulotomy ) . in cases with no - fluid , approximately 26.1% ( 6/23 ) of eyes needed manual completion of the capsulotomy and release of the microadhesions ( type ii capsulotomy ) . the incidence of residual adhesions and incomplete capsulotomies was significantly more in cases with release of white milky fluid ( p=0.003 ) . anterior capsular extension with radial tear was observed in one eye in fluid release group only ( p=0.42 ) . the mean circularity index was 0.9960.003 in the fluid group and 0.9970.003 in the no - fluid group ( p=0.1 ) . the mean diameter of the capsulorhexis was 5.30.3 mm in the fluid group and 5.40.4 mm in the no - fluid group ( p=0.7 , difference : 0.05 [ 95% ci : 0.3 , 0.2 ] ) . the mean circularity index was 0.880.04 in the fluid group and 0.940.04 in the no - fluid group ( p - value < 0.001 , difference : 0.05 [ 95% ci : 0.02 , 0.08 ] ) . inadequate coverage of iol by the capsulorhexis edge was noted in 15.8% ( 3/19 ) cases with fluid and 9.5% ( 2/21 ) cases with no - fluid ( p=0.65 ) . manual completion of the capsulotomy was needed in 76.5% ( 13/17 ) cases , of these 52.9% ( 9/17 ) had microadhesions ( type ii capsulotomy ) and 23.5% ( 4/17 ) had incomplete area of capsulotomy in 12 clock hours ( type iii capsulotomy ) . in cases with no - fluid , approximately 26.1% ( 6/23 ) of eyes needed manual completion of the capsulotomy and release of the microadhesions ( type ii capsulotomy ) . the mean circularity index was 0.9960.003 in the fluid group and 0.9970.003 in the no - fluid group ( p=0.1 ) . the mean diameter of the capsulorhexis was 5.30.3 mm in the fluid group and 5.40.4 mm in the no - fluid group ( p=0.7 , difference : 0.05 [ 95% ci : 0.3 , 0.2 ] ) . the mean circularity index was 0.880.04 in the fluid group and 0.940.04 in the no - fluid group ( p - value < 0.001 , difference : 0.05 [ 95% ci : 0.02 , 0.08 ] ) . inadequate coverage of iol by the capsulorhexis edge was noted in 15.8% ( 3/19 ) cases with fluid and 9.5% ( 2/21 ) cases with no - fluid ( p=0.65 ) . the absence of a red reflex , raised intralenticular pressure , and a fragile anterior capsule complicate the management of white cataracts , and an incomplete capsulorhexis has been reported in 3.85%28.3% cases of white cataracts.912,16 an ideal capsulorhexis is a well - centered circular opening with smooth margins and a slightly smaller diameter than the iol providing 360 iol coverage . inadequate coverage of iol may lead to posterior capsular opacification and change in the effective lens position,17 whereas an excessively small capsulorhexis may cause anterior capsular contraction.18 in our study , reproducible circular capsulotomies were achieved with the help of femtosecond laser , providing adequate iol coverage in 92.5% ( 37/40 ) of cases . the femtosecond laser - assisted capsulotomies may be of four types based on the pattern of completion.15 in our study , type i free - floating capsulotomies were achieved in 52.5% ( 21/40 ) cases . a significantly higher incidence of residual adhesions ( type ii and type iii capsulotomies ) was observed in the subgroup with release of white milky fluid after femtosecond laser delivery ( p<0.003 ) . the reason for microadhesions / incomplete capsulotomies was the explosive egress of white milky fluid on initiation of the capsulotomy , which obscures the visual field and hampers the subsequent delivery of the laser at the same plane . the release of the intralenticular pressure after initiation of the capsulotomy causes a slight change in the position of the anterior capsular plane and hence may result in the occurrence of incomplete capsulotomy . during the lifting of the capsulotomy flap with the microcapsulorhexis forceps , the areas obscured by the white milky fluid during femtosecond laser application should be carefully assessed , as they are likely to have microadhesions . in each of these cases , the free edge of the capsulotomy was grasped with the help of microcapsulorhexis forceps and a circumferential motion tracing the capsulotomy margins was performed , which was akin to the maneuver that one does while performing a manual capsulorhexis ( figure 1 , video s1 ) . despite the presence of microadhesions and incomplete treatment patterns , the continuity and circularity of the capsulotomies was achieved in 97.5% ( 39/40 ) of the eyes . in a case series of 1,300 cases of senile cataract undergoing flacs , 96% capsulotomies were free floating or had small areas of nonperforations.19 in our study , one case of anterior capsular extension with radial tear was noted during flacs . this is in contrast to the manual capsulorhexis where the release of fluid is associated with a subsequent decrease in the intralenticular pressure and more safety in completion of capsulorhexis . a two - stage femtosecond laser capsulotomy has also been described in six cases of intumescent white cataract ; however , remnant tissue bridges were present in one - third of the mini - capsulotomies.20 in cases undergoing conventional phacoemulsification , a multistep capsulorhexis was performed in 70% cases to achieve an adequately sized capsulorhexis . a previous study has demonstrated the safety and efficacy of femtosecond laser - assisted capsulotomy in 25 cases of white cataracts , with radial anterior tears in two eyes , an adherent tongue - like capsule adhesion in nine eyes , and an incomplete capsulotomy in three eyes.13 the total aspiration time and total surgical duration was longer in flacs , but the difference between the two groups was not statistically significant . a hybrid technique of ia using a combination of coaxial and bimanual ia hand pieces was frequently employed to aspirate the subincisional cortical matter in cases undergoing flacs , which may explain the longer aspiration times.14 to conclude , femtosecond laser creates single - step , circular , adequately sized capsulotomies and eliminates the difficulty associated with capsulorhexis in white cataracts . the release of white milky fluid during femtosecond laser delivery is the most important factor affecting the creation of a free - floating capsulotomy , and microadhesions and incomplete capsulotomies may be present in up to 47.5% cases of flacs . to the best of our knowledge , this is the first prospective study comparing conventional phacoemulsification with flacs in white cataracts in terms of intraoperative parameters and postoperative outcomes .

female sexual function is complex and affected by physical , psychological and social factors.1 the prevalence of female sexual dysfunction is high , ranging from 43% to 88%,2,3 and it may significantly affect self - esteem and quality of life . when chronic , it may lead to anxiety and depression , harm relationships , and cause problems in other aspects of life.1,4 moreover , the clinical effects of sexual dysfunction can be augmented by the intensity of the full range of climacteric symptomatology.5 diseases and factors such as aging , arterial hypertension , smoking and pelvic surgery have been associated with female sexual dysfunction.6 personality,7 lifestyle and culture - dependent variables should also be taken into consideration.8 although the epidemiology of male and female sexual function has been investigated in depth , the majority of studies continue to be confined to europe , the united states ( us ) and australasia.9 few studies have been carried out on the sexuality of climacteric women in latin american populations,1012 and data on this subject in women with high school or university educations is particularly sparse . the aim of this study was to collect information on the prevalence of sexual dysfunction and its associated factors in brazilian women of 40 to 65 years of age with eleven or more years of formal education . the target population was the female population of belo horizonte in the state of minas gerais , brazil , aged 4065 years , with at least 11 years of formal education , which consisted of 44,313 women in the year 2000 . the necessary minimum sample size for a similar study was calculated to be 377 women , based on the assumption that 43% of the female population experiences sexual dysfunction , with an expected difference of 5% between the sample and the general population and a type i error ( ) of 0.05.13 in the present study , which was limited to women who answered all five questions used to calculate the sexual dysfunction score , the sample size was recalculated to evaluate any possible loss of precision . a sample of 315 women is expected to result in an absolute difference of 5.5% . this cross - sectional , population - based study was carried out using a self - response questionnaire that was anonymously completed by participants at home between may and september 2005 in the city of belo horizonte , minas gerais , brazil . for the purposes of this study , the municipality of belo horizonte was stratified into nine regions . from these regions , the weighted area consisted of a geographical area that was considered as the primary sampling unit ( psu ) . each wa consisted of various census sectors.14 in each selected wa , five census sectors were randomly chosen ( secondary sampling unit ) . the variables of the sampling plan , strata and psu were included in the data analysis . the city of belo horizonte consists of 62 weighted areas ( wa ) , containing a total of 2,563 census sectors.15 all sectors were included in the randomization process . research assistants initiated the selection of women at each of the five randomly selected corners in each sector , guided by maps of the location . they went to each household and verified whether there were any brazilian - born women of 4065 years of age living in the home and whether they had at least 11 years of formal education . if there were eligible women living at that address , they were invited to participate in the study . if they agreed to participate , a questionnaire was left to be answered and a date was scheduled for the completed questionnaire to be collected . if the eligible women were not at home , they were contacted by telephone and , if they agreed to participate , the questionnaire was delivered to their home . completed questionnaires were collected by a messenger , placed in an unlabeled envelope , and put into a sealed post - box to be delivered to the principal investigator . when questionnaires were delivered to the principal investigator , the weighted area and the education level ( 11 years of schooling ) were noted to homogenize the response . a total of 420 questionnaires were distributed . the reasons given by the women for not participating were : lack of time , that they did not feel comfortable answering the questions , or that their husbands did not want them to participate in the study . thus , 378 questionnaires were filled out and delivered to principal investigator ; of these , 315 ( 83.3% ) contained answers to all questions used to calculate the sexual dysfunction score . the delivered questionnaires that returned incomplete thus , 315 middle - aged women took part in the present study . the questionnaire used in the study consisted of two parts . in the first part , the participants answered questions regarding their sociodemographic characteristics ( age , marital status , ethnic group , income , schooling and paid employment ) , clinical characteristics ( previous surgical history , body mass index , depression , arterial hypertension , diabetes , urinary incontinence , history of cancer , hot flashes , nervousness , insomnia , hormone therapy and self - perception of state of health ) , reproductive characteristics ( menopausal status , number of pregnancies ) and behavioral characteristics ( physical activity , smoking and presence of sexual partner ) . the study protocol was conducted in accordance with the helsinki declaration and approved by the internal review board of the institution . the instrument used to evaluate sexual dysfunction was based on the short personal experiences questionnaire ( speq).16 the original version of this instrument was made available by investigators at the university of melbourne , australia . the questionnaire was independently translated from its original english into brazilian portuguese by two translators fluent in both languages . next , the two versions were compared and a final version was obtained with the approval of both translators . this version was then tested in a group of 50 brazilian - born women of 4065 years of age with 11 years or more of schooling . to ensure cultural equivalence , all questions that generated doubt were once again adapted and tested until the questionnaire was completely comprehensible to all women in the pilot group . a final version of the questionnaire in brazilian portuguese was thus obtained . the variable sexual dysfunction was calculated from the mean of the sum of the scores of 1 ) sexual responsiveness , which evaluated pleasure in sexual activities ( graded from 1 to 6 , where 1 reflected an absence of pleasure and 6 maximum pleasure ) , excitation ( 16 ) and orgasm ( 16 ) ; 2 ) frequency of sexual activities ( 1=never , 2=less than once a week ; 3=once or twice a week , 4=several times a week , and 5=once a day or more ) ; and 3 ) libido ( 1=it never took place , 2=it took place less than once a week , 3=it took place once or twice a week , 4=it took place several times a week , 5= it took place once a day or more ) . a score 7 was considered indicative of sexual dysfunction and a score > 7 was considered indicative of no sexual dysfunction.16 age was dichotomized into < 50 years or 50 years of age . menopausal status was defined as premenopausal when the women had regular menstrual cycles or a menstrual cycle similar to the pattern that had been predominant throughout their reproductive life . women were considered to be in perimenopause if they had menstrual cycles during the previous 12 months , but with some alterations in their menstrual pattern . women were considered postmenopausal if their last menstrual period was at least 12 months prior to the interview.17 in women who been to hysterectomy , menopausal status was classified as follows : women aged 4044 years who had regular menstruation prior to hysterectomy were considered to be premenopausal ; women aged 4548 years who had irregular menstruation prior to hysterectomy were considered to be in perimenopause ; and women of more than 48 years of age or as well as those who had undergone hysterectomy with bilateral oophorectomy were considered to be postmenopausal , based on a previous report that the mean age at menopause in latin america is 48.6 years.18 marital status was dichotomized into married / living with a partner , or other ; ethnic group into white or non - white ; schooling into 11 years or more than 11 years of formal education ; family income into us $ 1300 or > us $ 1300 per month ; physical activity into none/ < 3 times a week or 3 times a week ; and number of pregnancies into > 2 or 2 . body mass index ( bmi ) was dichotomized into < 25 or 25 kg / m . paid employment was dichotomized into none/ 20 hours per week or > 20 hours per week . smoking was dichotomized into never smoked or current / past smoker . the presence or absence ( yes or no ) of the following variables was also investigated : depression , arterial hypertension , diabetes , urinary incontinence , history of cancer , hot flashes , nervousness , insomnia and the presence of a sexual partner in the previous month . self - perception of the state of health was classified as terrible / poor / average or good / excellent . a bivariate analysis was performed to evaluate sexual dysfunction as a function of the independent variables . the chi - square test was applied followed by yates correction or fisher s exact test.19 finally , poisson multiple regression analysis was applied to the model to calculate the prevalence ratio ( pr ) and the respective 95% confidence intervals ( 95%ci).20 the backward criterion strategy was used to select the variables.21 for this analysis , the strata and the cluster / smallest geographical unit of the sampling plan were used . stata software version 7.0 ( stata corporation , college station , texas , usa ) was used for the analysis . the criterion used for the inclusion of independent variables in multiple regression analysis consisted of a p - value of < 0.25 in bivariate analysis or in a simple logistic regression . the target population was the female population of belo horizonte in the state of minas gerais , brazil , aged 4065 years , with at least 11 years of formal education , which consisted of 44,313 women in the year 2000 . the necessary minimum sample size for a similar study was calculated to be 377 women , based on the assumption that 43% of the female population experiences sexual dysfunction , with an expected difference of 5% between the sample and the general population and a type i error ( ) of 0.05.13 in the present study , which was limited to women who answered all five questions used to calculate the sexual dysfunction score , the sample size was recalculated to evaluate any possible loss of precision . a sample of 315 women is expected to result in an absolute difference of 5.5% . this cross - sectional , population - based study was carried out using a self - response questionnaire that was anonymously completed by participants at home between may and september 2005 in the city of belo horizonte , minas gerais , brazil . for the purposes of this study , the municipality of belo horizonte was stratified into nine regions . from these regions , the weighted area consisted of a geographical area that was considered as the primary sampling unit ( psu ) . each wa consisted of various census sectors.14 in each selected wa , five census sectors were randomly chosen ( secondary sampling unit ) . the variables of the sampling plan , strata and psu were included in the data analysis . the city of belo horizonte consists of 62 weighted areas ( wa ) , containing a total of 2,563 census sectors.15 all sectors were included in the randomization process . research assistants initiated the selection of women at each of the five randomly selected corners in each sector , guided by maps of the location . they went to each household and verified whether there were any brazilian - born women of 4065 years of age living in the home and whether they had at least 11 years of formal education . if there were eligible women living at that address , they were invited to participate in the study . if they agreed to participate , a questionnaire was left to be answered and a date was scheduled for the completed questionnaire to be collected . if the eligible women were not at home , they were contacted by telephone and , if they agreed to participate , the questionnaire was delivered to their home . completed questionnaires were collected by a messenger , placed in an unlabeled envelope , and put into a sealed post - box to be delivered to the principal investigator . when questionnaires were delivered to the principal investigator , the weighted area and the education level ( 11 years of schooling ) the reasons given by the women for not participating were : lack of time , that they did not feel comfortable answering the questions , or that their husbands did not want them to participate in the study . thus , 378 questionnaires were filled out and delivered to principal investigator ; of these , 315 ( 83.3% ) contained answers to all questions used to calculate the sexual dysfunction score . the questionnaire used in the study consisted of two parts . in the first part , the participants answered questions regarding their sociodemographic characteristics ( age , marital status , ethnic group , income , schooling and paid employment ) , clinical characteristics ( previous surgical history , body mass index , depression , arterial hypertension , diabetes , urinary incontinence , history of cancer , hot flashes , nervousness , insomnia , hormone therapy and self - perception of state of health ) , reproductive characteristics ( menopausal status , number of pregnancies ) and behavioral characteristics ( physical activity , smoking and presence of sexual partner ) . the study protocol was conducted in accordance with the helsinki declaration and approved by the internal review board of the institution . the instrument used to evaluate sexual dysfunction was based on the short personal experiences questionnaire ( speq).16 the original version of this instrument was made available by investigators at the university of melbourne , australia . the questionnaire was independently translated from its original english into brazilian portuguese by two translators fluent in both languages . next , the two versions were compared and a final version was obtained with the approval of both translators . this version was then tested in a group of 50 brazilian - born women of 4065 years of age with 11 years or more of schooling . to ensure cultural equivalence , all questions that generated doubt were once again adapted and tested until the questionnaire was completely comprehensible to all women in the pilot group . the variable sexual dysfunction was calculated from the mean of the sum of the scores of 1 ) sexual responsiveness , which evaluated pleasure in sexual activities ( graded from 1 to 6 , where 1 reflected an absence of pleasure and 6 maximum pleasure ) , excitation ( 16 ) and orgasm ( 16 ) ; 2 ) frequency of sexual activities ( 1=never , 2=less than once a week ; 3=once or twice a week , 4=several times a week , and 5=once a day or more ) ; and 3 ) libido ( 1=it never took place , 2=it took place less than once a week , 3=it took place once or twice a week , 4=it took place several times a week , 5= it took place once a day or more ) . a score 7 was considered indicative of sexual dysfunction and a score > 7 was considered indicative of no sexual dysfunction.16 menopausal status was defined as premenopausal when the women had regular menstrual cycles or a menstrual cycle similar to the pattern that had been predominant throughout their reproductive life . women were considered to be in perimenopause if they had menstrual cycles during the previous 12 months , but with some alterations in their menstrual pattern . women were considered postmenopausal if their last menstrual period was at least 12 months prior to the interview.17 in women who been to hysterectomy , menopausal status was classified as follows : women aged 4044 years who had regular menstruation prior to hysterectomy were considered to be premenopausal ; women aged 4548 years who had irregular menstruation prior to hysterectomy were considered to be in perimenopause ; and women of more than 48 years of age or as well as those who had undergone hysterectomy with bilateral oophorectomy were considered to be postmenopausal , based on a previous report that the mean age at menopause in latin america is 48.6 years.18 marital status was dichotomized into married / living with a partner , or other ; ethnic group into white or non - white ; schooling into 11 years or more than 11 years of formal education ; family income into us $ 1300 or > us $ 1300 per month ; physical activity into none/ < 3 times a week or 3 times a week ; and number of pregnancies into > 2 or 2 . body mass index ( bmi ) was dichotomized into < 25 or 25 kg / m . paid employment was dichotomized into none/ 20 hours per week or > 20 hours per week . smoking was dichotomized into never smoked or current / past smoker . the presence or absence ( yes or no ) of the following variables was also investigated : depression , arterial hypertension , diabetes , urinary incontinence , history of cancer , hot flashes , nervousness , insomnia and the presence of a sexual partner in the previous month . self - perception of the state of health was classified as terrible / poor / average or good / excellent . a bivariate analysis was performed to evaluate sexual dysfunction as a function of the independent variables . the chi - square test was applied followed by yates correction or fisher s exact test.19 finally , poisson multiple regression analysis was applied to the model to calculate the prevalence ratio ( pr ) and the respective 95% confidence intervals ( 95%ci).20 the backward criterion strategy was used to select the variables.21 for this analysis , the strata and the cluster / smallest geographical unit of the sampling plan were used . stata software version 7.0 ( stata corporation , college station , texas , usa ) was used for the analysis . the criterion used for the inclusion of independent variables in multiple regression analysis consisted of a p - value of < 0.25 in bivariate analysis or in a simple logistic regression . in this sample , 46.7% of participants were 50 years of age or more ; 65.9% were married or living with a partner ; 70.8% reported having a sexual partner ; 50.8% stated that they had more than 11 years of schooling ; 59.5% had a family income us$1300 ; 51% had more than two children ; and 33.5% reported practicing physical activity regularly , three or more times a week ( data not presented as a table ) . the prevalence of sexual dysfunction among premenopausal women was 24.2% , perimenopausal women 37.3% and postmenopausal women 45.3% . the 315 women who answered the five questions comprising the sexual dysfunction variable were compared to the 63 who failed to answer these questions . women who were unmarried or not living with a partner ( p<0.001 ) and those who reported hypertension ( p=0.002 ) or urinary incontinence ( p=0.036 ) were more likely to answer all questions contained in the section on sexual dysfunction . in the bivariate analysis , women with greater probability of having sexual dysfunction ( score 7 ) had the following characteristics : they were in peri- or postmenopause ( p=0.007 ) , had a sedentary lifestyle ( p=0.019 ) , reported depression ( p=0.003 ) , arterial hypertension ( p<0.002 ) , diabetes ( p=0.046 ) , hot flashes ( p=0.009 ) , nervousness ( p=0.048 ) or insomnia ( p=0.003 ) , and had a poor perception of their own health ( p<0.002 ) , ( table 1 ) . multiple regression analysis showed that more advanced age ( pr = 1.04 ; 95%ci : 1.011.07 ) and the presence of hot flashes ( pr = 1.37 ; 95%ci : 1.041.80 ) were significantly associated with sexual dysfunction . conversely , women who felt well ( pr = 0.68 ; 95%ci : 0.520.88 ) and women with sexual partners ( pr = 0.47 ; 95%ci : 0.340.65 ) were less likely to have a score of 7 for sexual dysfunction ( table 2 ) . the objective of this study was to evaluate the prevalence of sexual dysfunction and to identify its associated factors in middle - aged , brazilian - born women . the profile of the population of the present study should be emphasized in view of the scarcity of population - based studies on female sexuality , particularly among middle - aged women . in the present study , overall sexual dysfunction was evaluated in women with or without a sexual partner . in a study carried out in a us population,22 kinsey ( 1948 ) showed that the search for sexual pleasure involves the mind and entire body of individuals , not only their genitals . given women s greater longevity , it is very probable that many will grow old alone ; however this does not imply the end of their sexuality or loss of their need for intimacy , touch and affection.23 the use of an internationally recognized questionnaire , the speq,16 which was adapted for use in brazil , is another factor that deserves particular mention . as direct interviews on sexual activity during menopause may lead to constraints in responses,24 it was important for the questionnaire to be self - response and anonymous . various measures were taken to ensure that the women selected would feel at ease to respond honestly to the questions . for instance , research assistants were selected who had a similar profile to that of the women in the study sample in order to encourage empathy , and reassurance was given to participants that all answers would remain confidential . in the present study , the prevalence of sexual dysfunction was 35.9% , increasing from the pre- to the postmenopause . these data are in agreement with findings from other studies carried out in different populations . in a prospective observational study , dennerstein et al 3 , 25 also found that from the beginning to the latter phase of the menopausal transition , the proportion of women with sexual dysfunction increased from 42% to 88% . in a study carried out in chile , castelo - branco et abdo et al.27 found at least one type of sexual dysfunction in 57.4% of women 41 years of age or older . nevertheless , in our sample , multiple analysis showed no association between menopausal status and sexual dysfunction . one possible explanation for this finding may be that in women with a higher educational level , the percentage affected by sexual dysfunction is lower.27 sexual dysfunction was not significantly associated with age , which was dichotomized into < 50 years and > 50 years for the bivariate analysis ; however , in the multiple regression analysis , when the variable was considered in a continuous manner , the correlation between sexual dysfunction and increasing age was significant . in a previous study13 carried out in this same sample population , albeit with a more general evaluation of sexuality , a decline in sexuality as a function of increased chronological age was also found . a longitudinal cohort study carried out in australia reported a significant negative effect of aging on the frequency of sexual activity , as well as on aspects of responsiveness ( sexual excitation , pleasure and orgasm).3,25 the women s international study of health and sexuality , a multinational , cross - sectional study carried out in europe and in the united states , reported a decline in all aspects of sexual function with aging.28 in a longitudinal study , ford et al 29 also reported that an increase in age was associated with poorer sexuality . castelo - branco et al26 evaluated 534 healthy , middle - aged chilean women and found that the prevalence of sexual dysfunction increased from 22% in the 4044-year old age - group to 66% in the 6064 year olds . hot flushes , depression , nervousness and insomnia were associated with scores 7 for sexual dysfunction in the present analysis . these results are in agreement with those of previous studies in which depression was associated with difficulties in vaginal lubrication in women in some regions of the world , whereas stress was associated with inability to achieve orgasm.30 other studies have also reported that psychological symptoms , stress and emotional problems may be related to a decline in sexual activity.31 it was also found that psychological symptoms were common in climacteric women and were associated with hot flashes . this observation connects psychological symptoms with the menopausal transition and suggests that their cause may be biological.32 hot flashes are known to constitute a triggering factor for insomnia , which , in turn , leads to a reduction in energy and to depression , which has a negative effect on sexual function.33 the women in the present sample who reported leading a sedentary lifestyle or who had a poor perception of their own health were more likely to be affected by sexual dysfunction . greendale et al34 reported that an increase in physical activity and satisfaction with life were related to improved sexual function . mental health,30,35 emotional well - being and positive self - image were associated with improved sexual function.30,35,36 on the other hand , additional studies have found correlations between chronic diseases and an increase in sexual dysfunction28,37 due to circulatory problems,38 neurological function , hormone balance or systemic health.39 it has been found that both an increase in systolic arterial pressure and the administration of beta blockers to treat hypertension may be detrimental to sexual function.40 the present analysis shows that arterial hypertension was associated with the occurrence of sexual dysfunction , although the use of medication for hypertension was not evaluated in this study . this finding is in agreement with other studies that reported that women with diabetes had a higher prevalence of sexual dysfunction compared to non - diabetic women.41,42 in the present study , women who had a sexual partner were less likely to have sexual dysfunction . this is consistent with other studies on middle - aged women conducted in different geographical locations , emphasizing the relevance of the sexual partner as a protective factor against sexual dysfunction.13,36,43 nevertheless , previous studies have also described the importance of feelings for the partner with respect to sexuality.44 these data suggest the need to carry out further studies to investigate , in addition to the simple presence of a partner , the quality of relationship of these couples . the questionnaires that were returned incomplete were not taken into consideration in the evaluation of sexual dysfunction . the group composed of unmarried women or those not living with a partner and who reported hypertension or urinary incontinence , was associated with answering the entire block of questions concerning sexual dysfunction . women with these characteristics who may have sexual dysfunction may be more motivated to answer all the questions . the data , which include information on hypertension , psychological symptoms and the use of hormone therapy , were obtained by participant self - report . although recall bias can not be ruled out , previous studies using sef - reports suggest a high validity of health information , indicating that women with higher education levels provide more reliable data.45 sexual dysfunction was found in more than one - third of women that were 4065 years of age with 11 years or more of formal education . informing women , giving them the tools necessary to change their own lifestyle , and treating climacteric symptoms and existing comorbidities may lead to improved sexuality and , consequently , to a better quality of life . future studies are required to evaluate the effects of hormones and other drugs on the sexuality of climacteric women .

objectiveto evaluate the prevalence of sexual dysfunction and its associated factors in middle - aged women with 11 years or more of formal education.methodsa cross - sectional , population - based study was carried out using an anonymous , self - response questionnaire . a total of 315 brazilian - born women , 4065 years of age with 11 years or more of schooling , participated in the study . the instrument used in the evaluation was based on the short personal experiences questionnaire . sexual dysfunction was calculated from the mean score of sexual responsiveness ( pleasure in sexual activities , excitation and orgasm ) , frequency of sexual activities and libido . sociodemographic and clinical factors were evaluated . poisson multiple regression analysis was carried out and the prevalence ratios with respective 95% confidence intervals ( 95%ci ) were calculated.resultsthe prevalence of sexual dysfunction was 35.9% among our study population . multiple regression analysis showed that sexual dysfunction was positively associated with older age ( prevalence ratios=1.04 ; 95%ci:1.011.07 ) and with the presence of hot flashes ( prevalence ratios=1.37 ; 95%ci:1.041.80 ) . having a sexual partner ( pr=0.47 ; 95%ci:0.340.65 ) and feeling well or excellent ( prevalence ratios= 0.68 ; 95%ci : 0.520.88 ) were factors associated with lower sexual dysfunction scores.conclusionssexual dysfunction was present in more than one - third of women that were 4065 years of age with 11 years or more of formal education . within that age group , older age and hot flashes were associated with higher sexual dysfunction scores , whereas feeling well and having a sexual partner were associated with better sexuality .

INTRODUCTION MATERIALS AND METHODS Sample size Subjects Evaluation of sexual dysfunction Dependent variable Independent variables Statistical analysis RESULTS DISCUSSION CONCLUSIONS

female sexual function is complex and affected by physical , psychological and social factors.1 the prevalence of female sexual dysfunction is high , ranging from 43% to 88%,2,3 and it may significantly affect self - esteem and quality of life . when chronic , it may lead to anxiety and depression , harm relationships , and cause problems in other aspects of life.1,4 moreover , the clinical effects of sexual dysfunction can be augmented by the intensity of the full range of climacteric symptomatology.5 diseases and factors such as aging , arterial hypertension , smoking and pelvic surgery have been associated with female sexual dysfunction.6 personality,7 lifestyle and culture - dependent variables should also be taken into consideration.8 although the epidemiology of male and female sexual function has been investigated in depth , the majority of studies continue to be confined to europe , the united states ( us ) and australasia.9 few studies have been carried out on the sexuality of climacteric women in latin american populations,1012 and data on this subject in women with high school or university educations is particularly sparse . the aim of this study was to collect information on the prevalence of sexual dysfunction and its associated factors in brazilian women of 40 to 65 years of age with eleven or more years of formal education . the target population was the female population of belo horizonte in the state of minas gerais , brazil , aged 4065 years , with at least 11 years of formal education , which consisted of 44,313 women in the year 2000 . the necessary minimum sample size for a similar study was calculated to be 377 women , based on the assumption that 43% of the female population experiences sexual dysfunction , with an expected difference of 5% between the sample and the general population and a type i error ( ) of 0.05.13 in the present study , which was limited to women who answered all five questions used to calculate the sexual dysfunction score , the sample size was recalculated to evaluate any possible loss of precision . this cross - sectional , population - based study was carried out using a self - response questionnaire that was anonymously completed by participants at home between may and september 2005 in the city of belo horizonte , minas gerais , brazil . the city of belo horizonte consists of 62 weighted areas ( wa ) , containing a total of 2,563 census sectors.15 all sectors were included in the randomization process . they went to each household and verified whether there were any brazilian - born women of 4065 years of age living in the home and whether they had at least 11 years of formal education . when questionnaires were delivered to the principal investigator , the weighted area and the education level ( 11 years of schooling ) were noted to homogenize the response . the reasons given by the women for not participating were : lack of time , that they did not feel comfortable answering the questions , or that their husbands did not want them to participate in the study . the delivered questionnaires that returned incomplete thus , 315 middle - aged women took part in the present study . the questionnaire used in the study consisted of two parts . in the first part , the participants answered questions regarding their sociodemographic characteristics ( age , marital status , ethnic group , income , schooling and paid employment ) , clinical characteristics ( previous surgical history , body mass index , depression , arterial hypertension , diabetes , urinary incontinence , history of cancer , hot flashes , nervousness , insomnia , hormone therapy and self - perception of state of health ) , reproductive characteristics ( menopausal status , number of pregnancies ) and behavioral characteristics ( physical activity , smoking and presence of sexual partner ) . the study protocol was conducted in accordance with the helsinki declaration and approved by the internal review board of the institution . the instrument used to evaluate sexual dysfunction was based on the short personal experiences questionnaire ( speq).16 the original version of this instrument was made available by investigators at the university of melbourne , australia . this version was then tested in a group of 50 brazilian - born women of 4065 years of age with 11 years or more of schooling . the variable sexual dysfunction was calculated from the mean of the sum of the scores of 1 ) sexual responsiveness , which evaluated pleasure in sexual activities ( graded from 1 to 6 , where 1 reflected an absence of pleasure and 6 maximum pleasure ) , excitation ( 16 ) and orgasm ( 16 ) ; 2 ) frequency of sexual activities ( 1=never , 2=less than once a week ; 3=once or twice a week , 4=several times a week , and 5=once a day or more ) ; and 3 ) libido ( 1=it never took place , 2=it took place less than once a week , 3=it took place once or twice a week , 4=it took place several times a week , 5= it took place once a day or more ) . a score 7 was considered indicative of sexual dysfunction and a score > 7 was considered indicative of no sexual dysfunction.16 age was dichotomized into < 50 years or 50 years of age . women were considered postmenopausal if their last menstrual period was at least 12 months prior to the interview.17 in women who been to hysterectomy , menopausal status was classified as follows : women aged 4044 years who had regular menstruation prior to hysterectomy were considered to be premenopausal ; women aged 4548 years who had irregular menstruation prior to hysterectomy were considered to be in perimenopause ; and women of more than 48 years of age or as well as those who had undergone hysterectomy with bilateral oophorectomy were considered to be postmenopausal , based on a previous report that the mean age at menopause in latin america is 48.6 years.18 marital status was dichotomized into married / living with a partner , or other ; ethnic group into white or non - white ; schooling into 11 years or more than 11 years of formal education ; family income into us $ 1300 or > us $ 1300 per month ; physical activity into none/ < 3 times a week or 3 times a week ; and number of pregnancies into > 2 or 2 . the presence or absence ( yes or no ) of the following variables was also investigated : depression , arterial hypertension , diabetes , urinary incontinence , history of cancer , hot flashes , nervousness , insomnia and the presence of a sexual partner in the previous month . a bivariate analysis was performed to evaluate sexual dysfunction as a function of the independent variables . the chi - square test was applied followed by yates correction or fisher s exact test.19 finally , poisson multiple regression analysis was applied to the model to calculate the prevalence ratio ( pr ) and the respective 95% confidence intervals ( 95%ci).20 the backward criterion strategy was used to select the variables.21 for this analysis , the strata and the cluster / smallest geographical unit of the sampling plan were used . the criterion used for the inclusion of independent variables in multiple regression analysis consisted of a p - value of < 0.25 in bivariate analysis or in a simple logistic regression . the target population was the female population of belo horizonte in the state of minas gerais , brazil , aged 4065 years , with at least 11 years of formal education , which consisted of 44,313 women in the year 2000 . the necessary minimum sample size for a similar study was calculated to be 377 women , based on the assumption that 43% of the female population experiences sexual dysfunction , with an expected difference of 5% between the sample and the general population and a type i error ( ) of 0.05.13 in the present study , which was limited to women who answered all five questions used to calculate the sexual dysfunction score , the sample size was recalculated to evaluate any possible loss of precision . this cross - sectional , population - based study was carried out using a self - response questionnaire that was anonymously completed by participants at home between may and september 2005 in the city of belo horizonte , minas gerais , brazil . the city of belo horizonte consists of 62 weighted areas ( wa ) , containing a total of 2,563 census sectors.15 all sectors were included in the randomization process . they went to each household and verified whether there were any brazilian - born women of 4065 years of age living in the home and whether they had at least 11 years of formal education . when questionnaires were delivered to the principal investigator , the weighted area and the education level ( 11 years of schooling ) the reasons given by the women for not participating were : lack of time , that they did not feel comfortable answering the questions , or that their husbands did not want them to participate in the study . the questionnaire used in the study consisted of two parts . in the first part , the participants answered questions regarding their sociodemographic characteristics ( age , marital status , ethnic group , income , schooling and paid employment ) , clinical characteristics ( previous surgical history , body mass index , depression , arterial hypertension , diabetes , urinary incontinence , history of cancer , hot flashes , nervousness , insomnia , hormone therapy and self - perception of state of health ) , reproductive characteristics ( menopausal status , number of pregnancies ) and behavioral characteristics ( physical activity , smoking and presence of sexual partner ) . the instrument used to evaluate sexual dysfunction was based on the short personal experiences questionnaire ( speq).16 the original version of this instrument was made available by investigators at the university of melbourne , australia . this version was then tested in a group of 50 brazilian - born women of 4065 years of age with 11 years or more of schooling . the variable sexual dysfunction was calculated from the mean of the sum of the scores of 1 ) sexual responsiveness , which evaluated pleasure in sexual activities ( graded from 1 to 6 , where 1 reflected an absence of pleasure and 6 maximum pleasure ) , excitation ( 16 ) and orgasm ( 16 ) ; 2 ) frequency of sexual activities ( 1=never , 2=less than once a week ; 3=once or twice a week , 4=several times a week , and 5=once a day or more ) ; and 3 ) libido ( 1=it never took place , 2=it took place less than once a week , 3=it took place once or twice a week , 4=it took place several times a week , 5= it took place once a day or more ) . a score 7 was considered indicative of sexual dysfunction and a score > 7 was considered indicative of no sexual dysfunction.16 menopausal status was defined as premenopausal when the women had regular menstrual cycles or a menstrual cycle similar to the pattern that had been predominant throughout their reproductive life . women were considered postmenopausal if their last menstrual period was at least 12 months prior to the interview.17 in women who been to hysterectomy , menopausal status was classified as follows : women aged 4044 years who had regular menstruation prior to hysterectomy were considered to be premenopausal ; women aged 4548 years who had irregular menstruation prior to hysterectomy were considered to be in perimenopause ; and women of more than 48 years of age or as well as those who had undergone hysterectomy with bilateral oophorectomy were considered to be postmenopausal , based on a previous report that the mean age at menopause in latin america is 48.6 years.18 marital status was dichotomized into married / living with a partner , or other ; ethnic group into white or non - white ; schooling into 11 years or more than 11 years of formal education ; family income into us $ 1300 or > us $ 1300 per month ; physical activity into none/ < 3 times a week or 3 times a week ; and number of pregnancies into > 2 or 2 . the presence or absence ( yes or no ) of the following variables was also investigated : depression , arterial hypertension , diabetes , urinary incontinence , history of cancer , hot flashes , nervousness , insomnia and the presence of a sexual partner in the previous month . the chi - square test was applied followed by yates correction or fisher s exact test.19 finally , poisson multiple regression analysis was applied to the model to calculate the prevalence ratio ( pr ) and the respective 95% confidence intervals ( 95%ci).20 the backward criterion strategy was used to select the variables.21 for this analysis , the strata and the cluster / smallest geographical unit of the sampling plan were used . in this sample , 46.7% of participants were 50 years of age or more ; 65.9% were married or living with a partner ; 70.8% reported having a sexual partner ; 50.8% stated that they had more than 11 years of schooling ; 59.5% had a family income us$1300 ; 51% had more than two children ; and 33.5% reported practicing physical activity regularly , three or more times a week ( data not presented as a table ) . the prevalence of sexual dysfunction among premenopausal women was 24.2% , perimenopausal women 37.3% and postmenopausal women 45.3% . women who were unmarried or not living with a partner ( p<0.001 ) and those who reported hypertension ( p=0.002 ) or urinary incontinence ( p=0.036 ) were more likely to answer all questions contained in the section on sexual dysfunction . in the bivariate analysis , women with greater probability of having sexual dysfunction ( score 7 ) had the following characteristics : they were in peri- or postmenopause ( p=0.007 ) , had a sedentary lifestyle ( p=0.019 ) , reported depression ( p=0.003 ) , arterial hypertension ( p<0.002 ) , diabetes ( p=0.046 ) , hot flashes ( p=0.009 ) , nervousness ( p=0.048 ) or insomnia ( p=0.003 ) , and had a poor perception of their own health ( p<0.002 ) , ( table 1 ) . multiple regression analysis showed that more advanced age ( pr = 1.04 ; 95%ci : 1.011.07 ) and the presence of hot flashes ( pr = 1.37 ; 95%ci : 1.041.80 ) were significantly associated with sexual dysfunction . conversely , women who felt well ( pr = 0.68 ; 95%ci : 0.520.88 ) and women with sexual partners ( pr = 0.47 ; 95%ci : 0.340.65 ) were less likely to have a score of 7 for sexual dysfunction ( table 2 ) . the objective of this study was to evaluate the prevalence of sexual dysfunction and to identify its associated factors in middle - aged , brazilian - born women . the profile of the population of the present study should be emphasized in view of the scarcity of population - based studies on female sexuality , particularly among middle - aged women . in the present study , overall sexual dysfunction was evaluated in women with or without a sexual partner . in a study carried out in a us population,22 kinsey ( 1948 ) showed that the search for sexual pleasure involves the mind and entire body of individuals , not only their genitals . for instance , research assistants were selected who had a similar profile to that of the women in the study sample in order to encourage empathy , and reassurance was given to participants that all answers would remain confidential . in the present study , the prevalence of sexual dysfunction was 35.9% , increasing from the pre- to the postmenopause . in a prospective observational study , dennerstein et al 3 , 25 also found that from the beginning to the latter phase of the menopausal transition , the proportion of women with sexual dysfunction increased from 42% to 88% . in a study carried out in chile , castelo - branco et abdo et al.27 found at least one type of sexual dysfunction in 57.4% of women 41 years of age or older . one possible explanation for this finding may be that in women with a higher educational level , the percentage affected by sexual dysfunction is lower.27 sexual dysfunction was not significantly associated with age , which was dichotomized into < 50 years and > 50 years for the bivariate analysis ; however , in the multiple regression analysis , when the variable was considered in a continuous manner , the correlation between sexual dysfunction and increasing age was significant . a longitudinal cohort study carried out in australia reported a significant negative effect of aging on the frequency of sexual activity , as well as on aspects of responsiveness ( sexual excitation , pleasure and orgasm).3,25 the women s international study of health and sexuality , a multinational , cross - sectional study carried out in europe and in the united states , reported a decline in all aspects of sexual function with aging.28 in a longitudinal study , ford et al 29 also reported that an increase in age was associated with poorer sexuality . castelo - branco et al26 evaluated 534 healthy , middle - aged chilean women and found that the prevalence of sexual dysfunction increased from 22% in the 4044-year old age - group to 66% in the 6064 year olds . hot flushes , depression , nervousness and insomnia were associated with scores 7 for sexual dysfunction in the present analysis . these results are in agreement with those of previous studies in which depression was associated with difficulties in vaginal lubrication in women in some regions of the world , whereas stress was associated with inability to achieve orgasm.30 other studies have also reported that psychological symptoms , stress and emotional problems may be related to a decline in sexual activity.31 it was also found that psychological symptoms were common in climacteric women and were associated with hot flashes . this observation connects psychological symptoms with the menopausal transition and suggests that their cause may be biological.32 hot flashes are known to constitute a triggering factor for insomnia , which , in turn , leads to a reduction in energy and to depression , which has a negative effect on sexual function.33 the women in the present sample who reported leading a sedentary lifestyle or who had a poor perception of their own health were more likely to be affected by sexual dysfunction . mental health,30,35 emotional well - being and positive self - image were associated with improved sexual function.30,35,36 on the other hand , additional studies have found correlations between chronic diseases and an increase in sexual dysfunction28,37 due to circulatory problems,38 neurological function , hormone balance or systemic health.39 it has been found that both an increase in systolic arterial pressure and the administration of beta blockers to treat hypertension may be detrimental to sexual function.40 the present analysis shows that arterial hypertension was associated with the occurrence of sexual dysfunction , although the use of medication for hypertension was not evaluated in this study . this finding is in agreement with other studies that reported that women with diabetes had a higher prevalence of sexual dysfunction compared to non - diabetic women.41,42 in the present study , women who had a sexual partner were less likely to have sexual dysfunction . this is consistent with other studies on middle - aged women conducted in different geographical locations , emphasizing the relevance of the sexual partner as a protective factor against sexual dysfunction.13,36,43 nevertheless , previous studies have also described the importance of feelings for the partner with respect to sexuality.44 these data suggest the need to carry out further studies to investigate , in addition to the simple presence of a partner , the quality of relationship of these couples . the questionnaires that were returned incomplete were not taken into consideration in the evaluation of sexual dysfunction . although recall bias can not be ruled out , previous studies using sef - reports suggest a high validity of health information , indicating that women with higher education levels provide more reliable data.45 sexual dysfunction was found in more than one - third of women that were 4065 years of age with 11 years or more of formal education .

female sexual function is complex and affected by physical , psychological and social factors.1 the prevalence of female sexual dysfunction is high , ranging from 43% to 88%,2,3 and it may significantly affect self - esteem and quality of life . when chronic , it may lead to anxiety and depression , harm relationships , and cause problems in other aspects of life.1,4 moreover , the clinical effects of sexual dysfunction can be augmented by the intensity of the full range of climacteric symptomatology.5 diseases and factors such as aging , arterial hypertension , smoking and pelvic surgery have been associated with female sexual dysfunction.6 personality,7 lifestyle and culture - dependent variables should also be taken into consideration.8 although the epidemiology of male and female sexual function has been investigated in depth , the majority of studies continue to be confined to europe , the united states ( us ) and australasia.9 few studies have been carried out on the sexuality of climacteric women in latin american populations,1012 and data on this subject in women with high school or university educations is particularly sparse . the aim of this study was to collect information on the prevalence of sexual dysfunction and its associated factors in brazilian women of 40 to 65 years of age with eleven or more years of formal education . the target population was the female population of belo horizonte in the state of minas gerais , brazil , aged 4065 years , with at least 11 years of formal education , which consisted of 44,313 women in the year 2000 . the necessary minimum sample size for a similar study was calculated to be 377 women , based on the assumption that 43% of the female population experiences sexual dysfunction , with an expected difference of 5% between the sample and the general population and a type i error ( ) of 0.05.13 in the present study , which was limited to women who answered all five questions used to calculate the sexual dysfunction score , the sample size was recalculated to evaluate any possible loss of precision . this cross - sectional , population - based study was carried out using a self - response questionnaire that was anonymously completed by participants at home between may and september 2005 in the city of belo horizonte , minas gerais , brazil . for the purposes of this study , the municipality of belo horizonte was stratified into nine regions . research assistants initiated the selection of women at each of the five randomly selected corners in each sector , guided by maps of the location . they went to each household and verified whether there were any brazilian - born women of 4065 years of age living in the home and whether they had at least 11 years of formal education . when questionnaires were delivered to the principal investigator , the weighted area and the education level ( 11 years of schooling ) were noted to homogenize the response . the delivered questionnaires that returned incomplete thus , 315 middle - aged women took part in the present study . in the first part , the participants answered questions regarding their sociodemographic characteristics ( age , marital status , ethnic group , income , schooling and paid employment ) , clinical characteristics ( previous surgical history , body mass index , depression , arterial hypertension , diabetes , urinary incontinence , history of cancer , hot flashes , nervousness , insomnia , hormone therapy and self - perception of state of health ) , reproductive characteristics ( menopausal status , number of pregnancies ) and behavioral characteristics ( physical activity , smoking and presence of sexual partner ) . the instrument used to evaluate sexual dysfunction was based on the short personal experiences questionnaire ( speq).16 the original version of this instrument was made available by investigators at the university of melbourne , australia . next , the two versions were compared and a final version was obtained with the approval of both translators . this version was then tested in a group of 50 brazilian - born women of 4065 years of age with 11 years or more of schooling . the variable sexual dysfunction was calculated from the mean of the sum of the scores of 1 ) sexual responsiveness , which evaluated pleasure in sexual activities ( graded from 1 to 6 , where 1 reflected an absence of pleasure and 6 maximum pleasure ) , excitation ( 16 ) and orgasm ( 16 ) ; 2 ) frequency of sexual activities ( 1=never , 2=less than once a week ; 3=once or twice a week , 4=several times a week , and 5=once a day or more ) ; and 3 ) libido ( 1=it never took place , 2=it took place less than once a week , 3=it took place once or twice a week , 4=it took place several times a week , 5= it took place once a day or more ) . women were considered postmenopausal if their last menstrual period was at least 12 months prior to the interview.17 in women who been to hysterectomy , menopausal status was classified as follows : women aged 4044 years who had regular menstruation prior to hysterectomy were considered to be premenopausal ; women aged 4548 years who had irregular menstruation prior to hysterectomy were considered to be in perimenopause ; and women of more than 48 years of age or as well as those who had undergone hysterectomy with bilateral oophorectomy were considered to be postmenopausal , based on a previous report that the mean age at menopause in latin america is 48.6 years.18 marital status was dichotomized into married / living with a partner , or other ; ethnic group into white or non - white ; schooling into 11 years or more than 11 years of formal education ; family income into us $ 1300 or > us $ 1300 per month ; physical activity into none/ < 3 times a week or 3 times a week ; and number of pregnancies into > 2 or 2 . the presence or absence ( yes or no ) of the following variables was also investigated : depression , arterial hypertension , diabetes , urinary incontinence , history of cancer , hot flashes , nervousness , insomnia and the presence of a sexual partner in the previous month . self - perception of the state of health was classified as terrible / poor / average or good / excellent . the chi - square test was applied followed by yates correction or fisher s exact test.19 finally , poisson multiple regression analysis was applied to the model to calculate the prevalence ratio ( pr ) and the respective 95% confidence intervals ( 95%ci).20 the backward criterion strategy was used to select the variables.21 for this analysis , the strata and the cluster / smallest geographical unit of the sampling plan were used . the criterion used for the inclusion of independent variables in multiple regression analysis consisted of a p - value of < 0.25 in bivariate analysis or in a simple logistic regression . the target population was the female population of belo horizonte in the state of minas gerais , brazil , aged 4065 years , with at least 11 years of formal education , which consisted of 44,313 women in the year 2000 . the necessary minimum sample size for a similar study was calculated to be 377 women , based on the assumption that 43% of the female population experiences sexual dysfunction , with an expected difference of 5% between the sample and the general population and a type i error ( ) of 0.05.13 in the present study , which was limited to women who answered all five questions used to calculate the sexual dysfunction score , the sample size was recalculated to evaluate any possible loss of precision . this cross - sectional , population - based study was carried out using a self - response questionnaire that was anonymously completed by participants at home between may and september 2005 in the city of belo horizonte , minas gerais , brazil . for the purposes of this study , the municipality of belo horizonte was stratified into nine regions . research assistants initiated the selection of women at each of the five randomly selected corners in each sector , guided by maps of the location . they went to each household and verified whether there were any brazilian - born women of 4065 years of age living in the home and whether they had at least 11 years of formal education . when questionnaires were delivered to the principal investigator , the weighted area and the education level ( 11 years of schooling ) the reasons given by the women for not participating were : lack of time , that they did not feel comfortable answering the questions , or that their husbands did not want them to participate in the study . in the first part , the participants answered questions regarding their sociodemographic characteristics ( age , marital status , ethnic group , income , schooling and paid employment ) , clinical characteristics ( previous surgical history , body mass index , depression , arterial hypertension , diabetes , urinary incontinence , history of cancer , hot flashes , nervousness , insomnia , hormone therapy and self - perception of state of health ) , reproductive characteristics ( menopausal status , number of pregnancies ) and behavioral characteristics ( physical activity , smoking and presence of sexual partner ) . the instrument used to evaluate sexual dysfunction was based on the short personal experiences questionnaire ( speq).16 the original version of this instrument was made available by investigators at the university of melbourne , australia . this version was then tested in a group of 50 brazilian - born women of 4065 years of age with 11 years or more of schooling . the variable sexual dysfunction was calculated from the mean of the sum of the scores of 1 ) sexual responsiveness , which evaluated pleasure in sexual activities ( graded from 1 to 6 , where 1 reflected an absence of pleasure and 6 maximum pleasure ) , excitation ( 16 ) and orgasm ( 16 ) ; 2 ) frequency of sexual activities ( 1=never , 2=less than once a week ; 3=once or twice a week , 4=several times a week , and 5=once a day or more ) ; and 3 ) libido ( 1=it never took place , 2=it took place less than once a week , 3=it took place once or twice a week , 4=it took place several times a week , 5= it took place once a day or more ) . a score 7 was considered indicative of sexual dysfunction and a score > 7 was considered indicative of no sexual dysfunction.16 menopausal status was defined as premenopausal when the women had regular menstrual cycles or a menstrual cycle similar to the pattern that had been predominant throughout their reproductive life . women were considered postmenopausal if their last menstrual period was at least 12 months prior to the interview.17 in women who been to hysterectomy , menopausal status was classified as follows : women aged 4044 years who had regular menstruation prior to hysterectomy were considered to be premenopausal ; women aged 4548 years who had irregular menstruation prior to hysterectomy were considered to be in perimenopause ; and women of more than 48 years of age or as well as those who had undergone hysterectomy with bilateral oophorectomy were considered to be postmenopausal , based on a previous report that the mean age at menopause in latin america is 48.6 years.18 marital status was dichotomized into married / living with a partner , or other ; ethnic group into white or non - white ; schooling into 11 years or more than 11 years of formal education ; family income into us $ 1300 or > us $ 1300 per month ; physical activity into none/ < 3 times a week or 3 times a week ; and number of pregnancies into > 2 or 2 . the presence or absence ( yes or no ) of the following variables was also investigated : depression , arterial hypertension , diabetes , urinary incontinence , history of cancer , hot flashes , nervousness , insomnia and the presence of a sexual partner in the previous month . self - perception of the state of health was classified as terrible / poor / average or good / excellent . the chi - square test was applied followed by yates correction or fisher s exact test.19 finally , poisson multiple regression analysis was applied to the model to calculate the prevalence ratio ( pr ) and the respective 95% confidence intervals ( 95%ci).20 the backward criterion strategy was used to select the variables.21 for this analysis , the strata and the cluster / smallest geographical unit of the sampling plan were used . the criterion used for the inclusion of independent variables in multiple regression analysis consisted of a p - value of < 0.25 in bivariate analysis or in a simple logistic regression . in this sample , 46.7% of participants were 50 years of age or more ; 65.9% were married or living with a partner ; 70.8% reported having a sexual partner ; 50.8% stated that they had more than 11 years of schooling ; 59.5% had a family income us$1300 ; 51% had more than two children ; and 33.5% reported practicing physical activity regularly , three or more times a week ( data not presented as a table ) . women who were unmarried or not living with a partner ( p<0.001 ) and those who reported hypertension ( p=0.002 ) or urinary incontinence ( p=0.036 ) were more likely to answer all questions contained in the section on sexual dysfunction . in the bivariate analysis , women with greater probability of having sexual dysfunction ( score 7 ) had the following characteristics : they were in peri- or postmenopause ( p=0.007 ) , had a sedentary lifestyle ( p=0.019 ) , reported depression ( p=0.003 ) , arterial hypertension ( p<0.002 ) , diabetes ( p=0.046 ) , hot flashes ( p=0.009 ) , nervousness ( p=0.048 ) or insomnia ( p=0.003 ) , and had a poor perception of their own health ( p<0.002 ) , ( table 1 ) . multiple regression analysis showed that more advanced age ( pr = 1.04 ; 95%ci : 1.011.07 ) and the presence of hot flashes ( pr = 1.37 ; 95%ci : 1.041.80 ) were significantly associated with sexual dysfunction . conversely , women who felt well ( pr = 0.68 ; 95%ci : 0.520.88 ) and women with sexual partners ( pr = 0.47 ; 95%ci : 0.340.65 ) were less likely to have a score of 7 for sexual dysfunction ( table 2 ) . the objective of this study was to evaluate the prevalence of sexual dysfunction and to identify its associated factors in middle - aged , brazilian - born women . the profile of the population of the present study should be emphasized in view of the scarcity of population - based studies on female sexuality , particularly among middle - aged women . in the present study , overall sexual dysfunction was evaluated in women with or without a sexual partner . given women s greater longevity , it is very probable that many will grow old alone ; however this does not imply the end of their sexuality or loss of their need for intimacy , touch and affection.23 the use of an internationally recognized questionnaire , the speq,16 which was adapted for use in brazil , is another factor that deserves particular mention . as direct interviews on sexual activity during menopause may lead to constraints in responses,24 it was important for the questionnaire to be self - response and anonymous . for instance , research assistants were selected who had a similar profile to that of the women in the study sample in order to encourage empathy , and reassurance was given to participants that all answers would remain confidential . in the present study , the prevalence of sexual dysfunction was 35.9% , increasing from the pre- to the postmenopause . in a prospective observational study , dennerstein et al 3 , 25 also found that from the beginning to the latter phase of the menopausal transition , the proportion of women with sexual dysfunction increased from 42% to 88% . in a study carried out in chile , castelo - branco et abdo et al.27 found at least one type of sexual dysfunction in 57.4% of women 41 years of age or older . one possible explanation for this finding may be that in women with a higher educational level , the percentage affected by sexual dysfunction is lower.27 sexual dysfunction was not significantly associated with age , which was dichotomized into < 50 years and > 50 years for the bivariate analysis ; however , in the multiple regression analysis , when the variable was considered in a continuous manner , the correlation between sexual dysfunction and increasing age was significant . in a previous study13 carried out in this same sample population , albeit with a more general evaluation of sexuality , a decline in sexuality as a function of increased chronological age was also found . a longitudinal cohort study carried out in australia reported a significant negative effect of aging on the frequency of sexual activity , as well as on aspects of responsiveness ( sexual excitation , pleasure and orgasm).3,25 the women s international study of health and sexuality , a multinational , cross - sectional study carried out in europe and in the united states , reported a decline in all aspects of sexual function with aging.28 in a longitudinal study , ford et al 29 also reported that an increase in age was associated with poorer sexuality . castelo - branco et al26 evaluated 534 healthy , middle - aged chilean women and found that the prevalence of sexual dysfunction increased from 22% in the 4044-year old age - group to 66% in the 6064 year olds . these results are in agreement with those of previous studies in which depression was associated with difficulties in vaginal lubrication in women in some regions of the world , whereas stress was associated with inability to achieve orgasm.30 other studies have also reported that psychological symptoms , stress and emotional problems may be related to a decline in sexual activity.31 it was also found that psychological symptoms were common in climacteric women and were associated with hot flashes . this observation connects psychological symptoms with the menopausal transition and suggests that their cause may be biological.32 hot flashes are known to constitute a triggering factor for insomnia , which , in turn , leads to a reduction in energy and to depression , which has a negative effect on sexual function.33 the women in the present sample who reported leading a sedentary lifestyle or who had a poor perception of their own health were more likely to be affected by sexual dysfunction . mental health,30,35 emotional well - being and positive self - image were associated with improved sexual function.30,35,36 on the other hand , additional studies have found correlations between chronic diseases and an increase in sexual dysfunction28,37 due to circulatory problems,38 neurological function , hormone balance or systemic health.39 it has been found that both an increase in systolic arterial pressure and the administration of beta blockers to treat hypertension may be detrimental to sexual function.40 the present analysis shows that arterial hypertension was associated with the occurrence of sexual dysfunction , although the use of medication for hypertension was not evaluated in this study . this finding is in agreement with other studies that reported that women with diabetes had a higher prevalence of sexual dysfunction compared to non - diabetic women.41,42 in the present study , women who had a sexual partner were less likely to have sexual dysfunction . this is consistent with other studies on middle - aged women conducted in different geographical locations , emphasizing the relevance of the sexual partner as a protective factor against sexual dysfunction.13,36,43 nevertheless , previous studies have also described the importance of feelings for the partner with respect to sexuality.44 these data suggest the need to carry out further studies to investigate , in addition to the simple presence of a partner , the quality of relationship of these couples . although recall bias can not be ruled out , previous studies using sef - reports suggest a high validity of health information , indicating that women with higher education levels provide more reliable data.45 sexual dysfunction was found in more than one - third of women that were 4065 years of age with 11 years or more of formal education .

urinary tract infection ( uti ) is the commonest bacterial infectious disease in community practice with a high rate of morbidity and financial cost . it has been estimated that 150 million people were infected with uti per annum worldwide which costing global economy more than 6 billion us dollars . the term cystitis has been used to define the lower uti infection and is characterized by symptoms such as dysuria , frequency , urgency , and suprapubic tenderness . the presence of the lower uti symptoms does not exclude the upper uti which is often present in most uti cases . the treatment of uti can be classified into uncomplicated and complicated on the basis of their choice of treatment . uti is more common in females than in males as female urethra structurally found less effective for preventing the bacterial entry . it may be due to the proximity of the genital tract and urethra and adherence of urothelial mucosa to the mucopolysaccharide lining . the other main factors which make females more prone to uti are pregnancy and sexual activity . in pregnancy , the physiological increase in plasma volume and decrease in urine concentration develop glycosuria in up to 70% women which ultimately leads to bacterial growth in urine . also in the nonpregnant state the uterus is situated over the bladder whereas in the pregnant state the enlarged uterus affects the urinary tract . sexual activity in females also increases the risk of urethra contamination as the bacteria could be pushed into the urethra during sexual intercourse as well as bacteria being massaged up the urethra into the bladder during child birth [ 11 , 12 ] . using a diaphragm also causes uti as it pushes against the urethra and makes the urethra unable to empty the bladder completely and the small concentration of urine left in the bladder leads to the growth of bacteria which ultimately causes uti . the spectrum of bacteria causing complicated uti is much broader than of those causing uncomplicated uti . however , the most commonly encountered microorganisms are gram negative bacteria including escherichia coli , citrobacter spp . , enterobacter aerogenes , pseudomonas aeruginosa , and proteus vulgaris whereas klebsiella spp . the infectious disease society of america ( idsa ) identified some microorganisms for new effective therapies . increasing drug resistance in uti needs regular monitoring of the antibiotic susceptibility of uropathogens in a particular area . various factors such as the type of uti ( complicated or uncomplicated ) , gender , age , and previous history of antibiotic therapy of each uti patient should also be considered to find out the correct global data on susceptibility . the distribution of antimicrobial susceptibility data of uti - causing microorganisms changes from time to time and from place to place . the susceptibility data provided by regional microbiology laboratories helps to choose the empirical choice of antimicrobials to treat uti ; however , these conditions are limited to complicated uti as the samples of uncomplicated uti are rarely sent to laboratories [ 16 , 17 ] . generally , the antimicrobial treatment is initiated before the laboratories results which may lead to the frequent misuse of antibiotics . the resistance pattern of community acquired uropathogens has not been extensively studied in india [ 1921 ] . to the best of our knowledge , no data regarding the bacterial resistance in utis from meerut district ( uttar pradesh ) , india , has been documented . since most utis are treated empirically , the criteria for the selection of antimicrobial agents should be determined on the basis of the most likely pathogen and its expected resistance pattern in a geographic area . therefore there is a need for periodic monitoring of etiologic agents of uti and their resistance pattern in the community . this study was undertaken in view of paucity of reports of utis in patients of meerut district ( uttar pradesh ) , india . the aim of the study is to determine the prevalence of uti in male and female patients as well as the effect of gender and age on its prevalence . the uti - causing microorganisms , their distribution among different ages and genders , and their antimicrobial susceptibility will also be determined . the study was carried out in the microbiology laboratory of the department of botany , meerut college , meerut ( uttar pradesh ) , india . the urine samples were collected from the opds ( outpatients departments ) section of three major hospitals ( meerut kidney hospital , pyarelal hospital , and jaswant rai hospital ) of meerut city . these sample collection sites were chosen as they mostly covered the urban area of the city . the duration of the study was one and a half year from july 2011 to january 2013 . the urine samples of 288 patients , comprised of 148 males and 140 females , who attended the outpatient departments ( opds ) of three hospitals and had clinical evidence of urinary tract infection , determined by treating physicians , were included in this study . patients with history of hospital admission a week before their presentation in opds were excluded from the study to rule out hospital - acquired infections . clean catch midstream urine was collected from each patient into a 20 ml calibrated sterile screw - capped universal container which was distributed to the patients . the specimens were labeled , transported to the laboratory , and analyzed within 6 hours . in each container boric acid ( 0.2 mg ) was added to prevent the growth of bacteria in urine samples . all patients were well instructed on how to collect sample aseptically prior to sample collection to avoid contaminations from urethra . the study was conducted after due ethical approval which was subjected to the hospital administrations . a calibrated loop method was used for the isolation of bacterial pathogens from urinary samples . a sterile 4.0 mm platinum wired calibrated loop was used which delivered 0.001 ml of urine . a loopful urine sample was plated on cystine - lactose - electrolyte deficient ( cled ) agar , macconkey agar , and blood agar medium ( hi media laboratories , mumbai , india ) . the inoculated plates were incubated at 37c for 24 h and for 48 h in negative cases . the number of isolated bacterial colonies was multiplied by 1000 for the estimation of bacterial load / ml of the urine sample . a specimen was considered positive for uti if an organism was cultured at a concentration of 10 cfu / ml or when an organism was cultured at a concentration of 10 cfu / ml and > 5 pus cells per high - power field were observed on microscopic examination of the urine . identification of bacterial isolates was done on the basis of their cultural and biochemical characteristics . gram negative bacteria were identified by the standard biochemical tests [ 14 , 23 ] and gram positive microorganisms were identified with the corresponding laboratory tests : catalase , coagulase , and mannitol test for staphylococcus aureus . identified and pure isolates were maintained in nutrient agar slants and incubated at 37c for 24 hrs . isolates were tested for antimicrobial susceptibility testing by the standard kirby bauer 's disc diffusion method . standard inoculums adjusted to 0.5 mcfarland was swabbed on mueller hinton agar and was allowed to soak for 2 to 5 minutes . mueller hinton agar plates were then incubated at 37c for 24 h. after 24 h the inhibition zones were measured and interpreted by the recommendations of clinical and laboratory standards . the following standard antibiotic discs were used for the isolates , ciprofloxacin ( cip ) , moxifloxacin ( mox ) , ofloxacin ( ofl ) , sparfloxacin ( spr ) , levofloxacin ( lev ) , nalidixic acid ( nal ) , gatifloxacin ( gtx ) , tobramycin ( tob ) , amikacin ( amk ) , gentamycin ( get ) , ceftazidime ( ctz ) , cefotaxime ( ctx ) , ceftriaxone ( cfx ) , imipenem ( imp ) , meropenem ( mrp ) , nitrofurantoin ( ntf ) , netillin ( ntl ) and co - trimoxazole ( cot ) . standard strains of e. coli ( atcc 25922 ) , s. aureus ( atcc 25923 ) , and p. aeruginosa ( atcc 27853 ) were used routinely in this study as control . the mean of triplicates was considered and standard error of mean was calculated by microsoft excel ver . the following formula was used for the calculation of mar index of antibiotics : mar index for an antibiotic = [ number of antibiotics resistant to the isolates/(number of antibiotics number of isolates ) ] . the data were analyzed using chi - square ( ) test , confidence interval ( ci ) , odds ratio ( or ) analysis , and student 's t - test for paired samples . relative risk and odds ratio were performed to compare the risk factors in the different groups of interest ( male and female patients ) , and the chi square test was conducted to find out the significant difference between the isolated uropathogens , infected male and female patients related to different age groups , and statistical comparisons for the mar indices group ; however , test for trend was conducted for antimicrobial resistance and sensitivity variables among all isolated uropathogens . a p value of < 0.05 was considered as statistically significant for all tests and at 95% level of confidence interval . all statistical tests were performed by statistical package for social sciences ( spss ) software , inc . 233 south wacker drive , 11th floor chicago , il 60606 - 6412 , usa , for windows , version 20 . the test for trend and graphs were prepared by graphpad prism software ( version 5.03 ) , inc . the overall prevalence of uti in both male and female patients was found to be 53.82% . total 155 urine samples showed the significant bacterial growth which were comprised of 52 ( 35.14% ) samples from males and 103 ( 73.57% ) from females . these results indicated that the prevalence of uti was higher in female patients than in males . the p value and the odds ratio showed a the significant variation between male and female patients ( table 1 ) . the highest susceptible age group of patients to uti was 48 years ( 63.51% ) followed by 2636 years ( 58.11% ) , 1525 years ( 54.55% ) , and 3747 years ( 39.19% ) . comparatively , however , more cases of uti were observed in females than in males in all age groups . the highest prevalence of uti in females was found in the age group of 2636 years ( 90.69% ) ; however in males the highest susceptible age group to uti was 48 years ( 71.15% ) . the chi square test showed statistically significant variations ( p < 0.05 ) at 95% level of confidence interval for the infected and not infected male and female patients variables among all age groups . for the infected and not infected male patients variable the chi - square test values were = 13.081 ; degree of freedom = 1 ; p = 0.000 and the values for infected and not infected female patients were = 31.114 ; degree of freedom = 1 ; p = 0.000 ( table 2 ) . the highest female to male ratio for the occurrence of uti was found in the age group of 1525 years ( 17 : 1 ) followed by 2636 years ( 9.75 : 1 ) , 3747 years ( 2.22 : 1 ) , and 48 years ( 0.27 : 1 ) . the test for trend results showed significant variations ( p < 0.05 ) between the female to male ratio variables in all age groups at 95% confidence interval level ( = 5.228 ; degree of freedom = 1 ; p = 0.0222 ) ( figure 1 ) . a total of 155 bacterial uropathogens comprised of 140 ( 90.32% ) gram negative and 15 ( 9.68% ) gram positive were isolated from positive urine samples . escherichia coli was found the dominant bacteria among all isolated uropathogens with the prevalence rate of 42.58% . the second most prevalent isolate was klebsiella pneumoniae ( 18.71% ) followed by pseudomonas aeruginosa ( 12.90% ) , staphylococcus aureus ( 9.68% ) , proteus spp . there was no statistically significant variation ( p > 0.05 ) was found among the isolates ( table 3 ) . out of 140 gram negative bacteria 50 ( 35.71% ) were isolated from males and 90 ( 64.29% ) were from female patients . only 2 ( 13.33% ) gram positive bacteria were isolated from male and 13 ( 86.67% ) were isolated from female patients . the highest number of gram positive and negative uropathogens ( 39 ) was found in the female patients of the age group 2636 years followed by 37 uropathogens which were isolated from the male patients with the age group of 48 years ( table 4 ) . the highest to lowest prevalence rate for the occurrence of different isolated uropathogens within the age groups were as follows : e. coli48 years ( 36.36% ) ; 1525 years ( 24.24% ) ; 2636 years ( 21.21% ) ; 3747 years ( 18.18% ) : k. pneumoniae1525 years ( 37.93% ) ; 2636 years ( 27.59% ) ; 48 years ( 24.14% ) ; 3747 years ( 10.34% ) : p. aeruginosa2636 years ( 35.00% ) ; 48 years ( 30.00% ) ; 3747 years ( 25.00% ) ; 1525 years ( 10.00% ) : proteus spp.3747 years ( 35.71% ) ; 48 years and 2636 years ( 28.57% ) ; 1525 years ( 7.14% ) : enterobacter spp.2636 years ( 45.45% ) ; 48 years and 2636 years ( 27.27% ) ; 3747 years ( 0.00% ) : s. aureus2636 years ( 33.33% ) ; 3747 years ( 26.67% ) ; 48 years and 1525 years ( 20.00% ) ( figure 2 ) . overall nal was found the most resistant drug as 122 ( 78.71% ) uropathogens were found resistant against nal . the second most resistant drug was ctz ( 71.61% ) followed by ctx ( 67.74% ) ; however , the most sensitive drug against all uropathogens was mrp ( 92.26% ) followed by imp ( 84.52% ) , lev , and ntl each showing 74.84% sensitivity ( figure 3 ) . the test for trend results showed a statistically significant variation ( p < 0.05 ) between the resistant and sensitive variables ( = 9.152 ; degree of freedom = 1 ; p = 0.0025 ) . tob was found the highest resistant drug against 96.97% e. coli followed by nal ( 90.91% ) and ctx ( 87.88% ) ; however , both carbapenems imp and mrp showed the highest sensitivity against 98.45% and 95.45% e. coli . 79.31% of k. pneumoniae were resistant against ctz and lev was found the most susceptible drug with the rate of 89.66% . in case of p. aeruginosa the highest resistant and susceptible antibiotics were spr ( 100% ) , and mrp ( 100% ) respectively . were resistant against cfx and 100% sensitive against both carbapenems ( imp and mrp ) . . showed 81.82% resistance against ntf ; however , all ( 100% ) were sensitive to ofl , spr , lev , imp , and mrp . all s. aureus ( 100% ) showed resistance against nal and ctx ; however , imp was found 100% sensitive followed by spr , cfx , and ntl ( each showed 93.33% sensitivity against s. aureus isolates ) ( table 5 ) . the results of the paired t - test showed that there was no statistical significance between e. coli resistant versus sensitive variables ( p = 0.876 ) , k. pneumoniae resistant versus sensitive variables ( p = 0.232 ) , p. aeruginosa resistant versus sensitive variables ( p = 0.950 ) , proteus spp . resistant versus sensitive variables ( p = 0.162 ) and s. aureus resistant versus sensitive variables ( p = 0.072 ) , however , enterobacter spp . showed the significant variations between resistant versus sensitive variables ( p = 0.000 ) . the highest mar index was found for nal ( 0.044 ) followed by ctz ( 0.039 ) and ctx ( 0.038 ) indicating that these antibiotics were highly resistant among all tested uropathogens ; however , the lowest mar index was found for both carbapenems mrp and imp which were 0.004 and 0.008 , respectively , indicating the highest sensitivity against uropathogens . the test results showed no statistically significant variation among the mar indices of all tested antibiotics ( = 1.556 ; degree of freedom = 15 ; p = 1.000 ) . this study provides valuable data to compare and monitor the status of antimicrobial resistance among uropathogens to improve efficient empirical treatment . increasing antimicrobial resistance has been documented globally [ 2733 ] . the prevalence of uti was found to be 53.82% in this study and this rate of prevalence is higher than in the other studies which accounts for 25.6% , 22% , 38.6% , 35.5% , 4.2% , 17.19% , 10.86% , 34.5% , and 36.68% in india ; however , the prevalence rate of uti in our study correlates with other studies done in south trinidad , and in the mexican population which showed such more highly significant uropathogens 49% and 97.3% , respectively . our study showed a high prevalence of uti in females ( 73.57% ) than in males ( 35.14% ) which correlates with other findings which revealed that the frequency of uti is greater in females as compared to males [ 6 , 30 , 4044 ] . the reason behind this high prevalence of uti in females is due to close proximity of the urethral meatus to the anus , shorter urethra , sexual intercourse , incontinence , and bad toilet [ 4547 ] . the occurrence of uti recorded among the elderly ( 48 years , 63.51% ) compared to young age patients ( 2637 years , 58.11% ; 1525 years , 54.55% ) and middle - age patients ( 3747 years , 39.19% ) in this study differs from the other studies done in kuwait and nigeria in which the highest incidence of uti was recorded among the age group 20 to 50 years ( 63.4 and 74.7% , resp . ) and lowest among the age group > 50 years ( 13.3 and 10.3% , resp . ) . however , our results agree with the study done in japan with a 20-year period in which a trend of increasing complicated uti was reported in elderly patients . in our study it was found that the elderly males ( 48 years ) had a higher incidence of uti ( 71.15% ) when compared with the elderly females ( 45.45% ) . this finding is similar to a study conducted at a tertiary care hospital in jaipur , rajasthan , india . the main cause behind this increasing incidence of uti with advancing age in males is due to prostate enlargement and neurogenic bladder . this factor is also reported by other authors whose studies showed that the prostate disease in males is responsible for the increase in incidence of uti and decrease in female : male ratio in patients above 50 years . females of the age group 2636 years were found more susceptible ( 90.69% ) to uti followed by 1525 years ( 82.93% ) , 3747 years ( 58.82% ) , and 48 years ( 45.45% ) . these findings correlate with other reports which showed that females are more prone to utis than males during adolescence and adulthood [ 12 , 18 , 20 , 44 , 5358 ] . the factors of this increasing incidence of uti in young age females are associated with high sexual activity , recent use of a diaphragm with spermicide , and a history of recurrent utis . the highest incidence of utis among female to male ratio was found in the age group of 1525 years ( 17 : 1 ) followed by 2636 years ( 9.75 : 1 ) , 3747 years ( 2.22 : 1 ) , and 48 years ( 0.27 : 1 ) . these findings differ from other reports [ 57 , 60 ] which stated a lower female to male ratio in neonates and young children . the prevalence rate of uti in boys depends on many factors including congenital malformations and uncircumcised genitalia which are often contaminated . in this study , the gram negative bacilli constituted 90.32% of the total bacterial isolates while gram positive cocci constituted 9.68% . escherichia coli ( 42.58% ) was found the most prevalent gram negative bacteria in the positive urine samples of uti . this result is consistent with reports from other studies [ 38 , 48 , 49 , 53 , 6163 ] but differs from the reports in which p. aeruginosa and klebsiella spp . other isolated bacteria from uti cases in this study were k. pneumoniae ( 18.71% ) , p. aeruginosa ( 12.90% ) , s. aureus ( 9.68% ) , proteus spp . these findings were not correlate with other reports in which p. aeruginosa was reported as the second most common bacterial isolate in uti studies in india and lafia , nigeria ; however , these results correlates with others in which klebsiella spp . was reported as the second most frequently isolated organism in uti [ 32 , 54 , 63 , 66 , 67 ] . the studies on uti in other places of the world also showed that e. coli and klebsiella spp . higher incidence of gram negative bacteria , related to enterobacteriaceae , in causing uti has many factors which are responsible for their attachment to the uroepithelium . in addition , they are able to colonize in the urogenital mucosa with adhesins , pili , fimbriae , and p-1 blood group phenotype receptor . in females of all age categories , e. coli is the most frequently isolated uropathogen which correlates with other studies [ 7173 ] but not with others which found that e. coli causes most male utis , followed by other enterobacteriaceae and enterococci [ 74 , 75 ] whereas proteus mirabilis was more frequently isolated in the younger female patients of uti and k. pneumoniae in the elderly patients . both carbepenems ( mrp and imp ) used in this study were found to be the most sensitive drugs against all isolated uropathogens . the sensitivity rate of carbepenems among uropathogens was as follows : e. coli ( mrp ; 95.45% and imp ; 98.89% ) , p. aeruginosa ( mrp ; 100% and imp ; 95.00% ) , proteus spp . ( mrp ; 100% and imp ; 100% ) , and s. aureus ( mrp ; 80% and imp ; 100% ) , followed by lev and ntl each of which showed 74.84% sensitivity , however , k. pneumonia did not show a high susceptibility to imp ( 24.14% ) but it was susceptible to mrp ( 86.21% ) . another study conducted in india showed that meropenem was highly sensitive against gram negative bacilli whereas cephalosporin showed highest resistance against gram negative rods . in other study , meropenem and imipenem were found to be 98% and 100% sensitive , respectively , against highly resistant gram negative bacilli . a study done in king fahd hospital , saudi arabia showed that meropenem was 95.8% sensitive followed by amikacin ( 93.7% ) and imipenem ( 91.71% ) against extended spectrum lactamase producing e. coli . tested fluoroquinolones in this study showed the highest resistance among uropathogens as in e. coli ; nal ( 90.91% ) : k. pneumoniae ; cip ( 79.31% ) , p. aeruginosa ; spr ( 100% ) , and s. aureus ; nal ( 100% ) ; however , iii generation cephalosporin showed the highest resistance in k. pneumoniae ; ctz ( 79.31% ) the proteus spp . ; . this high rate of resistance against fluoroquninolones was also suggested by other studies done in spain , europe , and iran [ 33 , 81 ] and also by other studies done in india [ 21 , 44 , 82 ] . another study done in spain also showed the reduced susceptibility of e. coli isolates from patients with uti to fluoroquinolones ( 16% ) . in several studies it has been shown that the highly prescribing habits of the physicians are the driving factor for the antibiotic resistance for this group of antibiotic [ 8385 ] . mcewen et al . found that 37% of physicians actually prescribe trimethoprim - sulphamethoxazole closely followed by fluoroquinolones ( 32% ) and the average duration of antibiotic therapy is 8.6 days in the united states which is the best example of this problem ; empiric use of fluoroquinolones should be restricted and founding the strategies against increasing resistance of pathogens to these antibiotics should be done . our finding about the fluoroquinolones did not correlate with others which showed that they were highly effective ( sensitive ) [ 11 , 55 , 64 , 87 , 88 ] . for these organisms , drugs with inhibitors like augmentin may be tried but such drugs should be reserved for the last line of treatment . the alarming finding in this study is the resistance to third - generation cephalosporin ; the highest resistance was seen against ctz ( 71.61% ) followed by ctx ( 67.74% ) among all uropathogens . the other possible explanation behind this situation is that the iii generation cephalosporin has been in use for a long period and must have been abused and over time organisms have developed resistant mechanisms due to changing their mode of action . the inappropriate usage of wide spectrum antibiotics , insufficient hygiene , immunosuppression , and a prolonged stay in the hospital are some other major etiological factors that elevate the chances of mdr infections . against the background of paucity of reports of uti in meerut city ( uttar pradesh ) , india , this is the first study conducted to determine the prevalence of uti , the effect of gender and age on its prevalence , and their susceptibility profile in the community of meerut city . this study provides valuable laboratory data to monitor the status of antimicrobial resistance among uropathogens and to improve treatment recommendations in a specific geographical region . the study also allows comparison of the situation in meerut with other regions within and outside the state as well as in the country .

urinary tract infection is one of the common infections in the indian community . distribution and susceptibility of uti - causing pathogens change according to time and place . this study was conducted to determine the distribution and antimicrobial susceptibility of uropathogens in the indian community as well as to determine the effect of gender and age on the etiology of bacterial uropathogens . clean catch midstream urine samples were collected from 288 patients of the age ranging from 15 to 48 years . antimicrobial susceptibility was performed on all isolated bacteria by kirby bauer 's disc diffusion method . the multiple antibiotic resistance ( mar ) index of each antibiotic was calculated . the uti prevalence was 53.82% in patients ; however , the prevalence was significantly higher in females than in males ( females : 73.57% ; males : 35.14% ; p = 0.000 ) . females within the age group of 2636 years and elderly males of 48 years showed higher prevalence of uti . gram negative bacteria ( 90.32% ) were found in high prevalence than gram positive ( 9.68% ) . escherichia coli ( 42.58% ) was the most prevalent gram negative isolate . nitrofurantoin ( 78.71% ) was found the most resistant drug among all uropathogens . tested carbapenems were found the most susceptible drug against isolated uropathogens which showed 92.26% and 84.52% susceptibility , respectively .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusion

urinary tract infection ( uti ) is the commonest bacterial infectious disease in community practice with a high rate of morbidity and financial cost . the treatment of uti can be classified into uncomplicated and complicated on the basis of their choice of treatment . uti is more common in females than in males as female urethra structurally found less effective for preventing the bacterial entry . also in the nonpregnant state the uterus is situated over the bladder whereas in the pregnant state the enlarged uterus affects the urinary tract . sexual activity in females also increases the risk of urethra contamination as the bacteria could be pushed into the urethra during sexual intercourse as well as bacteria being massaged up the urethra into the bladder during child birth [ 11 , 12 ] . however , the most commonly encountered microorganisms are gram negative bacteria including escherichia coli , citrobacter spp . increasing drug resistance in uti needs regular monitoring of the antibiotic susceptibility of uropathogens in a particular area . various factors such as the type of uti ( complicated or uncomplicated ) , gender , age , and previous history of antibiotic therapy of each uti patient should also be considered to find out the correct global data on susceptibility . the distribution of antimicrobial susceptibility data of uti - causing microorganisms changes from time to time and from place to place . the susceptibility data provided by regional microbiology laboratories helps to choose the empirical choice of antimicrobials to treat uti ; however , these conditions are limited to complicated uti as the samples of uncomplicated uti are rarely sent to laboratories [ 16 , 17 ] . generally , the antimicrobial treatment is initiated before the laboratories results which may lead to the frequent misuse of antibiotics . since most utis are treated empirically , the criteria for the selection of antimicrobial agents should be determined on the basis of the most likely pathogen and its expected resistance pattern in a geographic area . therefore there is a need for periodic monitoring of etiologic agents of uti and their resistance pattern in the community . this study was undertaken in view of paucity of reports of utis in patients of meerut district ( uttar pradesh ) , india . the aim of the study is to determine the prevalence of uti in male and female patients as well as the effect of gender and age on its prevalence . the uti - causing microorganisms , their distribution among different ages and genders , and their antimicrobial susceptibility will also be determined . the study was carried out in the microbiology laboratory of the department of botany , meerut college , meerut ( uttar pradesh ) , india . the urine samples were collected from the opds ( outpatients departments ) section of three major hospitals ( meerut kidney hospital , pyarelal hospital , and jaswant rai hospital ) of meerut city . the duration of the study was one and a half year from july 2011 to january 2013 . the urine samples of 288 patients , comprised of 148 males and 140 females , who attended the outpatient departments ( opds ) of three hospitals and had clinical evidence of urinary tract infection , determined by treating physicians , were included in this study . clean catch midstream urine was collected from each patient into a 20 ml calibrated sterile screw - capped universal container which was distributed to the patients . in each container boric acid ( 0.2 mg ) was added to prevent the growth of bacteria in urine samples . the study was conducted after due ethical approval which was subjected to the hospital administrations . the number of isolated bacterial colonies was multiplied by 1000 for the estimation of bacterial load / ml of the urine sample . identification of bacterial isolates was done on the basis of their cultural and biochemical characteristics . gram negative bacteria were identified by the standard biochemical tests [ 14 , 23 ] and gram positive microorganisms were identified with the corresponding laboratory tests : catalase , coagulase , and mannitol test for staphylococcus aureus . isolates were tested for antimicrobial susceptibility testing by the standard kirby bauer 's disc diffusion method . standard strains of e. coli ( atcc 25922 ) , s. aureus ( atcc 25923 ) , and p. aeruginosa ( atcc 27853 ) were used routinely in this study as control . the mean of triplicates was considered and standard error of mean was calculated by microsoft excel ver . relative risk and odds ratio were performed to compare the risk factors in the different groups of interest ( male and female patients ) , and the chi square test was conducted to find out the significant difference between the isolated uropathogens , infected male and female patients related to different age groups , and statistical comparisons for the mar indices group ; however , test for trend was conducted for antimicrobial resistance and sensitivity variables among all isolated uropathogens . the overall prevalence of uti in both male and female patients was found to be 53.82% . total 155 urine samples showed the significant bacterial growth which were comprised of 52 ( 35.14% ) samples from males and 103 ( 73.57% ) from females . these results indicated that the prevalence of uti was higher in female patients than in males . the highest susceptible age group of patients to uti was 48 years ( 63.51% ) followed by 2636 years ( 58.11% ) , 1525 years ( 54.55% ) , and 3747 years ( 39.19% ) . comparatively , however , more cases of uti were observed in females than in males in all age groups . the highest prevalence of uti in females was found in the age group of 2636 years ( 90.69% ) ; however in males the highest susceptible age group to uti was 48 years ( 71.15% ) . for the infected and not infected male patients variable the chi - square test values were = 13.081 ; degree of freedom = 1 ; p = 0.000 and the values for infected and not infected female patients were = 31.114 ; degree of freedom = 1 ; p = 0.000 ( table 2 ) . the highest female to male ratio for the occurrence of uti was found in the age group of 1525 years ( 17 : 1 ) followed by 2636 years ( 9.75 : 1 ) , 3747 years ( 2.22 : 1 ) , and 48 years ( 0.27 : 1 ) . the test for trend results showed significant variations ( p < 0.05 ) between the female to male ratio variables in all age groups at 95% confidence interval level ( = 5.228 ; degree of freedom = 1 ; p = 0.0222 ) ( figure 1 ) . a total of 155 bacterial uropathogens comprised of 140 ( 90.32% ) gram negative and 15 ( 9.68% ) gram positive were isolated from positive urine samples . escherichia coli was found the dominant bacteria among all isolated uropathogens with the prevalence rate of 42.58% . the second most prevalent isolate was klebsiella pneumoniae ( 18.71% ) followed by pseudomonas aeruginosa ( 12.90% ) , staphylococcus aureus ( 9.68% ) , proteus spp . there was no statistically significant variation ( p > 0.05 ) was found among the isolates ( table 3 ) . out of 140 gram negative bacteria 50 ( 35.71% ) were isolated from males and 90 ( 64.29% ) were from female patients . only 2 ( 13.33% ) gram positive bacteria were isolated from male and 13 ( 86.67% ) were isolated from female patients . the highest number of gram positive and negative uropathogens ( 39 ) was found in the female patients of the age group 2636 years followed by 37 uropathogens which were isolated from the male patients with the age group of 48 years ( table 4 ) . the highest to lowest prevalence rate for the occurrence of different isolated uropathogens within the age groups were as follows : e. coli48 years ( 36.36% ) ; 1525 years ( 24.24% ) ; 2636 years ( 21.21% ) ; 3747 years ( 18.18% ) : k. pneumoniae1525 years ( 37.93% ) ; 2636 years ( 27.59% ) ; 48 years ( 24.14% ) ; 3747 years ( 10.34% ) : p. aeruginosa2636 years ( 35.00% ) ; 48 years ( 30.00% ) ; 3747 years ( 25.00% ) ; 1525 years ( 10.00% ) : proteus spp.3747 years ( 35.71% ) ; 48 years and 2636 years ( 28.57% ) ; 1525 years ( 7.14% ) : enterobacter spp.2636 years ( 45.45% ) ; 48 years and 2636 years ( 27.27% ) ; 3747 years ( 0.00% ) : s. aureus2636 years ( 33.33% ) ; 3747 years ( 26.67% ) ; 48 years and 1525 years ( 20.00% ) ( figure 2 ) . overall nal was found the most resistant drug as 122 ( 78.71% ) uropathogens were found resistant against nal . the second most resistant drug was ctz ( 71.61% ) followed by ctx ( 67.74% ) ; however , the most sensitive drug against all uropathogens was mrp ( 92.26% ) followed by imp ( 84.52% ) , lev , and ntl each showing 74.84% sensitivity ( figure 3 ) . the test for trend results showed a statistically significant variation ( p < 0.05 ) between the resistant and sensitive variables ( = 9.152 ; degree of freedom = 1 ; p = 0.0025 ) . tob was found the highest resistant drug against 96.97% e. coli followed by nal ( 90.91% ) and ctx ( 87.88% ) ; however , both carbapenems imp and mrp showed the highest sensitivity against 98.45% and 95.45% e. coli . 79.31% of k. pneumoniae were resistant against ctz and lev was found the most susceptible drug with the rate of 89.66% . showed 81.82% resistance against ntf ; however , all ( 100% ) were sensitive to ofl , spr , lev , imp , and mrp . all s. aureus ( 100% ) showed resistance against nal and ctx ; however , imp was found 100% sensitive followed by spr , cfx , and ntl ( each showed 93.33% sensitivity against s. aureus isolates ) ( table 5 ) . the results of the paired t - test showed that there was no statistical significance between e. coli resistant versus sensitive variables ( p = 0.876 ) , k. pneumoniae resistant versus sensitive variables ( p = 0.232 ) , p. aeruginosa resistant versus sensitive variables ( p = 0.950 ) , proteus spp . resistant versus sensitive variables ( p = 0.162 ) and s. aureus resistant versus sensitive variables ( p = 0.072 ) , however , enterobacter spp . showed the significant variations between resistant versus sensitive variables ( p = 0.000 ) . the highest mar index was found for nal ( 0.044 ) followed by ctz ( 0.039 ) and ctx ( 0.038 ) indicating that these antibiotics were highly resistant among all tested uropathogens ; however , the lowest mar index was found for both carbapenems mrp and imp which were 0.004 and 0.008 , respectively , indicating the highest sensitivity against uropathogens . the test results showed no statistically significant variation among the mar indices of all tested antibiotics ( = 1.556 ; degree of freedom = 15 ; p = 1.000 ) . the prevalence of uti was found to be 53.82% in this study and this rate of prevalence is higher than in the other studies which accounts for 25.6% , 22% , 38.6% , 35.5% , 4.2% , 17.19% , 10.86% , 34.5% , and 36.68% in india ; however , the prevalence rate of uti in our study correlates with other studies done in south trinidad , and in the mexican population which showed such more highly significant uropathogens 49% and 97.3% , respectively . our study showed a high prevalence of uti in females ( 73.57% ) than in males ( 35.14% ) which correlates with other findings which revealed that the frequency of uti is greater in females as compared to males [ 6 , 30 , 4044 ] . the reason behind this high prevalence of uti in females is due to close proximity of the urethral meatus to the anus , shorter urethra , sexual intercourse , incontinence , and bad toilet [ 4547 ] . the occurrence of uti recorded among the elderly ( 48 years , 63.51% ) compared to young age patients ( 2637 years , 58.11% ; 1525 years , 54.55% ) and middle - age patients ( 3747 years , 39.19% ) in this study differs from the other studies done in kuwait and nigeria in which the highest incidence of uti was recorded among the age group 20 to 50 years ( 63.4 and 74.7% , resp . ) and lowest among the age group > 50 years ( 13.3 and 10.3% , resp . ) however , our results agree with the study done in japan with a 20-year period in which a trend of increasing complicated uti was reported in elderly patients . in our study it was found that the elderly males ( 48 years ) had a higher incidence of uti ( 71.15% ) when compared with the elderly females ( 45.45% ) . the main cause behind this increasing incidence of uti with advancing age in males is due to prostate enlargement and neurogenic bladder . this factor is also reported by other authors whose studies showed that the prostate disease in males is responsible for the increase in incidence of uti and decrease in female : male ratio in patients above 50 years . females of the age group 2636 years were found more susceptible ( 90.69% ) to uti followed by 1525 years ( 82.93% ) , 3747 years ( 58.82% ) , and 48 years ( 45.45% ) . the highest incidence of utis among female to male ratio was found in the age group of 1525 years ( 17 : 1 ) followed by 2636 years ( 9.75 : 1 ) , 3747 years ( 2.22 : 1 ) , and 48 years ( 0.27 : 1 ) . the prevalence rate of uti in boys depends on many factors including congenital malformations and uncircumcised genitalia which are often contaminated . in this study , the gram negative bacilli constituted 90.32% of the total bacterial isolates while gram positive cocci constituted 9.68% . escherichia coli ( 42.58% ) was found the most prevalent gram negative bacteria in the positive urine samples of uti . other isolated bacteria from uti cases in this study were k. pneumoniae ( 18.71% ) , p. aeruginosa ( 12.90% ) , s. aureus ( 9.68% ) , proteus spp . these findings were not correlate with other reports in which p. aeruginosa was reported as the second most common bacterial isolate in uti studies in india and lafia , nigeria ; however , these results correlates with others in which klebsiella spp . higher incidence of gram negative bacteria , related to enterobacteriaceae , in causing uti has many factors which are responsible for their attachment to the uroepithelium . in addition , they are able to colonize in the urogenital mucosa with adhesins , pili , fimbriae , and p-1 blood group phenotype receptor . in females of all age categories , e. coli is the most frequently isolated uropathogen which correlates with other studies [ 7173 ] but not with others which found that e. coli causes most male utis , followed by other enterobacteriaceae and enterococci [ 74 , 75 ] whereas proteus mirabilis was more frequently isolated in the younger female patients of uti and k. pneumoniae in the elderly patients . both carbepenems ( mrp and imp ) used in this study were found to be the most sensitive drugs against all isolated uropathogens . ( mrp ; 100% and imp ; 100% ) , and s. aureus ( mrp ; 80% and imp ; 100% ) , followed by lev and ntl each of which showed 74.84% sensitivity , however , k. pneumonia did not show a high susceptibility to imp ( 24.14% ) but it was susceptible to mrp ( 86.21% ) . in other study , meropenem and imipenem were found to be 98% and 100% sensitive , respectively , against highly resistant gram negative bacilli . tested fluoroquinolones in this study showed the highest resistance among uropathogens as in e. coli ; nal ( 90.91% ) : k. pneumoniae ; cip ( 79.31% ) , p. aeruginosa ; spr ( 100% ) , and s. aureus ; nal ( 100% ) ; however , iii generation cephalosporin showed the highest resistance in k. pneumoniae ; ctz ( 79.31% ) the proteus spp . in several studies it has been shown that the highly prescribing habits of the physicians are the driving factor for the antibiotic resistance for this group of antibiotic [ 8385 ] . found that 37% of physicians actually prescribe trimethoprim - sulphamethoxazole closely followed by fluoroquinolones ( 32% ) and the average duration of antibiotic therapy is 8.6 days in the united states which is the best example of this problem ; empiric use of fluoroquinolones should be restricted and founding the strategies against increasing resistance of pathogens to these antibiotics should be done . our finding about the fluoroquinolones did not correlate with others which showed that they were highly effective ( sensitive ) [ 11 , 55 , 64 , 87 , 88 ] . the alarming finding in this study is the resistance to third - generation cephalosporin ; the highest resistance was seen against ctz ( 71.61% ) followed by ctx ( 67.74% ) among all uropathogens . the inappropriate usage of wide spectrum antibiotics , insufficient hygiene , immunosuppression , and a prolonged stay in the hospital are some other major etiological factors that elevate the chances of mdr infections . against the background of paucity of reports of uti in meerut city ( uttar pradesh ) , india , this is the first study conducted to determine the prevalence of uti , the effect of gender and age on its prevalence , and their susceptibility profile in the community of meerut city . the study also allows comparison of the situation in meerut with other regions within and outside the state as well as in the country .

the term cystitis has been used to define the lower uti infection and is characterized by symptoms such as dysuria , frequency , urgency , and suprapubic tenderness . the presence of the lower uti symptoms does not exclude the upper uti which is often present in most uti cases . the treatment of uti can be classified into uncomplicated and complicated on the basis of their choice of treatment . uti is more common in females than in males as female urethra structurally found less effective for preventing the bacterial entry . it may be due to the proximity of the genital tract and urethra and adherence of urothelial mucosa to the mucopolysaccharide lining . in pregnancy , the physiological increase in plasma volume and decrease in urine concentration develop glycosuria in up to 70% women which ultimately leads to bacterial growth in urine . also in the nonpregnant state the uterus is situated over the bladder whereas in the pregnant state the enlarged uterus affects the urinary tract . sexual activity in females also increases the risk of urethra contamination as the bacteria could be pushed into the urethra during sexual intercourse as well as bacteria being massaged up the urethra into the bladder during child birth [ 11 , 12 ] . using a diaphragm also causes uti as it pushes against the urethra and makes the urethra unable to empty the bladder completely and the small concentration of urine left in the bladder leads to the growth of bacteria which ultimately causes uti . increasing drug resistance in uti needs regular monitoring of the antibiotic susceptibility of uropathogens in a particular area . various factors such as the type of uti ( complicated or uncomplicated ) , gender , age , and previous history of antibiotic therapy of each uti patient should also be considered to find out the correct global data on susceptibility . the susceptibility data provided by regional microbiology laboratories helps to choose the empirical choice of antimicrobials to treat uti ; however , these conditions are limited to complicated uti as the samples of uncomplicated uti are rarely sent to laboratories [ 16 , 17 ] . to the best of our knowledge , no data regarding the bacterial resistance in utis from meerut district ( uttar pradesh ) , india , has been documented . since most utis are treated empirically , the criteria for the selection of antimicrobial agents should be determined on the basis of the most likely pathogen and its expected resistance pattern in a geographic area . this study was undertaken in view of paucity of reports of utis in patients of meerut district ( uttar pradesh ) , india . the aim of the study is to determine the prevalence of uti in male and female patients as well as the effect of gender and age on its prevalence . the study was carried out in the microbiology laboratory of the department of botany , meerut college , meerut ( uttar pradesh ) , india . the urine samples were collected from the opds ( outpatients departments ) section of three major hospitals ( meerut kidney hospital , pyarelal hospital , and jaswant rai hospital ) of meerut city . the urine samples of 288 patients , comprised of 148 males and 140 females , who attended the outpatient departments ( opds ) of three hospitals and had clinical evidence of urinary tract infection , determined by treating physicians , were included in this study . a specimen was considered positive for uti if an organism was cultured at a concentration of 10 cfu / ml or when an organism was cultured at a concentration of 10 cfu / ml and > 5 pus cells per high - power field were observed on microscopic examination of the urine . gram negative bacteria were identified by the standard biochemical tests [ 14 , 23 ] and gram positive microorganisms were identified with the corresponding laboratory tests : catalase , coagulase , and mannitol test for staphylococcus aureus . the following standard antibiotic discs were used for the isolates , ciprofloxacin ( cip ) , moxifloxacin ( mox ) , ofloxacin ( ofl ) , sparfloxacin ( spr ) , levofloxacin ( lev ) , nalidixic acid ( nal ) , gatifloxacin ( gtx ) , tobramycin ( tob ) , amikacin ( amk ) , gentamycin ( get ) , ceftazidime ( ctz ) , cefotaxime ( ctx ) , ceftriaxone ( cfx ) , imipenem ( imp ) , meropenem ( mrp ) , nitrofurantoin ( ntf ) , netillin ( ntl ) and co - trimoxazole ( cot ) . standard strains of e. coli ( atcc 25922 ) , s. aureus ( atcc 25923 ) , and p. aeruginosa ( atcc 27853 ) were used routinely in this study as control . the following formula was used for the calculation of mar index of antibiotics : mar index for an antibiotic = [ number of antibiotics resistant to the isolates/(number of antibiotics number of isolates ) ] . relative risk and odds ratio were performed to compare the risk factors in the different groups of interest ( male and female patients ) , and the chi square test was conducted to find out the significant difference between the isolated uropathogens , infected male and female patients related to different age groups , and statistical comparisons for the mar indices group ; however , test for trend was conducted for antimicrobial resistance and sensitivity variables among all isolated uropathogens . the overall prevalence of uti in both male and female patients was found to be 53.82% . total 155 urine samples showed the significant bacterial growth which were comprised of 52 ( 35.14% ) samples from males and 103 ( 73.57% ) from females . these results indicated that the prevalence of uti was higher in female patients than in males . the p value and the odds ratio showed a the significant variation between male and female patients ( table 1 ) . the highest susceptible age group of patients to uti was 48 years ( 63.51% ) followed by 2636 years ( 58.11% ) , 1525 years ( 54.55% ) , and 3747 years ( 39.19% ) . comparatively , however , more cases of uti were observed in females than in males in all age groups . the highest prevalence of uti in females was found in the age group of 2636 years ( 90.69% ) ; however in males the highest susceptible age group to uti was 48 years ( 71.15% ) . the chi square test showed statistically significant variations ( p < 0.05 ) at 95% level of confidence interval for the infected and not infected male and female patients variables among all age groups . for the infected and not infected male patients variable the chi - square test values were = 13.081 ; degree of freedom = 1 ; p = 0.000 and the values for infected and not infected female patients were = 31.114 ; degree of freedom = 1 ; p = 0.000 ( table 2 ) . the highest female to male ratio for the occurrence of uti was found in the age group of 1525 years ( 17 : 1 ) followed by 2636 years ( 9.75 : 1 ) , 3747 years ( 2.22 : 1 ) , and 48 years ( 0.27 : 1 ) . the test for trend results showed significant variations ( p < 0.05 ) between the female to male ratio variables in all age groups at 95% confidence interval level ( = 5.228 ; degree of freedom = 1 ; p = 0.0222 ) ( figure 1 ) . a total of 155 bacterial uropathogens comprised of 140 ( 90.32% ) gram negative and 15 ( 9.68% ) gram positive were isolated from positive urine samples . escherichia coli was found the dominant bacteria among all isolated uropathogens with the prevalence rate of 42.58% . the highest number of gram positive and negative uropathogens ( 39 ) was found in the female patients of the age group 2636 years followed by 37 uropathogens which were isolated from the male patients with the age group of 48 years ( table 4 ) . the highest to lowest prevalence rate for the occurrence of different isolated uropathogens within the age groups were as follows : e. coli48 years ( 36.36% ) ; 1525 years ( 24.24% ) ; 2636 years ( 21.21% ) ; 3747 years ( 18.18% ) : k. pneumoniae1525 years ( 37.93% ) ; 2636 years ( 27.59% ) ; 48 years ( 24.14% ) ; 3747 years ( 10.34% ) : p. aeruginosa2636 years ( 35.00% ) ; 48 years ( 30.00% ) ; 3747 years ( 25.00% ) ; 1525 years ( 10.00% ) : proteus spp.3747 years ( 35.71% ) ; 48 years and 2636 years ( 28.57% ) ; 1525 years ( 7.14% ) : enterobacter spp.2636 years ( 45.45% ) ; 48 years and 2636 years ( 27.27% ) ; 3747 years ( 0.00% ) : s. aureus2636 years ( 33.33% ) ; 3747 years ( 26.67% ) ; 48 years and 1525 years ( 20.00% ) ( figure 2 ) . the second most resistant drug was ctz ( 71.61% ) followed by ctx ( 67.74% ) ; however , the most sensitive drug against all uropathogens was mrp ( 92.26% ) followed by imp ( 84.52% ) , lev , and ntl each showing 74.84% sensitivity ( figure 3 ) . the test for trend results showed a statistically significant variation ( p < 0.05 ) between the resistant and sensitive variables ( = 9.152 ; degree of freedom = 1 ; p = 0.0025 ) . tob was found the highest resistant drug against 96.97% e. coli followed by nal ( 90.91% ) and ctx ( 87.88% ) ; however , both carbapenems imp and mrp showed the highest sensitivity against 98.45% and 95.45% e. coli . 79.31% of k. pneumoniae were resistant against ctz and lev was found the most susceptible drug with the rate of 89.66% . in case of p. aeruginosa the highest resistant and susceptible antibiotics were spr ( 100% ) , and mrp ( 100% ) respectively . all s. aureus ( 100% ) showed resistance against nal and ctx ; however , imp was found 100% sensitive followed by spr , cfx , and ntl ( each showed 93.33% sensitivity against s. aureus isolates ) ( table 5 ) . the results of the paired t - test showed that there was no statistical significance between e. coli resistant versus sensitive variables ( p = 0.876 ) , k. pneumoniae resistant versus sensitive variables ( p = 0.232 ) , p. aeruginosa resistant versus sensitive variables ( p = 0.950 ) , proteus spp . the highest mar index was found for nal ( 0.044 ) followed by ctz ( 0.039 ) and ctx ( 0.038 ) indicating that these antibiotics were highly resistant among all tested uropathogens ; however , the lowest mar index was found for both carbapenems mrp and imp which were 0.004 and 0.008 , respectively , indicating the highest sensitivity against uropathogens . the test results showed no statistically significant variation among the mar indices of all tested antibiotics ( = 1.556 ; degree of freedom = 15 ; p = 1.000 ) . this study provides valuable data to compare and monitor the status of antimicrobial resistance among uropathogens to improve efficient empirical treatment . the prevalence of uti was found to be 53.82% in this study and this rate of prevalence is higher than in the other studies which accounts for 25.6% , 22% , 38.6% , 35.5% , 4.2% , 17.19% , 10.86% , 34.5% , and 36.68% in india ; however , the prevalence rate of uti in our study correlates with other studies done in south trinidad , and in the mexican population which showed such more highly significant uropathogens 49% and 97.3% , respectively . our study showed a high prevalence of uti in females ( 73.57% ) than in males ( 35.14% ) which correlates with other findings which revealed that the frequency of uti is greater in females as compared to males [ 6 , 30 , 4044 ] . the reason behind this high prevalence of uti in females is due to close proximity of the urethral meatus to the anus , shorter urethra , sexual intercourse , incontinence , and bad toilet [ 4547 ] . the occurrence of uti recorded among the elderly ( 48 years , 63.51% ) compared to young age patients ( 2637 years , 58.11% ; 1525 years , 54.55% ) and middle - age patients ( 3747 years , 39.19% ) in this study differs from the other studies done in kuwait and nigeria in which the highest incidence of uti was recorded among the age group 20 to 50 years ( 63.4 and 74.7% , resp . ) however , our results agree with the study done in japan with a 20-year period in which a trend of increasing complicated uti was reported in elderly patients . in our study it was found that the elderly males ( 48 years ) had a higher incidence of uti ( 71.15% ) when compared with the elderly females ( 45.45% ) . this factor is also reported by other authors whose studies showed that the prostate disease in males is responsible for the increase in incidence of uti and decrease in female : male ratio in patients above 50 years . females of the age group 2636 years were found more susceptible ( 90.69% ) to uti followed by 1525 years ( 82.93% ) , 3747 years ( 58.82% ) , and 48 years ( 45.45% ) . the factors of this increasing incidence of uti in young age females are associated with high sexual activity , recent use of a diaphragm with spermicide , and a history of recurrent utis . the highest incidence of utis among female to male ratio was found in the age group of 1525 years ( 17 : 1 ) followed by 2636 years ( 9.75 : 1 ) , 3747 years ( 2.22 : 1 ) , and 48 years ( 0.27 : 1 ) . in this study , the gram negative bacilli constituted 90.32% of the total bacterial isolates while gram positive cocci constituted 9.68% . other isolated bacteria from uti cases in this study were k. pneumoniae ( 18.71% ) , p. aeruginosa ( 12.90% ) , s. aureus ( 9.68% ) , proteus spp . these findings were not correlate with other reports in which p. aeruginosa was reported as the second most common bacterial isolate in uti studies in india and lafia , nigeria ; however , these results correlates with others in which klebsiella spp . higher incidence of gram negative bacteria , related to enterobacteriaceae , in causing uti has many factors which are responsible for their attachment to the uroepithelium . in females of all age categories , e. coli is the most frequently isolated uropathogen which correlates with other studies [ 7173 ] but not with others which found that e. coli causes most male utis , followed by other enterobacteriaceae and enterococci [ 74 , 75 ] whereas proteus mirabilis was more frequently isolated in the younger female patients of uti and k. pneumoniae in the elderly patients . the sensitivity rate of carbepenems among uropathogens was as follows : e. coli ( mrp ; 95.45% and imp ; 98.89% ) , p. aeruginosa ( mrp ; 100% and imp ; 95.00% ) , proteus spp . ( mrp ; 100% and imp ; 100% ) , and s. aureus ( mrp ; 80% and imp ; 100% ) , followed by lev and ntl each of which showed 74.84% sensitivity , however , k. pneumonia did not show a high susceptibility to imp ( 24.14% ) but it was susceptible to mrp ( 86.21% ) . a study done in king fahd hospital , saudi arabia showed that meropenem was 95.8% sensitive followed by amikacin ( 93.7% ) and imipenem ( 91.71% ) against extended spectrum lactamase producing e. coli . tested fluoroquinolones in this study showed the highest resistance among uropathogens as in e. coli ; nal ( 90.91% ) : k. pneumoniae ; cip ( 79.31% ) , p. aeruginosa ; spr ( 100% ) , and s. aureus ; nal ( 100% ) ; however , iii generation cephalosporin showed the highest resistance in k. pneumoniae ; ctz ( 79.31% ) the proteus spp . another study done in spain also showed the reduced susceptibility of e. coli isolates from patients with uti to fluoroquinolones ( 16% ) . in several studies it has been shown that the highly prescribing habits of the physicians are the driving factor for the antibiotic resistance for this group of antibiotic [ 8385 ] . found that 37% of physicians actually prescribe trimethoprim - sulphamethoxazole closely followed by fluoroquinolones ( 32% ) and the average duration of antibiotic therapy is 8.6 days in the united states which is the best example of this problem ; empiric use of fluoroquinolones should be restricted and founding the strategies against increasing resistance of pathogens to these antibiotics should be done . the alarming finding in this study is the resistance to third - generation cephalosporin ; the highest resistance was seen against ctz ( 71.61% ) followed by ctx ( 67.74% ) among all uropathogens . the other possible explanation behind this situation is that the iii generation cephalosporin has been in use for a long period and must have been abused and over time organisms have developed resistant mechanisms due to changing their mode of action . against the background of paucity of reports of uti in meerut city ( uttar pradesh ) , india , this is the first study conducted to determine the prevalence of uti , the effect of gender and age on its prevalence , and their susceptibility profile in the community of meerut city . this study provides valuable laboratory data to monitor the status of antimicrobial resistance among uropathogens and to improve treatment recommendations in a specific geographical region .

no clear etiology has been associated with most ependymomas to date . unlike many other cancers for which existing familial cancer syndromes provided important clues for our initial understanding of tumorigenic mechanisms , there are few known familial ependymoma syndromes . we do know , however , that there is increased incidence of spinal intramedullary ependymomas in patients with neurofibromatosis type 2 ( nf2),.the nf2 gene is located on chromosome 22q , which is frequently lost in patients with spinal ependymomas. however , many of these tumors , especially those that occur intracranially , do not harbor nf2 mutations . thus , despite that the nf2 gene may be important in the formation of some spinal ependymomas , it is probably not the critical tumor suppressor gene on chromosome 22q that is involved in sporadic intracranial ependymoma tumorigenesis. ependymoma has also been reported in patients with li - fraumeni syndrome , i.e. germline mutation of the tp53 tumor suppressor gene , but such occurrences as well as somatic mutations of tp53 in sporadic ependymomas are rare , thus diminishing the role of p53 in ependymoma tumorigenesis . there has been one report of a patient with turcot syndrome , i.e. germline mutation of the adenomatous polyposis coli ( apc ) gene , whose loss of function activates the wnt pathway and predisposes the patient to colorectal cancer , who developed multiple ependymomas located intracranially and spinally , . both intracranial and spinal ependymomas have also been observed in patients with the multiple endocrine neoplasia type i ( men1 ) syndrome. however , the role of the apc gene / wnt signaling activation and that of men1 in sporadic ependymoma tumorigenesis remain unknown . furthermore , there are a few families with increased ependymoma incidence but without any currently known familial cancer syndromes. two such families have loss of 22q but lack nf2 mutation , further suggesting the presence of another crucial tumor suppressor gene at that chromosomal region , . dna sequences similar to sv40 virus and the virus - encoded large t - antigen have also been found in some ependymomas. furthermore , ependymoma can be induced in rodents through intracerebral inoculation of the sv40 virus , . nevertheless , several studies have disqualified the sv40 tumor virus as a causative agent of ependymoma. the strongest opposing argument is based on epidemiologic studies that showed no increase in the incidence of ependymoma and other cancers in the years following the massive introduction of sv40-contaminated polio vaccines into the human population , . to date , knowledge of whether sv40 virus contributes to ependymoma tumorigenesis remains unknown ; and if it does , the oncogenic pathways on which it acts remain to be elucidated . over the years , cytogenetic studies using karyotyping and comparative genomic hybridization ( cgh ) have reported numerous broad chromosomal abnormalities in ependymoma . results varied considerably among early studies largely due to their small sample sizes and the variations among sample sets in terms of patient age and anatomical tumor location . in fact , increasing evidence supports that ependymomas are heterogeneous and can be classified as distinct disease subtypes based on patient age , anatomical tumor location , and genetic alterations , , . frequently observed genomic anomalies in pediatric ependymomas include loss of chromosomes 1p , 2 , 3 , 6/6q , 9p , 13q , 17 , and 22 as well as gains of 1q , 5 , 7 , 8 , 9 , 11 , 18 , and 20 , with the gain of 1q occurring in over 20% of cases being the most common . in adult ependymomas , chromosomes 6 , 10 , 13q , 14q , 16 , and 22/22q are frequently lost whereas chromosomes 2 , 5 , 7 , 9 , 12 , 18 , and x are gained , with gains of 7 and 9 and loss of 22q being the most frequently observed , though with each occurring in only 30% of cases or less . location - specific genomic anomalies observed in intracranial versus spinal ependymomas roughly correspond to those seen in children versus adults , as most pediatric ependymomas occur intracranially whereas adult cases predominantly occur within the spinal cord . aside from genomic gains and losses , cytogenetic studies have also identified translocations within the ependymoma genome , often involving chromosomes 1 , 11 , and 22. adult ependymomas have been observed to display more frequent and broader chromosomal aberrations than pediatric tumors . based on a meta - analysis of all cgh studies performed on more than 300 primary ependymomas , kilday et al . calculated that there are on average 7.5 and 3.8 genomic anomalies per adult and pediatric tumor , respectively . this finding is reinforced given that over 40% of pediatric ependymomas exhibit balanced genetic profiles , whereas a balanced genomic profile is observed in less than 10% of adult cases , , . interestingly , the large number of genomic aberrations often seen in adult spinal ependymomas is associated with tumors of lower histological grades and favorable patient outcome , , . furthermore , according to the cgh analysis done by dyer et al . on pediatric intracranial ependymomas , tumors can be subdivided into three distinct subgroups based on the number of chromosomal anomalies detected per tumor . tumors with a balanced genetic profile make up the balanced group , which is significantly associated with an infant age at diagnosis . the second structural group shows few and mainly partial genomic imbalances . lastly , the third numerical group exhibits 13 or more primarily whole chromosome imbalances similar to those often seen in adult ependymomas . these subdivisions are significantly associated with prognosis , with the numerical group demonstrating the best patient outcome and the structural group doing the worst . consistent with this observation , almost all recurrent ependymomas exhibit genetic profiles characteristic of the structural group , , . despite the identification of the aforementioned common genomic gains and losses in ependymomas and their cytogenetic profile - based stratification , few insights into the oncogenes , tumor suppressors , and molecular pathways responsible for the development of ependymoma could be obtained from these findings . furthermore , specific genetic events could not be identified as prognostic markers for this disease at only chromosome - level resolution . these chromosome - level aberrations are broad and typically span numerous genes , making it difficult to discriminate driver genetic events from passenger events . recently , array cgh ( acgh ) has been adopted by the research community to fine - map copy number variations in cancer at much higher resolutions . the list of genes within the common regions of amplification or deletion identified using acgh can be further narrowed through correlation with their expression levels . this permits the discovery of candidate driver genes for ependymoma development , with putative oncogenes and tumor suppressor genes exhibiting copy number - driven expression . indeed , the advances in microarray and next generation sequencing technologies have permitted the examination of ependymoma genetics in terms of copy number variations and gene expression levels in much greater detail . irrespective of anatomical tumor location or patient age , monosomy 22 and allelic losses on chromosome 22q have been found in numerous studies to be the most common genetic abnormalities in sporadic ependymoma , with frequencies ranging from 26 % to 71% , . initial quests for tumor suppressor genes present on 22q focused on nf2 located at 22q12 ; however , nf2 mutation is not associated with the majority of ependymoma cases.another potential tumor suppressor gene is hsnf5/ini1 at 22q11.23 . kraus et al . found no mutations or homozygous deletions of this gene in a series of 53 ependymomas , and this gene has not been shown to be silenced by dna promoter methylation . mapping of deletions and translocation breakpoints on 22q using high - resolution techniques revealed 22pter22q11.2 , 22q11 , 22q11.2112.2 , and 22q13.113.3 to be the hotspots where the elusive tumor suppressor gene is likely to be found,. within the frequently deleted region 22q12.3q13.33 , karakoula et al . found rac2 and c22orf2 to be deleted in 38% and 32% of the 47 pediatric intracranial ependymomas analyzed , respectively . in over 60% of these ependymomas , c22orf2 was found to be transcriptionally inactive , indicating its potential importance in the development of pediatric intracranial ependymomas . loss of rac2 , on the other hand , was shown to be a prognostic factor significantly associated with shorter overall survival in patients younger than 2 years . using gene expression microarray technology , suarez - merino et al . found the transcripts of four genes mapping to 22q12.322q13.33 , namely c22orf2 , as identified by karakoula et al . mentioned above , fbx7 , cbx7 , and sbf1 , to be under - expressed in pediatric ependymomas as compared to normal brain controls . allelic loss of one of these genes , cbx7 located at 22q13.1 , could be detected in 55% of ependymoma cases . interestingly , cbx7 controls cellular lifespan through regulating both the p16/rb and the arf / p53 pathways . the role of these pathways in ependymoma is unclear , though their deregulation is central to many types of cancer , including gliomas . furthermore , deletion and hypermethylation of cdkn2a / p16 at 9q21.3 and rb at 13q14.2 have been reported in ependymomas. in the same study by suarez - merino et al . schip-1 is known to interact with the nf2 gene product merlin , and their interaction is regulated by conformational changes in merlin induced by post - translational modifications , alternative splicing , or mutations . furthermore , by integrating the genomic and expression profiles of 24 primary intracranial ependymomas , modena et al . identified down - regulation of the sult4a1 gene located at 22q13.3 . in pediatric intracranial ependymomas , importantly , this genetic aberration is preferentially associated with tumors in the posterior fossa location in children and with anaplastic histological features . it is also a significant predictor of tumor aggressiveness and poor patient outcome,,,,,. interestingly , 1q gain is occasionally the only observable alteration , with few other chromosome imbalances detected in ependymomas , , , ; yet , in some cases , it marks tumor recurrence , . this suggests the presence of genes located on 1q that may be involved in the initiation , progression , and/or therapeutic resistance of ependymoma . thus , efforts have been made to determine the critical region on chromosome 1q for the identification of these crucial genes . reported a minimal overlapping region with high - copy amplification at 1q2431 in pediatric ependymomas . subsequently , an acgh study done on 49 sporadic intracranial ependymomas by mendrzyk et al . identified two commonly gained regions on 1q , one at 1q21.323.1 and another at 1q31.131.3 . they also found that gains of 1q21.132.1 were correlated with tumor recurrence and identified the gain of 1q25 as an independent prognostic marker for significantly lower recurrence - free or overall survival rate . additionally , they identified dusp12 , found to be over - expressed in all their tested samples , as a candidate gene located at 1q23.3 . the mrna level of dusp12 correlates with that of cyclin d1 throughout the cell cycle , suggesting its role in regulating cell division and potentially in neoplastic transformation , . dusp12 was also found to be important for cell survival in response to heat - shock - induced cell death , which further supports its proposed oncogenic function . gene expression analyses correlated with copy number variations have since uncovered additional candidate oncogenes located within 1q2132 , including laminin , prelp , hspa6 , gac1 , chi3l1 , tpr , jtb , shc1 , and s100a10 and other s100 family members , , , , . among these , gac1 amplification - driven over - expression has also been implicated in the pathogenesis of other malignant gliomas , suggesting its likely importance in ependymoma development . in addition to chromosome 22q loss and 1q gain , other commonly identified chromosomal aberrations include deletion of chromosomes 6q and 9 and gain of chromosome 7 , notably the region from 7q11.2322.1 , which is associated almost exclusively with spinal ependymomas , , . candidate oncogenes proposed by analyzing recurrent gains on chromosome 7 include egfr ( epidermal growth factor receptor ) at 7p11.2 , twist1 and hdac9 at 7p21.1 , and arhgef5 at 7q34 , egfr in particular exhibits frequent gains and high - level amplifications in intracranial ependymomas , and its over - expression predicts poor patient outcome . loss of chromosome 6q is found mostly in infratentorial tumors , whereas deletions on chromosome 9 occur more frequently in supratentorial tumors , , , . with microsatellite analysis , loh hotspots on chromosome 6 were determined to be 6q1516 , 6q2122.1 , and 6q24.325.3 , which were further limited to 6q24.3 and 6q25.225.3 , . locus 6q25.3 , containing the snx9 and synj2 genes , was found to be the most frequently deleted . however , loss of 6q25.3 was a favorable prognostic marker for overall survival of patients with anaplastic intracranial ependymomas , as the deletion of the snx9 and synj2 genes , which are known to regulate cell migration and invasion , could inhibit tumor progression . furthermore , the polyamine biosynthesis gene amd1 and the cyclin - dependent kinase cdk11 , both located at 6q21 , as well as the tumor suppressor gene sash1 at 6q24.3 were found to be under - expressed by suarez - merino et al . which is also frequently deleted in patients with ependymomas , homozygous deletion spanning the cdkn2a locus at 9q21.3 has been detected and is a characteristic of anaplastic supratentorial tumors , the molecular staging system developed by korshunov et al . highlighted that cdkn2a deletion together with young age at diagnosis and gain of 1q comprise the most reliable independent indicators of unfavorable patient outcome . in contrast , gains of chromosomes 9 , 15q , and 18 and loss of chromosome 6 are features indicating excellent chance of survival . furthermore , detection of the expression of p14 protein by immunohistochemistry in 103 intracranial ependymomas revealed that decreased p14 expression is associated with tumor aggressiveness in terms of higher tumor grade , elevated growth fraction , and p53 protein accumulation . closely examined the aberrations on chromosome 9 in both adult and pediatric ependymomas and identified 9p21.122.3 and 9q31.333.2 to be the potential tumor suppressor genes located within 9q31.333.2 include dbc1 , which is frequently deleted in bladder cancer and also exhibits markedly reduced mrna expression in gliomas , ; dec1 , whose down - regulation driven by copy number loss is frequently seen in esophageal cancer and contributes to tumor cell motility , ; lpar1 , which is known to mediate cell proliferation , differentiation , and migration among other functions ; and txn , which inhibits apoptosis and enhances drug resistance in cancer cells , . other frequently occurring regions of genomic imbalances have been revealed by profiling the ependymoma genome at high resolution,,,,, and are summarized in table 1 . among these imbalances , the combined presence of 6p22-pter and 13q14.3-qter losses predicted significantly reduced survival in intracranial pediatric ependymomas . puget et al . found that gains of 1q and 9qter and loss of 6q occurred more often in recurrent tumors . interestingly , the specific 9qter region linked to tumor recurrence is associated with posterior fossa ependymomas , whereas chromosome 9 deletion is usually associated with supratentorial ependymomas . candidate oncogenes and tumor suppressor genes proposed based on these copy number variation hotspots,,, are listed in table 2 . among the putative oncogenes in ependymoma are notch1 , notch4 , and jag1 , which are two of the membrane receptors and one of the ligands , respectively , of the notch signaling pathway , suggesting the involvement of notch signaling in ependymoma tumorigenesis . furthermore , recurrent gains at 5p15.33 , which includes the human telomerase reverse transcriptase ( htert ) gene , were validated by immunohistochemistry . elevated htert expression has been shown to be associated with ependymoma progression and recurrence and is currently the most important predictor of survival for pediatric intracranial ependymomas independent of other clinicopathologic prognostic features,. furthermore , htert expression relates with telomerase activity . recently , wong et al . proposed telomerase inhibition as a novel therapy for ependymoma after demonstrating its effects on reducing ependymoma cell viability by increasing dna damage , decreasing proliferation , and increasing apoptosis . in addition to fine - mapping genomic aberrations to identify candidate genes involved in ependymoma development , profiling studies have also been used to divide ependymomas into distinct subgroups that correlate with tumor location . categorized these tumors into three molecularly distinct subgroups that correlate with the anatomical location of the tumor , namely the supratentorial region , the posterior fossa , or the spine . although ependymomas from these different anatomical regions are histologically indistinguishable , they are in fact molecularly distinct diseases that should be separately examined to determine the genetic events involved in tumorigenesis and progression , as well as prognostic factors and patient outcome . according to the results of their acgh experiment , taylor et al . found that cdkn2a deletion occurred in > 90% of supratentorial ependymomas but was rare in tumors from other regions of the central nervous system ( cns ) . furthermore , posterior fossa ependymomas could be further classified into three subgroups : tumors harboring multiple concurrent dna amplifications , tumors with chromosome 1q gain , and tumors exhibiting a balanced genomic profile . although acgh analyses have considerably advanced our understanding of the genetic events in ependymoma tumorigenesis , almost half of ependymomas present a balanced acgh profile , making it imperative to interrogate alternative mechanisms of gene regulation . epigenetics in the form of promoter dna ( cpg ) hypermethylation is an important route by which transcriptional inactivation can be achieved and , as in other cancers , likely plays a significant role in silencing tumor suppressor genes involved in ependymoma development . unfortunately , epigenetic studies on ependymoma have been limited to candidate gene approaches , with the genes in question selected based on their roles as tumor suppressor genes and methylation status in other malignancies . therefore investigated the methylation status of the hypermethylated in cancer 1 ( hic-1 ) putative tumor suppressor gene , which exhibits hypermethylation and loss of expression in various tumors such as medulloblastoma and gliomas . they detected hic-1 hypermethylation and down - regulation in 83% and 81% of ependymomas , respectively , and found that hic-1 hypermethylation was significantly correlated with nonspinal localization and pediatric age . the ras association domain family 1 isoform a ( rassf1a ) gene has also been found , independent of clinical and histological subtype , to be frequently silenced by methylation in ependymoma , with an incidence of 86% . rassf1a is a recently well - recognized tumor suppressor gene whose inactivation through promoter methylation is implicated in the development of many human cancers . rna interference experiments have shown that down - regulation of rassf1a , an effector of ras , results in loss of cell cycle control , enhanced genetic instability and cell motility , and resistance to k - ras and tumor necrosis factor ( tnf)-induced apoptosis . identified the trail apoptosis pathway - related genes casp8 , tfrsf10c , tfrsf10d , and tnfrsf10c to be methylated in ependymoma , with incidences of 30% , 9.5% , 36.4% , and 9.5% , respectively . other commonly methylated genes in ependymoma include dapk , thbs1 , timp3 , tp73 , mgmt , gstp1 , cdkn2a , fhit , rarb , blu , and mcj , with incidence ranging from 10% to 57% , , , , . gene expression profiling employs microarray technology to capture gene expression levels of thousands of genes simultaneously . integration of gene expression with copy number data allows one to determine the genes demonstrating copy number - driven expression as putative oncogenes and tumor suppressor genes . moreover , by applying ontological analysis on the gene expression profiles , it is possible to uncover those aberrant cellular processes and pathways that contribute to ependymoma . using microarray - based gene expression profiling to compare ependymoma with normal brain controls , suarez - merino et al . identified 112 abnormally expressed genes in ependymoma . genes with increased expression included the oncogene wnt5a , tp53 homologue tp63 , and several cell cycle , proliferation , adhesion , and extracellular matrix genes such as the transcription factor zic1 , the angiogenesis factor vegf , and fibronectin 1 ( fn1 ) . other putative oncogenes identified in this study that have been implicated in other cancers are col4a1 , ibp2 , hox7 , wee1 , and gac1 . genes that were found to be down - regulated included the a / f2-interacting gene schip-1 , the apc - associated gene eb1 , and genes that are involved in vesicle trafficking and recycling such as npc1 , rab40b , tj2 , and sh3gl3 . consistent with ependymoma subgroups based on acgh profiles , ependymoma gene expression profiles are significantly associated with tumor location , patient age at disease onset , grade , and retrospective risk for relapse , , , . found that supratentorial ependymomas expressed markedly elevated levels of members of the ephb - ephrin ( ephb2/3/4 and ephrin a3/4 ) and notch ( jagged 1/2 ) signaling pathways , as well as genes involved in cell cycle regulation ( cyclin b2/d1/g2 , cdk2/4 , and cdkn1c/2c ) . on the other hand , the highly expressed genes that distinguished posterior fossa ependymomas were inhibitors of differentiation ( id1/2/4 ) and members of the aquaporin family ( aqp1/3/4 ) . spinal ependymomas are characterized by the up - regulation of multiple homeobox ( hox ) family members ( hoxa7/9 , hoxb6/7 , and hoxc6/10 ) and insulin - like growth factor 1 ( igf1 ) . subsequently , gene expression profiling studies performed by modena et al . and palm et al . confirmed that intracranial ependymomas are indeed characterized by high expression levels of genes involved in notch signaling and that spinal ependymomas are defined by over - expression of numerous hox genes . additionally , up - regulation of the sonic hedgehog ( shh ) and bone morphogenetic protein ( bmp ) pathway members were also evident in intracranial ependymomas , . deregulated notch signaling , which is crucial for neural development , is believed to play a significant role in ependymoma tumorigenesis , especially at the supratentorial location , since oncogenesis is thought to mirror normal development gone awry . in addition to over - expression of the notch ligands jagged 1/2 shown by taylor et al . , there is consistent up - regulation of the notch receptors ( notch1/2 ) , ligands ( jagged 1/2 and dll1/3 ) , and target genes ( hes1/5 , hey2 , c - myc , and erbb2 ) , whereas fbxw7 , the major repressor of the notch pathway , is consistently down - regulated , , , . in an early study , missense mutations of notch1 , either in the heterodimerization domain c or the transactivation domain , were detected in 8.3% of pediatric intracranial ependymomas from the posterior fossa , thus making notch1 the first oncogene found to be mutated in ependymomas . these mutations cause the notch1 receptor to be constitutively active in a ligand - independent manner . moreover , inhibition of notch signaling with -secretase inhibitor gsi - ix impaired ependymoma primary cell culture growth . gilbertson et al . further demonstrated that high - level expression of erbb receptors ( erbb2/4 ) , which are direct targets of notch signaling , could be found in over 75% of pediatric ependymomas and were significantly correlated to tumor proliferative activity as measured by the ki-67 labeling index . functional studies proved that activating erbb receptor signaling in short - term ependymoma cell cultures resulted in akt phosphorylation and cell proliferation , which could be effectively blocked in a dose - dependent manner with an inhibitor of erbb2 tyrosine kinase activity . coincidentally , we have recently learned that the sv40 virus , which was thought to be a causative agent for ependymoma , can in fact induce oncogenic transformation of human mesothelial cells through direct induction of notch1 over - expression . currently , neither cell lines nor animal models are available to elucidate the sequential events in ependymoma development . thus , researchers have compared the gene expression profiles of low grade versus high grade ependymomas and primary tumors versus recurrent tumors to better understand the molecular genetics of ependymoma progression . palm et al . revealed that who grade 3 anaplastic ependymomas differed from grade 2 tumors by the over - expression of genes implicated in wnt/-catenin signaling activation , cell cycle regulation / cell proliferation ( cyclin - dependent kinases cdk2/4 , cell division cycle proteins cdc25a/25b/25c/2 , and minimal chromosome maintenance proteins mcm2/3/5/6/7 ) , apoptosis ( tumor necrosis factor super family members tnfrsf11a/21 and caspases casp1/4 ) , angiogenesis ( vegf , vegfr2 , vegfb , tnip2 , and doc2 ) , and remodeling of adherens junctions through e - cadherin destruction ( met , mn23h1 , caveolin , rab5/7 gtpases ) , as well as up - regulation of the transcription factors e2f1 and dp1 ( tfdp1 ) . wnt signaling activation in grade 3 ependymomas is indicated by the over - expression of wnt ligand ( wnt11 ) , frizzled receptors ( fzd2/5/8 ) , and dishevelled genes ( dvl2/3 ) . furthermore , increased expression was detected for -catenin ( ctnnb1 ) and its associated transcription factor tcf3 and the wnt target genes birc5 , ccnd1 , fosl1 , c - myc , and tp53 . similarly , to comprehend the molecular mechanisms underlying ependymoma recurrence , peyre et al . performed a dual - color gene expression microarray analysis on 17 tumors at diagnosis co - hybridized with 27 corresponding tumors at first or subsequent relapses they identified 87 genes collectively as the expression signature of ependymoma recurrence . like the gene expression characteristics of high grade ependymomas noted by palm et al . , the signature of ependymoma recurrence was also marked by wnt pathway activation with over - expression of sfrp1 , sfrp2 , fzd2 , fzd8 , and wnt10b . other frequently over - expressed genes in recurrent ependymomas included cd133 , members of the notch signaling pathway , and genes involved in the kinetochore ( kif14 , kif11 , kif1c , kif2c , prc1 , bub1b , zwint , aspm , kntc2 , and cenpf ) . the genes that were significantly down - regulated were metallothionein genes ( mt1l , mt1 g , mt1e , mt1x , mt1b , mt2a , mts ) , with reduced expression in up to 80% of recurrences , and genes involved in the immune system ( cxcl5 , cx3cl1 , traf3ip2 , itgbl1 , serping1 , ift20 , entpd3 , hp , and hpr ) . the importance of immune function in hindering ependymoma progression and recurrence was also recognized through the study by donson et al .. their ontological analysis on gene expression profiles from pediatric ependymomas correlated with clinical outcome revealed that the up - regulation of immune function - related genes was associated with non - recurrent ependymomas and a longer time to progression in recurrent ependymomas . in addition , increased infiltration of cd4 t cells were observed by immunohistochemistry in non - recurrent ependymoma samples . furthermore , like the primary ependymomas which can be subgrouped based on location , peyre et al . found that supratentorial versus infratentorial ependymomas showed distinct changes in expression profile at recurrence . recurrent supratentorial ependymomas were characterized by the up - regulation of genes related to cytoskeleton organization ( gelsolin , sema5a , contactin-1 , sarcoglycan , villin - like , scinderin ) and extracellular matrix - cell interactions ( gliomedin , extl1 , galectin-9 , desmuslin , tetranectin , versican , col21a1 , col16a1 , cxcl12 ) , which are functionally involved in the mesenchymal transition . infratentorial ependymoma recurrences , on the other hand , were associated with over - expression of ribosomal protein genes , which are markers of oncogenic transformation in many human tumor types , . one of the key questions to answer in the field of cancer research is to determine the normal cell type that gives rise to a particular malignancy . this is a crucial step towards functionally identifying the successive oncogenic events leading to tumor onset and progression , which would be indispensable for developing targeted therapies and finding keys to prevention . growing evidence suggests that tumor subgroups may arise due to deregulation of cell signaling pathways involved in normal development of different precursor cell populations . thus , the unique gene expression signatures of ependymoma subgroups might provide insight into their cells of origin . indeed , taylor et al . showed that the signature genes which characterize supratentorial , posterior fossa , and spinal ependymomas are expressed in the matching regions in the developing cns of embryonic mice . moreover , many of these signature genes are members of signaling pathways that modulate neural precursor cell proliferation and differentiation in the corresponding regions of the cns , . this confirmed the hypothesis that subgroups of ependymoma either maintain or recapitulate the developmental expression profiles of anatomically restricted progenitor cells , which were then identified to be radial glial cells ( rgcs).taylor et al . further demonstrated that rgcs are likely the cells of origin for ependymoma by isolating a rare population of self - renewing and multipotent cancer stem cells from fresh samples of ependymoma . these cancer stem cells exhibited bipolar morphology resembling rgcs , expressed the rgc immunophenotype cd133/ nestin/ rc2/brain lipid - binding protein ( blbp ) , and were both required and sufficient to recapitulate the original tumor when transplanted into immunocompromised mice . rgcs are a pivotal cell type in the developing cns of all vertebrates and are a specific group of neural stem cells . they serve as ubiquitous precursors that generate neurons and glia , as guide cells for subsequent neuronal migration , and as key elements in patterning and region - specific differentiation of the cns . studies have also shown that ependymal cells , from which ependymoma arises , are derived from rgcs during embryogenesis . genetic mutations in rgcs may therefore lead to their transformation into cancer stem cells of pediatric ependymomas , . since supratentorial ependymomas are characterized by elevated expression of members of the notch and ephb - ephrin signaling pathways , it is likely that over - activation of these pathways may induce neoplastic transformation of rgcs in the cerebral subventricular zone . likewise , up - regulation of the hox family of transcription factors may be involved in spinal ependymoma development by transforming rgcs in the spinal region . furthermore , there is evidence that rg - like cells are present not only during development but also persist in the adult cns , specifically in the subventricular zone and the spinal cord . thus , these rg - like cells may serve as the cells of origin for adult ependymomas. recently , johnson et al . catalogued dna copy number alterations among 204 tumor samples , which is the largest cohort of ependymomas ever examined at the highest resolution . they further generated mrna and microrna expression profiles for 83 and 64 of these tumors , respectively . these profiles segregated ependymomas by cns location and unmasked additional subgroups among supratentorial , posterior fossa , and spinal ependymomas . to test that distinct subgroups of ependymoma might arise due to oncogenic transformation of regionally and developmentally restricted populations of rgcs by characteristic genetic mutations , the gene expression profile of a subset of human supratentorial ependymomas was matched with that of embryonic cerebral rgcs taken from ink4a / arf ( cdkn2a)-null mice , as the cdkn2a locus is frequently deleted from human supratentorial ependymomas . these embryonic cerebral ink4a / arf ( cdkn2a)-null rgcs were first transduced with ephb2 , which has been shown to be focally amplified in a subgroup - specific manner and to exhibit copy number - driven over - expression in supratentorial ependymomas , and were subsequently implanted into the cerebrum of immunocompromised mice . this established the first highly penetrant ( over 70% incidence ) murine allograft model of supratentorial ependymoma that accurately recapitulates the histological features and gene expression profile of the human tumor . comparative gene expression analysis of matched mouse and human tumors revealed deregulation of genes in neural differentiation and maintenance , particularly ion transport and synaptogenesis , thus highlighting the importance of these events in the formation of this ependymoma subgroup . thus , this study provided functional confirmation that ependymoma variants indeed arise from their matched populations of rgcs transformed with the subgroup - specific mutations . over the past decade , research has significantly advanced our knowledge on the molecular genetics of ependymoma . early cytogenetic studies identified broad chromosomal gains and losses , with loss of 22q being the most common . nf2 is recognized as a putative tumor suppressor gene in spinal ependymomas based on mutational analysis and increased incidence of ependymoma in patients with nf2 familial syndrome . it is , however , rarely mutated in pediatric intracranial ependymomas , for which much effort is still being made in identifying the elusive tumor suppressor gene(s ) on chromosome 22q . other common genomic imbalances include gain of 1q and losses of 6q and 9 in intracranial ependymomas , and gain of 7 in spinal ependymomas , among many others . with the advent of acgh technology permitting the identification of genomic imbalances at much greater resolution , it became possible to uncover putative oncogenes and tumor suppressor genes , such as htert and cdkn2a , respectively , by testing candidates found in focal regions of amplifications and deletions for copy number - driven expression . importantly , over the years , ependymoma tumor heterogeneity has become progressively more appreciated at the genetic level and can be subgrouped based on chromosomal abnormalities , acgh , and , recently , gene expression profiles . it is now generally recognized that ependymomas from different regions of the cns , i.e. the supratentorium , posterior fossa , and the spinal cord , are genetically distinct diseases marked by unique gene expression signatures , indicating the deregulation of different developmental pathways involved in tumorigenesis . supratentorial ependymomas are characterized by notch and ephb - ephrin signaling , whereas spinal ependymomas show specific over - expression of hox family transcription factors . furthermore , comparing the expression profiles of ependymomas at first diagnosis versus at relapse and at low grade versus high grade revealed that ependymoma recurrence and progression likely result from the up - regulation of wnt signaling and down - regulation of immune function - related genes . recently , rcgs at various locations throughout the cns have been identified to be the cells of origin for the corresponding ependymoma subgroups , as illustrated in figure 1 . the notion that subgroups of ependymoma arise from regionally and developmentally distinct rgcs that have undergone transformation by subgroup - specific genetic mutations was further confirmed functionally in the case of supratentorial ependymomas . despite these achievements in ependymoma research , greater progress is still urgently needed if we are to realize the ultimate goal of improving clinical outcome for patients . with newly developed microarray platforms able to detect copy number changes and gene expressions at even higher resolution , next - generation sequencing technologies and high - throughput techniques for unbiased epigenetic profiling posterior fossa ependymomas in particular deserve our attention , as they frequently occur in children of very young age , and complete surgical resection is often difficult to achieve owing to the involvement of multiple cranial nerves and branches of the vertebrobasilar arterial system at this location . in addition , up to half of posterior fossa ependymomas present a balanced genomic profile , making the identification of genetic events contributing to their tumorigenesis especially challenging . consequently , it is important to examine the genetics of posterior fossa ependymomas at a greater resolution to identify very focal amplifications and deletions , as well as to concentrate on decoding its epigenome . candidate oncogenes and tumor suppressor genes discovered to date should be promptly assessed for their diagnostic and therapeutic potential , with the aim to effectively translate our knowledge from laboratory to clinic . at present , however , ependymoma research is severely hampered by the lack of in vitro and in vivo systems to functionally examine the genetic events identified through acgh and gene expression studies that potentially contribute to ependymoma development . indeed , the identification of rgcs as the cells of origin for ependymoma was a significant breakthrough towards mapping out the pathogenic mechanisms of ependymoma . similar to what has been done for one subset of supratentorial ependymomas , the next step will be to identify the distinct populations of rgcs for all ependymoma variants and functionally determine the subgroup - specific driver mutations necessary for transforming corresponding rgcs to ependymoma . this approach will allow us to functionally identify the key genetic events involved in the initiation and progression of all ependymoma subgroups , as well as to model these subgroups in vitro and in vivo . unlike end - stage tumor samples which provide little information on the chronology and relative importance of the uncovered genetic events in the process of ependymoma pathogenesis , these functional models will be instrumental in deciphering the pathogenic mechanisms of the ependymoma subgroups , as well as in uncovering and verifying potential targets for therapy .

brain tumors are the leading cause of cancer death in children , with ependymoma being the third most common and posing a significant clinical burden . its mechanism of pathogenesis , reliable prognostic indicators , and effective treatments other than surgical resection have all remained elusive . until recently , ependymoma research was hindered by the small number of tumors available for study , low resolution of cytogenetic techniques , and lack of cell lines and animal models . ependymoma heterogeneity , which manifests as variations in tumor location , patient age , histological grade , and clinical behavior , together with the observation of a balanced genomic profile in up to 50% of cases , presents additional challenges in understanding the development and progression of this disease . despite these difficulties , we have made significant headway in the past decade in identifying the genetic alterations and pathways involved in ependymoma tumorigenesis through collaborative efforts and the application of microarray - based genetic ( copy number ) and transcriptome profiling platforms . genetic characterization of ependymoma unraveled distinct mrna - defined subclasses and led to the identification of radial glial cells as its cell type of origin . this review summarizes our current knowledge in the molecular genetics of ependymoma and proposes future research directions necessary to further advance this field .

Familial Syndromes and Risk Factors Cytogenetic Abnormalities Molecular Genetic Aberrations Epigenetics Gene Expression Profiles Cells of Origin of Ependymoma Conclusion and Future Directions

thus , despite that the nf2 gene may be important in the formation of some spinal ependymomas , it is probably not the critical tumor suppressor gene on chromosome 22q that is involved in sporadic intracranial ependymoma tumorigenesis. germline mutation of the tp53 tumor suppressor gene , but such occurrences as well as somatic mutations of tp53 in sporadic ependymomas are rare , thus diminishing the role of p53 in ependymoma tumorigenesis . the strongest opposing argument is based on epidemiologic studies that showed no increase in the incidence of ependymoma and other cancers in the years following the massive introduction of sv40-contaminated polio vaccines into the human population , . results varied considerably among early studies largely due to their small sample sizes and the variations among sample sets in terms of patient age and anatomical tumor location . in fact , increasing evidence supports that ependymomas are heterogeneous and can be classified as distinct disease subtypes based on patient age , anatomical tumor location , and genetic alterations , , . frequently observed genomic anomalies in pediatric ependymomas include loss of chromosomes 1p , 2 , 3 , 6/6q , 9p , 13q , 17 , and 22 as well as gains of 1q , 5 , 7 , 8 , 9 , 11 , 18 , and 20 , with the gain of 1q occurring in over 20% of cases being the most common . in adult ependymomas , chromosomes 6 , 10 , 13q , 14q , 16 , and 22/22q are frequently lost whereas chromosomes 2 , 5 , 7 , 9 , 12 , 18 , and x are gained , with gains of 7 and 9 and loss of 22q being the most frequently observed , though with each occurring in only 30% of cases or less . this finding is reinforced given that over 40% of pediatric ependymomas exhibit balanced genetic profiles , whereas a balanced genomic profile is observed in less than 10% of adult cases , , . these subdivisions are significantly associated with prognosis , with the numerical group demonstrating the best patient outcome and the structural group doing the worst . despite the identification of the aforementioned common genomic gains and losses in ependymomas and their cytogenetic profile - based stratification , few insights into the oncogenes , tumor suppressors , and molecular pathways responsible for the development of ependymoma could be obtained from these findings . recently , array cgh ( acgh ) has been adopted by the research community to fine - map copy number variations in cancer at much higher resolutions . this permits the discovery of candidate driver genes for ependymoma development , with putative oncogenes and tumor suppressor genes exhibiting copy number - driven expression . indeed , the advances in microarray and next generation sequencing technologies have permitted the examination of ependymoma genetics in terms of copy number variations and gene expression levels in much greater detail . irrespective of anatomical tumor location or patient age , monosomy 22 and allelic losses on chromosome 22q have been found in numerous studies to be the most common genetic abnormalities in sporadic ependymoma , with frequencies ranging from 26 % to 71% , . initial quests for tumor suppressor genes present on 22q focused on nf2 located at 22q12 ; however , nf2 mutation is not associated with the majority of ependymoma cases.another potential tumor suppressor gene is hsnf5/ini1 at 22q11.23 . found no mutations or homozygous deletions of this gene in a series of 53 ependymomas , and this gene has not been shown to be silenced by dna promoter methylation . in over 60% of these ependymomas , c22orf2 was found to be transcriptionally inactive , indicating its potential importance in the development of pediatric intracranial ependymomas . schip-1 is known to interact with the nf2 gene product merlin , and their interaction is regulated by conformational changes in merlin induced by post - translational modifications , alternative splicing , or mutations . this suggests the presence of genes located on 1q that may be involved in the initiation , progression , and/or therapeutic resistance of ependymoma . thus , efforts have been made to determine the critical region on chromosome 1q for the identification of these crucial genes . gene expression analyses correlated with copy number variations have since uncovered additional candidate oncogenes located within 1q2132 , including laminin , prelp , hspa6 , gac1 , chi3l1 , tpr , jtb , shc1 , and s100a10 and other s100 family members , , , , . among these , gac1 amplification - driven over - expression has also been implicated in the pathogenesis of other malignant gliomas , suggesting its likely importance in ependymoma development . with microsatellite analysis , loh hotspots on chromosome 6 were determined to be 6q1516 , 6q2122.1 , and 6q24.325.3 , which were further limited to 6q24.3 and 6q25.225.3 , . furthermore , detection of the expression of p14 protein by immunohistochemistry in 103 intracranial ependymomas revealed that decreased p14 expression is associated with tumor aggressiveness in terms of higher tumor grade , elevated growth fraction , and p53 protein accumulation . closely examined the aberrations on chromosome 9 in both adult and pediatric ependymomas and identified 9p21.122.3 and 9q31.333.2 to be the potential tumor suppressor genes located within 9q31.333.2 include dbc1 , which is frequently deleted in bladder cancer and also exhibits markedly reduced mrna expression in gliomas , ; dec1 , whose down - regulation driven by copy number loss is frequently seen in esophageal cancer and contributes to tumor cell motility , ; lpar1 , which is known to mediate cell proliferation , differentiation , and migration among other functions ; and txn , which inhibits apoptosis and enhances drug resistance in cancer cells , . among the putative oncogenes in ependymoma are notch1 , notch4 , and jag1 , which are two of the membrane receptors and one of the ligands , respectively , of the notch signaling pathway , suggesting the involvement of notch signaling in ependymoma tumorigenesis . in addition to fine - mapping genomic aberrations to identify candidate genes involved in ependymoma development , profiling studies have also been used to divide ependymomas into distinct subgroups that correlate with tumor location . although ependymomas from these different anatomical regions are histologically indistinguishable , they are in fact molecularly distinct diseases that should be separately examined to determine the genetic events involved in tumorigenesis and progression , as well as prognostic factors and patient outcome . furthermore , posterior fossa ependymomas could be further classified into three subgroups : tumors harboring multiple concurrent dna amplifications , tumors with chromosome 1q gain , and tumors exhibiting a balanced genomic profile . although acgh analyses have considerably advanced our understanding of the genetic events in ependymoma tumorigenesis , almost half of ependymomas present a balanced acgh profile , making it imperative to interrogate alternative mechanisms of gene regulation . epigenetics in the form of promoter dna ( cpg ) hypermethylation is an important route by which transcriptional inactivation can be achieved and , as in other cancers , likely plays a significant role in silencing tumor suppressor genes involved in ependymoma development . the ras association domain family 1 isoform a ( rassf1a ) gene has also been found , independent of clinical and histological subtype , to be frequently silenced by methylation in ependymoma , with an incidence of 86% . rassf1a is a recently well - recognized tumor suppressor gene whose inactivation through promoter methylation is implicated in the development of many human cancers . identified the trail apoptosis pathway - related genes casp8 , tfrsf10c , tfrsf10d , and tnfrsf10c to be methylated in ependymoma , with incidences of 30% , 9.5% , 36.4% , and 9.5% , respectively . other commonly methylated genes in ependymoma include dapk , thbs1 , timp3 , tp73 , mgmt , gstp1 , cdkn2a , fhit , rarb , blu , and mcj , with incidence ranging from 10% to 57% , , , , . genes that were found to be down - regulated included the a / f2-interacting gene schip-1 , the apc - associated gene eb1 , and genes that are involved in vesicle trafficking and recycling such as npc1 , rab40b , tj2 , and sh3gl3 . consistent with ependymoma subgroups based on acgh profiles , ependymoma gene expression profiles are significantly associated with tumor location , patient age at disease onset , grade , and retrospective risk for relapse , , , . found that supratentorial ependymomas expressed markedly elevated levels of members of the ephb - ephrin ( ephb2/3/4 and ephrin a3/4 ) and notch ( jagged 1/2 ) signaling pathways , as well as genes involved in cell cycle regulation ( cyclin b2/d1/g2 , cdk2/4 , and cdkn1c/2c ) . spinal ependymomas are characterized by the up - regulation of multiple homeobox ( hox ) family members ( hoxa7/9 , hoxb6/7 , and hoxc6/10 ) and insulin - like growth factor 1 ( igf1 ) . deregulated notch signaling , which is crucial for neural development , is believed to play a significant role in ependymoma tumorigenesis , especially at the supratentorial location , since oncogenesis is thought to mirror normal development gone awry . in an early study , missense mutations of notch1 , either in the heterodimerization domain c or the transactivation domain , were detected in 8.3% of pediatric intracranial ependymomas from the posterior fossa , thus making notch1 the first oncogene found to be mutated in ependymomas . further demonstrated that high - level expression of erbb receptors ( erbb2/4 ) , which are direct targets of notch signaling , could be found in over 75% of pediatric ependymomas and were significantly correlated to tumor proliferative activity as measured by the ki-67 labeling index . coincidentally , we have recently learned that the sv40 virus , which was thought to be a causative agent for ependymoma , can in fact induce oncogenic transformation of human mesothelial cells through direct induction of notch1 over - expression . currently , neither cell lines nor animal models are available to elucidate the sequential events in ependymoma development . thus , researchers have compared the gene expression profiles of low grade versus high grade ependymomas and primary tumors versus recurrent tumors to better understand the molecular genetics of ependymoma progression . revealed that who grade 3 anaplastic ependymomas differed from grade 2 tumors by the over - expression of genes implicated in wnt/-catenin signaling activation , cell cycle regulation / cell proliferation ( cyclin - dependent kinases cdk2/4 , cell division cycle proteins cdc25a/25b/25c/2 , and minimal chromosome maintenance proteins mcm2/3/5/6/7 ) , apoptosis ( tumor necrosis factor super family members tnfrsf11a/21 and caspases casp1/4 ) , angiogenesis ( vegf , vegfr2 , vegfb , tnip2 , and doc2 ) , and remodeling of adherens junctions through e - cadherin destruction ( met , mn23h1 , caveolin , rab5/7 gtpases ) , as well as up - regulation of the transcription factors e2f1 and dp1 ( tfdp1 ) . wnt signaling activation in grade 3 ependymomas is indicated by the over - expression of wnt ligand ( wnt11 ) , frizzled receptors ( fzd2/5/8 ) , and dishevelled genes ( dvl2/3 ) . furthermore , increased expression was detected for -catenin ( ctnnb1 ) and its associated transcription factor tcf3 and the wnt target genes birc5 , ccnd1 , fosl1 , c - myc , and tp53 . , the signature of ependymoma recurrence was also marked by wnt pathway activation with over - expression of sfrp1 , sfrp2 , fzd2 , fzd8 , and wnt10b . other frequently over - expressed genes in recurrent ependymomas included cd133 , members of the notch signaling pathway , and genes involved in the kinetochore ( kif14 , kif11 , kif1c , kif2c , prc1 , bub1b , zwint , aspm , kntc2 , and cenpf ) . the genes that were significantly down - regulated were metallothionein genes ( mt1l , mt1 g , mt1e , mt1x , mt1b , mt2a , mts ) , with reduced expression in up to 80% of recurrences , and genes involved in the immune system ( cxcl5 , cx3cl1 , traf3ip2 , itgbl1 , serping1 , ift20 , entpd3 , hp , and hpr ) . recurrent supratentorial ependymomas were characterized by the up - regulation of genes related to cytoskeleton organization ( gelsolin , sema5a , contactin-1 , sarcoglycan , villin - like , scinderin ) and extracellular matrix - cell interactions ( gliomedin , extl1 , galectin-9 , desmuslin , tetranectin , versican , col21a1 , col16a1 , cxcl12 ) , which are functionally involved in the mesenchymal transition . one of the key questions to answer in the field of cancer research is to determine the normal cell type that gives rise to a particular malignancy . this is a crucial step towards functionally identifying the successive oncogenic events leading to tumor onset and progression , which would be indispensable for developing targeted therapies and finding keys to prevention . growing evidence suggests that tumor subgroups may arise due to deregulation of cell signaling pathways involved in normal development of different precursor cell populations . showed that the signature genes which characterize supratentorial , posterior fossa , and spinal ependymomas are expressed in the matching regions in the developing cns of embryonic mice . this confirmed the hypothesis that subgroups of ependymoma either maintain or recapitulate the developmental expression profiles of anatomically restricted progenitor cells , which were then identified to be radial glial cells ( rgcs).taylor et al . rgcs are a pivotal cell type in the developing cns of all vertebrates and are a specific group of neural stem cells . likewise , up - regulation of the hox family of transcription factors may be involved in spinal ependymoma development by transforming rgcs in the spinal region . to test that distinct subgroups of ependymoma might arise due to oncogenic transformation of regionally and developmentally restricted populations of rgcs by characteristic genetic mutations , the gene expression profile of a subset of human supratentorial ependymomas was matched with that of embryonic cerebral rgcs taken from ink4a / arf ( cdkn2a)-null mice , as the cdkn2a locus is frequently deleted from human supratentorial ependymomas . these embryonic cerebral ink4a / arf ( cdkn2a)-null rgcs were first transduced with ephb2 , which has been shown to be focally amplified in a subgroup - specific manner and to exhibit copy number - driven over - expression in supratentorial ependymomas , and were subsequently implanted into the cerebrum of immunocompromised mice . over the past decade , research has significantly advanced our knowledge on the molecular genetics of ependymoma . early cytogenetic studies identified broad chromosomal gains and losses , with loss of 22q being the most common . it is , however , rarely mutated in pediatric intracranial ependymomas , for which much effort is still being made in identifying the elusive tumor suppressor gene(s ) on chromosome 22q . with the advent of acgh technology permitting the identification of genomic imbalances at much greater resolution , it became possible to uncover putative oncogenes and tumor suppressor genes , such as htert and cdkn2a , respectively , by testing candidates found in focal regions of amplifications and deletions for copy number - driven expression . importantly , over the years , ependymoma tumor heterogeneity has become progressively more appreciated at the genetic level and can be subgrouped based on chromosomal abnormalities , acgh , and , recently , gene expression profiles . the supratentorium , posterior fossa , and the spinal cord , are genetically distinct diseases marked by unique gene expression signatures , indicating the deregulation of different developmental pathways involved in tumorigenesis . recently , rcgs at various locations throughout the cns have been identified to be the cells of origin for the corresponding ependymoma subgroups , as illustrated in figure 1 . despite these achievements in ependymoma research , greater progress is still urgently needed if we are to realize the ultimate goal of improving clinical outcome for patients . with newly developed microarray platforms able to detect copy number changes and gene expressions at even higher resolution , next - generation sequencing technologies and high - throughput techniques for unbiased epigenetic profiling posterior fossa ependymomas in particular deserve our attention , as they frequently occur in children of very young age , and complete surgical resection is often difficult to achieve owing to the involvement of multiple cranial nerves and branches of the vertebrobasilar arterial system at this location . in addition , up to half of posterior fossa ependymomas present a balanced genomic profile , making the identification of genetic events contributing to their tumorigenesis especially challenging . at present , however , ependymoma research is severely hampered by the lack of in vitro and in vivo systems to functionally examine the genetic events identified through acgh and gene expression studies that potentially contribute to ependymoma development . indeed , the identification of rgcs as the cells of origin for ependymoma was a significant breakthrough towards mapping out the pathogenic mechanisms of ependymoma . this approach will allow us to functionally identify the key genetic events involved in the initiation and progression of all ependymoma subgroups , as well as to model these subgroups in vitro and in vivo . unlike end - stage tumor samples which provide little information on the chronology and relative importance of the uncovered genetic events in the process of ependymoma pathogenesis , these functional models will be instrumental in deciphering the pathogenic mechanisms of the ependymoma subgroups , as well as in uncovering and verifying potential targets for therapy .

we do know , however , that there is increased incidence of spinal intramedullary ependymomas in patients with neurofibromatosis type 2 ( nf2),.the nf2 gene is located on chromosome 22q , which is frequently lost in patients with spinal ependymomas. thus , despite that the nf2 gene may be important in the formation of some spinal ependymomas , it is probably not the critical tumor suppressor gene on chromosome 22q that is involved in sporadic intracranial ependymoma tumorigenesis. germline mutation of the tp53 tumor suppressor gene , but such occurrences as well as somatic mutations of tp53 in sporadic ependymomas are rare , thus diminishing the role of p53 in ependymoma tumorigenesis . germline mutation of the adenomatous polyposis coli ( apc ) gene , whose loss of function activates the wnt pathway and predisposes the patient to colorectal cancer , who developed multiple ependymomas located intracranially and spinally , . however , the role of the apc gene / wnt signaling activation and that of men1 in sporadic ependymoma tumorigenesis remain unknown . the strongest opposing argument is based on epidemiologic studies that showed no increase in the incidence of ependymoma and other cancers in the years following the massive introduction of sv40-contaminated polio vaccines into the human population , . frequently observed genomic anomalies in pediatric ependymomas include loss of chromosomes 1p , 2 , 3 , 6/6q , 9p , 13q , 17 , and 22 as well as gains of 1q , 5 , 7 , 8 , 9 , 11 , 18 , and 20 , with the gain of 1q occurring in over 20% of cases being the most common . in adult ependymomas , chromosomes 6 , 10 , 13q , 14q , 16 , and 22/22q are frequently lost whereas chromosomes 2 , 5 , 7 , 9 , 12 , 18 , and x are gained , with gains of 7 and 9 and loss of 22q being the most frequently observed , though with each occurring in only 30% of cases or less . location - specific genomic anomalies observed in intracranial versus spinal ependymomas roughly correspond to those seen in children versus adults , as most pediatric ependymomas occur intracranially whereas adult cases predominantly occur within the spinal cord . despite the identification of the aforementioned common genomic gains and losses in ependymomas and their cytogenetic profile - based stratification , few insights into the oncogenes , tumor suppressors , and molecular pathways responsible for the development of ependymoma could be obtained from these findings . initial quests for tumor suppressor genes present on 22q focused on nf2 located at 22q12 ; however , nf2 mutation is not associated with the majority of ependymoma cases.another potential tumor suppressor gene is hsnf5/ini1 at 22q11.23 . gene expression analyses correlated with copy number variations have since uncovered additional candidate oncogenes located within 1q2132 , including laminin , prelp , hspa6 , gac1 , chi3l1 , tpr , jtb , shc1 , and s100a10 and other s100 family members , , , , . among these , gac1 amplification - driven over - expression has also been implicated in the pathogenesis of other malignant gliomas , suggesting its likely importance in ependymoma development . in addition to chromosome 22q loss and 1q gain , other commonly identified chromosomal aberrations include deletion of chromosomes 6q and 9 and gain of chromosome 7 , notably the region from 7q11.2322.1 , which is associated almost exclusively with spinal ependymomas , , . candidate oncogenes proposed by analyzing recurrent gains on chromosome 7 include egfr ( epidermal growth factor receptor ) at 7p11.2 , twist1 and hdac9 at 7p21.1 , and arhgef5 at 7q34 , egfr in particular exhibits frequent gains and high - level amplifications in intracranial ependymomas , and its over - expression predicts poor patient outcome . however , loss of 6q25.3 was a favorable prognostic marker for overall survival of patients with anaplastic intracranial ependymomas , as the deletion of the snx9 and synj2 genes , which are known to regulate cell migration and invasion , could inhibit tumor progression . furthermore , the polyamine biosynthesis gene amd1 and the cyclin - dependent kinase cdk11 , both located at 6q21 , as well as the tumor suppressor gene sash1 at 6q24.3 were found to be under - expressed by suarez - merino et al . which is also frequently deleted in patients with ependymomas , homozygous deletion spanning the cdkn2a locus at 9q21.3 has been detected and is a characteristic of anaplastic supratentorial tumors , the molecular staging system developed by korshunov et al . furthermore , detection of the expression of p14 protein by immunohistochemistry in 103 intracranial ependymomas revealed that decreased p14 expression is associated with tumor aggressiveness in terms of higher tumor grade , elevated growth fraction , and p53 protein accumulation . closely examined the aberrations on chromosome 9 in both adult and pediatric ependymomas and identified 9p21.122.3 and 9q31.333.2 to be the potential tumor suppressor genes located within 9q31.333.2 include dbc1 , which is frequently deleted in bladder cancer and also exhibits markedly reduced mrna expression in gliomas , ; dec1 , whose down - regulation driven by copy number loss is frequently seen in esophageal cancer and contributes to tumor cell motility , ; lpar1 , which is known to mediate cell proliferation , differentiation , and migration among other functions ; and txn , which inhibits apoptosis and enhances drug resistance in cancer cells , . among the putative oncogenes in ependymoma are notch1 , notch4 , and jag1 , which are two of the membrane receptors and one of the ligands , respectively , of the notch signaling pathway , suggesting the involvement of notch signaling in ependymoma tumorigenesis . elevated htert expression has been shown to be associated with ependymoma progression and recurrence and is currently the most important predictor of survival for pediatric intracranial ependymomas independent of other clinicopathologic prognostic features,. although ependymomas from these different anatomical regions are histologically indistinguishable , they are in fact molecularly distinct diseases that should be separately examined to determine the genetic events involved in tumorigenesis and progression , as well as prognostic factors and patient outcome . epigenetics in the form of promoter dna ( cpg ) hypermethylation is an important route by which transcriptional inactivation can be achieved and , as in other cancers , likely plays a significant role in silencing tumor suppressor genes involved in ependymoma development . unfortunately , epigenetic studies on ependymoma have been limited to candidate gene approaches , with the genes in question selected based on their roles as tumor suppressor genes and methylation status in other malignancies . therefore investigated the methylation status of the hypermethylated in cancer 1 ( hic-1 ) putative tumor suppressor gene , which exhibits hypermethylation and loss of expression in various tumors such as medulloblastoma and gliomas . the ras association domain family 1 isoform a ( rassf1a ) gene has also been found , independent of clinical and histological subtype , to be frequently silenced by methylation in ependymoma , with an incidence of 86% . rna interference experiments have shown that down - regulation of rassf1a , an effector of ras , results in loss of cell cycle control , enhanced genetic instability and cell motility , and resistance to k - ras and tumor necrosis factor ( tnf)-induced apoptosis . identified the trail apoptosis pathway - related genes casp8 , tfrsf10c , tfrsf10d , and tnfrsf10c to be methylated in ependymoma , with incidences of 30% , 9.5% , 36.4% , and 9.5% , respectively . other commonly methylated genes in ependymoma include dapk , thbs1 , timp3 , tp73 , mgmt , gstp1 , cdkn2a , fhit , rarb , blu , and mcj , with incidence ranging from 10% to 57% , , , , . genes with increased expression included the oncogene wnt5a , tp53 homologue tp63 , and several cell cycle , proliferation , adhesion , and extracellular matrix genes such as the transcription factor zic1 , the angiogenesis factor vegf , and fibronectin 1 ( fn1 ) . genes that were found to be down - regulated included the a / f2-interacting gene schip-1 , the apc - associated gene eb1 , and genes that are involved in vesicle trafficking and recycling such as npc1 , rab40b , tj2 , and sh3gl3 . consistent with ependymoma subgroups based on acgh profiles , ependymoma gene expression profiles are significantly associated with tumor location , patient age at disease onset , grade , and retrospective risk for relapse , , , . found that supratentorial ependymomas expressed markedly elevated levels of members of the ephb - ephrin ( ephb2/3/4 and ephrin a3/4 ) and notch ( jagged 1/2 ) signaling pathways , as well as genes involved in cell cycle regulation ( cyclin b2/d1/g2 , cdk2/4 , and cdkn1c/2c ) . on the other hand , the highly expressed genes that distinguished posterior fossa ependymomas were inhibitors of differentiation ( id1/2/4 ) and members of the aquaporin family ( aqp1/3/4 ) . spinal ependymomas are characterized by the up - regulation of multiple homeobox ( hox ) family members ( hoxa7/9 , hoxb6/7 , and hoxc6/10 ) and insulin - like growth factor 1 ( igf1 ) . deregulated notch signaling , which is crucial for neural development , is believed to play a significant role in ependymoma tumorigenesis , especially at the supratentorial location , since oncogenesis is thought to mirror normal development gone awry . , there is consistent up - regulation of the notch receptors ( notch1/2 ) , ligands ( jagged 1/2 and dll1/3 ) , and target genes ( hes1/5 , hey2 , c - myc , and erbb2 ) , whereas fbxw7 , the major repressor of the notch pathway , is consistently down - regulated , , , . in an early study , missense mutations of notch1 , either in the heterodimerization domain c or the transactivation domain , were detected in 8.3% of pediatric intracranial ependymomas from the posterior fossa , thus making notch1 the first oncogene found to be mutated in ependymomas . further demonstrated that high - level expression of erbb receptors ( erbb2/4 ) , which are direct targets of notch signaling , could be found in over 75% of pediatric ependymomas and were significantly correlated to tumor proliferative activity as measured by the ki-67 labeling index . functional studies proved that activating erbb receptor signaling in short - term ependymoma cell cultures resulted in akt phosphorylation and cell proliferation , which could be effectively blocked in a dose - dependent manner with an inhibitor of erbb2 tyrosine kinase activity . thus , researchers have compared the gene expression profiles of low grade versus high grade ependymomas and primary tumors versus recurrent tumors to better understand the molecular genetics of ependymoma progression . revealed that who grade 3 anaplastic ependymomas differed from grade 2 tumors by the over - expression of genes implicated in wnt/-catenin signaling activation , cell cycle regulation / cell proliferation ( cyclin - dependent kinases cdk2/4 , cell division cycle proteins cdc25a/25b/25c/2 , and minimal chromosome maintenance proteins mcm2/3/5/6/7 ) , apoptosis ( tumor necrosis factor super family members tnfrsf11a/21 and caspases casp1/4 ) , angiogenesis ( vegf , vegfr2 , vegfb , tnip2 , and doc2 ) , and remodeling of adherens junctions through e - cadherin destruction ( met , mn23h1 , caveolin , rab5/7 gtpases ) , as well as up - regulation of the transcription factors e2f1 and dp1 ( tfdp1 ) . furthermore , increased expression was detected for -catenin ( ctnnb1 ) and its associated transcription factor tcf3 and the wnt target genes birc5 , ccnd1 , fosl1 , c - myc , and tp53 . other frequently over - expressed genes in recurrent ependymomas included cd133 , members of the notch signaling pathway , and genes involved in the kinetochore ( kif14 , kif11 , kif1c , kif2c , prc1 , bub1b , zwint , aspm , kntc2 , and cenpf ) . the genes that were significantly down - regulated were metallothionein genes ( mt1l , mt1 g , mt1e , mt1x , mt1b , mt2a , mts ) , with reduced expression in up to 80% of recurrences , and genes involved in the immune system ( cxcl5 , cx3cl1 , traf3ip2 , itgbl1 , serping1 , ift20 , entpd3 , hp , and hpr ) . the importance of immune function in hindering ependymoma progression and recurrence was also recognized through the study by donson et al .. their ontological analysis on gene expression profiles from pediatric ependymomas correlated with clinical outcome revealed that the up - regulation of immune function - related genes was associated with non - recurrent ependymomas and a longer time to progression in recurrent ependymomas . recurrent supratentorial ependymomas were characterized by the up - regulation of genes related to cytoskeleton organization ( gelsolin , sema5a , contactin-1 , sarcoglycan , villin - like , scinderin ) and extracellular matrix - cell interactions ( gliomedin , extl1 , galectin-9 , desmuslin , tetranectin , versican , col21a1 , col16a1 , cxcl12 ) , which are functionally involved in the mesenchymal transition . infratentorial ependymoma recurrences , on the other hand , were associated with over - expression of ribosomal protein genes , which are markers of oncogenic transformation in many human tumor types , . further demonstrated that rgcs are likely the cells of origin for ependymoma by isolating a rare population of self - renewing and multipotent cancer stem cells from fresh samples of ependymoma . these cancer stem cells exhibited bipolar morphology resembling rgcs , expressed the rgc immunophenotype cd133/ nestin/ rc2/brain lipid - binding protein ( blbp ) , and were both required and sufficient to recapitulate the original tumor when transplanted into immunocompromised mice . since supratentorial ependymomas are characterized by elevated expression of members of the notch and ephb - ephrin signaling pathways , it is likely that over - activation of these pathways may induce neoplastic transformation of rgcs in the cerebral subventricular zone . to test that distinct subgroups of ependymoma might arise due to oncogenic transformation of regionally and developmentally restricted populations of rgcs by characteristic genetic mutations , the gene expression profile of a subset of human supratentorial ependymomas was matched with that of embryonic cerebral rgcs taken from ink4a / arf ( cdkn2a)-null mice , as the cdkn2a locus is frequently deleted from human supratentorial ependymomas . these embryonic cerebral ink4a / arf ( cdkn2a)-null rgcs were first transduced with ephb2 , which has been shown to be focally amplified in a subgroup - specific manner and to exhibit copy number - driven over - expression in supratentorial ependymomas , and were subsequently implanted into the cerebrum of immunocompromised mice . comparative gene expression analysis of matched mouse and human tumors revealed deregulation of genes in neural differentiation and maintenance , particularly ion transport and synaptogenesis , thus highlighting the importance of these events in the formation of this ependymoma subgroup . with the advent of acgh technology permitting the identification of genomic imbalances at much greater resolution , it became possible to uncover putative oncogenes and tumor suppressor genes , such as htert and cdkn2a , respectively , by testing candidates found in focal regions of amplifications and deletions for copy number - driven expression . furthermore , comparing the expression profiles of ependymomas at first diagnosis versus at relapse and at low grade versus high grade revealed that ependymoma recurrence and progression likely result from the up - regulation of wnt signaling and down - regulation of immune function - related genes . with newly developed microarray platforms able to detect copy number changes and gene expressions at even higher resolution , next - generation sequencing technologies and high - throughput techniques for unbiased epigenetic profiling posterior fossa ependymomas in particular deserve our attention , as they frequently occur in children of very young age , and complete surgical resection is often difficult to achieve owing to the involvement of multiple cranial nerves and branches of the vertebrobasilar arterial system at this location . at present , however , ependymoma research is severely hampered by the lack of in vitro and in vivo systems to functionally examine the genetic events identified through acgh and gene expression studies that potentially contribute to ependymoma development . similar to what has been done for one subset of supratentorial ependymomas , the next step will be to identify the distinct populations of rgcs for all ependymoma variants and functionally determine the subgroup - specific driver mutations necessary for transforming corresponding rgcs to ependymoma . this approach will allow us to functionally identify the key genetic events involved in the initiation and progression of all ependymoma subgroups , as well as to model these subgroups in vitro and in vivo . unlike end - stage tumor samples which provide little information on the chronology and relative importance of the uncovered genetic events in the process of ependymoma pathogenesis , these functional models will be instrumental in deciphering the pathogenic mechanisms of the ependymoma subgroups , as well as in uncovering and verifying potential targets for therapy .

fe(ii)-oxidizing bacteria ( feob ) gain energy from the chemical oxidation of fe(ii ) coupled to reduction of oxygen or nitrate or using light energy coupled to reduction of co2 , e.g. , anoxygenic photosynthesis . at the near neutral ph of many surface waters , the oxidation of fe(ii ) for that reason , cyanobacteria , which generate oxygen as a result of oxygenic photosynthesis , can act as indirect fe(ii)-oxidizing bacteria where anoxic and fe(ii)-containing waters encounter to sunlit surface environments . the contribution of cyanobacteria to fe(ii ) oxidation has been quantitatively addressed in fe(ii)-rich hot spring environments and in benthic photosynthetic communities living at the sediment water interface . although the modern oceans are predominantly oxygenated to great depths , promoting the speciation of iron as ferric [ fe(iii ) ] rather than ferrous [ fe(ii ) ] , fe(ii ) may be increasingly mobilized out of sediments and stabilized in the marine water column due to expanding low - oxygen conditions in so - called oxygen minimum zones ( omz ) . where omz intersect with the photic zone , fe(ii ) oxidation by planktonic oxygen - producing cyanobacteria could contribute to the marine iron cycle . furthermore , anoxic and fe(ii)-rich bottom waters are a pervasive feature of oceans in the precambrian era [ before about 500 million years ( my ) ago ] at a time when oxygen was building up in the surface oceans as a result of cyanobacteria and other oxygenic phototrophs . therefore , redox interfaces between anoxic and fe(ii)-containing waters and photosynthetically produced oxygen were likely common throughout much of earth s history . iron redox processes fractionate the naturally occurring isotopes of iron dependent on their mass ( e.g. , fe , fe , fe , and fe ) , such that the quantitative contribution of biotic and abiotic iron cycling at the earth s surface may be recorded in sediments composed of iron - rich minerals . due to the large fractionations between fe(ii ) and fe(iii ) species , fe(ii ) oxidation generally produces a solid iron phase that is enriched in heavy isotopes of iron relative to aqueous fe(ii ) , regardless of the mechanism of oxidation . this makes it difficult to parse the contribution of enzymatic fe(ii)-oxidizing bacteria from abiotic fe(ii ) oxidation , not to mention indirect fe(ii ) oxidation by oxygen - producing cyanobacteria by using iron isotopes . however , subtle differences in the mechanism of oxidation and precipitation , and in the characteristics of the iron minerals or phases ( e.g. , mineralogy , particle size , or presence of impurities ) formed , can influence the overall fractionation between aqueous fe(ii ) and iron minerals . furthermore , the role of cyanobacteria in direct or indirect redox cycling of iron at the cell surface is increasingly recognized and may be associated with distinct isotope fractionation . therefore , detailed mechanistic studies of iron isotope fractionation during different pathways of fe(ii ) oxidation are warranted and may help to define isotopic , mineralogical , or microscopic signatures associated with certain biological processes . furthermore , the isotopic composition of iron minerals is known to be modified by electron and atom exchange between aqueous fe(ii ) and fe(iii ) ( oxyhdr)oxide minerals . these processes have been most effectively characterized under reducing conditions , when a supply of aqueous fe(ii ) is produced by , for instance , microbial fe(iii ) reduction . however , at fe(ii)o2 interfaces with a flux of aqueous fe(ii ) , electron and atom - exchange could also occur on newly formed fe(iii ) ( oxyhydr)oxide minerals . although the effect of some organics as well as si and low ph on blocking electron and atom exchange have been investigated , the effect of cell surfaces and microbially produced organics on this reaction and via blocking sites on fe(iii ) minerals , particularly in an oxidizing system , are not known . in this contribution , we tracked the iron isotope composition of different pools of iron during fe(ii ) oxidation by the marine planktonic cyanobacterium synechococcus pcc 7002 . several prior studies have characterized the interaction of this oxygen - producing strain with fe(ii ) , which gives us a body of work to aid in interpreting the nature of different iron phases in the system , and their mechanism of transformation . additional microscopy and mineral characterization in this study are used to build the picture of how iron is processed during indirect fe(ii ) oxidation resulting from oxygenic photosynthesis . the results have implications for understanding the reactivity of iron minerals as well as identifying isotopic signatures associated with biological activity . synechococcus pcc 7002 was routinely cultivated on ph 6.8 marine phototroph ( mp ) medium containing 6 mg l ferric ammonium citrate as the fe(iii ) source at 24 c under an irradiance of 12.8 mol photons m s from a standard 40w tungsten light bulb as measured by a li-250a light probe ( li - cor , inc . ) . for fe(ii ) fe(ii ) amendments were added from a sterile , anoxic fecl2 stock solution , and the medium was filtered twice through a 0.22 m filter in an anoxic glovebox ( 100% n2 ) , separated by 48 h incubations at 4 c to ensure that all fe(ii ) precipitated as carbonate and phosphate minerals with growth media components were removed . the final fe(ii ) concentration in the medium after filtration was 2 mm as measured by the spectrophotometric ferrozine assay . a log - phase culture of synechococcus pcc 7002 grown with ferric ammonium citrate was degassed for 5 min with sterile n2/co2 ( 90%:10% ) and inoculated into the 2 mm fe(ii)-containing medium to a final concentration of 5 10 cells ml . glass media bottles were acid washed in 1 m hcl for 24 h and then soaked in fresh ultrapure water ( resistivity of 18.2 m cm ) for two successive 24 h treatments before use . all anoxic bottles were closed with butyl rubber stoppers that had been washed in 1 n hcl for 24 h and then thrice boiled in ultrapure water . this concentration of 2 mm fe(ii ) was chosen for experiments because a freshly inoculated culture of synechococcus pcc 7002 took about 10 days to oxidize this , during which time we could sample sufficiently often to have resolution on the evolution of the isotopic composition of different iron pools . despite the fact that this strain grows more slowly at 2 mm fe(ii ) than at lower fe(ii ) concentrations , due to fe(ii ) toxicity , sufficient growth did occur to fully oxidize all fe(ii ) . although this concentration is at the upper end of fe(ii ) concentrations in modern sunlit environments , it is within the range documented for environments where cyanobacteria have been documented as having a role in fe(ii ) oxidation . during fe(ii ) oxidation , which lasted about 10 days , volumes of 2 ml were repeatedly removed with a syringe from the bottles of two contemporaneous replicate experiments ( bottles 1 and 2 ) in an anoxic glovebox . before extracting , the bottles were shaken to yield a homogeneous slurry of iron precipitates . the aliquots were subsequently centrifuged for 10 min at 16 000 g , and the supernatants were filtered through a nylon 0.22 m centrifuge tube filter ( costar spin - x , corning , international ) to yield particle - free aqueous fe(ii ) , henceforth fe(ii)aq . the solids were washed with anoxic ultrapure water to remove any loosely bound iron . a second wash utilized anoxic 0.5 m sodium acetate ( adjusted to ph 4.85 using acetic acid ) to recover sorbed iron ( fenaac ) from the solids ( 24 h incubation in the dark ) . the concentrations of fe(ii ) and total iron in the four different iron fractions were measured with the ferrozine assay . the fe(ii ) in the fe(ii)aq , water wash , and fenaac was stabilized in a final concentration of anoxic 1 m hcl prior to measurements . fe(iii ) was determined as the difference between fe(ii ) measurement and total iron measurements ( after reduction of iron by hydroxylamine hydrochloride ) . purification of the fe(ii)aq , fenaac , and feppt fractions was performed in positively pressured clean laboratories of the isotope geochemistry group at the university of tuebingen under conditions and with reagents that have previously been described . the concentrations of iron in the water washes of feppt were below the detection limit of the ferrozine assay ( < 0.01 mm , 0.56 g ml ) , and so , these samples were not purified . sample aliquots of the separated iron fractions containing 5 g of iron were purified for iron isotope measurements using anion exchange chromatography according to prior methodology . an adequate amount of fe fe double spike was added to the samples prior to fe purification to ensure accurate correction of the instrumental mass bias and possible fe isotope fractionation during anion chromatography caused by the organic matrix of the samples . iron isotope analyses were performed on the thermofisher scientific neptuneplus multicollector inductively coupled plasma mass spectrometer ( mc - icp - ms ) of the isotope geochemistry group of the university of tuebingen . polyatomic interferences , such as arn on fe or aro on fe were resolved using the high mass - resolution mode ( 16 m slit ) . the four iron isotope beams were simultaneously detected with 90 integration cycles at 8 s each during the runs . background corrections for sample signals were based on on - peak - zero measurements on the pure analyte solution ( 0.3 m hno3 ) run before and after each sample . iron isotope data are reported relative to the isotopically certified international reference material irmm-014 ( institute for reference materials and measurements in gent , belgium ) using the -notation : the results are reported in units of per mil ( ) . the reproducibility of the double - spike measuring method as determined by repeated fe measurements of the irmm-014 reference material in between sample runs was 0.00 0.032 ( 2sd ; n = 18 ) . interspersed measurements of our in - house iron standard , hanfe , yielded fe = 0.282 0.039 ( 2sd ; n = 12 ) , which is in excellent agreement with previously published values of 0.28 0.05 ( 2sd ; n = 19 ) and 0.279 0.030 ( 2sd ; ( f ) of fe(ii ) remaining were utilized to determine the isotopic enrichment factor ( fe ) using the following equations:12 fe(ii)aq-0 indicates the fe(ii)aq at the beginning of the experiment . the isotopic fractionation fe ( ) is related to fe by the equation:3 the fitting parameters were determined by minimizing the sum of values . data were also fit by linear regression , with slope and intercept determined by minimizing the sum of values . high energy synchrotron x - ray scattering experiments were performed on the solid , dry products of fe(ii ) oxidation by synechococcus pcc 7002 at the advanced photon source at argonne national laboratory , beamline 11-id - b . 5 mm fe(ii ) , freeze - dried , and washed three times with millipore water to remove excess salt . cells of synechococcus pcc 7002 were grown under similar conditions as described above until the initial ca . mineral aggregates were imaged by confocal laser scanning microscopy ( clsm ; leica spe , mannheim , germany ) . a 635 nm laser was used for excitation autofluorescence of synechococcus pcc 7002 , with a maximum emission peak at 660 nm ( detected range of emission , 640700 nm ) . the fe(iii ) minerals were visualized using the reflection signal of the 488 nm laser . alexa dye conjugates were screened in order to optimize visualization of exopolysaccharides ( eps ) without overlap with the autofluorescence emission maximum of the pigments of synechococcus pcc 7002 ( 660 nm ) . alexa 488 ( maximum emission peak at 520 nm ) was chosen because sba bound to the eps in higher amounts , resulting in brighter fluorescence than the other lectins screened [ wheat germ agglutinin alexa fluor 555 conjugate ( wga-555 ) and lectin pna from arachis hypogaea ( peanut ) , alex fluor 568 conjugate ( pna-568 ) ] . brighter fluorescence at lower laser power was observed with sba-488 , which binds terminal - and -n - acetylgalactosamine and galactopyranosyl residues , compared to wga-555 and pna-568 , which are specific to sialic acid and n - acetylglucosaminyl residues and terminal -galactose , respectively . a turn - on type selective probe for fluorescent labeling of dissolved , sorbed , or ligand - bound fe(iii ) was previously used to visualize the relationship of fe(iii ) synechococcus pcc 7002 cells and minerals from this same incubation . because of spectral overlap , the lectin and fe(iii)-binding probe could not be combined in a single experiment here , and therefore , we compare new results to prior data . the auto - quant deconvolution algorithm implemented in the leica las af software was applied to blind deconvolute the 3d image stacks . the spatial relationships of species detected using fluorescence dyes and cell autofluorescence in clsm image stacks were analyzed using scatterj , a plugin for correlation analysis of species - specific maps for use in imagej and fiji . the fe(ii)aq values from two replicated experiments evolved from an initial value near 0 to lighter values during oxidation ( table 1 , figure 1 ) . the fe(ii)aq fraction , measured after the sample was centrifuged and filtered , consisted of only fe(ii ) . all iron concentration and speciation data ( measured by ferrozine ) the first feppt samples analyzed , at about 40% fe(ii ) oxidized , had feppt of about 2 , trending toward 0 at 100% fe(ii ) oxidized . the speciation of feppt , which was measured after washing with water and sodium acetate , consisted of predominantly fe(iii ) , with generally < 10% fe(ii ) . iron in the water wash of the precipitates was below the detection limit of the ferrozine assay ( < 0.01 mm , 0.56 g ml ) . the sodium acetate wash removed sorbed iron , which contained both fe(ii ) and fe(iii ) . the fenaac fraction represented 1020% of total iron in the system after feppt began to form . the fenaac had an intermediate isotopic composition between fe(ii)aq and feppt but was variable and generally lighter than 0. ( a ) bottle 1 and ( b ) bottle 2 are biological replicates of the fe(ii ) oxidation experiment with synechococcus pcc 7002 . green circles are fe(ii)aq data ; orange squares are feppt data ; blue diamonds are fenaac data . the solid green lines are the rayleigh fits of the fe(ii)aq data , with an fe for fe(ii)aq of 1.79 ( a ) to 2.15 ( b ) . the solid orange lines are the rayleigh fits of the feppt data , with fe for feppt of 2.44 ( a ) and 2.98 ( b ) . samples from b2 and e2 had anomalous values for fenaac and feaq , respectively . these samples were most likely lost after drying due to electrostatic charging of the teflon beakers . the fast reaction between fe(ii ) and oxygen favors the heavy isotopes of iron in the resulting fe(iii ) minerals that precipitate . abiotic and biotic fe(ii ) oxidation both follow this trend , resulting in fe of 24 between aqueous fe(ii ) and fe(iii ) minerals , with minerals enriched in heavy isotopes . our fe for fe(ii)aq ( 1.792.15 for bottles 1 and 2 , respectively ; table 1 ) , determined from a rayleigh fit of the fe(ii)aq data , is on the low end of this range , similar to what was previously documented for fe(ii ) oxidation by anoxygenic phototrophs . the rayleigh fit of the feppt data from both replicates resulted in fe of 2.44 and 2.98 , larger than that attained for the fe(ii)aq data ( table 1 ) and within the literature range . prior explanation for the offset in fe between these two fractions is that , following precipitation , the feppt underwent partial equilibration with another phase of iron in the system , possibly a ligand - bound or sorbed fe(iii ) phase or fe(ii)aq . below , we use our mineralogical and microscopic characterizations of the experiment to explore possible exchange processes in this system . the third , quantitatively significant fraction of iron in the system in addition to fe(ii)aq and feppt was fenaac ( up to 18% of total fe ) . the fenaac data had an intermediate isotopic composition between fe(ii)aq and feppt , from 0.10 to 0.73 throughout the experiment , and contained both fe(ii ) and fe(iii ) ( supplementary table 1 ) . the presence of fe(iii ) in fenaac has previously been observed in fe(ii ) oxidation experiments with anoxygenic phototrophs but not with nitrate - dependent fe(ii)-oxidizing bacteria . the fenaac must have been sorbed onto one of the surfaces , either feppt or cells , on the basis of its extraction with sodium acetate . in order to infer whether equilibration processes were occurring between fenaac and feppt , it is necessary to know ( 1 ) where fenaac was in our experiments and ( 2 ) what type of iron species [ i.e. , fe(ii ) or fe(iii ) ] that it was . our use of a lectin - binding dye in confocal microscopy documents that eps was also forming during fe(ii ) oxidation with synechococcus pcc 7002 ( figure 3 ) . we can use this data set to first determine whether eps was important in binding / sorbing iron extracted as fenaac and then to determine whether iron was associated with the surface of cells and/or feppt . an overlay of figure 3a c , which show the location of cells , eps , and feppt , indicates that eps is colocalized with feppt ( figure 3d ) . the correlation analysis in figure 3e implies there is no spatial overlap of eps with cells . in previous work with synechococcus pcc 7002 under identical growth conditions as in figure 3 , a fluorescent sensor for soluble or ligand - bound fe(iii ) was used in clsm , and fluorescence was localized directly at the synechococccus pcc 7002 cell surfaces . while spectral interferences prevented us from simultaneously labeling eps and fe(iii ) in our current clsm experiments , we can infer from comparing our data set with the previously published one that there was fe(iii ) sorbed to the surface of cells but not eps or feppt . in support of this , eps is expected to stay with the aqueous phase during filtration through a 0.2 m filter or be washed off of feppt in the water wash . we did not detect any fe(iii ) in the fe(ii)aq fraction or measure any detectable iron in the water wash . from these results , we exclude eps as having a major role in binding soluble fe(iii ) in the current system . previous experiments with synechococcus pcc 7002 cells demonstrated that sorption to cells is a major fate for aqueous iron , although the oxidation state of sorbed iron was not determined in those experiments , so we can not rule out that some fe(ii ) was also sorbed to cells . however , sorption onto cells has previously been documented as a fate for aqueous iron with diverse cyanobacteria , with fe(iii ) more commonly detected at the cell surface than fe(ii ) , via attachment of fe o fe polymers to phosphoryl groups , strengthening the inferences made from clsm that synechococcus pcc 7002 cells sorbed fe(iii ) . ( a ) x - ray diffraction ( xrd ) pattern obtained from x - ray total scattering data of the feppt phase after complete fe(ii ) oxidation , freeze - drying , and water washing . the indexed reflections for lepidocrocite ( lp ) and goethite ( gt ) are shown . ( b ) a 3-component linear combination fit of 58% ferrihydrite , 22% goethite , and 20% lepidocrocite ( supplementary table 4 ) . ( a ) autofluorescent cells , ( b ) stained with the lectin - binding dye sba-488 , ( c ) the reflection signal from fe(iii ) minerals , and ( d ) an overlay of ( a c ) . correlation plot of the fluorescence intensity in individual pixels from ( e ) autofluorescence ( a ) vs sba-488 ( b ) and ( f ) sba-488 ( b ) vs fe(iii ) minerals ( c ) . this analysis demonstrates that eps , which is bound by sba-488 , is coating fe(iii ) minerals but is not spatially associated with cells . the other surface in our experiments that could have sorbed iron extracted as fenaac was feppt . the three techniques we used to address mineralogy indicate that our feppt was a mixture of 58% ferrihydrite , 22% goethite , and 20% lepidocrocite ( figure 2 ) , and ferrihydrite was likely the predominant mineral present during the experiments ( see supporting information ) . minerals such as ferrihydrite and goethite , similar to what was present in our experiments , can sorb fe(ii ) . both fe(ii ) and fe(iii ) were detected in the fenaac ( supplementary table 1 ) , raising the possibility that fe(iii ) was extracted from the mineral . however , we verified that no fe(iii ) was extracted from synthetic ferrihydrite with our 0.5 m sodium acetate solution prior to beginning experiments ( data not shown ) , consistent with previous reports that used a 1 m sodium acetate solution . a further inference in support of sorbed fe(ii ) being extracted from feppt by sodium acetate is that feppt still contained some fe(ii ) after extraction , as measured by ferrozine ( supplementary table 1 ) . we take this as evidence that sorbed iron associated with the mineral was predominantly fe(ii ) , although we can not exclude that some fe(iii ) may also be sorbed to the mineral surface . we observed evidence for three reactions in our experiments that are essential for understanding the observed fractionations of iron isotopes , and these are summarized in figure 4 . they are ( 1 ) fe(ii ) oxidation to fe(iii ) , which forms feppt , ( 2 ) sorption of fe(iii ) to cells , and ( 3 ) sorption of fe(ii ) to feppt . these observations fit a two step - model of fe(ii ) oxidation , where fe(ii ) is oxidized and undergoes rapid isotopic equilibration with a pool of fe(iii ) , which then precipitates as fe(iii ) minerals . we suggest , however , that in our experiments , feppt undergoes subsequent partial equilibration with fe(ii)aq . controls on the overall iron isotope fractionation in the system are ( 1 ) fe(ii ) oxidation and precipitation of fe(iii ) as feppt ; ( 2 ) sorption of fe(iii ) to cells ; ( 3 ) partial equilibrium atom and electron exchange after sorption of fe(ii)aq to feppt . ( 1 ) generates feppt ( solid orange line ) that is 23 heavier than fe(ii)aq ( solid green line ) . ( 2 ) produces sorbed fe(iii ) on cells with an estimated equilibrium fefenaac - fe(ii)aq of 1.84. ( 3 ) produces fe(ii ) sorbed on goethite with an estimated fefenaac - fe(ii)aq of 0.8. the resulting fenaac predicted from ( 2 ) and ( 3 ) are denoted by the light blue diamonds . the fitting of our fe(ii)aq and feppt with rayleigh equations representing isolation of the feppt pool from fe(ii)aq after precipitation and a linear equation representing complete isotopic equilibrium are helpful in interpreting the fractionation mechanisms taking place . the larger values for linear fits of all data as compared to rayleigh fits indicate that complete isotopic equilibrium between fe(ii)aq and feppt is not occurring during fe(ii ) oxidation and precipitation ( table 1 ) . the smaller fe values for rayleigh fits of the fe(ii)aq ( 1.79 and 2.15 as compared to 2.44 and 2.98 for feppt ) are on the order of fractionation observed in other biological fe(ii ) oxidation experiments in batch at circumneutral ph : 1.5 , 1.52 , and 12. such small net fractionations have been noted when the presence of significant quantities of sorbed or ligand - bound fe(iii ) has been detected or observed . up to a few percent of total iron was found as fe(iii ) in the fenaac fraction ( supplementary table 1 ) . on the basis of detection of iron sorbed to cells with a dye that is specific for an aqueous or ligand - bound fe(iii ) , we suggest that this fe(iii ) could equilibrate with fe(ii)aq . in experiments with synechococcus sp . cells at ph 6 , added fe(ii ) [ which was adsorbed as fe(iii ) ] was 1.84 heavier than aqueous fe(ii ) , and equilibrium with fe(ii)aq was inferred from the data . this fractionation is very similar to our fe values derived from rayleigh fits of fe(ii)aq ( 1.79 and 2.15 ) . because of the similar type of organism that we used , it is likely that equilibrium fractionation between fe(ii)aq and fe(iii ) sorbed to cells is a relevant process in our experiments . while the feppt data are not well fit by a linear model representing complete equilibrium exchange with fe(ii)aq , the range of the fe determined from rayleigh fits of the two feppt data sets ( 2.44 and 2.98 ) is of the same magnitude expected for equilibrium between feppt and fe(ii)aq . wu et al . inferred a feferrihydrite - fe(ii)aq ( where feferrihydrite - fe(ii)aq = feferrihydrite fefe(ii)aq ) of 3.2 , while beard et al . and frierdich et al . reported a fegoethite - fe(ii)aq of 1.1. considering the 58% ferrihydrite , 22% goethite , and 20% lepidocrocite in our precipitates as determined from x - ray scattering ( figure 2 ) , the equilibrium fefeppt - fe(ii)aq for our minerals could range from 2.3 to 2.7 , depending on whether the assumed fractionation for lepidocrocite is the same as for goethite or ferrihydrite , respectively . the larger fe we calculate for our second data set ( 2.98 ) may reflect that ferrihydrite , with a larger fe value , was likely the mineral present during active fe(ii ) oxidation ( see supporting information ) . in our batch experiments , fe(ii)aq may continue to react with feppt , given their proximity and the time frame of experiments ( 10 days ) . sorption of fe(ii ) on feppt provides a likely mechanism for partial isotope equilibrium and is supported by our detection of fe(ii ) in feppt ( supplementary table 1 ) . sorption of fe(ii ) is an important pathway in recrystallization of fe(iii ) ( oxyhydr)oxide minerals , particularly ferrihydrite . during this process , equilibrium atom and electron exchange occur between sorbed fe(ii ) and fe(iii ) minerals , with complete equilibrium attained within 2 weeks for goethite , for instance ( a similar time frame as our 10 day experiment ) . in this model , fe(ii ) sorbs to fe(iii ) minerals and donates an electron into the bulk mineral structure , adding to the fe(iii ) mineral , and causing the desorption of a newly produced fe(ii ) from the mineral . this is also consistent with the shifts in mineralogy we see during the course of oxidation ( see supporting information ) . we do not see evidence for complete equilibrium between fe(ii)aq and feppt , given the poor linear fit of feppt ( table 1 ) . atom and electron exchange between feppt and sorbed fe(ii ) is expected to be diminished in the presence of organic compounds . our clsm data indicate that eps is colocalized to minerals ( figure 2 ) . it is possible that atom and electron exchange can still occur when fe(iii ) minerals are coprecipitated with organics , as retardation of this process seems to result from blockage of surface sites when organics coat already formed fe(iii ) minerals , or if long chain carbon molecules are present . therefore , we suggest that only partial atom and electron exchange occurred in our system as a result of the eps coating the feppt . during atom exchange , the fractionation when fe(ii)aq sorbs onto goethite varies among experiments . one study reports sorbed fe(ii ) is 0.73 heavier than fe(ii)aq , and another reported sorbed fe(ii ) was 1.24 heavier . crosby et al . directly measured sorbed fe(ii ) extracted with sodium acetate during microbial fe(iii ) reduction experiments , which was just 0.3 heavier than fe(ii)aq for experiments using hematite and up to 0.8 heavier for experiments using goethite as the sorbing surface . our fefenaac - fe(ii)aq ranged from 0.45 to 2.66 , which is much larger and more variable than the experiments of crosby et al . , which may be in part because our fenaac includes both fe(ii ) and fe(iii ) . another factor is that , in our experiments , the less crystalline mineral ferrihydrite was likely the sorbing surface present during experiments ( see supplementary figures 1 and 2 and supporting information ) . several studies have noted a trend of larger fractionations during sorption to less crystalline minerals or higher surface area minerals . the feppt in our experiments had a surface area of 122.1 m g. we calculated fenaac considering that the fe(iii ) fraction of fenaac should be 1.84 heavier than fe(ii)aq due to adsorption of fe(iii ) at cell surfaces , and the sorbed fe(ii ) fraction of fenaac should be at least 0.8 heavier than fe(ii)aq . the calculated fenaac is a good model of our actual fenaac values ( figure 4 ) . this calculation supports the model presented here , in which fe(ii ) is oxidized to fe(iii ) , which is sorbed onto cells and equilibrates with fe(ii)aq , and full equilibration of fe(ii)aq with feppt via atom and electron exchange is hindered by the presence of eps on feppt . our experiments provide evidence that iron isotope fractionation during microbially influenced fe(ii ) oxidation by cyanobacteria is not a simple reaction , controlled only by the abiotic oxidation of fe(ii ) with oxygen and rapid precipitation of fe(iii ) at circumneutral ph . multiple secondary processes generate a significant fraction of sorbed iron that is isotopically distinct from either residual fe(ii)aq or feppt , and subsequent equilibration between the iron pools can further modify the isotopic composition of these phases . our data indicate that sorption of fe(iii ) at cell surfaces likely further fractionates the fe(ii)aq pool . in addition , abiotic sorption of fe(ii ) to fe(iii ) mineral surfaces can also fractionate fe(ii)aq , through equilibrium atom and electron exchange subsequent to fe(ii ) sorption , despite the presence of eps . follow - up experiments could investigate atom and electron exchange in this system . specifically , isotopically enriched fe(ii)aq solutions mixed with preformed cells and minerals would be useful for monitoring atom exchange between the fe(ii)aq and feppt . the processes and phases described here can overprint the anticipated fractionations and compositions of fe(iii ) minerals and organic - associated iron present in the environment and can challenge interpretation of the genesis of fe(iii ) minerals in the geological record , where the residual fe(ii)aq pool is no longer present . although iron atom and electron exchange has received the most attention as a process relevant to fe(iii)-reducing systems where fe(ii ) is in contact with fe(iii ) minerals , our data suggest this process could also be relevant in environments where fe(ii ) is abundant during fe(ii ) oxidation and fe(iii ) mineral formation . this includes oxidizing environments with high enough fluxes of fe(ii ) for fe(ii ) to persist even in the face of rapid oxidation , such as fe(ii)-rich springs or seeps and marine upwelling zones that tap ferruginous bottom waters , past or present . furthermore , our work documents atom and electron exchange in the presence of iron minerals whose formation pathways are biologically induced and when organic phases coat iron minerals . finally , iron redox cycling and sorption of iron at the surface of cyanobacteria may be an important component of modern and ancient aquatic iron cycling , and our work highlights the effect of such processes on iron isotope systematics .

in this study , we couple iron isotope analysis to microscopic and mineralogical investigation of iron speciation during circumneutral fe(ii ) oxidation and fe(iii ) precipitation with photosynthetically produced oxygen . in the presence of the cyanobacterium synechococcus pcc 7002 , aqueous fe(ii ) ( fe(ii)aq ) is oxidized and precipitated as amorphous fe(iii ) oxyhydroxide minerals ( iron precipitates , feppt ) , with distinct isotopic fractionation ( 56fe ) values determined from fitting the 56fe(ii)aq ( 1.79 and 2.15 ) and the 56feppt ( 2.44 and 2.98 ) data trends from two replicate experiments . additional fe(ii ) and fe(iii ) phases were detected using microscopy and chemical extractions and likely represent fe(ii ) and fe(iii ) sorbed to minerals and cells . the iron desorbed with sodium acetate ( fenaac ) yielded heavier 56fe compositions than fe(ii)aq . modeling of the fractionation during fe(iii ) sorption to cells and fe(ii ) sorption to feppt , combined with equilibration of sorbed iron and with fe(ii)aq using published fractionation factors , is consistent with our resulting 56fenaac . the 56feppt data trend is inconsistent with complete equilibrium exchange with fe(ii)aq . because of this and our detection of microbially excreted organics ( e.g. , exopolysaccharides ) coating feppt in our microscopic analysis , we suggest that electron and atom exchange is partially suppressed in this system by biologically produced organics . these results indicate that cyanobacteria influence the fate and composition of iron in sunlit environments via their role in fe(ii ) oxidation through o2 production , the capacity of their cell surfaces to sorb iron , and the interaction of secreted organics with fe(iii ) minerals .

Introduction Experimental Section Results and Discussion

due to the large fractionations between fe(ii ) and fe(iii ) species , fe(ii ) oxidation generally produces a solid iron phase that is enriched in heavy isotopes of iron relative to aqueous fe(ii ) , regardless of the mechanism of oxidation . however , subtle differences in the mechanism of oxidation and precipitation , and in the characteristics of the iron minerals or phases ( e.g. , mineralogy , particle size , or presence of impurities ) formed , can influence the overall fractionation between aqueous fe(ii ) and iron minerals . therefore , detailed mechanistic studies of iron isotope fractionation during different pathways of fe(ii ) oxidation are warranted and may help to define isotopic , mineralogical , or microscopic signatures associated with certain biological processes . furthermore , the isotopic composition of iron minerals is known to be modified by electron and atom exchange between aqueous fe(ii ) and fe(iii ) ( oxyhdr)oxide minerals . however , at fe(ii)o2 interfaces with a flux of aqueous fe(ii ) , electron and atom - exchange could also occur on newly formed fe(iii ) ( oxyhydr)oxide minerals . although the effect of some organics as well as si and low ph on blocking electron and atom exchange have been investigated , the effect of cell surfaces and microbially produced organics on this reaction and via blocking sites on fe(iii ) minerals , particularly in an oxidizing system , are not known . in this contribution , we tracked the iron isotope composition of different pools of iron during fe(ii ) oxidation by the marine planktonic cyanobacterium synechococcus pcc 7002 . several prior studies have characterized the interaction of this oxygen - producing strain with fe(ii ) , which gives us a body of work to aid in interpreting the nature of different iron phases in the system , and their mechanism of transformation . additional microscopy and mineral characterization in this study are used to build the picture of how iron is processed during indirect fe(ii ) oxidation resulting from oxygenic photosynthesis . for fe(ii ) fe(ii ) amendments were added from a sterile , anoxic fecl2 stock solution , and the medium was filtered twice through a 0.22 m filter in an anoxic glovebox ( 100% n2 ) , separated by 48 h incubations at 4 c to ensure that all fe(ii ) precipitated as carbonate and phosphate minerals with growth media components were removed . this concentration of 2 mm fe(ii ) was chosen for experiments because a freshly inoculated culture of synechococcus pcc 7002 took about 10 days to oxidize this , during which time we could sample sufficiently often to have resolution on the evolution of the isotopic composition of different iron pools . although this concentration is at the upper end of fe(ii ) concentrations in modern sunlit environments , it is within the range documented for environments where cyanobacteria have been documented as having a role in fe(ii ) oxidation . the aliquots were subsequently centrifuged for 10 min at 16 000 g , and the supernatants were filtered through a nylon 0.22 m centrifuge tube filter ( costar spin - x , corning , international ) to yield particle - free aqueous fe(ii ) , henceforth fe(ii)aq . a second wash utilized anoxic 0.5 m sodium acetate ( adjusted to ph 4.85 using acetic acid ) to recover sorbed iron ( fenaac ) from the solids ( 24 h incubation in the dark ) . the concentrations of fe(ii ) and total iron in the four different iron fractions were measured with the ferrozine assay . the concentrations of iron in the water washes of feppt were below the detection limit of the ferrozine assay ( < 0.01 mm , 0.56 g ml ) , and so , these samples were not purified . interspersed measurements of our in - house iron standard , hanfe , yielded fe = 0.282 0.039 ( 2sd ; n = 12 ) , which is in excellent agreement with previously published values of 0.28 0.05 ( 2sd ; n = 19 ) and 0.279 0.030 ( 2sd ; ( f ) of fe(ii ) remaining were utilized to determine the isotopic enrichment factor ( fe ) using the following equations:12 fe(ii)aq-0 indicates the fe(ii)aq at the beginning of the experiment . a 635 nm laser was used for excitation autofluorescence of synechococcus pcc 7002 , with a maximum emission peak at 660 nm ( detected range of emission , 640700 nm ) . a turn - on type selective probe for fluorescent labeling of dissolved , sorbed , or ligand - bound fe(iii ) was previously used to visualize the relationship of fe(iii ) synechococcus pcc 7002 cells and minerals from this same incubation . because of spectral overlap , the lectin and fe(iii)-binding probe could not be combined in a single experiment here , and therefore , we compare new results to prior data . the speciation of feppt , which was measured after washing with water and sodium acetate , consisted of predominantly fe(iii ) , with generally < 10% fe(ii ) . the sodium acetate wash removed sorbed iron , which contained both fe(ii ) and fe(iii ) . ( a ) bottle 1 and ( b ) bottle 2 are biological replicates of the fe(ii ) oxidation experiment with synechococcus pcc 7002 . the solid green lines are the rayleigh fits of the fe(ii)aq data , with an fe for fe(ii)aq of 1.79 ( a ) to 2.15 ( b ) . the solid orange lines are the rayleigh fits of the feppt data , with fe for feppt of 2.44 ( a ) and 2.98 ( b ) . the fast reaction between fe(ii ) and oxygen favors the heavy isotopes of iron in the resulting fe(iii ) minerals that precipitate . abiotic and biotic fe(ii ) oxidation both follow this trend , resulting in fe of 24 between aqueous fe(ii ) and fe(iii ) minerals , with minerals enriched in heavy isotopes . our fe for fe(ii)aq ( 1.792.15 for bottles 1 and 2 , respectively ; table 1 ) , determined from a rayleigh fit of the fe(ii)aq data , is on the low end of this range , similar to what was previously documented for fe(ii ) oxidation by anoxygenic phototrophs . the rayleigh fit of the feppt data from both replicates resulted in fe of 2.44 and 2.98 , larger than that attained for the fe(ii)aq data ( table 1 ) and within the literature range . prior explanation for the offset in fe between these two fractions is that , following precipitation , the feppt underwent partial equilibration with another phase of iron in the system , possibly a ligand - bound or sorbed fe(iii ) phase or fe(ii)aq . the fenaac data had an intermediate isotopic composition between fe(ii)aq and feppt , from 0.10 to 0.73 throughout the experiment , and contained both fe(ii ) and fe(iii ) ( supplementary table 1 ) . the presence of fe(iii ) in fenaac has previously been observed in fe(ii ) oxidation experiments with anoxygenic phototrophs but not with nitrate - dependent fe(ii)-oxidizing bacteria . in order to infer whether equilibration processes were occurring between fenaac and feppt , it is necessary to know ( 1 ) where fenaac was in our experiments and ( 2 ) what type of iron species [ i.e. in previous work with synechococcus pcc 7002 under identical growth conditions as in figure 3 , a fluorescent sensor for soluble or ligand - bound fe(iii ) was used in clsm , and fluorescence was localized directly at the synechococccus pcc 7002 cell surfaces . while spectral interferences prevented us from simultaneously labeling eps and fe(iii ) in our current clsm experiments , we can infer from comparing our data set with the previously published one that there was fe(iii ) sorbed to the surface of cells but not eps or feppt . from these results , we exclude eps as having a major role in binding soluble fe(iii ) in the current system . previous experiments with synechococcus pcc 7002 cells demonstrated that sorption to cells is a major fate for aqueous iron , although the oxidation state of sorbed iron was not determined in those experiments , so we can not rule out that some fe(ii ) was also sorbed to cells . however , sorption onto cells has previously been documented as a fate for aqueous iron with diverse cyanobacteria , with fe(iii ) more commonly detected at the cell surface than fe(ii ) , via attachment of fe o fe polymers to phosphoryl groups , strengthening the inferences made from clsm that synechococcus pcc 7002 cells sorbed fe(iii ) . ( a ) x - ray diffraction ( xrd ) pattern obtained from x - ray total scattering data of the feppt phase after complete fe(ii ) oxidation , freeze - drying , and water washing . correlation plot of the fluorescence intensity in individual pixels from ( e ) autofluorescence ( a ) vs sba-488 ( b ) and ( f ) sba-488 ( b ) vs fe(iii ) minerals ( c ) . both fe(ii ) and fe(iii ) were detected in the fenaac ( supplementary table 1 ) , raising the possibility that fe(iii ) was extracted from the mineral . however , we verified that no fe(iii ) was extracted from synthetic ferrihydrite with our 0.5 m sodium acetate solution prior to beginning experiments ( data not shown ) , consistent with previous reports that used a 1 m sodium acetate solution . we take this as evidence that sorbed iron associated with the mineral was predominantly fe(ii ) , although we can not exclude that some fe(iii ) may also be sorbed to the mineral surface . they are ( 1 ) fe(ii ) oxidation to fe(iii ) , which forms feppt , ( 2 ) sorption of fe(iii ) to cells , and ( 3 ) sorption of fe(ii ) to feppt . these observations fit a two step - model of fe(ii ) oxidation , where fe(ii ) is oxidized and undergoes rapid isotopic equilibration with a pool of fe(iii ) , which then precipitates as fe(iii ) minerals . controls on the overall iron isotope fractionation in the system are ( 1 ) fe(ii ) oxidation and precipitation of fe(iii ) as feppt ; ( 2 ) sorption of fe(iii ) to cells ; ( 3 ) partial equilibrium atom and electron exchange after sorption of fe(ii)aq to feppt . ( 3 ) produces fe(ii ) sorbed on goethite with an estimated fefenaac - fe(ii)aq of 0.8. the resulting fenaac predicted from ( 2 ) and ( 3 ) are denoted by the light blue diamonds . the larger values for linear fits of all data as compared to rayleigh fits indicate that complete isotopic equilibrium between fe(ii)aq and feppt is not occurring during fe(ii ) oxidation and precipitation ( table 1 ) . the smaller fe values for rayleigh fits of the fe(ii)aq ( 1.79 and 2.15 as compared to 2.44 and 2.98 for feppt ) are on the order of fractionation observed in other biological fe(ii ) oxidation experiments in batch at circumneutral ph : 1.5 , 1.52 , and 12. such small net fractionations have been noted when the presence of significant quantities of sorbed or ligand - bound fe(iii ) has been detected or observed . on the basis of detection of iron sorbed to cells with a dye that is specific for an aqueous or ligand - bound fe(iii ) , we suggest that this fe(iii ) could equilibrate with fe(ii)aq . cells at ph 6 , added fe(ii ) [ which was adsorbed as fe(iii ) ] was 1.84 heavier than aqueous fe(ii ) , and equilibrium with fe(ii)aq was inferred from the data . this fractionation is very similar to our fe values derived from rayleigh fits of fe(ii)aq ( 1.79 and 2.15 ) . because of the similar type of organism that we used , it is likely that equilibrium fractionation between fe(ii)aq and fe(iii ) sorbed to cells is a relevant process in our experiments . while the feppt data are not well fit by a linear model representing complete equilibrium exchange with fe(ii)aq , the range of the fe determined from rayleigh fits of the two feppt data sets ( 2.44 and 2.98 ) is of the same magnitude expected for equilibrium between feppt and fe(ii)aq . reported a fegoethite - fe(ii)aq of 1.1. considering the 58% ferrihydrite , 22% goethite , and 20% lepidocrocite in our precipitates as determined from x - ray scattering ( figure 2 ) , the equilibrium fefeppt - fe(ii)aq for our minerals could range from 2.3 to 2.7 , depending on whether the assumed fractionation for lepidocrocite is the same as for goethite or ferrihydrite , respectively . the larger fe we calculate for our second data set ( 2.98 ) may reflect that ferrihydrite , with a larger fe value , was likely the mineral present during active fe(ii ) oxidation ( see supporting information ) . during this process , equilibrium atom and electron exchange occur between sorbed fe(ii ) and fe(iii ) minerals , with complete equilibrium attained within 2 weeks for goethite , for instance ( a similar time frame as our 10 day experiment ) . in this model , fe(ii ) sorbs to fe(iii ) minerals and donates an electron into the bulk mineral structure , adding to the fe(iii ) mineral , and causing the desorption of a newly produced fe(ii ) from the mineral . atom and electron exchange between feppt and sorbed fe(ii ) is expected to be diminished in the presence of organic compounds . therefore , we suggest that only partial atom and electron exchange occurred in our system as a result of the eps coating the feppt . one study reports sorbed fe(ii ) is 0.73 heavier than fe(ii)aq , and another reported sorbed fe(ii ) was 1.24 heavier . directly measured sorbed fe(ii ) extracted with sodium acetate during microbial fe(iii ) reduction experiments , which was just 0.3 heavier than fe(ii)aq for experiments using hematite and up to 0.8 heavier for experiments using goethite as the sorbing surface . the feppt in our experiments had a surface area of 122.1 m g. we calculated fenaac considering that the fe(iii ) fraction of fenaac should be 1.84 heavier than fe(ii)aq due to adsorption of fe(iii ) at cell surfaces , and the sorbed fe(ii ) fraction of fenaac should be at least 0.8 heavier than fe(ii)aq . this calculation supports the model presented here , in which fe(ii ) is oxidized to fe(iii ) , which is sorbed onto cells and equilibrates with fe(ii)aq , and full equilibration of fe(ii)aq with feppt via atom and electron exchange is hindered by the presence of eps on feppt . our experiments provide evidence that iron isotope fractionation during microbially influenced fe(ii ) oxidation by cyanobacteria is not a simple reaction , controlled only by the abiotic oxidation of fe(ii ) with oxygen and rapid precipitation of fe(iii ) at circumneutral ph . multiple secondary processes generate a significant fraction of sorbed iron that is isotopically distinct from either residual fe(ii)aq or feppt , and subsequent equilibration between the iron pools can further modify the isotopic composition of these phases . in addition , abiotic sorption of fe(ii ) to fe(iii ) mineral surfaces can also fractionate fe(ii)aq , through equilibrium atom and electron exchange subsequent to fe(ii ) sorption , despite the presence of eps . the processes and phases described here can overprint the anticipated fractionations and compositions of fe(iii ) minerals and organic - associated iron present in the environment and can challenge interpretation of the genesis of fe(iii ) minerals in the geological record , where the residual fe(ii)aq pool is no longer present . although iron atom and electron exchange has received the most attention as a process relevant to fe(iii)-reducing systems where fe(ii ) is in contact with fe(iii ) minerals , our data suggest this process could also be relevant in environments where fe(ii ) is abundant during fe(ii ) oxidation and fe(iii ) mineral formation . furthermore , our work documents atom and electron exchange in the presence of iron minerals whose formation pathways are biologically induced and when organic phases coat iron minerals .

at the near neutral ph of many surface waters , the oxidation of fe(ii ) for that reason , cyanobacteria , which generate oxygen as a result of oxygenic photosynthesis , can act as indirect fe(ii)-oxidizing bacteria where anoxic and fe(ii)-containing waters encounter to sunlit surface environments . the contribution of cyanobacteria to fe(ii ) oxidation has been quantitatively addressed in fe(ii)-rich hot spring environments and in benthic photosynthetic communities living at the sediment water interface . although the modern oceans are predominantly oxygenated to great depths , promoting the speciation of iron as ferric [ fe(iii ) ] rather than ferrous [ fe(ii ) ] , fe(ii ) may be increasingly mobilized out of sediments and stabilized in the marine water column due to expanding low - oxygen conditions in so - called oxygen minimum zones ( omz ) . furthermore , anoxic and fe(ii)-rich bottom waters are a pervasive feature of oceans in the precambrian era [ before about 500 million years ( my ) ago ] at a time when oxygen was building up in the surface oceans as a result of cyanobacteria and other oxygenic phototrophs . , fe , fe , fe , and fe ) , such that the quantitative contribution of biotic and abiotic iron cycling at the earth s surface may be recorded in sediments composed of iron - rich minerals . due to the large fractionations between fe(ii ) and fe(iii ) species , fe(ii ) oxidation generally produces a solid iron phase that is enriched in heavy isotopes of iron relative to aqueous fe(ii ) , regardless of the mechanism of oxidation . however , subtle differences in the mechanism of oxidation and precipitation , and in the characteristics of the iron minerals or phases ( e.g. furthermore , the role of cyanobacteria in direct or indirect redox cycling of iron at the cell surface is increasingly recognized and may be associated with distinct isotope fractionation . therefore , detailed mechanistic studies of iron isotope fractionation during different pathways of fe(ii ) oxidation are warranted and may help to define isotopic , mineralogical , or microscopic signatures associated with certain biological processes . furthermore , the isotopic composition of iron minerals is known to be modified by electron and atom exchange between aqueous fe(ii ) and fe(iii ) ( oxyhdr)oxide minerals . although the effect of some organics as well as si and low ph on blocking electron and atom exchange have been investigated , the effect of cell surfaces and microbially produced organics on this reaction and via blocking sites on fe(iii ) minerals , particularly in an oxidizing system , are not known . in this contribution , we tracked the iron isotope composition of different pools of iron during fe(ii ) oxidation by the marine planktonic cyanobacterium synechococcus pcc 7002 . several prior studies have characterized the interaction of this oxygen - producing strain with fe(ii ) , which gives us a body of work to aid in interpreting the nature of different iron phases in the system , and their mechanism of transformation . synechococcus pcc 7002 was routinely cultivated on ph 6.8 marine phototroph ( mp ) medium containing 6 mg l ferric ammonium citrate as the fe(iii ) source at 24 c under an irradiance of 12.8 mol photons m s from a standard 40w tungsten light bulb as measured by a li-250a light probe ( li - cor , inc . ) purification of the fe(ii)aq , fenaac , and feppt fractions was performed in positively pressured clean laboratories of the isotope geochemistry group at the university of tuebingen under conditions and with reagents that have previously been described . the concentrations of iron in the water washes of feppt were below the detection limit of the ferrozine assay ( < 0.01 mm , 0.56 g ml ) , and so , these samples were not purified . interspersed measurements of our in - house iron standard , hanfe , yielded fe = 0.282 0.039 ( 2sd ; n = 12 ) , which is in excellent agreement with previously published values of 0.28 0.05 ( 2sd ; n = 19 ) and 0.279 0.030 ( 2sd ; ( f ) of fe(ii ) remaining were utilized to determine the isotopic enrichment factor ( fe ) using the following equations:12 fe(ii)aq-0 indicates the fe(ii)aq at the beginning of the experiment . high energy synchrotron x - ray scattering experiments were performed on the solid , dry products of fe(ii ) oxidation by synechococcus pcc 7002 at the advanced photon source at argonne national laboratory , beamline 11-id - b . alexa dye conjugates were screened in order to optimize visualization of exopolysaccharides ( eps ) without overlap with the autofluorescence emission maximum of the pigments of synechococcus pcc 7002 ( 660 nm ) . alexa 488 ( maximum emission peak at 520 nm ) was chosen because sba bound to the eps in higher amounts , resulting in brighter fluorescence than the other lectins screened [ wheat germ agglutinin alexa fluor 555 conjugate ( wga-555 ) and lectin pna from arachis hypogaea ( peanut ) , alex fluor 568 conjugate ( pna-568 ) ] . brighter fluorescence at lower laser power was observed with sba-488 , which binds terminal - and -n - acetylgalactosamine and galactopyranosyl residues , compared to wga-555 and pna-568 , which are specific to sialic acid and n - acetylglucosaminyl residues and terminal -galactose , respectively . a turn - on type selective probe for fluorescent labeling of dissolved , sorbed , or ligand - bound fe(iii ) was previously used to visualize the relationship of fe(iii ) synechococcus pcc 7002 cells and minerals from this same incubation . the spatial relationships of species detected using fluorescence dyes and cell autofluorescence in clsm image stacks were analyzed using scatterj , a plugin for correlation analysis of species - specific maps for use in imagej and fiji . all iron concentration and speciation data ( measured by ferrozine ) the first feppt samples analyzed , at about 40% fe(ii ) oxidized , had feppt of about 2 , trending toward 0 at 100% fe(ii ) oxidized . abiotic and biotic fe(ii ) oxidation both follow this trend , resulting in fe of 24 between aqueous fe(ii ) and fe(iii ) minerals , with minerals enriched in heavy isotopes . our fe for fe(ii)aq ( 1.792.15 for bottles 1 and 2 , respectively ; table 1 ) , determined from a rayleigh fit of the fe(ii)aq data , is on the low end of this range , similar to what was previously documented for fe(ii ) oxidation by anoxygenic phototrophs . the rayleigh fit of the feppt data from both replicates resulted in fe of 2.44 and 2.98 , larger than that attained for the fe(ii)aq data ( table 1 ) and within the literature range . prior explanation for the offset in fe between these two fractions is that , following precipitation , the feppt underwent partial equilibration with another phase of iron in the system , possibly a ligand - bound or sorbed fe(iii ) phase or fe(ii)aq . the third , quantitatively significant fraction of iron in the system in addition to fe(ii)aq and feppt was fenaac ( up to 18% of total fe ) . the fenaac data had an intermediate isotopic composition between fe(ii)aq and feppt , from 0.10 to 0.73 throughout the experiment , and contained both fe(ii ) and fe(iii ) ( supplementary table 1 ) . the presence of fe(iii ) in fenaac has previously been observed in fe(ii ) oxidation experiments with anoxygenic phototrophs but not with nitrate - dependent fe(ii)-oxidizing bacteria . our use of a lectin - binding dye in confocal microscopy documents that eps was also forming during fe(ii ) oxidation with synechococcus pcc 7002 ( figure 3 ) . we can use this data set to first determine whether eps was important in binding / sorbing iron extracted as fenaac and then to determine whether iron was associated with the surface of cells and/or feppt . an overlay of figure 3a c , which show the location of cells , eps , and feppt , indicates that eps is colocalized with feppt ( figure 3d ) . in previous work with synechococcus pcc 7002 under identical growth conditions as in figure 3 , a fluorescent sensor for soluble or ligand - bound fe(iii ) was used in clsm , and fluorescence was localized directly at the synechococccus pcc 7002 cell surfaces . while spectral interferences prevented us from simultaneously labeling eps and fe(iii ) in our current clsm experiments , we can infer from comparing our data set with the previously published one that there was fe(iii ) sorbed to the surface of cells but not eps or feppt . previous experiments with synechococcus pcc 7002 cells demonstrated that sorption to cells is a major fate for aqueous iron , although the oxidation state of sorbed iron was not determined in those experiments , so we can not rule out that some fe(ii ) was also sorbed to cells . however , sorption onto cells has previously been documented as a fate for aqueous iron with diverse cyanobacteria , with fe(iii ) more commonly detected at the cell surface than fe(ii ) , via attachment of fe o fe polymers to phosphoryl groups , strengthening the inferences made from clsm that synechococcus pcc 7002 cells sorbed fe(iii ) . ( a ) x - ray diffraction ( xrd ) pattern obtained from x - ray total scattering data of the feppt phase after complete fe(ii ) oxidation , freeze - drying , and water washing . ( a ) autofluorescent cells , ( b ) stained with the lectin - binding dye sba-488 , ( c ) the reflection signal from fe(iii ) minerals , and ( d ) an overlay of ( a c ) . correlation plot of the fluorescence intensity in individual pixels from ( e ) autofluorescence ( a ) vs sba-488 ( b ) and ( f ) sba-488 ( b ) vs fe(iii ) minerals ( c ) . the three techniques we used to address mineralogy indicate that our feppt was a mixture of 58% ferrihydrite , 22% goethite , and 20% lepidocrocite ( figure 2 ) , and ferrihydrite was likely the predominant mineral present during the experiments ( see supporting information ) . however , we verified that no fe(iii ) was extracted from synthetic ferrihydrite with our 0.5 m sodium acetate solution prior to beginning experiments ( data not shown ) , consistent with previous reports that used a 1 m sodium acetate solution . we take this as evidence that sorbed iron associated with the mineral was predominantly fe(ii ) , although we can not exclude that some fe(iii ) may also be sorbed to the mineral surface . we observed evidence for three reactions in our experiments that are essential for understanding the observed fractionations of iron isotopes , and these are summarized in figure 4 . they are ( 1 ) fe(ii ) oxidation to fe(iii ) , which forms feppt , ( 2 ) sorption of fe(iii ) to cells , and ( 3 ) sorption of fe(ii ) to feppt . controls on the overall iron isotope fractionation in the system are ( 1 ) fe(ii ) oxidation and precipitation of fe(iii ) as feppt ; ( 2 ) sorption of fe(iii ) to cells ; ( 3 ) partial equilibrium atom and electron exchange after sorption of fe(ii)aq to feppt . the fitting of our fe(ii)aq and feppt with rayleigh equations representing isolation of the feppt pool from fe(ii)aq after precipitation and a linear equation representing complete isotopic equilibrium are helpful in interpreting the fractionation mechanisms taking place . the smaller fe values for rayleigh fits of the fe(ii)aq ( 1.79 and 2.15 as compared to 2.44 and 2.98 for feppt ) are on the order of fractionation observed in other biological fe(ii ) oxidation experiments in batch at circumneutral ph : 1.5 , 1.52 , and 12. such small net fractionations have been noted when the presence of significant quantities of sorbed or ligand - bound fe(iii ) has been detected or observed . on the basis of detection of iron sorbed to cells with a dye that is specific for an aqueous or ligand - bound fe(iii ) , we suggest that this fe(iii ) could equilibrate with fe(ii)aq . because of the similar type of organism that we used , it is likely that equilibrium fractionation between fe(ii)aq and fe(iii ) sorbed to cells is a relevant process in our experiments . while the feppt data are not well fit by a linear model representing complete equilibrium exchange with fe(ii)aq , the range of the fe determined from rayleigh fits of the two feppt data sets ( 2.44 and 2.98 ) is of the same magnitude expected for equilibrium between feppt and fe(ii)aq . reported a fegoethite - fe(ii)aq of 1.1. considering the 58% ferrihydrite , 22% goethite , and 20% lepidocrocite in our precipitates as determined from x - ray scattering ( figure 2 ) , the equilibrium fefeppt - fe(ii)aq for our minerals could range from 2.3 to 2.7 , depending on whether the assumed fractionation for lepidocrocite is the same as for goethite or ferrihydrite , respectively . in this model , fe(ii ) sorbs to fe(iii ) minerals and donates an electron into the bulk mineral structure , adding to the fe(iii ) mineral , and causing the desorption of a newly produced fe(ii ) from the mineral . it is possible that atom and electron exchange can still occur when fe(iii ) minerals are coprecipitated with organics , as retardation of this process seems to result from blockage of surface sites when organics coat already formed fe(iii ) minerals , or if long chain carbon molecules are present . the feppt in our experiments had a surface area of 122.1 m g. we calculated fenaac considering that the fe(iii ) fraction of fenaac should be 1.84 heavier than fe(ii)aq due to adsorption of fe(iii ) at cell surfaces , and the sorbed fe(ii ) fraction of fenaac should be at least 0.8 heavier than fe(ii)aq . this calculation supports the model presented here , in which fe(ii ) is oxidized to fe(iii ) , which is sorbed onto cells and equilibrates with fe(ii)aq , and full equilibration of fe(ii)aq with feppt via atom and electron exchange is hindered by the presence of eps on feppt . our experiments provide evidence that iron isotope fractionation during microbially influenced fe(ii ) oxidation by cyanobacteria is not a simple reaction , controlled only by the abiotic oxidation of fe(ii ) with oxygen and rapid precipitation of fe(iii ) at circumneutral ph . multiple secondary processes generate a significant fraction of sorbed iron that is isotopically distinct from either residual fe(ii)aq or feppt , and subsequent equilibration between the iron pools can further modify the isotopic composition of these phases . in addition , abiotic sorption of fe(ii ) to fe(iii ) mineral surfaces can also fractionate fe(ii)aq , through equilibrium atom and electron exchange subsequent to fe(ii ) sorption , despite the presence of eps . the processes and phases described here can overprint the anticipated fractionations and compositions of fe(iii ) minerals and organic - associated iron present in the environment and can challenge interpretation of the genesis of fe(iii ) minerals in the geological record , where the residual fe(ii)aq pool is no longer present . although iron atom and electron exchange has received the most attention as a process relevant to fe(iii)-reducing systems where fe(ii ) is in contact with fe(iii ) minerals , our data suggest this process could also be relevant in environments where fe(ii ) is abundant during fe(ii ) oxidation and fe(iii ) mineral formation . this includes oxidizing environments with high enough fluxes of fe(ii ) for fe(ii ) to persist even in the face of rapid oxidation , such as fe(ii)-rich springs or seeps and marine upwelling zones that tap ferruginous bottom waters , past or present . finally , iron redox cycling and sorption of iron at the surface of cyanobacteria may be an important component of modern and ancient aquatic iron cycling , and our work highlights the effect of such processes on iron isotope systematics .

the photochemistry of ru(ii ) complexes plays an important role in fields that include photochemotherapy ( pct ) , molecular devices and switches , and solar energy conversion . ru(ii)polypyridyl complexes are commonly employed in these schemes due to their relatively strong absorption throughout the ultraviolet and visible spectral regions , chemical stability in solution , and their long excited state lifetimes and reactivity that is inaccessible in the ground state . the population of the metal - to - ligand charge transfer ( mlct ) excited state in these complexes following absorption of a photon is known to undergo fast intersystem crossing to the corresponding mlct state ; in [ ru(bpy)3 ] ( bpy = 2,2-bipyridine ) within 1540 fs . the mlct state can decay via radiative or nonradiative processes to the ground state or may populate thermally accessible triplet ligand field ( lf ) state(s ) . because the lf state exhibits ru l( * ) character ( l = ligand ) , it can be tuned and exploited to promote efficient ligand dissociation . complexes related to [ ru(tpy)(bpy)(l ) ] ( tpy = 2,2:2,6-terpyridine ; l = monodentate ligand ) have been employed for applications such as light - activated drug delivery and photocatalysis . for example , the photoisomerization from s - bound to o - bound dmso for a series of [ ru(tpy)(l)(dmso ) ] complexes ( dmso = dimethyl sulfoxide , l = bidentate ligand ) has been used for potential applications in information storage because the compounds are photochromic . in these systems , the s o isomerization proceeds with a quantum yield ( so ) dependent on the -donor strength of the atom positioned trans to the dmso ligand , with values of 0.25(1 ) , 0.024(1 ) , and 0.007(1 ) for the complexes with l = pic ( picolinate ) , bpy , and tmen ( n , the irradiation of the related complex [ ru(tpy)(bpy)(ch3cn ) ] with visible light in ch3cn in the presence of 1 m pyridine produces [ ru(tpy)(bpy)(py ) ] ( py = pyridine ) with = 0.0013 ( irr = 464 nm ) . an important point of interest is that the photorelease of pyridine in [ ru(tpy)(bpy)(py ) ] is a significantly less efficient process because pyridine forms a stronger bond with ru(ii ) , such that monodentate ligand dissociation in the excited state is less favorable in the py complex than in the corresponding ch3cn system . this difference is evident in experiments that show that the irradiation of [ ru(tpy)(bpy)(l ) ] ( l = py , ch3cn ) in dmf to generate [ ru(tpy)(bpy)(dmf ) ] . this process occurs with < 10 for l = py but is orders of magnitude more efficient for l = ch3cn , with = 0.006 ( irr = 436 nm ) under similar experimental conditions . due to the inefficiency and exhaustive photolysis required , applications involving the photodissociation of pyridine - containing ligands coordinated to ru(ii ) has been largely impractical to date . the addition of steric bulk to the ligand set in ru(ii ) complexes provides a means to distort the pseudo - octahedral geometry around the metal , which has been shown to enhance the efficiency of the excited state ligand exchange . the synthesis of [ ru(tpy)(nn)(py ) ] complexes with steric bulk on the bidentate nn ligand has been published , although the photoinduced pyridine exchange in these motifs remains largely unexplored . related work on a series of sterically bulky complexes for the photoinduced release of nitriles in the presence of pyridine was reported . the irradiation of [ ru(bu - tpy)(phen)(meobn ) ] ( bu - tpy = 4-(3,5-ditertiobutyl)phenyl-2,2,6,2-terpyridine ; meobn = 2,6-dimethoxybenzonitrile ; phen = 1,10-phenanthroline ) with 476 nm promotes the exchange of meobn with pyridine to produce [ ru(bu - tpy)(phen)(py ) ] . a 20-fold increase in efficiency of this process was observed when the analogous [ ru(bu - tpy)(me2phen)(meobn ) ] complex was used under similar experimental conditions , which contains the sterically bulky me2phen ( me2phen = 2,9-dimethyl-1,10-phenanthroline ) ligand . in addition , the release of pyridine from [ ru(ttpy)(me2bpy)(py ) ] ( ttpy = 4-tolyl-2,2:6,2-terpyridine , me2bpy = 6,6-dimethyl-2,2-bipyridine ) in ch3cn to produce [ ru(ttpy)(me2bpy)(ch3cn ) ] was reported , although few details were provided . the present work focuses on the investigation of two new complexes related to [ ru(tpy)(bpy)(py ) ] ( 1 ) , but with additional steric bulk on the bidentate ligand , [ ru(tpy)(me2bpy)(py ) ] ( 2 ) and [ ru(tpy)(biq)(py ) ] ( 3 ; biq = 2,2-biquinoline ) . the ch3 substituents on 2 are positioned toward the center of the molecule to strain the pseudo - octahedral geometry around the metal for efficient pyridine exchange . in addition to the steric demands of the biq ligand in 3 , the low - lying biq acceptor orbitals serve to red shift the mlct absorption maximum , an important factor in pct to achieve tissue penetration . a spectroscopic analysis of the photolysis of 13 in ch3cn and h2o are provided , and the quantum yields of pyridine exchange are discussed in relation to the distortion induced by sterically demanding bidentate ligands as determined from x - ray crystal structures and theoretical calculations . schematic representation of the [ ru(tpy)(nn)(py ) ] complexes with the structures of the bidentate nn ligands bpy , me2bpy , and biq in 1 , 2 , and 3 , respectively . pyridine was acquired from mallinckrodt chemicals , ammonium hexafluorophosphate and deuterated acetone were purchased from aldrich , diethyl ether and acetonitrile were obtained from fisher scientific , and 200 proof ethanol was purchased from decon laboratories . the complexes [ ru(tpy)(me2bpy)cl](pf6 ) , [ ru(tpy)(biq)cl](pf6 ) , and [ ru(tpy)(bpy)(py)](pf6)2 ( 1 ) [ ru(tpy)(me2bpy)cl](pf6 ) ( 0.040 g , 0.057 mmol ) and an excess of pyridine ( 1 ml ) in 10 ml of ethanol and 10 ml of h2o were heated at reflux for 16 h. the reaction mixture was cooled to room temperature , and the ethanol was removed under reduced pressure . ( 0.1 g ) was added to the reaction mixture to induce precipitation , and the orange solid was collected by vacuum filtration . the product was washed with 30 ml of h2o and 30 ml of diethyl ether ( 0.045 g , 90% yield ) . the cl salt was obtained by ion exchange with an amberlite column and eluted with methanol . h nmr ( 400 mhz ) in ( cd3)2co , ppm ( splitting , integration ) : 8.78 ( dd , 3h ) , 8.70 ( m , 2h ) , 8.52 ( m , 1h ) , 8.45 ( ddd , 2h ) , 8.32 ( q , 2h ) , 8.21 ( td , 2h ) , 7.88 ( m , 2h ) , 7.81 ( m , 3h ) , 7.68 ( ddd , 2h ) , 7.19 ( dd , 2h ) , 7.07 ( d , 1h ) , 2.12 ( s , 3h ) , 1.59 ( s , 3h ) . [ ru(tpy)(biq)(py)](pf6)2 was prepared following the above procedure and substituting [ ru(tpy)(biq)cl](pf6 ) ( 0.03 g , 0.039 mmol ) for [ ru(tpy)(me2bpy)cl](pf6 ) ( 0.033 g , 88% yield ) . h nmr ( 400 mhz ) in ( cd3)2co , ppm ( splitting , integration ) : 9.27 ( d , 1h ) , 9.12 ( dd , 1h ) , 9.02 ( d , 1h ) , 8.94 ( d , 2h ) , 8.73 ( m , 2h ) , 8.55 ( m , 1h ) , 8.43 ( m , 2h ) , 8.30 ( ddd , 2h ) , 8.13 ( td , 2h ) , 7.92 ( m , 3h ) , 7.76 ( m , 2h ) , 7.54 ( m , 3h ) , 7.42 ( m , 1h ) , 7.35 ( m , 2h ) , 7.11 ( dd , 2h ) , 6.89 ( dd , 1h ) . electronic absorption spectroscopy was performed with a hewlett - packard 8453 diode array spectrophotometer . a 150 w xe arc lamp ( ushio ) in a milliarc lamp housing unit with an lps-220 power supply and an lps-221 igniter ( pti ) was used for photolysis experiments . the appropriate irradiation wavelengths were selected with a bandpass filter ( thorlabs ) and long - pass filter ( cvi melles griot ) . h nmr spectroscopy of 13 was performed in acetone - d6 , and the resonances were referenced to the residual acetone signal . electronic absorption spectroscopy was measured in acetone , ch3cn , and h2o at room temperature in a 1 1 cm quart cuvette . in the h2o photolysis experiments , the samples were absorbance matched at 600 nm ( a = 0.075 ) so that the solutions absorb a similar quantity of photons . the quantum yields ( ) for pyridine dissociation were determined in ch3cn with an irradiation wavelength of 500 nm . the rate of moles reacted at early irradiation times was determined by monitoring the decrease in the mlct absorption maximum as a function of time . the photon flux of the lamp with a 435 nm long - pass filter and a 500 nm bandpass filter was determined using ferrioxalate actinometry as previously described in detail , resulting in a flux of 5.06 0.31 mol photons / min . single crystals of 2 and 3 were isolated as rod - like and chunk - like red crystals , respectively , and handled under a pool of fluorinated oil . examination of the diffraction pattern was done on a nonius kappa ccd diffractometer with mo k radiation . data integration was performed with denzo , and scaling and merging of the data were done with scalepack . full - matrix least - squares refinements based on f were performed in shelxl-13 , as incorporated in the wingx package . for each methyl group , the hydrogen atoms were added at calculated positions using a riding model with u(h ) = 1.5ueq ( bonded carbon atom ) . the rest of the hydrogen atoms were included in the model at calculated positions using a riding model with u(h ) = 1.2ueq ( bonded atom ) . the structure of 2 had a disordered diethyl ether molecule that had crystallized on an inversion center . to model this disorder , each atom was assigned an occupancy of 50% , which allowed the symmetry operator to generate the second half of the molecule . the atoms were left in their isotropic state as modeling them anisotropically led to an unstable refinement . modeling of this residual electron density was not straightforward , and despite several attempts at refinement , the solvent molecules remained unstable . the residual density was removed from the model using the squeeze protocol in platon , which ultimately removed 67 electrons from a solvent accessible void of 196 and corresponds to diethyl ether , a solvent used in crystallization . the cif file in the supporting information provides a description of how 2 was modeled , and table s1 ( supporting information ) provides crystallographic data collection parameters for 2 and 3 . calculations were performed with density functional theory ( dft ) using the gaussian 09 program . the b3lyp functional along with the 6 - 31 g * basis set for h , c , and n and the sdd energy consistent pseudopotentials were used for ru . optimization of full geometries was carried out with the respective programs , and orbital analysis was performed in gaussview version 3.09 . following optimization of the molecular structures , frequency analysis was performed to ensure the existence of local minima on the potential energy surfaces . electronic absorption singlet to singlet transitions were calculated using time - dependent dft ( td - dft ) methods with the polarizable continuum model ( pcm ) that mimicked the solvation effect of ch3cn in gaussian 09 . the electronic absorption spectra of the series of complexes [ ru(tpy)(nn)(py ) ] , where nn = bpy ( 1 ) , me2bpy ( 2 ) , and biq ( 3 ) , are shown in figure 2 . in general , the spectra of 13 feature * transitions associated with tpy and the nn bidentate ligands in the uv region and ru(d ) tpy / nn( * ) mlct transitions in the visible range , both of which are typical of ru(ii)polypyridyl complexes . in the case of 1 and 2 , the lowest energy mlct transitions are centered at 468 nm ( = 8120 m cm ) and 471 nm ( = 8020 m cm ) in acetone , respectively . the reported absorption maximum of 1 in ch3cn of 467 nm is in sound agreement with the present observations . in contrast , the lowest energy band of 3 is red - shifted compared to those of 1 and 2 , with a maximum at 530 nm ( = 9020 m cm ) . the stabilized biq * orbitals relative to those of bpy , me2bpy , and tpy shift the mlct absorption to lower energy . the lowest energy absorption peaks in 1 and 2 have contributions from both ru bpy / me2bpy and ru tpy mlct transitions , and the red shift observed for 3 clearly indicates that the lowest energy transition is ru biq mlct due to stabilized biq * orbitals relative to those of bpy , me2bpy , and tpy . for comparison , the lowest energy ru(ii ) biq transition of [ ru(bpy)2(biq ) ] is centered at 525 nm in etoh / meoh solution , similar to the maximum of 3 ( figure 2 ) . the stronger absorption of 3 in the low energy tail ( > 600 nm ) represents a promising feature for the application of the complex as a photoactivated drug delivery vehicle because low energy light ( 600900 nm ) is necessary to penetrate tissue for targeted drug delivery . electronic absorption spectra of 1 ( black ) , 2 ( red ) , and 3 ( blue ) in acetone . the photosubstitution of pyridine in 13 with ch3cn was investigated to compare the efficiency of ligand exchange as a function of the identity of the bidentate ligand . in the dark , 13 do not undergo ligand exchange in ch3cn ( figures s1s3 , supporting information ) , but clear changes in the electronic absorption spectra are observed for 2 and 3 upon irradiation with visible light in ch3cn , resulting in a blue shift in the mlct maximum from 470 to 454 nm in 2 ( figure 3a ) and from 529 to 513 nm for 3 ( figure 3b ) . these shifts correspond to 750 and 590 cm for 2 and 3 , respectively , which are similar to the 610 cm difference between the absorption maxima of 1 and [ ru(tpy)(bpy)(ncch3 ) ] . the resulting spectra are consistent with the substitution of pyridine with a ch3cn ligand to form [ ru(tpy)(me2bpy)(ncch3 ) ] and [ ru(tpy)(biq)(ncch3 ) ] , respectively . this ligand exchange occurs quite rapidly , within 2 and 4 min of irradiation for 2 and 3 , respectively ( irr > 395 nm ) . the multiple isosbestic points observed for each complex as a function of irradiation time , at 385 , 415 , and 462 nm for 2 and at 392 , 425 , 457 , and 513 nm for 3 , indicate the formation of a single photoproduct in each reaction . very little change is observed in the spectrum of 1 on the same time scale ( figure s4 , supporting information ) . changes in the electronic absorption spectroscopy in ch3cn with irr > 395 nm of ( a ) 50 m 2 for tirr = 0 , 0.25 , 0.5 , 1 , and 2 min and ( b ) 40 m 3 for tirr = 0 , 0.25 , 0.5 , 1 , 2 , and 4 min . the quantum yield ( 500 ) of pyridine dissociation to form the corresponding [ ru(tpy)(nn)(ncch3 ) ] species was determined to be 0.16(1 ) and 0.03(1 ) for 2 and 3 ( irr = 500 nm ) , respectively , whereas the analogous pyridine exchange in 1 occurs with 500 < 0.0001 ( table 1 ) . the low efficiency measured for 1 is consistent with the reported quantum yield for dmf substitution of < 10 upon irradiation ( irr = 436 nm ) . it should be noted that the quantum yields observed for 2 and 3 are remarkably large for the photodissociation of a pyridine ligand with low energy visible light . for [ ru(bpy)2(py)2 ] , the photoinduced ligand exchange of one py ligand with cl in ch3cn was reported to take place with 436 = 0.0059 , and the introduction of ch3 groups to produce [ ru(4,4-me2bpy)2(py)2 ] ( 4,4-me2bpy = 4,4-dimethyl-2,2-bipyridine ) resulted in 436 = 0.025 under similar experimental conditions . the 4.2-fold increase in ligand exchange quantum yield is primarily a result of electronic effects from the electron - donating ch3 substituents . an electronic effect from the ch3 groups in 2 is expected , but the significantly larger enhancement for 2 relative to 1 measured in the present work ( > 1600-fold ; table 1 ) indicates that the steric effects of the bulky nn ligand influence the photoreactivity to a much greater extent than electronic effects alone . moreover , the efficiency of the reaction measured for 2 is 4-fold greater than the cl ligand exchange reported for [ ru(tpy)(py)3 ] and cis-[ru(tpy)(py)2cl ] ( irr = 436 nm ) . measured in acetone at room temperature . measured in ch3cn . irradiation of 2 and 3 with low energy light ( > 590 nm ) also promotes pyridine substitution in aqueous solution . the photolysis of 2 and 3 in h2o results in a red shift of the mlct transition , consistent with substitution of the pyridine ligand with a solvent h2o molecule to form the corresponding [ ru(tpy)(nn)(oh2 ) ] complex . the changes in the electronic absorption spectra of 2 and 3 as a function of irradiation time are displayed in figure 4a , b , respectively . photolysis of 2 in h2o shifts the mlct absorption from 473 to 485 nm with an isosbestic point at 483 nm , resulting in a spectrum that is in sound agreement with that reported for [ ru(tpy)(me2bpy)(oh2 ) ] . a similar trend is observed for 3 , in which the mlct absorption shifts from 530 to 548 nm with isosbestic points at 408 and 540 nm , and the spectrum of the photoproduct is consistent with that of [ ru(tpy)(biq)(oh2 ) ] . although this conversion for 2 and 3 occurs on a relatively short time scale ( within 30 and 100 min , respectively ) , no spectral changes are observed for 1 after photolysis for 2 h ( figure s5 , supporting information ) . in contrast , complexes 2 and 3 do not undergo ligand substitution in water when kept in the dark ( figures s6 and s7 , supporting information ) . the large differences in the efficiency of pyridine photodissociation for 2 and 3 relative to 1 in water are consistent with the analogous ch3cn experiments . changes in the electronic absorption spectroscopy in h2o with irr > 590 nm of ( a ) 94 m 2 for tirr = 0 , 1 , 3 , 7 , 15 , and 30 min and ( b ) 62 m 3 for tirr = 0 , 3 , 7 , 15 , 25 , 45 , 75 , and 100 min . structural analysis of 13 provides insight into the factors governing the enhanced ligand exchange of 2 and 3 compared to 1 . the numbering scheme for the atoms of interest is depicted in figure 5 and those for all the atoms in 2 and 3 appear in figure s8 ( supporting information ) , the experimental and calculated ru n bond distances are listed in table 2 , and the experimental and calculated n ru the crystal structures of 2 and 3 are shown in figure 5a , b , respectively , and that of 1 was previously reported ; the experimental data for all three complexes are provided in tables 2 and 3 for comparison . the structures predicted by dft calculations agree well with the structures determined by x - ray crystallography , although the bond distances are calculated to be 0.030.06 longer than the experimental bond lengths . similar differences between experimental and theoretical bond distances have also been reported for related complexes . n bond distances involving the pyridine and tpy ligands are relatively unperturbed upon addition of steric bulk on the bidentate ligand . this effect was previously observed in the monodentate and bidentate bond distances of [ ru(tpy)(phen)(ncch3 ) ] and [ ru(tpy)(me2phen)(ncch3 ) ] ( me2phen = 2,9-dimethyl-1,10-phenanthroline ) . it is evident from table 2 that the addition of steric bulk on nn results in an increase in ru n(5 ) bond lengths associated with the bidentate ligand by 0.040.05 and 0.010.02 , respectively , in both 2 and 3 relative to those of 1 . ortep plots of ( a ) 2 and ( b ) 3 ( light blue = ruthenium , dark blue = nitrogen , gray = carbon ) drawn at 50% probability with selected atom numbers ; solvent , hydrogens and counteranions removed for clarity . experimental values from ref ( 24 ) . the strained pseudo - octahedral geometry around the ruthenium center is further highlighted by select bond angle distortions ( table 3 ) . the bite angle of the tpy ligand distorts the bond angles from the 90 ( between cis positions ) and 180 ( between trans positions ) of an ideal octahedral complex . this distortion is evident in the angles associated with the coordinated pyridine units on the tpy ligand in 1 , determined to be 79.7 for n(1)ru n(2 ) and n(2)ru these angles remain relatively constant in all three complexes , 13 , indicating that the bonding of the tridentate tpy ligand to the ruthenium metal is not affected by the steric bulk on the bidentate ligand . in all three complexes , the planes defined by the three ru n bonds of the bidentate ligands are nearly orthogonal , as evidenced by the corresponding n(5)ru n(1,3 ) and n(4)ru n(13 ) angles , which range from 87.3 to 105.5 ( average = 95.3 ) , and n(2)ru n(5 ) , determined to be 171.9 in 1 ( table 3 ) . similarly , the n(5)ru n(1,3 ) and n(4)ru n(13 ) angles in 2 range from 85.6 to 101.6 , and from 83.5 to 102.3 in 3 . n(5 ) angles were measured to be 179.1 and 175.6 in 2 and 3 , respectively . although the ru n bond angles and distances do not exhibit significant variation in 13 , distortion from planarity and significant tilting is observed in the bidentate ligands due to steric hindrance in 2 and 3 . the angle describing the orientation of the me2bpy ligand relative to tpy in 2 , defined by a plane containing the n(4 ) , n(5 ) , c(20 ) , and c(23 ) atoms of me2bpy ( figure 5 ) and a plane containing the n(1 ) , n(2 ) , and n(3 ) atoms in the tpy ligand , was determined to be 67.87. the corresponding angle in 3 was 61.89 , using the biq n(4 ) , n(5 ) , c(28 ) , and c(31 ) atoms to define the plane . for comparison , only a small distortion from the octahedral geometry is observed between the corresponding atoms in the bpy and tpy ligands in 1 , 83.34. therefore , an increase in the tpy nn angle of 15.47 in 2 and 21.45 in 3 are observed relative to the tpy these distortions are also evident in the calculated structures of 13 shown in figure 6a c . optimized structures predicted by dft calculations viewed along the axis containing the n(2)ru n(5 ) bonds for ( a ) 1 , ( b ) 2 , and ( c ) 3 , such that the plane of the tpy ligand is horizontal and that of the bidentate ligand is vertical with respect to the page . moreover , the structures of 13 reveal a tilt of the bulky bidentate ligands of 2 and 3 that is not present in 1 . in the predicted and experimental structures of 2 and 3 , the bidentate ligands are tilted relative to the position of the bpy ligand in 1 , as the ch3 groups of me2bpy and the quinoline ligands are oriented toward the n(3 ) atom on tpy . to compare the tilting of the bidentate ligands in 2 and 3 , one plane was defined by the ru atom and the n4 and n5 atoms , and a second plane was defined by four atoms on each bidentate ligand ( figure s9 , supporting information ) . in 2 , the latter were n4 , n5 , c20 , and c23 on me2bpy and in 3 the n4 , n5 , c28 , and c31 atoms of biq were used to define the plane . the resulting tilt angles in the crystal structures of 2 and 3 are + 20.97 and + 25.88 , respectively , which are similar to the calculated values of + 21 in 2 and + 23 in 3 . in contrast , a much smaller tilt is calculated for bpy in 1 and in the opposite direction , 6 , with a comparable value in the crystal structure , 6.75 , as expected from its less sterically demanding structure . overall , the me2bpy and biq ligands are more tilted away from the n(4)ru n(5 ) plane than bpy in 1 by 27 and 32 in 2 and 3 , respectively . a similar effect was noted in [ ru(bpy)2(dmdppz ) ] ( dmdppz = 3,6-dimethyldipyridylphenazine ) , [ ru(biq)2(phen ) ] , and [ ru(bu - tpy)(me2phen)(meobn ) ] in which the dmdppz , biq , and me2phen ligands tilt approximately 15 , 20 , and 23 , respectively , due to the steric constraints from the bulky ligands . another major structural difference measured and calculated for 2 and 3 relative to 1 can be found in the tilt of pyridine toward the n(1 ) and n(2 ) atoms of the tpy ligand and its rotation about the ru n(13 ) bond angles listed in table 3 for 2 and 3 show that the pyridine ligand is significantly tilted toward the portion of tpy ligand bearing the n(1 ) and n(2 ) atoms and away from the n(3 ) atom . n(6 ) and n(2)ru n(6 ) angles in the crystal structures of 2 and 3 ranged from 84.45 to 87.84 , and are smaller than the same angles in 1 , 92.6 and 91.0 , respectively . in contrast , the n(3)ru n(6 ) angles in 2 and 3 , 94.17 and 91.81 , respectively , are larger than the 89.11 determined for 1 . n(4,5 ) bond angles are largely unaffected by the nature of the bidentate ligand . the optimized geometries of the three complexes shown in figure 6 provide a visual comparison of the deviations from the predicted 90 n(6)ru n(13 ) angle in 2 and 3 relative to 1 ; this tilt of the pyridine ligand in 2 and 3 is expected to weaken its -bonding and -bonding with the metal relative to 1 . in addition , both the crystal structure and the calculations show that the rotation of the pyridine about its ru n bond deviates significantly in 2 and 3 as compared to that in 1 due to steric constraints imparted by the me2bpy and biq ligands . this rotation in the former is clearly evident in the views provided in figure 6 and is described by the n(2)ru n(6)c(39 ) dihedral angle in 3 , where c(32 ) and c(32 ) are the carbon atoms on pyridine pointing toward n(3 ) on the tpy ligand in each complex . in the crystal structure of 1 , this dihedral angle is 128.49 , a geometry that is expected to provide good orbital overlap for -back - bonding to the metal . this angle is much larger than the 56.66 and 45.57 dihedral angles in 2 and 3 , respectively , such that overlap between the * orbitals of the py group and filled metal t2g - type orbitals for -back - bonding is decreased relative to 1 . the dihedral angles measured in the calculated structures , 114.36 , 59.89 , and 52.78 for 1 , 2 , and 3 , respectively , are in good agreement with those from the crystal structures . this rotation of pyridine , taken together with its tilting , is expected to further weaken the ru(ii)py -back - bonding in 2 and 3 . the distorted geometries of 2 and 3 relative to 1 are important for efficient pyridine dissociation . similar distortions in [ ru(bpy)2(dmdppz ) ] , [ ru(biq)2(phen ) ] , and [ ru(bu - tpy)(me2phen)(meobn ) ] are believed to result in enhanced photoinduced ligand exchange of bulky bidentate ligands . on the basis of the electronic absorption spectroscopy , the significantly distorted geometry for 2 lowers the energy of the lf state and weakens the ru py -bond and -back - bonding , resulting in greater relative population of the lf state and enhanced ligand dissociation . the geometric distortions are similar for 2 and 3 , but the lf state in 3 is also predicted to be stabilized relative to that of 1 . however , the mlct state of 3 is lower in energy than that of 2 due to the lower - lying biq orbitals relative to me2bpy , such that the energy difference between the lowest energy mlct state and the dissociative lf state(s ) is greater in the former . the larger mlct - lf energy gap is expected to result in lower thermal population of the lf state in 3 , consistent with the observed lower quantum yield for pyridine dissociation . additionally , the more distorted geometry of the bound pyridine in 2 as compared to 3 may result in smaller overlap in the bonding orbitals in the former , facilitating more efficient pyridine dissociation for 2 in the excited state . photoinduced pyridine dissociation with greatly enhanced efficiencies over the previously reported 1 was achieved with the bulky bidentate ligands me2bpy in 2 and biq in 3 . in ch3cn solution , pyridine is replaced by a solvent molecule with 500 = 0.16(1 ) and 0.033(1 ) for 2 and 3 , respectively , whereas ligand exchange is much less efficient for 1 ( 500 < 0.0001 ) . although pyridine dissociation is less efficient for 3 than 2 , the red - shifted absorption of 3 is beneficial in developing complexes for drug delivery as red light is optimal for pct . to this end , low energy light ( > 590 nm ) was shown to promote pyridine dissociation in aqueous solution for 2 and 3 on time scales that are inaccessible with 1 . the x - ray crystal structures and theoretical calculations for the three complexes depict significantly more distorted geometries for 2 and 3 compared to 1 due to steric hindrance between the pyridine ligand and the bulky substituent . n(6 ) bond distances are largely unaffected by the addition of steric bulk , suggesting that differences in photoreactivity are influenced by bond angle distortions . these results are important for design considerations for ru(ii ) complexes to be used as potential pct agents , molecular switches / devices , and catalysts . a detailed investigation into the excited state processes involved in pyridine dissociation from ru(ii ) complexes with sterically demanding ligands is ongoing .

the introduction of steric bulk to the bidentate ligand in [ ru(tpy)(bpy)(py)]2 + ( 1 ; tpy = 2,2:2,6-terpyridine ; bpy = 2,2-bipyridine ; py = pyridine ) to provide [ ru(tpy)(me2bpy)(py)]2 + ( 2 ; me2bpy = 6,6-dimethyl-2,2-bipyridine ) and [ ru(tpy)(biq)(py)]2 + ( 3 ; biq = 2,2-biquinoline ) facilitates photoinduced dissociation of pyridine with visible light . upon irradiation of 2 and 3 in ch3cn ( irr = 500 nm ) , ligand exchange occurs to produce the corresponding [ ru(tpy)(nn)(ncch3)]2 + ( nn = me2bpy , biq ) complex with quantum yields , 500 , of 0.16(1 ) and 0.033(1 ) for 2 and 3 , respectively . these values represent an increase in efficiency of the reaction by 23 orders of magnitude as compared to that of 1 , 500 < 0.0001 , under similar experimental conditions . the photolysis of 2 and 3 in h2o with low energy light to produce [ ru(tpy)(nn)(oh2)]2 + ( nn = me2bpy , biq ) also proceeds rapidly ( irr > 590 nm ) . complexes 13 are stable in the dark in both ch3cn and h2o under similar experimental conditions . x - ray crystal structures and theoretical calculations highlight significant distortion of the planes of the bidentate ligands in 2 and 3 relative to that of 1 . the crystallographic dihedral angles defined by the bidentate ligand , me2bpy in 2 and biq in 3 , and the tpy ligand were determined to be 67.87 and 61.89 , respectively , whereas only a small distortion from the octahedral geometry is observed between bpy and tpy in 1 , 83.34. the steric bulk afforded by me2bpy and biq also result in major distortions of the pyridine ligand in 2 and 3 , respectively , relative to 1 , which are believed to weaken its -bonding and -back - bonding to the metal and play a crucial role in the efficiency of the photoinduced ligand exchange . the ability of 2 and 3 to undergo ligand exchange with irr > 590 nm makes them potential candidates to build photochemotherapeutic agents for the delivery of drugs with pyridine binding groups .

Introduction Experimental Section Results and Discussion Conclusions

the population of the metal - to - ligand charge transfer ( mlct ) excited state in these complexes following absorption of a photon is known to undergo fast intersystem crossing to the corresponding mlct state ; in [ ru(bpy)3 ] ( bpy = 2,2-bipyridine ) within 1540 fs . in these systems , the s o isomerization proceeds with a quantum yield ( so ) dependent on the -donor strength of the atom positioned trans to the dmso ligand , with values of 0.25(1 ) , 0.024(1 ) , and 0.007(1 ) for the complexes with l = pic ( picolinate ) , bpy , and tmen ( n , the irradiation of the related complex [ ru(tpy)(bpy)(ch3cn ) ] with visible light in ch3cn in the presence of 1 m pyridine produces [ ru(tpy)(bpy)(py ) ] ( py = pyridine ) with = 0.0013 ( irr = 464 nm ) . this process occurs with < 10 for l = py but is orders of magnitude more efficient for l = ch3cn , with = 0.006 ( irr = 436 nm ) under similar experimental conditions . the addition of steric bulk to the ligand set in ru(ii ) complexes provides a means to distort the pseudo - octahedral geometry around the metal , which has been shown to enhance the efficiency of the excited state ligand exchange . a 20-fold increase in efficiency of this process was observed when the analogous [ ru(bu - tpy)(me2phen)(meobn ) ] complex was used under similar experimental conditions , which contains the sterically bulky me2phen ( me2phen = 2,9-dimethyl-1,10-phenanthroline ) ligand . in addition , the release of pyridine from [ ru(ttpy)(me2bpy)(py ) ] ( ttpy = 4-tolyl-2,2:6,2-terpyridine , me2bpy = 6,6-dimethyl-2,2-bipyridine ) in ch3cn to produce [ ru(ttpy)(me2bpy)(ch3cn ) ] was reported , although few details were provided . the present work focuses on the investigation of two new complexes related to [ ru(tpy)(bpy)(py ) ] ( 1 ) , but with additional steric bulk on the bidentate ligand , [ ru(tpy)(me2bpy)(py ) ] ( 2 ) and [ ru(tpy)(biq)(py ) ] ( 3 ; biq = 2,2-biquinoline ) . in addition to the steric demands of the biq ligand in 3 , the low - lying biq acceptor orbitals serve to red shift the mlct absorption maximum , an important factor in pct to achieve tissue penetration . a spectroscopic analysis of the photolysis of 13 in ch3cn and h2o are provided , and the quantum yields of pyridine exchange are discussed in relation to the distortion induced by sterically demanding bidentate ligands as determined from x - ray crystal structures and theoretical calculations . schematic representation of the [ ru(tpy)(nn)(py ) ] complexes with the structures of the bidentate nn ligands bpy , me2bpy , and biq in 1 , 2 , and 3 , respectively . the complexes [ ru(tpy)(me2bpy)cl](pf6 ) , [ ru(tpy)(biq)cl](pf6 ) , and [ ru(tpy)(bpy)(py)](pf6)2 ( 1 ) [ ru(tpy)(me2bpy)cl](pf6 ) ( 0.040 g , 0.057 mmol ) and an excess of pyridine ( 1 ml ) in 10 ml of ethanol and 10 ml of h2o were heated at reflux for 16 h. the reaction mixture was cooled to room temperature , and the ethanol was removed under reduced pressure . the electronic absorption spectra of the series of complexes [ ru(tpy)(nn)(py ) ] , where nn = bpy ( 1 ) , me2bpy ( 2 ) , and biq ( 3 ) , are shown in figure 2 . the lowest energy absorption peaks in 1 and 2 have contributions from both ru bpy / me2bpy and ru tpy mlct transitions , and the red shift observed for 3 clearly indicates that the lowest energy transition is ru biq mlct due to stabilized biq * orbitals relative to those of bpy , me2bpy , and tpy . the stronger absorption of 3 in the low energy tail ( > 600 nm ) represents a promising feature for the application of the complex as a photoactivated drug delivery vehicle because low energy light ( 600900 nm ) is necessary to penetrate tissue for targeted drug delivery . the photosubstitution of pyridine in 13 with ch3cn was investigated to compare the efficiency of ligand exchange as a function of the identity of the bidentate ligand . in the dark , 13 do not undergo ligand exchange in ch3cn ( figures s1s3 , supporting information ) , but clear changes in the electronic absorption spectra are observed for 2 and 3 upon irradiation with visible light in ch3cn , resulting in a blue shift in the mlct maximum from 470 to 454 nm in 2 ( figure 3a ) and from 529 to 513 nm for 3 ( figure 3b ) . these shifts correspond to 750 and 590 cm for 2 and 3 , respectively , which are similar to the 610 cm difference between the absorption maxima of 1 and [ ru(tpy)(bpy)(ncch3 ) ] . this ligand exchange occurs quite rapidly , within 2 and 4 min of irradiation for 2 and 3 , respectively ( irr > 395 nm ) . the quantum yield ( 500 ) of pyridine dissociation to form the corresponding [ ru(tpy)(nn)(ncch3 ) ] species was determined to be 0.16(1 ) and 0.03(1 ) for 2 and 3 ( irr = 500 nm ) , respectively , whereas the analogous pyridine exchange in 1 occurs with 500 < 0.0001 ( table 1 ) . it should be noted that the quantum yields observed for 2 and 3 are remarkably large for the photodissociation of a pyridine ligand with low energy visible light . for [ ru(bpy)2(py)2 ] , the photoinduced ligand exchange of one py ligand with cl in ch3cn was reported to take place with 436 = 0.0059 , and the introduction of ch3 groups to produce [ ru(4,4-me2bpy)2(py)2 ] ( 4,4-me2bpy = 4,4-dimethyl-2,2-bipyridine ) resulted in 436 = 0.025 under similar experimental conditions . an electronic effect from the ch3 groups in 2 is expected , but the significantly larger enhancement for 2 relative to 1 measured in the present work ( > 1600-fold ; table 1 ) indicates that the steric effects of the bulky nn ligand influence the photoreactivity to a much greater extent than electronic effects alone . moreover , the efficiency of the reaction measured for 2 is 4-fold greater than the cl ligand exchange reported for [ ru(tpy)(py)3 ] and cis-[ru(tpy)(py)2cl ] ( irr = 436 nm ) . irradiation of 2 and 3 with low energy light ( > 590 nm ) also promotes pyridine substitution in aqueous solution . the photolysis of 2 and 3 in h2o results in a red shift of the mlct transition , consistent with substitution of the pyridine ligand with a solvent h2o molecule to form the corresponding [ ru(tpy)(nn)(oh2 ) ] complex . the large differences in the efficiency of pyridine photodissociation for 2 and 3 relative to 1 in water are consistent with the analogous ch3cn experiments . changes in the electronic absorption spectroscopy in h2o with irr > 590 nm of ( a ) 94 m 2 for tirr = 0 , 1 , 3 , 7 , 15 , and 30 min and ( b ) 62 m 3 for tirr = 0 , 3 , 7 , 15 , 25 , 45 , 75 , and 100 min . the numbering scheme for the atoms of interest is depicted in figure 5 and those for all the atoms in 2 and 3 appear in figure s8 ( supporting information ) , the experimental and calculated ru n bond distances are listed in table 2 , and the experimental and calculated n ru the crystal structures of 2 and 3 are shown in figure 5a , b , respectively , and that of 1 was previously reported ; the experimental data for all three complexes are provided in tables 2 and 3 for comparison . it is evident from table 2 that the addition of steric bulk on nn results in an increase in ru n(5 ) bond lengths associated with the bidentate ligand by 0.040.05 and 0.010.02 , respectively , in both 2 and 3 relative to those of 1 . this distortion is evident in the angles associated with the coordinated pyridine units on the tpy ligand in 1 , determined to be 79.7 for n(1)ru n(2 ) and n(2)ru these angles remain relatively constant in all three complexes , 13 , indicating that the bonding of the tridentate tpy ligand to the ruthenium metal is not affected by the steric bulk on the bidentate ligand . in all three complexes , the planes defined by the three ru n bonds of the bidentate ligands are nearly orthogonal , as evidenced by the corresponding n(5)ru n(1,3 ) and n(4)ru n(13 ) angles , which range from 87.3 to 105.5 ( average = 95.3 ) , and n(2)ru n(5 ) , determined to be 171.9 in 1 ( table 3 ) . although the ru n bond angles and distances do not exhibit significant variation in 13 , distortion from planarity and significant tilting is observed in the bidentate ligands due to steric hindrance in 2 and 3 . the angle describing the orientation of the me2bpy ligand relative to tpy in 2 , defined by a plane containing the n(4 ) , n(5 ) , c(20 ) , and c(23 ) atoms of me2bpy ( figure 5 ) and a plane containing the n(1 ) , n(2 ) , and n(3 ) atoms in the tpy ligand , was determined to be 67.87. the corresponding angle in 3 was 61.89 , using the biq n(4 ) , n(5 ) , c(28 ) , and c(31 ) atoms to define the plane . for comparison , only a small distortion from the octahedral geometry is observed between the corresponding atoms in the bpy and tpy ligands in 1 , 83.34. therefore , an increase in the tpy nn angle of 15.47 in 2 and 21.45 in 3 are observed relative to the tpy these distortions are also evident in the calculated structures of 13 shown in figure 6a c . optimized structures predicted by dft calculations viewed along the axis containing the n(2)ru n(5 ) bonds for ( a ) 1 , ( b ) 2 , and ( c ) 3 , such that the plane of the tpy ligand is horizontal and that of the bidentate ligand is vertical with respect to the page . in the predicted and experimental structures of 2 and 3 , the bidentate ligands are tilted relative to the position of the bpy ligand in 1 , as the ch3 groups of me2bpy and the quinoline ligands are oriented toward the n(3 ) atom on tpy . to compare the tilting of the bidentate ligands in 2 and 3 , one plane was defined by the ru atom and the n4 and n5 atoms , and a second plane was defined by four atoms on each bidentate ligand ( figure s9 , supporting information ) . the resulting tilt angles in the crystal structures of 2 and 3 are + 20.97 and + 25.88 , respectively , which are similar to the calculated values of + 21 in 2 and + 23 in 3 . overall , the me2bpy and biq ligands are more tilted away from the n(4)ru n(5 ) plane than bpy in 1 by 27 and 32 in 2 and 3 , respectively . a similar effect was noted in [ ru(bpy)2(dmdppz ) ] ( dmdppz = 3,6-dimethyldipyridylphenazine ) , [ ru(biq)2(phen ) ] , and [ ru(bu - tpy)(me2phen)(meobn ) ] in which the dmdppz , biq , and me2phen ligands tilt approximately 15 , 20 , and 23 , respectively , due to the steric constraints from the bulky ligands . another major structural difference measured and calculated for 2 and 3 relative to 1 can be found in the tilt of pyridine toward the n(1 ) and n(2 ) atoms of the tpy ligand and its rotation about the ru n(13 ) bond angles listed in table 3 for 2 and 3 show that the pyridine ligand is significantly tilted toward the portion of tpy ligand bearing the n(1 ) and n(2 ) atoms and away from the n(3 ) atom . n(6 ) and n(2)ru n(6 ) angles in the crystal structures of 2 and 3 ranged from 84.45 to 87.84 , and are smaller than the same angles in 1 , 92.6 and 91.0 , respectively . in contrast , the n(3)ru n(6 ) angles in 2 and 3 , 94.17 and 91.81 , respectively , are larger than the 89.11 determined for 1 . the optimized geometries of the three complexes shown in figure 6 provide a visual comparison of the deviations from the predicted 90 n(6)ru n(13 ) angle in 2 and 3 relative to 1 ; this tilt of the pyridine ligand in 2 and 3 is expected to weaken its -bonding and -bonding with the metal relative to 1 . in addition , both the crystal structure and the calculations show that the rotation of the pyridine about its ru n bond deviates significantly in 2 and 3 as compared to that in 1 due to steric constraints imparted by the me2bpy and biq ligands . this rotation in the former is clearly evident in the views provided in figure 6 and is described by the n(2)ru n(6)c(39 ) dihedral angle in 3 , where c(32 ) and c(32 ) are the carbon atoms on pyridine pointing toward n(3 ) on the tpy ligand in each complex . in the crystal structure of 1 , this dihedral angle is 128.49 , a geometry that is expected to provide good orbital overlap for -back - bonding to the metal . this angle is much larger than the 56.66 and 45.57 dihedral angles in 2 and 3 , respectively , such that overlap between the * orbitals of the py group and filled metal t2g - type orbitals for -back - bonding is decreased relative to 1 . the dihedral angles measured in the calculated structures , 114.36 , 59.89 , and 52.78 for 1 , 2 , and 3 , respectively , are in good agreement with those from the crystal structures . similar distortions in [ ru(bpy)2(dmdppz ) ] , [ ru(biq)2(phen ) ] , and [ ru(bu - tpy)(me2phen)(meobn ) ] are believed to result in enhanced photoinduced ligand exchange of bulky bidentate ligands . the geometric distortions are similar for 2 and 3 , but the lf state in 3 is also predicted to be stabilized relative to that of 1 . however , the mlct state of 3 is lower in energy than that of 2 due to the lower - lying biq orbitals relative to me2bpy , such that the energy difference between the lowest energy mlct state and the dissociative lf state(s ) is greater in the former . additionally , the more distorted geometry of the bound pyridine in 2 as compared to 3 may result in smaller overlap in the bonding orbitals in the former , facilitating more efficient pyridine dissociation for 2 in the excited state . photoinduced pyridine dissociation with greatly enhanced efficiencies over the previously reported 1 was achieved with the bulky bidentate ligands me2bpy in 2 and biq in 3 . in ch3cn solution , pyridine is replaced by a solvent molecule with 500 = 0.16(1 ) and 0.033(1 ) for 2 and 3 , respectively , whereas ligand exchange is much less efficient for 1 ( 500 < 0.0001 ) . to this end , low energy light ( > 590 nm ) was shown to promote pyridine dissociation in aqueous solution for 2 and 3 on time scales that are inaccessible with 1 . the x - ray crystal structures and theoretical calculations for the three complexes depict significantly more distorted geometries for 2 and 3 compared to 1 due to steric hindrance between the pyridine ligand and the bulky substituent .

ru(ii)polypyridyl complexes are commonly employed in these schemes due to their relatively strong absorption throughout the ultraviolet and visible spectral regions , chemical stability in solution , and their long excited state lifetimes and reactivity that is inaccessible in the ground state . the population of the metal - to - ligand charge transfer ( mlct ) excited state in these complexes following absorption of a photon is known to undergo fast intersystem crossing to the corresponding mlct state ; in [ ru(bpy)3 ] ( bpy = 2,2-bipyridine ) within 1540 fs . for example , the photoisomerization from s - bound to o - bound dmso for a series of [ ru(tpy)(l)(dmso ) ] complexes ( dmso = dimethyl sulfoxide , l = bidentate ligand ) has been used for potential applications in information storage because the compounds are photochromic . in these systems , the s o isomerization proceeds with a quantum yield ( so ) dependent on the -donor strength of the atom positioned trans to the dmso ligand , with values of 0.25(1 ) , 0.024(1 ) , and 0.007(1 ) for the complexes with l = pic ( picolinate ) , bpy , and tmen ( n , the irradiation of the related complex [ ru(tpy)(bpy)(ch3cn ) ] with visible light in ch3cn in the presence of 1 m pyridine produces [ ru(tpy)(bpy)(py ) ] ( py = pyridine ) with = 0.0013 ( irr = 464 nm ) . an important point of interest is that the photorelease of pyridine in [ ru(tpy)(bpy)(py ) ] is a significantly less efficient process because pyridine forms a stronger bond with ru(ii ) , such that monodentate ligand dissociation in the excited state is less favorable in the py complex than in the corresponding ch3cn system . the addition of steric bulk to the ligand set in ru(ii ) complexes provides a means to distort the pseudo - octahedral geometry around the metal , which has been shown to enhance the efficiency of the excited state ligand exchange . a 20-fold increase in efficiency of this process was observed when the analogous [ ru(bu - tpy)(me2phen)(meobn ) ] complex was used under similar experimental conditions , which contains the sterically bulky me2phen ( me2phen = 2,9-dimethyl-1,10-phenanthroline ) ligand . the present work focuses on the investigation of two new complexes related to [ ru(tpy)(bpy)(py ) ] ( 1 ) , but with additional steric bulk on the bidentate ligand , [ ru(tpy)(me2bpy)(py ) ] ( 2 ) and [ ru(tpy)(biq)(py ) ] ( 3 ; biq = 2,2-biquinoline ) . in addition to the steric demands of the biq ligand in 3 , the low - lying biq acceptor orbitals serve to red shift the mlct absorption maximum , an important factor in pct to achieve tissue penetration . a spectroscopic analysis of the photolysis of 13 in ch3cn and h2o are provided , and the quantum yields of pyridine exchange are discussed in relation to the distortion induced by sterically demanding bidentate ligands as determined from x - ray crystal structures and theoretical calculations . schematic representation of the [ ru(tpy)(nn)(py ) ] complexes with the structures of the bidentate nn ligands bpy , me2bpy , and biq in 1 , 2 , and 3 , respectively . electronic absorption singlet to singlet transitions were calculated using time - dependent dft ( td - dft ) methods with the polarizable continuum model ( pcm ) that mimicked the solvation effect of ch3cn in gaussian 09 . the electronic absorption spectra of the series of complexes [ ru(tpy)(nn)(py ) ] , where nn = bpy ( 1 ) , me2bpy ( 2 ) , and biq ( 3 ) , are shown in figure 2 . in general , the spectra of 13 feature * transitions associated with tpy and the nn bidentate ligands in the uv region and ru(d ) tpy / nn( * ) mlct transitions in the visible range , both of which are typical of ru(ii)polypyridyl complexes . the lowest energy absorption peaks in 1 and 2 have contributions from both ru bpy / me2bpy and ru tpy mlct transitions , and the red shift observed for 3 clearly indicates that the lowest energy transition is ru biq mlct due to stabilized biq * orbitals relative to those of bpy , me2bpy , and tpy . the stronger absorption of 3 in the low energy tail ( > 600 nm ) represents a promising feature for the application of the complex as a photoactivated drug delivery vehicle because low energy light ( 600900 nm ) is necessary to penetrate tissue for targeted drug delivery . the photosubstitution of pyridine in 13 with ch3cn was investigated to compare the efficiency of ligand exchange as a function of the identity of the bidentate ligand . in the dark , 13 do not undergo ligand exchange in ch3cn ( figures s1s3 , supporting information ) , but clear changes in the electronic absorption spectra are observed for 2 and 3 upon irradiation with visible light in ch3cn , resulting in a blue shift in the mlct maximum from 470 to 454 nm in 2 ( figure 3a ) and from 529 to 513 nm for 3 ( figure 3b ) . the multiple isosbestic points observed for each complex as a function of irradiation time , at 385 , 415 , and 462 nm for 2 and at 392 , 425 , 457 , and 513 nm for 3 , indicate the formation of a single photoproduct in each reaction . changes in the electronic absorption spectroscopy in ch3cn with irr > 395 nm of ( a ) 50 m 2 for tirr = 0 , 0.25 , 0.5 , 1 , and 2 min and ( b ) 40 m 3 for tirr = 0 , 0.25 , 0.5 , 1 , 2 , and 4 min . the quantum yield ( 500 ) of pyridine dissociation to form the corresponding [ ru(tpy)(nn)(ncch3 ) ] species was determined to be 0.16(1 ) and 0.03(1 ) for 2 and 3 ( irr = 500 nm ) , respectively , whereas the analogous pyridine exchange in 1 occurs with 500 < 0.0001 ( table 1 ) . for [ ru(bpy)2(py)2 ] , the photoinduced ligand exchange of one py ligand with cl in ch3cn was reported to take place with 436 = 0.0059 , and the introduction of ch3 groups to produce [ ru(4,4-me2bpy)2(py)2 ] ( 4,4-me2bpy = 4,4-dimethyl-2,2-bipyridine ) resulted in 436 = 0.025 under similar experimental conditions . an electronic effect from the ch3 groups in 2 is expected , but the significantly larger enhancement for 2 relative to 1 measured in the present work ( > 1600-fold ; table 1 ) indicates that the steric effects of the bulky nn ligand influence the photoreactivity to a much greater extent than electronic effects alone . moreover , the efficiency of the reaction measured for 2 is 4-fold greater than the cl ligand exchange reported for [ ru(tpy)(py)3 ] and cis-[ru(tpy)(py)2cl ] ( irr = 436 nm ) . the photolysis of 2 and 3 in h2o results in a red shift of the mlct transition , consistent with substitution of the pyridine ligand with a solvent h2o molecule to form the corresponding [ ru(tpy)(nn)(oh2 ) ] complex . photolysis of 2 in h2o shifts the mlct absorption from 473 to 485 nm with an isosbestic point at 483 nm , resulting in a spectrum that is in sound agreement with that reported for [ ru(tpy)(me2bpy)(oh2 ) ] . a similar trend is observed for 3 , in which the mlct absorption shifts from 530 to 548 nm with isosbestic points at 408 and 540 nm , and the spectrum of the photoproduct is consistent with that of [ ru(tpy)(biq)(oh2 ) ] . changes in the electronic absorption spectroscopy in h2o with irr > 590 nm of ( a ) 94 m 2 for tirr = 0 , 1 , 3 , 7 , 15 , and 30 min and ( b ) 62 m 3 for tirr = 0 , 3 , 7 , 15 , 25 , 45 , 75 , and 100 min . the numbering scheme for the atoms of interest is depicted in figure 5 and those for all the atoms in 2 and 3 appear in figure s8 ( supporting information ) , the experimental and calculated ru n bond distances are listed in table 2 , and the experimental and calculated n ru the crystal structures of 2 and 3 are shown in figure 5a , b , respectively , and that of 1 was previously reported ; the experimental data for all three complexes are provided in tables 2 and 3 for comparison . this effect was previously observed in the monodentate and bidentate bond distances of [ ru(tpy)(phen)(ncch3 ) ] and [ ru(tpy)(me2phen)(ncch3 ) ] ( me2phen = 2,9-dimethyl-1,10-phenanthroline ) . it is evident from table 2 that the addition of steric bulk on nn results in an increase in ru n(5 ) bond lengths associated with the bidentate ligand by 0.040.05 and 0.010.02 , respectively , in both 2 and 3 relative to those of 1 . the bite angle of the tpy ligand distorts the bond angles from the 90 ( between cis positions ) and 180 ( between trans positions ) of an ideal octahedral complex . this distortion is evident in the angles associated with the coordinated pyridine units on the tpy ligand in 1 , determined to be 79.7 for n(1)ru n(2 ) and n(2)ru these angles remain relatively constant in all three complexes , 13 , indicating that the bonding of the tridentate tpy ligand to the ruthenium metal is not affected by the steric bulk on the bidentate ligand . in all three complexes , the planes defined by the three ru n bonds of the bidentate ligands are nearly orthogonal , as evidenced by the corresponding n(5)ru n(1,3 ) and n(4)ru n(13 ) angles , which range from 87.3 to 105.5 ( average = 95.3 ) , and n(2)ru n(5 ) , determined to be 171.9 in 1 ( table 3 ) . although the ru n bond angles and distances do not exhibit significant variation in 13 , distortion from planarity and significant tilting is observed in the bidentate ligands due to steric hindrance in 2 and 3 . the angle describing the orientation of the me2bpy ligand relative to tpy in 2 , defined by a plane containing the n(4 ) , n(5 ) , c(20 ) , and c(23 ) atoms of me2bpy ( figure 5 ) and a plane containing the n(1 ) , n(2 ) , and n(3 ) atoms in the tpy ligand , was determined to be 67.87. the corresponding angle in 3 was 61.89 , using the biq n(4 ) , n(5 ) , c(28 ) , and c(31 ) atoms to define the plane . for comparison , only a small distortion from the octahedral geometry is observed between the corresponding atoms in the bpy and tpy ligands in 1 , 83.34. therefore , an increase in the tpy nn angle of 15.47 in 2 and 21.45 in 3 are observed relative to the tpy these distortions are also evident in the calculated structures of 13 shown in figure 6a c . optimized structures predicted by dft calculations viewed along the axis containing the n(2)ru n(5 ) bonds for ( a ) 1 , ( b ) 2 , and ( c ) 3 , such that the plane of the tpy ligand is horizontal and that of the bidentate ligand is vertical with respect to the page . in the predicted and experimental structures of 2 and 3 , the bidentate ligands are tilted relative to the position of the bpy ligand in 1 , as the ch3 groups of me2bpy and the quinoline ligands are oriented toward the n(3 ) atom on tpy . to compare the tilting of the bidentate ligands in 2 and 3 , one plane was defined by the ru atom and the n4 and n5 atoms , and a second plane was defined by four atoms on each bidentate ligand ( figure s9 , supporting information ) . in 2 , the latter were n4 , n5 , c20 , and c23 on me2bpy and in 3 the n4 , n5 , c28 , and c31 atoms of biq were used to define the plane . the resulting tilt angles in the crystal structures of 2 and 3 are + 20.97 and + 25.88 , respectively , which are similar to the calculated values of + 21 in 2 and + 23 in 3 . in contrast , a much smaller tilt is calculated for bpy in 1 and in the opposite direction , 6 , with a comparable value in the crystal structure , 6.75 , as expected from its less sterically demanding structure . a similar effect was noted in [ ru(bpy)2(dmdppz ) ] ( dmdppz = 3,6-dimethyldipyridylphenazine ) , [ ru(biq)2(phen ) ] , and [ ru(bu - tpy)(me2phen)(meobn ) ] in which the dmdppz , biq , and me2phen ligands tilt approximately 15 , 20 , and 23 , respectively , due to the steric constraints from the bulky ligands . another major structural difference measured and calculated for 2 and 3 relative to 1 can be found in the tilt of pyridine toward the n(1 ) and n(2 ) atoms of the tpy ligand and its rotation about the ru n(13 ) bond angles listed in table 3 for 2 and 3 show that the pyridine ligand is significantly tilted toward the portion of tpy ligand bearing the n(1 ) and n(2 ) atoms and away from the n(3 ) atom . n(6 ) and n(2)ru n(6 ) angles in the crystal structures of 2 and 3 ranged from 84.45 to 87.84 , and are smaller than the same angles in 1 , 92.6 and 91.0 , respectively . the optimized geometries of the three complexes shown in figure 6 provide a visual comparison of the deviations from the predicted 90 n(6)ru n(13 ) angle in 2 and 3 relative to 1 ; this tilt of the pyridine ligand in 2 and 3 is expected to weaken its -bonding and -bonding with the metal relative to 1 . in addition , both the crystal structure and the calculations show that the rotation of the pyridine about its ru n bond deviates significantly in 2 and 3 as compared to that in 1 due to steric constraints imparted by the me2bpy and biq ligands . this rotation in the former is clearly evident in the views provided in figure 6 and is described by the n(2)ru n(6)c(39 ) dihedral angle in 3 , where c(32 ) and c(32 ) are the carbon atoms on pyridine pointing toward n(3 ) on the tpy ligand in each complex . this angle is much larger than the 56.66 and 45.57 dihedral angles in 2 and 3 , respectively , such that overlap between the * orbitals of the py group and filled metal t2g - type orbitals for -back - bonding is decreased relative to 1 . on the basis of the electronic absorption spectroscopy , the significantly distorted geometry for 2 lowers the energy of the lf state and weakens the ru py -bond and -back - bonding , resulting in greater relative population of the lf state and enhanced ligand dissociation . however , the mlct state of 3 is lower in energy than that of 2 due to the lower - lying biq orbitals relative to me2bpy , such that the energy difference between the lowest energy mlct state and the dissociative lf state(s ) is greater in the former . additionally , the more distorted geometry of the bound pyridine in 2 as compared to 3 may result in smaller overlap in the bonding orbitals in the former , facilitating more efficient pyridine dissociation for 2 in the excited state . the x - ray crystal structures and theoretical calculations for the three complexes depict significantly more distorted geometries for 2 and 3 compared to 1 due to steric hindrance between the pyridine ligand and the bulky substituent .

this study was approved by the institutional review board ( irb ) of the georgia regents university , and informed consent was obtained from every subject or his / her legally authorized representative . the sex and age distributions , age of onset for the patients , presence or absence of a first - degree relative with the disease , duration of diabetes , complications of diabetes , as well as genetic risk information for subjects have been summarized in supplementary table 1 . peripheral blood ( 2.5 ml ) after 2 h at room temperature , the tubes were frozen at 80c and total rna was extracted within a few weeks . an aliquot of total rna for each sample was arrayed in 96-well plates and then converted to cdna using a high - capacity cdna reverse transcription kit ( applied biosystems ) . the cdna products were diluted , and an aliquot of cdna equivalent to 10 ng of total rna was used for quantitative real - time pcr performed using ready - to - use taqman gene expression assays from applied biosystems . a total of 1,850 cdna samples were arrayed in five 384-well plates , each plate containing 185 t1d and 185 autoantibody - negative ( abn ) samples in addition to a serial dilution of a common cdna sample for the establishment of a standard curve . a total of 24 assays , including 18 target genes , 3 negative control genes , and 3 reference or housekeeping genes , were analyzed by real - time pcr using taqman assay ( applied biosystems ) ( supplementary table 2 ) . real - time pcr was performed in 384-well plates with the abi 7900ht fast real - time pcr system . standard thermal cycling conditions ( 10 min at 95c , 40 cycles for 15 s at 95c , and 1 min at 60c ) were used for all genes . cycle threshold ( ct ) values and quantity ( q ) values , calculated from ct values based on a standard curve , for each test gene were obtained for each sample using the sds2.3 software . all statistical analyses were performed using the r language and environment for statistical computing ( r version 2.12.1 ; r foundation for statistical computing , www.r-project.org ) . there was a small proportion ( < 5% ) of cdna samples that failed to amplify for each gene , and those data were removed before statistical analyses . plate - plate variations were normalized by using a standard curve on each 384-well plate . after selection and validation , the combination of gapdh , b2 m , and abl genes was used as reference genes for normalization . the pairwise correlation between individual gene expression levels was computed using the pearson correlation coefficient . linear regression of gene expression level with age as a covariate on data stratified by sex and disease status was completed to assess the effect of age on the expression level of each studied gene . since there was a significant effect of age and sex on the expression levels of many genes , case - control matching was performed with respect to age and sex using the matching r package ( 9 ) . to estimate the relative risk of diabetes at different gene expression levels the odds ratios ( ors ) and 95% cis were computed for each gene . to investigate the association between gene expression levels and the diabetes complications , the samples from the t1d patients with a particular complication conditional logistic regression was performed to estimate the relative risk of a particular complication at different expression levels . for analyses using gene expression as a continuous variable , the data were log transformed and scaled to unit sd . for analyses using gene expression level as categorical variables , the values of low or high were assigned using the middle value ( median ) in control subjects as a cutoff point . this study was approved by the institutional review board ( irb ) of the georgia regents university , and informed consent was obtained from every subject or his / her legally authorized representative . the sex and age distributions , age of onset for the patients , presence or absence of a first - degree relative with the disease , duration of diabetes , complications of diabetes , as well as genetic risk information for subjects have been summarized in supplementary table 1 . after 2 h at room temperature , the tubes were frozen at 80c and total rna was extracted within a few weeks . an aliquot of total rna for each sample was arrayed in 96-well plates and then converted to cdna using a high - capacity cdna reverse transcription kit ( applied biosystems ) . the cdna products were diluted , and an aliquot of cdna equivalent to 10 ng of total rna was used for quantitative real - time pcr performed using ready - to - use taqman gene expression assays from applied biosystems . a total of 1,850 cdna samples were arrayed in five 384-well plates , each plate containing 185 t1d and 185 autoantibody - negative ( abn ) samples in addition to a serial dilution of a common cdna sample for the establishment of a standard curve . a total of 24 assays , including 18 target genes , 3 negative control genes , and 3 reference or housekeeping genes , were analyzed by real - time pcr using taqman assay ( applied biosystems ) ( supplementary table 2 ) . real - time pcr was performed in 384-well plates with the abi 7900ht fast real - time pcr system . standard thermal cycling conditions ( 10 min at 95c , 40 cycles for 15 s at 95c , and 1 min at 60c ) were used for all genes . cycle threshold ( ct ) values and quantity ( q ) values , calculated from ct values based on a standard curve , for each test gene were obtained for each sample using the sds2.3 software . all statistical analyses were performed using the r language and environment for statistical computing ( r version 2.12.1 ; r foundation for statistical computing , www.r-project.org ) . there was a small proportion ( < 5% ) of cdna samples that failed to amplify for each gene , and those data were removed before statistical analyses . plate - plate variations were normalized by using a standard curve on each 384-well plate . after selection and validation , the combination of gapdh , b2 m , and abl genes was used as reference genes for normalization . the pairwise correlation between individual gene expression levels was computed using the pearson correlation coefficient . linear regression of gene expression level with age as a covariate on data stratified by sex and disease status was completed to assess the effect of age on the expression level of each studied gene . since there was a significant effect of age and sex on the expression levels of many genes , case - control matching was performed with respect to age and sex using the matching r package ( 9 ) . to estimate the relative risk of diabetes at different gene expression levels the odds ratios ( ors ) and 95% cis were computed for each gene . to investigate the association between gene expression levels and the diabetes complications , the samples from the t1d patients with a particular complication conditional logistic regression was performed to estimate the relative risk of a particular complication at different expression levels . for analyses using gene expression as a continuous variable , the data were log transformed and scaled to unit sd . for analyses using gene expression level as categorical variables , the values of low or high were assigned using the middle value ( median ) in control subjects as a cutoff point . fourteen genes were selected for validation studies based on our previous study ( 2 ) , and these genes showed higher expression in both t1d and autoantibody - positive groups as compared with the abn control group . four other genes ( tgfb1 , nfkb1 , foxp3 , and il12a ) were selected based on their potential relevance to t1d . three genes ( tsc22d3 , rpl17 , and eef1b2 ) that were not significant on our microarray dataset were included as negative controls . all genes were analyzed in a large sample set that included 928 t1d patients and 922 abn control subjects using taqman assays . , we determined whether sex and sampling age of the subjects were potential confounding factors . data for each gene were divided into four subgroups based on sex and disease phenotype . there were significant correlations with age in both phenotypic groups and for both sexes for several genes ( data not shown ) . in order to accurately assess the gene expression associated with t1d the ors per sd increment of gene expression levels were first computed using expression data as continuous variable ( table 1 ) . these analyses suggest that eight genes ( tgfb1 , sell , psmb3 , cd74 , s100a8 , s100a9 , and il12a ) were significantly upregulated and three genes ( gnly , psma4 , and smad7 ) were significantly downregulated in t1d patients . conditional logistic regression analysis was also performed using expression values as categorical variable after assigning each subject into low- or high - expression subgroups using the mean expression levels observed in abn control subjects . the results are highly consistent with the analysis using expression levels as continuous data ( table 1 ) . we also compared gene expression differences between juvenile - onset ( n = 692 ) and adult - onset t1d patients ( n = 232 ) , but no significant difference was found ( data not shown ) . the three negative control genes were analyzed using the same approaches , and no significant differences were found for these genes ( data not shown ) . box plots for gene expression levels ( data were log transformed ) in different subject groups including abn , t1d without any complication ( t1d - nocomp ) , t1d with at least one complication ( anycomp ) , and t1d with one specific complication ( neuropathy , htn , retinopathy , nephropathy , or cad ) . gene expression changes in t1d compared with abn ( n = 922 ) the t1d group is a heterogeneous set of subjects as some have developed complications such as neuropathy , retinopathy , nephropathy , coronary artery disease ( cad ) , and hypertension ( htn ) . as the expression levels for some genes may be associated with diabetes complications , it is important to delineate the gene expression differences caused by hyperglycemia or the specific types of complications . among the 928 t1d patients in our dataset , 238 patients had at least one diabetes complication . to exclude expression differences caused by diabetes complications , we compared expression levels between 922 abn control subjects and 407 t1d patients without any complications ( table 1 ) . in the first analysis , each t1d patient without complication is matched with an abn control for age and sex ; the expression data were analyzed as continuous variables using logistic regression . the results indicate that the seven of eight genes significantly upregulated in the entire dataset ( except for s100a9 ) were also significantly higher in t1d patients without complications compared with control subjects . additionally , the three genes shown to be downregulated in the entire t1d cohort were consistently downregulated in the t1d subset without complications ( table 1 ) . conditional logistic regression analysis using expression values as categorical variable also revealed similar findings ( table 1 ) . we next examined gene expression changes associated with complications seen in t1d patients . in this dataset , there were 119 patients with hypertension , 59 patients with neuropathy , 70 patients with retinopathy , 35 patients with cad , and 26 patients with nephropathy . before examining the gene expression changes in different diabetes complications , we determined whether t1d duration is a potential confounding factor since t1d duration is significantly longer for all complication groups ( supplementary table 3 ) . we performed regression analysis of gene expression level with t1d duration as covariate ( supplementary table 4 ) . there were significant correlations with t1d duration in both sexes for four genes ( s100a9 , s100a8 , psma4 , and il12a ) and significant correlation in only the male group for four genes ( rnf31 , foxp3 , mfng , and nfkb1 ) . since t1d duration and sex are confounding factors for most genes , age , t1d duration , and sex matching was then performed using a multivariate and propensity score matching software ( 9 ) . each t1d patient with a specific complication was paired with one or two of the closest t1d patient(s ) without any complications . the comparisons of expression levels between complication and noncomplication groups are shown as a box plot for each complication ( fig . conditional logistic regression analysis was performed to first assess changes associated with diabetes complication using all 238 patients with at least one complication . four genes ( s100a8 , s100a9 , sell , and mnda ) were significantly higher in the t1d patients with any one kind of complication ( any - complication group ) compared with the no - complication group , whereas four other genes ( smad7 , psma4 , mfng , and gnly ) were significantly lower in t1d with complication than t1d without complication ( table 2 ) . to exclude the possibility that the gene expression changes result from the treatment for secondary complications , we further examined gene expression differences between patients that were on treatment with specific drugs and those without the treatment . as the numbers of patients on individual drugs were very small , we grouped the treatments into four major categories : hypertension , blood cholesterol , hypothyroidism , and psychological disorders . our results suggest that the expression differences observed in this study were not due to treatment for diabetes complications ( data not shown ) . box plots for gene expression levels ( data were log transformed ) in t1d patients without any complication and t1d patients with different complication groups : at least one of complications ( anycomp ) , neuropathy , htn , retinopathy , nephropathy , and cad . comp , complications ; n , no for complications ; y , yes for complications . gene expressions associated with t1d complications we further examined these eight genes in five patient subsets with specific complications ( neuropathy , hypertension , retinopathy , nephropathy , and cad ) . patients with neuropathy showed significant changes for all eight genes , especially with the four upregulated genes . for example , subjects with higher s100a8 expression are 7.9 times more likely to have neuropathy . t1d patients with neuropathy are also much more likely to have higher gene expression levels for s100a9 , sell , and mnda ( or 4.8 , 4.9 , and 5.2 , respectively ) . patients with hypertension also showed significant changes for seven of the eight genes , including all four upregulated genes ( s100a8 , s100a9 , sell , and mnda ) ( table 2 ) . although statistical significance was not reached , the four upregulated genes were also higher in patients with other examined complications , including cad , retinopathy , and nephropathy , suggesting that these genes are probably implicated in all diabetes complications . finally , we examined pairwise correlation of gene expression levels for all 18 genes , and the results are graphically presented in fig . clustering of the genes based on the expression correlation values revealed a main cluster of proinflammatory genes ( s100a8 , s100a9 , mnda , and sell ) significantly upregulated in t1d and its complications . tgfb1 , nfkb1 , and rnf10 are also loosely grouped within this cluster , and these genes , especially tgfb1 , are increased in t1d compared with control subjects . pairwise correlations between gene expression levels for 18 studied genes in different subject groups : abn , t1d without any complication ( t1d - nocomp ) , t1d with at least one complication ( anycomp ) , and t1d with neuropathy . the squares indicate the high correlation between s100a8 , s100a9 , mnda , and sell . fourteen genes were selected for validation studies based on our previous study ( 2 ) , and these genes showed higher expression in both t1d and autoantibody - positive groups as compared with the abn control group . four other genes ( tgfb1 , nfkb1 , foxp3 , and il12a ) were selected based on their potential relevance to t1d . three genes ( tsc22d3 , rpl17 , and eef1b2 ) that were not significant on our microarray dataset were included as negative controls . all genes were analyzed in a large sample set that included 928 t1d patients and 922 abn control subjects using taqman assays . , we determined whether sex and sampling age of the subjects were potential confounding factors . data for each gene were divided into four subgroups based on sex and disease phenotype . there were significant correlations with age in both phenotypic groups and for both sexes for several genes ( data not shown ) . in order to accurately assess the gene expression associated with t1d the ors per sd increment of gene expression levels were first computed using expression data as continuous variable ( table 1 ) . these analyses suggest that eight genes ( tgfb1 , sell , psmb3 , cd74 , s100a8 , s100a9 , and il12a ) were significantly upregulated and three genes ( gnly , psma4 , and smad7 ) were significantly downregulated in t1d patients . conditional logistic regression analysis was also performed using expression values as categorical variable after assigning each subject into low- or high - expression subgroups using the mean expression levels observed in abn control subjects . the results are highly consistent with the analysis using expression levels as continuous data ( table 1 ) . we also compared gene expression differences between juvenile - onset ( n = 692 ) and adult - onset t1d patients ( n = 232 ) , but no significant difference was found ( data not shown ) . the three negative control genes were analyzed using the same approaches , and no significant differences were found for these genes ( data not shown ) . box plots for gene expression levels ( data were log transformed ) in different subject groups including abn , t1d without any complication ( t1d - nocomp ) , t1d with at least one complication ( anycomp ) , and t1d with one specific complication ( neuropathy , htn , retinopathy , nephropathy , or cad ) . gene expression changes in t1d compared with abn ( n = 922 ) the t1d group is a heterogeneous set of subjects as some have developed complications such as neuropathy , retinopathy , nephropathy , coronary artery disease ( cad ) , and hypertension ( htn ) . as the expression levels for some genes may be associated with diabetes complications , it is important to delineate the gene expression differences caused by hyperglycemia or the specific types of complications . among the 928 t1d patients in our dataset , 238 patients had at least one diabetes complication . to exclude expression differences caused by diabetes complications , we compared expression levels between 922 abn control subjects and 407 t1d patients without any complications ( table 1 ) . in the first analysis , each t1d patient without complication is matched with an abn control for age and sex ; the expression data were analyzed as continuous variables using logistic regression . the results indicate that the seven of eight genes significantly upregulated in the entire dataset ( except for s100a9 ) were also significantly higher in t1d patients without complications compared with control subjects . additionally , the three genes shown to be downregulated in the entire t1d cohort were consistently downregulated in the t1d subset without complications ( table 1 ) . conditional logistic regression analysis using expression values as categorical variable also revealed similar findings ( table 1 ) . we next examined gene expression changes associated with complications seen in t1d patients . in this dataset , there were 119 patients with hypertension , 59 patients with neuropathy , 70 patients with retinopathy , 35 patients with cad , and 26 patients with nephropathy . before examining the gene expression changes in different diabetes complications , we determined whether t1d duration is a potential confounding factor since t1d duration is significantly longer for all complication groups ( supplementary table 3 ) . we performed regression analysis of gene expression level with t1d duration as covariate ( supplementary table 4 ) . there were significant correlations with t1d duration in both sexes for four genes ( s100a9 , s100a8 , psma4 , and il12a ) and significant correlation in only the male group for four genes ( rnf31 , foxp3 , mfng , and nfkb1 ) . since t1d duration and sex are confounding factors for most genes , age , t1d duration , and sex matching was then performed using a multivariate and propensity score matching software ( 9 ) . each t1d patient with a specific complication was paired with one or two of the closest t1d patient(s ) without any complications . the comparisons of expression levels between complication and noncomplication groups are shown as a box plot for each complication ( fig . conditional logistic regression analysis was performed to first assess changes associated with diabetes complication using all 238 patients with at least one complication . four genes ( s100a8 , s100a9 , sell , and mnda ) were significantly higher in the t1d patients with any one kind of complication ( any - complication group ) compared with the no - complication group , whereas four other genes ( smad7 , psma4 , mfng , and gnly ) were significantly lower in t1d with complication than t1d without complication ( table 2 ) . to exclude the possibility that the gene expression changes result from the treatment for secondary complications , we further examined gene expression differences between patients that were on treatment with specific drugs and those without the treatment . as the numbers of patients on individual drugs were very small , we grouped the treatments into four major categories : hypertension , blood cholesterol , hypothyroidism , and psychological disorders . our results suggest that the expression differences observed in this study were not due to treatment for diabetes complications ( data not shown ) . box plots for gene expression levels ( data were log transformed ) in t1d patients without any complication and t1d patients with different complication groups : at least one of complications ( anycomp ) , neuropathy , htn , retinopathy , nephropathy , and cad . comp , complications ; n , no for complications ; y , yes for complications . gene expressions associated with t1d complications we further examined these eight genes in five patient subsets with specific complications ( neuropathy , hypertension , retinopathy , nephropathy , and cad ) . patients with neuropathy showed significant changes for all eight genes , especially with the four upregulated genes . for example , subjects with higher s100a8 expression are 7.9 times more likely to have neuropathy . t1d patients with neuropathy are also much more likely to have higher gene expression levels for s100a9 , sell , and mnda ( or 4.8 , 4.9 , and 5.2 , respectively ) . patients with hypertension also showed significant changes for seven of the eight genes , including all four upregulated genes ( s100a8 , s100a9 , sell , and mnda ) ( table 2 ) . although statistical significance was not reached , the four upregulated genes were also higher in patients with other examined complications , including cad , retinopathy , and nephropathy , suggesting that these genes are probably implicated in all diabetes complications . finally , we examined pairwise correlation of gene expression levels for all 18 genes , and the results are graphically presented in fig . clustering of the genes based on the expression correlation values revealed a main cluster of proinflammatory genes ( s100a8 , s100a9 , mnda , and sell ) significantly upregulated in t1d and its complications . tgfb1 , nfkb1 , and rnf10 are also loosely grouped within this cluster , and these genes , especially tgfb1 , are increased in t1d compared with control subjects . pairwise correlations between gene expression levels for 18 studied genes in different subject groups : abn , t1d without any complication ( t1d - nocomp ) , t1d with at least one complication ( anycomp ) , and t1d with neuropathy . the squares indicate the high correlation between s100a8 , s100a9 , mnda , and sell . this is to date the largest - scale study on gene expression profiles in human t1d patients . the large sample set of close to 2,000 individuals was especially informative for the gene expression profiles in control subjects and t1d patients with and without diabetes complications . since expression data were generated on multiple plates , normalization through the use of a standard curve for each gene in each 384-well plate was critical to ensure consistency across plates . furthermore , the use of multiple validated reference genes , rather than a single gene , improved the normalization of the rna concentration across all samples . this well - designed and adequately powered study allowed us to confirm gene expression differences between t1d and control subjects initially suggested by microarray experiments . we also demonstrated that gene expression is significantly different between abn control subjects and t1d patients without any diabetes complications . the genes with a higher expression in t1d are implicated in immune function , such as inflammation ( s100a8 , s100a9 , mnda , and il12-a ) , immune regulation / promotion ( tgfb1 and sell ) , and antigen processing and presentation ( cd74 and psmb3 ) . our findings strongly support the notion that chronic inflammation contributes to t1d development ( 1012 ) . upregulation of genes involved in antigen processing and presentation may occur in response to the activation and proliferation of autoreactive pathogenic t cells in t1d patients . two previous studies with small sample sizes ( 13,14 ) reported a decreased tgfb1 level in t1d patients ; however , this study with a very large sample size revealed a significantly higher expression of tgfb1 in t1d patients . it induces immune tolerance by acting as a potent immune suppressor through the inhibition of proliferation , differentiation , activation , and effector function of immune cells ( 1517 ) . parodoxically , it also promotes immune reaction by being a potent chemoattractant for neutrophils and promoting inflammation . in addition , it can induce differentiation into the anti - inflammatory regulatory t cells ( 1820 ) or the proinflammatory th17 cells ( 21 ) depending on the context . although the protective effects of tgfb1 have been documented in various autoimmune diseases , including t1d , the increased tgfb1 gene expression in t1d shown in the current study suggests that tgfb1 may mainly play a pathogenic role in t1d as a proinflammatory mediator . interestingly , the expression of an inhibitor of tgfb1 signaling , smad7 , showed a decreased level of expression in t1d as compared with healthy control subjects . smad7 encodes a nuclear protein that binds the e3 ubiquitin ligase smurf2 and inhibits tgfb1 signaling in many ways ( 22 ) . overexpression of smad7 has been shown to antagonize tgfb1-mediated fibrosis , carcinogenesis , and inflammation ( 23 ) . more interestingly , it has been reported that tgfb1 and smad7 play a major role in diabetic nephropathy ( 24 ) . in this study , smad7 is indeed reduced in t1d patients with complications compared with patients without any complications . the most noteworthy finding of this study is the identification of a set of genes associated with diabetes complications . relative to the comparison between t1d patients and control subjects , the sample sizes for the study on specific complications are smaller and the results should be viewed with more caution . however , several strategies used in this study ensure that the findings are most likely robust . first , we started by comparing t1d patients without any complication ( n = 407 ) and t1d patients with any complication ( n = 238 ) . only those genes showing significant differences in this relatively large dataset second , many of the genes are altered in multiple complications , further strengthening evidence for the findings . for example , the most prominent genes are two inflammatory genes , s100a8 and s100a9 , both being strongly associated with complications , especially neuropathy ( or 7.9 and 4.8 ) and hypertension ( 2.7 and 2.2 ) . these two genes may also be increased in retinopathy ( or 1.9 and 1.4 ) and nephropathy ( 3.6 and 1.3 ) , even though statistical significance was not reached due to small sample size . cad is a major macrovascular complication , and neuropathy , nephropathy , and retinopathy are major microvascular complications ( 25 ) . although the pathogenesis of diabetic angiopathy is incompletely understood , patients with one complication often present with a second one , suggesting the existence of common risk factors and pathogenic mechanisms . endothelial dysfunction , a common change among t1d patients , represents a major link between vascular complications ( 26 ) . s100a8 and s100a9 may play important roles in different pathways leading to endothelial dysfunction initiated by inflammation and/or hyperglycemia , two hallmarks of t1d patients ( fig . 4 ) . s100a8/a9 , also referred to as mrp8/14 , are two calcium - binding proteins primarily expressed in cells of myeloid origin , particularly in monocytes and neutrophils . as a marker of monocyte and neutrophil activation , the expression and secretion of the s100a8/a9 heterodimer could be induced by inflammatory cytokines such as il1 ( 27 ) . other inflammatory cytokines such as il6 and tnf- also had positive associations with s100a8/a9 and diabetic vascular complications ( 28 ) . in one pathway , s100a8/a9 stimulation can cause a rapid increase of cd11b expression on the monocyte surface , accounting for the increase of trans - endothelial migratory activity of monocytes and hence vascular endothelium changes ( 29 ) . in another pathway , s100a8/9 protein can bind specifically to and induce a specific inflammatory response in human vascular endothelial cells by increasing the transcription of proinflammatory chemokines and adhesion molecules ( 30 ) . more interestingly , the proinflammatory endothelial response to s100a8/a9 can be increased by advanced glycation end products ( ages ) since s100a8/a9 can work as ligands of rage ( receptor for age ) and have effects on the rage - nfkb mediated induction of proinflammatory gene expression ( 31 ) . studied genes involved in pathways leading to endothelial dysfunction . another myeloid cell related gene , mnda , was found as having highly correlated expression with s100a8/s100a9 and was also increased in neuropathy and hypertension ( or 5.2 and 2.5 ) . the myeloid cell nuclear differentiation antigen ( mnda ) is detected only in the nuclei of cells of the granulocyte - monocyte lineage and plays a role in the cell - specific response to interferon ( 32 ) . l - selectin ( sell ) is a cell surface adhesion molecule that plays a critical role in facilitating leukocyte migration from blood vessels to secondary lymphoid organ and sites of local inflammation . although there are controversial reports about the role of l - selectin in the development of autoimmune diabetes in nod mice ( 3335 ) , a positive correlation between the serum level of soluble l - selectin and t1d , diabetic retinopathy , atherosclerosis , and arterial hypertension was reported in human studies ( 3638 ) . the percentage of t cells expressing l - selectin was significantly increased in t1d patients and in t1d patients with arterial hypertension as well . consistent with these findings , we provided convincing evidence that l - selectin expression in peripheral blood mononuclear cells is higher in t1d patients ( or 1.4 ) , especially those patients with diabetic neuropathy ( 4.9 ) and hypertension ( 1.6 ) . the examination of a pairwise correlation of gene expression revealed coordinated upregulation of multiple proinflammatory genes in myeloid cells . s100a8 , s100a9 , and mnda are likely examples of the cluster of myeloid cell - related genes associated with t1d and diabetes complications . it will therefore be important to examine the cellular functions and pathogenic roles of myeloid cells in the development of t1d and its complications in future studies .

objectiveour previous gene expression microarray studies identified a number of genes differentially expressed in patients with type 1 diabetes ( t1d ) and islet autoantibody - positive subjects . this study was designed to validate these gene expression changes in t1d patients and to identify gene expression changes in diabetes complications.research desigh and methodswe performed high - throughput real - time rt - pcr to validate gene expression changes in peripheral blood mononuclear cells ( pbmcs ) from a large sample set of 928 t1d patients and 922 control subjects.resultsof the 18 genes analyzed here , eight genes ( s100a8 , s100a9 , mnda , sell , tgfb1 , psmb3 , cd74 , and il12a ) had higher expression and three genes ( gnly , psma4 , and smad7 ) had lower expression in t1d patients compared with control subjects , indicating that genes involved in inflammation , immune regulation , and antigen processing and presentation are significantly altered in pbmcs from t1d patients . furthermore , one adhesion molecule ( sell ) and three inflammatory genes mainly expressed by myeloid cells ( s100a8 , s100a9 , and mnda ) were significantly higher in t1d patients with complications ( odds ratio [ or ] 1.32.6 , adjusted p value = 0.005108 ) , especially those patients with neuropathy ( or 4.87.9 , adjusted p value < 0.005).conclusionsthese findings suggest that inflammatory mediators secreted mainly by myeloid cells are implicated in t1d and its complications .

RESEARCH DESIGN AND METHODS Human subjects and samples High-throughput real-time RT-PCR Statistical analyses RESULTS Gene expression changes associated with diabetes Gene expression changes associated with diabetes complications CONCLUSIONS

this study was approved by the institutional review board ( irb ) of the georgia regents university , and informed consent was obtained from every subject or his / her legally authorized representative . the cdna products were diluted , and an aliquot of cdna equivalent to 10 ng of total rna was used for quantitative real - time pcr performed using ready - to - use taqman gene expression assays from applied biosystems . a total of 1,850 cdna samples were arrayed in five 384-well plates , each plate containing 185 t1d and 185 autoantibody - negative ( abn ) samples in addition to a serial dilution of a common cdna sample for the establishment of a standard curve . a total of 24 assays , including 18 target genes , 3 negative control genes , and 3 reference or housekeeping genes , were analyzed by real - time pcr using taqman assay ( applied biosystems ) ( supplementary table 2 ) . to investigate the association between gene expression levels and the diabetes complications , the samples from the t1d patients with a particular complication conditional logistic regression was performed to estimate the relative risk of a particular complication at different expression levels . this study was approved by the institutional review board ( irb ) of the georgia regents university , and informed consent was obtained from every subject or his / her legally authorized representative . the cdna products were diluted , and an aliquot of cdna equivalent to 10 ng of total rna was used for quantitative real - time pcr performed using ready - to - use taqman gene expression assays from applied biosystems . a total of 24 assays , including 18 target genes , 3 negative control genes , and 3 reference or housekeeping genes , were analyzed by real - time pcr using taqman assay ( applied biosystems ) ( supplementary table 2 ) . real - time pcr was performed in 384-well plates with the abi 7900ht fast real - time pcr system . to investigate the association between gene expression levels and the diabetes complications , the samples from the t1d patients with a particular complication conditional logistic regression was performed to estimate the relative risk of a particular complication at different expression levels . for analyses using gene expression level as categorical variables , the values of low or high were assigned using the middle value ( median ) in control subjects as a cutoff point . fourteen genes were selected for validation studies based on our previous study ( 2 ) , and these genes showed higher expression in both t1d and autoantibody - positive groups as compared with the abn control group . four other genes ( tgfb1 , nfkb1 , foxp3 , and il12a ) were selected based on their potential relevance to t1d . three genes ( tsc22d3 , rpl17 , and eef1b2 ) that were not significant on our microarray dataset were included as negative controls . all genes were analyzed in a large sample set that included 928 t1d patients and 922 abn control subjects using taqman assays . these analyses suggest that eight genes ( tgfb1 , sell , psmb3 , cd74 , s100a8 , s100a9 , and il12a ) were significantly upregulated and three genes ( gnly , psma4 , and smad7 ) were significantly downregulated in t1d patients . we also compared gene expression differences between juvenile - onset ( n = 692 ) and adult - onset t1d patients ( n = 232 ) , but no significant difference was found ( data not shown ) . the three negative control genes were analyzed using the same approaches , and no significant differences were found for these genes ( data not shown ) . box plots for gene expression levels ( data were log transformed ) in different subject groups including abn , t1d without any complication ( t1d - nocomp ) , t1d with at least one complication ( anycomp ) , and t1d with one specific complication ( neuropathy , htn , retinopathy , nephropathy , or cad ) . gene expression changes in t1d compared with abn ( n = 922 ) the t1d group is a heterogeneous set of subjects as some have developed complications such as neuropathy , retinopathy , nephropathy , coronary artery disease ( cad ) , and hypertension ( htn ) . as the expression levels for some genes may be associated with diabetes complications , it is important to delineate the gene expression differences caused by hyperglycemia or the specific types of complications . among the 928 t1d patients in our dataset , 238 patients had at least one diabetes complication . to exclude expression differences caused by diabetes complications , we compared expression levels between 922 abn control subjects and 407 t1d patients without any complications ( table 1 ) . the results indicate that the seven of eight genes significantly upregulated in the entire dataset ( except for s100a9 ) were also significantly higher in t1d patients without complications compared with control subjects . we next examined gene expression changes associated with complications seen in t1d patients . in this dataset , there were 119 patients with hypertension , 59 patients with neuropathy , 70 patients with retinopathy , 35 patients with cad , and 26 patients with nephropathy . before examining the gene expression changes in different diabetes complications , we determined whether t1d duration is a potential confounding factor since t1d duration is significantly longer for all complication groups ( supplementary table 3 ) . there were significant correlations with t1d duration in both sexes for four genes ( s100a9 , s100a8 , psma4 , and il12a ) and significant correlation in only the male group for four genes ( rnf31 , foxp3 , mfng , and nfkb1 ) . four genes ( s100a8 , s100a9 , sell , and mnda ) were significantly higher in the t1d patients with any one kind of complication ( any - complication group ) compared with the no - complication group , whereas four other genes ( smad7 , psma4 , mfng , and gnly ) were significantly lower in t1d with complication than t1d without complication ( table 2 ) . our results suggest that the expression differences observed in this study were not due to treatment for diabetes complications ( data not shown ) . box plots for gene expression levels ( data were log transformed ) in t1d patients without any complication and t1d patients with different complication groups : at least one of complications ( anycomp ) , neuropathy , htn , retinopathy , nephropathy , and cad . gene expressions associated with t1d complications we further examined these eight genes in five patient subsets with specific complications ( neuropathy , hypertension , retinopathy , nephropathy , and cad ) . patients with neuropathy showed significant changes for all eight genes , especially with the four upregulated genes . t1d patients with neuropathy are also much more likely to have higher gene expression levels for s100a9 , sell , and mnda ( or 4.8 , 4.9 , and 5.2 , respectively ) . patients with hypertension also showed significant changes for seven of the eight genes , including all four upregulated genes ( s100a8 , s100a9 , sell , and mnda ) ( table 2 ) . although statistical significance was not reached , the four upregulated genes were also higher in patients with other examined complications , including cad , retinopathy , and nephropathy , suggesting that these genes are probably implicated in all diabetes complications . finally , we examined pairwise correlation of gene expression levels for all 18 genes , and the results are graphically presented in fig . clustering of the genes based on the expression correlation values revealed a main cluster of proinflammatory genes ( s100a8 , s100a9 , mnda , and sell ) significantly upregulated in t1d and its complications . tgfb1 , nfkb1 , and rnf10 are also loosely grouped within this cluster , and these genes , especially tgfb1 , are increased in t1d compared with control subjects . pairwise correlations between gene expression levels for 18 studied genes in different subject groups : abn , t1d without any complication ( t1d - nocomp ) , t1d with at least one complication ( anycomp ) , and t1d with neuropathy . the squares indicate the high correlation between s100a8 , s100a9 , mnda , and sell . fourteen genes were selected for validation studies based on our previous study ( 2 ) , and these genes showed higher expression in both t1d and autoantibody - positive groups as compared with the abn control group . four other genes ( tgfb1 , nfkb1 , foxp3 , and il12a ) were selected based on their potential relevance to t1d . three genes ( tsc22d3 , rpl17 , and eef1b2 ) that were not significant on our microarray dataset were included as negative controls . all genes were analyzed in a large sample set that included 928 t1d patients and 922 abn control subjects using taqman assays . these analyses suggest that eight genes ( tgfb1 , sell , psmb3 , cd74 , s100a8 , s100a9 , and il12a ) were significantly upregulated and three genes ( gnly , psma4 , and smad7 ) were significantly downregulated in t1d patients . we also compared gene expression differences between juvenile - onset ( n = 692 ) and adult - onset t1d patients ( n = 232 ) , but no significant difference was found ( data not shown ) . box plots for gene expression levels ( data were log transformed ) in different subject groups including abn , t1d without any complication ( t1d - nocomp ) , t1d with at least one complication ( anycomp ) , and t1d with one specific complication ( neuropathy , htn , retinopathy , nephropathy , or cad ) . gene expression changes in t1d compared with abn ( n = 922 ) the t1d group is a heterogeneous set of subjects as some have developed complications such as neuropathy , retinopathy , nephropathy , coronary artery disease ( cad ) , and hypertension ( htn ) . as the expression levels for some genes may be associated with diabetes complications , it is important to delineate the gene expression differences caused by hyperglycemia or the specific types of complications . among the 928 t1d patients in our dataset , 238 patients had at least one diabetes complication . to exclude expression differences caused by diabetes complications , we compared expression levels between 922 abn control subjects and 407 t1d patients without any complications ( table 1 ) . the results indicate that the seven of eight genes significantly upregulated in the entire dataset ( except for s100a9 ) were also significantly higher in t1d patients without complications compared with control subjects . we next examined gene expression changes associated with complications seen in t1d patients . in this dataset , there were 119 patients with hypertension , 59 patients with neuropathy , 70 patients with retinopathy , 35 patients with cad , and 26 patients with nephropathy . before examining the gene expression changes in different diabetes complications , we determined whether t1d duration is a potential confounding factor since t1d duration is significantly longer for all complication groups ( supplementary table 3 ) . there were significant correlations with t1d duration in both sexes for four genes ( s100a9 , s100a8 , psma4 , and il12a ) and significant correlation in only the male group for four genes ( rnf31 , foxp3 , mfng , and nfkb1 ) . four genes ( s100a8 , s100a9 , sell , and mnda ) were significantly higher in the t1d patients with any one kind of complication ( any - complication group ) compared with the no - complication group , whereas four other genes ( smad7 , psma4 , mfng , and gnly ) were significantly lower in t1d with complication than t1d without complication ( table 2 ) . to exclude the possibility that the gene expression changes result from the treatment for secondary complications , we further examined gene expression differences between patients that were on treatment with specific drugs and those without the treatment . our results suggest that the expression differences observed in this study were not due to treatment for diabetes complications ( data not shown ) . box plots for gene expression levels ( data were log transformed ) in t1d patients without any complication and t1d patients with different complication groups : at least one of complications ( anycomp ) , neuropathy , htn , retinopathy , nephropathy , and cad . gene expressions associated with t1d complications we further examined these eight genes in five patient subsets with specific complications ( neuropathy , hypertension , retinopathy , nephropathy , and cad ) . patients with neuropathy showed significant changes for all eight genes , especially with the four upregulated genes . t1d patients with neuropathy are also much more likely to have higher gene expression levels for s100a9 , sell , and mnda ( or 4.8 , 4.9 , and 5.2 , respectively ) . patients with hypertension also showed significant changes for seven of the eight genes , including all four upregulated genes ( s100a8 , s100a9 , sell , and mnda ) ( table 2 ) . although statistical significance was not reached , the four upregulated genes were also higher in patients with other examined complications , including cad , retinopathy , and nephropathy , suggesting that these genes are probably implicated in all diabetes complications . finally , we examined pairwise correlation of gene expression levels for all 18 genes , and the results are graphically presented in fig . clustering of the genes based on the expression correlation values revealed a main cluster of proinflammatory genes ( s100a8 , s100a9 , mnda , and sell ) significantly upregulated in t1d and its complications . tgfb1 , nfkb1 , and rnf10 are also loosely grouped within this cluster , and these genes , especially tgfb1 , are increased in t1d compared with control subjects . pairwise correlations between gene expression levels for 18 studied genes in different subject groups : abn , t1d without any complication ( t1d - nocomp ) , t1d with at least one complication ( anycomp ) , and t1d with neuropathy . the squares indicate the high correlation between s100a8 , s100a9 , mnda , and sell . this is to date the largest - scale study on gene expression profiles in human t1d patients . the large sample set of close to 2,000 individuals was especially informative for the gene expression profiles in control subjects and t1d patients with and without diabetes complications . this well - designed and adequately powered study allowed us to confirm gene expression differences between t1d and control subjects initially suggested by microarray experiments . we also demonstrated that gene expression is significantly different between abn control subjects and t1d patients without any diabetes complications . the genes with a higher expression in t1d are implicated in immune function , such as inflammation ( s100a8 , s100a9 , mnda , and il12-a ) , immune regulation / promotion ( tgfb1 and sell ) , and antigen processing and presentation ( cd74 and psmb3 ) . upregulation of genes involved in antigen processing and presentation may occur in response to the activation and proliferation of autoreactive pathogenic t cells in t1d patients . two previous studies with small sample sizes ( 13,14 ) reported a decreased tgfb1 level in t1d patients ; however , this study with a very large sample size revealed a significantly higher expression of tgfb1 in t1d patients . it induces immune tolerance by acting as a potent immune suppressor through the inhibition of proliferation , differentiation , activation , and effector function of immune cells ( 1517 ) . although the protective effects of tgfb1 have been documented in various autoimmune diseases , including t1d , the increased tgfb1 gene expression in t1d shown in the current study suggests that tgfb1 may mainly play a pathogenic role in t1d as a proinflammatory mediator . interestingly , the expression of an inhibitor of tgfb1 signaling , smad7 , showed a decreased level of expression in t1d as compared with healthy control subjects . in this study , smad7 is indeed reduced in t1d patients with complications compared with patients without any complications . the most noteworthy finding of this study is the identification of a set of genes associated with diabetes complications . relative to the comparison between t1d patients and control subjects , the sample sizes for the study on specific complications are smaller and the results should be viewed with more caution . first , we started by comparing t1d patients without any complication ( n = 407 ) and t1d patients with any complication ( n = 238 ) . for example , the most prominent genes are two inflammatory genes , s100a8 and s100a9 , both being strongly associated with complications , especially neuropathy ( or 7.9 and 4.8 ) and hypertension ( 2.7 and 2.2 ) . these two genes may also be increased in retinopathy ( or 1.9 and 1.4 ) and nephropathy ( 3.6 and 1.3 ) , even though statistical significance was not reached due to small sample size . more interestingly , the proinflammatory endothelial response to s100a8/a9 can be increased by advanced glycation end products ( ages ) since s100a8/a9 can work as ligands of rage ( receptor for age ) and have effects on the rage - nfkb mediated induction of proinflammatory gene expression ( 31 ) . studied genes involved in pathways leading to endothelial dysfunction . another myeloid cell related gene , mnda , was found as having highly correlated expression with s100a8/s100a9 and was also increased in neuropathy and hypertension ( or 5.2 and 2.5 ) . l - selectin ( sell ) is a cell surface adhesion molecule that plays a critical role in facilitating leukocyte migration from blood vessels to secondary lymphoid organ and sites of local inflammation . although there are controversial reports about the role of l - selectin in the development of autoimmune diabetes in nod mice ( 3335 ) , a positive correlation between the serum level of soluble l - selectin and t1d , diabetic retinopathy , atherosclerosis , and arterial hypertension was reported in human studies ( 3638 ) . the percentage of t cells expressing l - selectin was significantly increased in t1d patients and in t1d patients with arterial hypertension as well . consistent with these findings , we provided convincing evidence that l - selectin expression in peripheral blood mononuclear cells is higher in t1d patients ( or 1.4 ) , especially those patients with diabetic neuropathy ( 4.9 ) and hypertension ( 1.6 ) . the examination of a pairwise correlation of gene expression revealed coordinated upregulation of multiple proinflammatory genes in myeloid cells . s100a8 , s100a9 , and mnda are likely examples of the cluster of myeloid cell - related genes associated with t1d and diabetes complications . it will therefore be important to examine the cellular functions and pathogenic roles of myeloid cells in the development of t1d and its complications in future studies .

the sex and age distributions , age of onset for the patients , presence or absence of a first - degree relative with the disease , duration of diabetes , complications of diabetes , as well as genetic risk information for subjects have been summarized in supplementary table 1 . linear regression of gene expression level with age as a covariate on data stratified by sex and disease status was completed to assess the effect of age on the expression level of each studied gene . since there was a significant effect of age and sex on the expression levels of many genes , case - control matching was performed with respect to age and sex using the matching r package ( 9 ) . to estimate the relative risk of diabetes at different gene expression levels the odds ratios ( ors ) and 95% cis were computed for each gene . to investigate the association between gene expression levels and the diabetes complications , the samples from the t1d patients with a particular complication conditional logistic regression was performed to estimate the relative risk of a particular complication at different expression levels . the sex and age distributions , age of onset for the patients , presence or absence of a first - degree relative with the disease , duration of diabetes , complications of diabetes , as well as genetic risk information for subjects have been summarized in supplementary table 1 . the cdna products were diluted , and an aliquot of cdna equivalent to 10 ng of total rna was used for quantitative real - time pcr performed using ready - to - use taqman gene expression assays from applied biosystems . a total of 24 assays , including 18 target genes , 3 negative control genes , and 3 reference or housekeeping genes , were analyzed by real - time pcr using taqman assay ( applied biosystems ) ( supplementary table 2 ) . linear regression of gene expression level with age as a covariate on data stratified by sex and disease status was completed to assess the effect of age on the expression level of each studied gene . since there was a significant effect of age and sex on the expression levels of many genes , case - control matching was performed with respect to age and sex using the matching r package ( 9 ) . to estimate the relative risk of diabetes at different gene expression levels the odds ratios ( ors ) and 95% cis were computed for each gene . to investigate the association between gene expression levels and the diabetes complications , the samples from the t1d patients with a particular complication conditional logistic regression was performed to estimate the relative risk of a particular complication at different expression levels . fourteen genes were selected for validation studies based on our previous study ( 2 ) , and these genes showed higher expression in both t1d and autoantibody - positive groups as compared with the abn control group . three genes ( tsc22d3 , rpl17 , and eef1b2 ) that were not significant on our microarray dataset were included as negative controls . there were significant correlations with age in both phenotypic groups and for both sexes for several genes ( data not shown ) . in order to accurately assess the gene expression associated with t1d the ors per sd increment of gene expression levels were first computed using expression data as continuous variable ( table 1 ) . these analyses suggest that eight genes ( tgfb1 , sell , psmb3 , cd74 , s100a8 , s100a9 , and il12a ) were significantly upregulated and three genes ( gnly , psma4 , and smad7 ) were significantly downregulated in t1d patients . we also compared gene expression differences between juvenile - onset ( n = 692 ) and adult - onset t1d patients ( n = 232 ) , but no significant difference was found ( data not shown ) . the three negative control genes were analyzed using the same approaches , and no significant differences were found for these genes ( data not shown ) . box plots for gene expression levels ( data were log transformed ) in different subject groups including abn , t1d without any complication ( t1d - nocomp ) , t1d with at least one complication ( anycomp ) , and t1d with one specific complication ( neuropathy , htn , retinopathy , nephropathy , or cad ) . gene expression changes in t1d compared with abn ( n = 922 ) the t1d group is a heterogeneous set of subjects as some have developed complications such as neuropathy , retinopathy , nephropathy , coronary artery disease ( cad ) , and hypertension ( htn ) . as the expression levels for some genes may be associated with diabetes complications , it is important to delineate the gene expression differences caused by hyperglycemia or the specific types of complications . among the 928 t1d patients in our dataset , 238 patients had at least one diabetes complication . to exclude expression differences caused by diabetes complications , we compared expression levels between 922 abn control subjects and 407 t1d patients without any complications ( table 1 ) . in the first analysis , each t1d patient without complication is matched with an abn control for age and sex ; the expression data were analyzed as continuous variables using logistic regression . the results indicate that the seven of eight genes significantly upregulated in the entire dataset ( except for s100a9 ) were also significantly higher in t1d patients without complications compared with control subjects . additionally , the three genes shown to be downregulated in the entire t1d cohort were consistently downregulated in the t1d subset without complications ( table 1 ) . in this dataset , there were 119 patients with hypertension , 59 patients with neuropathy , 70 patients with retinopathy , 35 patients with cad , and 26 patients with nephropathy . before examining the gene expression changes in different diabetes complications , we determined whether t1d duration is a potential confounding factor since t1d duration is significantly longer for all complication groups ( supplementary table 3 ) . there were significant correlations with t1d duration in both sexes for four genes ( s100a9 , s100a8 , psma4 , and il12a ) and significant correlation in only the male group for four genes ( rnf31 , foxp3 , mfng , and nfkb1 ) . since t1d duration and sex are confounding factors for most genes , age , t1d duration , and sex matching was then performed using a multivariate and propensity score matching software ( 9 ) . conditional logistic regression analysis was performed to first assess changes associated with diabetes complication using all 238 patients with at least one complication . four genes ( s100a8 , s100a9 , sell , and mnda ) were significantly higher in the t1d patients with any one kind of complication ( any - complication group ) compared with the no - complication group , whereas four other genes ( smad7 , psma4 , mfng , and gnly ) were significantly lower in t1d with complication than t1d without complication ( table 2 ) . to exclude the possibility that the gene expression changes result from the treatment for secondary complications , we further examined gene expression differences between patients that were on treatment with specific drugs and those without the treatment . as the numbers of patients on individual drugs were very small , we grouped the treatments into four major categories : hypertension , blood cholesterol , hypothyroidism , and psychological disorders . our results suggest that the expression differences observed in this study were not due to treatment for diabetes complications ( data not shown ) . box plots for gene expression levels ( data were log transformed ) in t1d patients without any complication and t1d patients with different complication groups : at least one of complications ( anycomp ) , neuropathy , htn , retinopathy , nephropathy , and cad . gene expressions associated with t1d complications we further examined these eight genes in five patient subsets with specific complications ( neuropathy , hypertension , retinopathy , nephropathy , and cad ) . t1d patients with neuropathy are also much more likely to have higher gene expression levels for s100a9 , sell , and mnda ( or 4.8 , 4.9 , and 5.2 , respectively ) . patients with hypertension also showed significant changes for seven of the eight genes , including all four upregulated genes ( s100a8 , s100a9 , sell , and mnda ) ( table 2 ) . although statistical significance was not reached , the four upregulated genes were also higher in patients with other examined complications , including cad , retinopathy , and nephropathy , suggesting that these genes are probably implicated in all diabetes complications . finally , we examined pairwise correlation of gene expression levels for all 18 genes , and the results are graphically presented in fig . clustering of the genes based on the expression correlation values revealed a main cluster of proinflammatory genes ( s100a8 , s100a9 , mnda , and sell ) significantly upregulated in t1d and its complications . tgfb1 , nfkb1 , and rnf10 are also loosely grouped within this cluster , and these genes , especially tgfb1 , are increased in t1d compared with control subjects . pairwise correlations between gene expression levels for 18 studied genes in different subject groups : abn , t1d without any complication ( t1d - nocomp ) , t1d with at least one complication ( anycomp ) , and t1d with neuropathy . fourteen genes were selected for validation studies based on our previous study ( 2 ) , and these genes showed higher expression in both t1d and autoantibody - positive groups as compared with the abn control group . in order to accurately assess the gene expression associated with t1d the ors per sd increment of gene expression levels were first computed using expression data as continuous variable ( table 1 ) . these analyses suggest that eight genes ( tgfb1 , sell , psmb3 , cd74 , s100a8 , s100a9 , and il12a ) were significantly upregulated and three genes ( gnly , psma4 , and smad7 ) were significantly downregulated in t1d patients . we also compared gene expression differences between juvenile - onset ( n = 692 ) and adult - onset t1d patients ( n = 232 ) , but no significant difference was found ( data not shown ) . box plots for gene expression levels ( data were log transformed ) in different subject groups including abn , t1d without any complication ( t1d - nocomp ) , t1d with at least one complication ( anycomp ) , and t1d with one specific complication ( neuropathy , htn , retinopathy , nephropathy , or cad ) . gene expression changes in t1d compared with abn ( n = 922 ) the t1d group is a heterogeneous set of subjects as some have developed complications such as neuropathy , retinopathy , nephropathy , coronary artery disease ( cad ) , and hypertension ( htn ) . as the expression levels for some genes may be associated with diabetes complications , it is important to delineate the gene expression differences caused by hyperglycemia or the specific types of complications . to exclude expression differences caused by diabetes complications , we compared expression levels between 922 abn control subjects and 407 t1d patients without any complications ( table 1 ) . the results indicate that the seven of eight genes significantly upregulated in the entire dataset ( except for s100a9 ) were also significantly higher in t1d patients without complications compared with control subjects . additionally , the three genes shown to be downregulated in the entire t1d cohort were consistently downregulated in the t1d subset without complications ( table 1 ) . in this dataset , there were 119 patients with hypertension , 59 patients with neuropathy , 70 patients with retinopathy , 35 patients with cad , and 26 patients with nephropathy . before examining the gene expression changes in different diabetes complications , we determined whether t1d duration is a potential confounding factor since t1d duration is significantly longer for all complication groups ( supplementary table 3 ) . there were significant correlations with t1d duration in both sexes for four genes ( s100a9 , s100a8 , psma4 , and il12a ) and significant correlation in only the male group for four genes ( rnf31 , foxp3 , mfng , and nfkb1 ) . since t1d duration and sex are confounding factors for most genes , age , t1d duration , and sex matching was then performed using a multivariate and propensity score matching software ( 9 ) . four genes ( s100a8 , s100a9 , sell , and mnda ) were significantly higher in the t1d patients with any one kind of complication ( any - complication group ) compared with the no - complication group , whereas four other genes ( smad7 , psma4 , mfng , and gnly ) were significantly lower in t1d with complication than t1d without complication ( table 2 ) . to exclude the possibility that the gene expression changes result from the treatment for secondary complications , we further examined gene expression differences between patients that were on treatment with specific drugs and those without the treatment . as the numbers of patients on individual drugs were very small , we grouped the treatments into four major categories : hypertension , blood cholesterol , hypothyroidism , and psychological disorders . our results suggest that the expression differences observed in this study were not due to treatment for diabetes complications ( data not shown ) . box plots for gene expression levels ( data were log transformed ) in t1d patients without any complication and t1d patients with different complication groups : at least one of complications ( anycomp ) , neuropathy , htn , retinopathy , nephropathy , and cad . gene expressions associated with t1d complications we further examined these eight genes in five patient subsets with specific complications ( neuropathy , hypertension , retinopathy , nephropathy , and cad ) . t1d patients with neuropathy are also much more likely to have higher gene expression levels for s100a9 , sell , and mnda ( or 4.8 , 4.9 , and 5.2 , respectively ) . patients with hypertension also showed significant changes for seven of the eight genes , including all four upregulated genes ( s100a8 , s100a9 , sell , and mnda ) ( table 2 ) . although statistical significance was not reached , the four upregulated genes were also higher in patients with other examined complications , including cad , retinopathy , and nephropathy , suggesting that these genes are probably implicated in all diabetes complications . finally , we examined pairwise correlation of gene expression levels for all 18 genes , and the results are graphically presented in fig . clustering of the genes based on the expression correlation values revealed a main cluster of proinflammatory genes ( s100a8 , s100a9 , mnda , and sell ) significantly upregulated in t1d and its complications . pairwise correlations between gene expression levels for 18 studied genes in different subject groups : abn , t1d without any complication ( t1d - nocomp ) , t1d with at least one complication ( anycomp ) , and t1d with neuropathy . the large sample set of close to 2,000 individuals was especially informative for the gene expression profiles in control subjects and t1d patients with and without diabetes complications . the genes with a higher expression in t1d are implicated in immune function , such as inflammation ( s100a8 , s100a9 , mnda , and il12-a ) , immune regulation / promotion ( tgfb1 and sell ) , and antigen processing and presentation ( cd74 and psmb3 ) . our findings strongly support the notion that chronic inflammation contributes to t1d development ( 1012 ) . upregulation of genes involved in antigen processing and presentation may occur in response to the activation and proliferation of autoreactive pathogenic t cells in t1d patients . two previous studies with small sample sizes ( 13,14 ) reported a decreased tgfb1 level in t1d patients ; however , this study with a very large sample size revealed a significantly higher expression of tgfb1 in t1d patients . it induces immune tolerance by acting as a potent immune suppressor through the inhibition of proliferation , differentiation , activation , and effector function of immune cells ( 1517 ) . although the protective effects of tgfb1 have been documented in various autoimmune diseases , including t1d , the increased tgfb1 gene expression in t1d shown in the current study suggests that tgfb1 may mainly play a pathogenic role in t1d as a proinflammatory mediator . interestingly , the expression of an inhibitor of tgfb1 signaling , smad7 , showed a decreased level of expression in t1d as compared with healthy control subjects . relative to the comparison between t1d patients and control subjects , the sample sizes for the study on specific complications are smaller and the results should be viewed with more caution . for example , the most prominent genes are two inflammatory genes , s100a8 and s100a9 , both being strongly associated with complications , especially neuropathy ( or 7.9 and 4.8 ) and hypertension ( 2.7 and 2.2 ) . although the pathogenesis of diabetic angiopathy is incompletely understood , patients with one complication often present with a second one , suggesting the existence of common risk factors and pathogenic mechanisms . as a marker of monocyte and neutrophil activation , the expression and secretion of the s100a8/a9 heterodimer could be induced by inflammatory cytokines such as il1 ( 27 ) . in one pathway , s100a8/a9 stimulation can cause a rapid increase of cd11b expression on the monocyte surface , accounting for the increase of trans - endothelial migratory activity of monocytes and hence vascular endothelium changes ( 29 ) . in another pathway , s100a8/9 protein can bind specifically to and induce a specific inflammatory response in human vascular endothelial cells by increasing the transcription of proinflammatory chemokines and adhesion molecules ( 30 ) . more interestingly , the proinflammatory endothelial response to s100a8/a9 can be increased by advanced glycation end products ( ages ) since s100a8/a9 can work as ligands of rage ( receptor for age ) and have effects on the rage - nfkb mediated induction of proinflammatory gene expression ( 31 ) . the myeloid cell nuclear differentiation antigen ( mnda ) is detected only in the nuclei of cells of the granulocyte - monocyte lineage and plays a role in the cell - specific response to interferon ( 32 ) . l - selectin ( sell ) is a cell surface adhesion molecule that plays a critical role in facilitating leukocyte migration from blood vessels to secondary lymphoid organ and sites of local inflammation . although there are controversial reports about the role of l - selectin in the development of autoimmune diabetes in nod mice ( 3335 ) , a positive correlation between the serum level of soluble l - selectin and t1d , diabetic retinopathy , atherosclerosis , and arterial hypertension was reported in human studies ( 3638 ) . the percentage of t cells expressing l - selectin was significantly increased in t1d patients and in t1d patients with arterial hypertension as well . consistent with these findings , we provided convincing evidence that l - selectin expression in peripheral blood mononuclear cells is higher in t1d patients ( or 1.4 ) , especially those patients with diabetic neuropathy ( 4.9 ) and hypertension ( 1.6 ) . it will therefore be important to examine the cellular functions and pathogenic roles of myeloid cells in the development of t1d and its complications in future studies .

delirium is an extremely common problem occurring in over 50% of older , hospitalized patients , and is associated with both a decline in functional ability and increased morbidity and mortality . consequently , it has been referred to as being an independent marker of functional decline. in addition , the development of delirium is associated with higher rates of falling , contributing to poor clinical outcomes . currently , the management for delirium is multifactorial and should include the treatment of reversible conditions and addressing specific patient and care factors which may be contributing to the delirium . a recognized component of this approach , both for the prevention and in the management of delirium , involves the employment of constant observation ( co ) ( also known as special observation ) . the concept of co has been around for some time , and associated definitions have ranged from an intervention in which continuous one - to - one monitoring is used to assure the safety and well - being of an individual patient or others , to direct observation of patients for the purpose of providing a safer environment for the patient. however , a more practical description for co uses co for the provision of one - to - one observation by a constant observer ( a dedicated person employed to sit with the patient ) . once mostly reserved for use within the psychiatric setting for patients at high risk of self - harm or suicide , the role of co has diversified and is now most commonly implemented for the management of delirious patients . the term sitter is used to refer to the person providing constant observation . whilst numerous different terminologies exist for these constant observers ( i.e. , patient sitters , volunteers , client attendants , patient attendants , companions ) , for the purpose of this review they will be referred to as sitters . therefore , our primary objective for this literature review is to provide an overview of the current role of the sitter within the management of delirium in hospitalized patients by first addressing the current understanding of the role for sitters , the type of person employed in this role , indications for current usage , and the frequency they are used . secondary objectives include the identification of key training requirements required for the sitter role and establishment of the cost - effectiveness of their role within delirium management . a literature search was performed of medline , cochrane database of systematic reviews , and pubmed from the years 1960 to october 2011 . the following search terms were used : sitter / patient sitter / companion / nurses aides / special care aides , confusion / delirium / acute confusional state , responsibilities / roles , constant observation / observation , outcomes , intervention , cost / cost analysis . a large number of search terms were used in order to ensure completeness , with identification and inclusion of all potential studies . inclusion criteria required the articles ( and abstracts ) to be in the english language and contain relevant research involving at least one of the following : the roles of the sitter , people used for the sitter role , current usage of sitters , indications / assessment and/or tools / evaluation for sitter use , education / training requirements / pre - requisitions required for the role , costs associated with sitter use , sitter use / role within delirium setting including non - pharmalogical intervention models for delirium management . one additional article addressing accuracy of nursing staff for detecting delirium was also included due to the significant impact this could have for sitter usage . one reviewer was responsible for data collection , data analysis , and performing the literature review . quality assessment was made based on : study design ( and blinding ) , sample size , methodology , and validity of results . from a total of 147 citations initially identified , 50 full - text manuscripts were reviewed and 37 articles were included in the final analysis . a meta - analysis was not conducted due to clinical heterogeneity between included articles . instead , one of the first reports documenting sitter use was in 1985 as part of a nursing intervention for post - operative orthopedic patients , in which a nurse visitor was employed to provide consistent contact with patient . whilst their presence may not have significantly influenced the rate or duration of delirium , it does provide evidence supporting the use of patient sitters in the management of delirium . currently , there is no guidance available regarding who should be used in the sitter role , and no evaluation has been performed to assess the impact that the type of person in this role has upon the patient s clinical outcome ; as a result the type of sitter used is determined by the local health region . the current literature describes a variety of people having been used in the sitter role . the use of skilled nursing staff or nurses aides as sitters has been well - described . whilst the experience and training they could offer is vast , their use is significantly restricted by an associated high - cost expenditure combined with limited staffing levels . family members or other relatives are used quite often , inadvertently , as sitters by health - care staff , which is supported by a study that looked at family participation in patient care in the hospital setting and showed their involvement ranging from direct provision of patient care to companionship . one study based in australia used volunteers in the combined role of observers and companions for patients who had been highlighted as being at a high risk of falling . these volunteers remained with them in the room and were involved in patient interaction , in addition to alerting nursing staff any time patients were identified as being about to fall . whilst the results from this study showed significant reductions in patient fall rates , limited availability of these volunteers , combined with significant restrictions imposed from occupational health and safety regulations on their role , may limit their use in this capacity . although once used for observation of high - risk psychiatric patients , the use of sitters is now most commonly employed as part of the management for delirious patients . another established , albeit less frequently recognized role , is the provision of companionship for patients in palliative care . alternative uses for the sitter role have been seen as part of a preventative strategy for wandering patients and in fall prevention . an alternative role for the sitter has included their use for rooming with patients . this is when a family member or close friend stays in the patient s room , and has been shown to be effective for elderly orthopaedic patients , as described by one study on post - operative elderly orthopaedic patients to assess its clinical impact on delirium rates . although hospital length of stay and duration of delirium were not significantly reduced , it was shown to be feasible and may possibly be most beneficial for the acutely admitted elderly population . more recently , successful sitter use was shown in the role of a rehabilitation patient companion on an acute brain injury unit in an attempt to reduce costs and improve patient and nurse satisfaction . an absence of national guidelines or regulations governing sitter use has inevitably led to a lack of specific criteria ( including indications ) for hiring sitters , resulting in decisions being made at the local level . this lack of standardized criteria regulating the use of sitters and the absence of formal guidelines ( provincial and federally ) has highlighted an important deficit in the system that requires addressing . one study based in the uk looked at sitters currently employed in the palliative care setting and identified the reasons why sitters got involved and showed ( indirectly ) that higher sitter turnover was linked with inadequate or insufficient training , which supports the need for adequate training for the role . a quality improvement project was done to assess the effectiveness of a one - hour educational program aimed at sitters ( and nurses ) which addressed key issues including policy and procedure , risk assessment , and symptom recognition , combined with self - learning . in addition to showing improvement through the comparisons of pre- and post - test results , it was also shown to be cost - effective , which supports the use of similar interventions in the provision of sufficient training for sitters . the use of sitters is dependent on the ability of nurses or health - care staff effectively recognizing patients requiring constant observation . however , one study , which addressed the recognition by nursing staff of patients with features of delirium by assessing nursing documentation , showed poor documentation of delirium features . thus , a failure to recognize such patients will inevitably affect the efficiency surrounding the use of sitters . to help with identification of patients who would benefit from sitter use , and aid and improve sitter requests , an assessment tool , the patient attendant assessment tool ( paat ) was created . using the paat , an improvement was seen in completion of the requisition forms , hiring of sitters , and in actual sitter usage , which was accompanied by reduction in restraint use . the use of sitters as part of the multi - interventional approach for delirium has been well established in the hospital elderly life program ( help ) , initially described by inouye et al . this model used rigorously trained volunteers as part of an interdisciplinary approach to reduce the incidence and rates of delirium . the success of the program was based , in part , on the use of well - trained volunteers for delirium management . additional support for the use of sitters is provided from the results of the revive study , aimed at the prevention and reduction of delirium using a multi - interventional approach through the use of trained volunteers to provide patient interaction and re - orientation . the results revealed a reduction in the incidence and duration of delirium , together with a reduced frequency of falls , thus supporting the importance of this approach . in addition , it was associated with a reduced length of stay in hospital , providing indirectly evidence for the use of sitters for cost - saving purposes . this model is based on placing all patients who require constant or very close observation in close proximity to one another , either in a single room or unit in the presence of nursing staff , which provides for continuous or close monitoring for these patients . with the implementation of these safe units , the use of sitters was decreased significantly , in addition to a reduction in restraint use . one study that specifically focused on sitter costs stated median sitter costs as being $ 772 compared to $ 2397 for high sitter use . the interpretation of this information , however , is difficult due to the terminology used for average and high sitter usage for which there is a paucity of data . certain factors have been associated with higher rates of sitter usage and thus higher sitter costs , which include understaffing of units with registered nurses and certain patient characteristics . this same study proposed that increasing the number of hours worked by skilled nurses or by patient care assistants may reduce use of sitters and thus lead to lower sitter use and lower sitter costs . the cost - effectiveness of a patient sitter program was addressed using outcomes which included the frequency of patient falls and patient satisfaction . the results from this study showed only a marginal benefit was gained for both patient satisfaction and fall rates . in this study , the cost for a typical sitter shift was $ 160 , which , when adjusted for the ( slight ) benefit seen in fall rates and patient satisfaction , revealed a net cost expense of $ 156.24 . the help program , aimed at reducing the incidence and duration of delirium in hospitalized elderly patients , reduced hospital costs substantially , and has the potential to be replicated in alternative settings from that in which it was first described , possibly making it the most cost - effective way for employing sitters . however , a cochrane review looking at the cost - effectiveness of the implementation of similar programs has suggested that whilst this may reduce both incidence of delirium along with costs for intermediate risk patients , there was no significant benefit seen in the potential benefit offered by sitters is tremendous , especially considering the provision of constant observation on a busy acute medical ward . the one - to - one monitoring that can be given is invaluable , and provides a safety net for the nursing staff for high risk patients . this is supported by one study which showed the presence of sitters acted as a significant motivator and improved nurses confidence in ensuring the provision of adequate patient care . the results from the revive study provided evidence of the potential benefit that trained volunteers could offer with patient interaction , whilst providing additional benefits in terms of reduced numbers of nursing assistants required , as well as hospital cost savings . whilst the role of sitters for use with certain patient population has previously been established , evidence for the cost - effectiveness of sitter usage for patients with delirium compared to traditional management is conflicting . although the use of sitters may reduce restraint use , their impact on patient fall rates ( a major patient safety outcome measure ) is marginal at best , and not clinically significant . one study assessed the use of sitters for patients deemed at high risk of falling and patients in a psychiatric crisis . by identification of patient volume , current sitter usage , and an assessment of demand , the study authors concluded that the use of constant observation with sitters was an ineffective and costly way of attempting to improve patient care . the study further noted that it was more beneficial to identify high - risk patients on admission ( using assessment tools ) , and to train and employ the hospital s own health - care staff as sitters ( using a good quality sitter education program ) rather than use an external agency . one of the first reports documenting sitter use was in 1985 as part of a nursing intervention for post - operative orthopedic patients , in which a nurse visitor was employed to provide consistent contact with patient . whilst their presence may not have significantly influenced the rate or duration of delirium , it does provide evidence supporting the use of patient sitters in the management of delirium . currently , there is no guidance available regarding who should be used in the sitter role , and no evaluation has been performed to assess the impact that the type of person in this role has upon the patient s clinical outcome ; as a result the type of sitter used is determined by the local health region . the current literature describes a variety of people having been used in the sitter role . the use of skilled nursing staff or nurses aides as sitters has been well - described . whilst the experience and training they could offer is vast , their use is significantly restricted by an associated high - cost expenditure combined with limited staffing levels . family members or other relatives are used quite often , inadvertently , as sitters by health - care staff , which is supported by a study that looked at family participation in patient care in the hospital setting and showed their involvement ranging from direct provision of patient care to companionship . one study based in australia used volunteers in the combined role of observers and companions for patients who had been highlighted as being at a high risk of falling . these volunteers remained with them in the room and were involved in patient interaction , in addition to alerting nursing staff any time patients were identified as being about to fall . whilst the results from this study showed significant reductions in patient fall rates , limited availability of these volunteers , combined with significant restrictions imposed from occupational health and safety regulations on their role , may limit their use in this capacity . although once used for observation of high - risk psychiatric patients , the use of sitters is now most commonly employed as part of the management for delirious patients . another established , albeit less frequently recognized role , is the provision of companionship for patients in palliative care . alternative uses for the sitter role have been seen as part of a preventative strategy for wandering patients and in fall prevention . an alternative role for the sitter has included their use for rooming with patients . this is when a family member or close friend stays in the patient s room , and has been shown to be effective for elderly orthopaedic patients , as described by one study on post - operative elderly orthopaedic patients to assess its clinical impact on delirium rates . although hospital length of stay and duration of delirium were not significantly reduced , it was shown to be feasible and may possibly be most beneficial for the acutely admitted elderly population . more recently , successful sitter use was shown in the role of a rehabilitation patient companion on an acute brain injury unit in an attempt to reduce costs and improve patient and nurse satisfaction . an absence of national guidelines or regulations governing sitter use has inevitably led to a lack of specific criteria ( including indications ) for hiring sitters , resulting in decisions being made at the local level . this lack of standardized criteria regulating the use of sitters and the absence of formal guidelines ( provincial and federally ) has highlighted an important deficit in the system that requires addressing . one study based in the uk looked at sitters currently employed in the palliative care setting and identified the reasons why sitters got involved and showed ( indirectly ) that higher sitter turnover was linked with inadequate or insufficient training , which supports the need for adequate training for the role . a quality improvement project was done to assess the effectiveness of a one - hour educational program aimed at sitters ( and nurses ) which addressed key issues including policy and procedure , risk assessment , and symptom recognition , combined with self - learning . in addition to showing improvement through the comparisons of pre- and post - test results , it was also shown to be cost - effective , which supports the use of similar interventions in the provision of sufficient training for sitters . the use of sitters is dependent on the ability of nurses or health - care staff effectively recognizing patients requiring constant observation . however , one study , which addressed the recognition by nursing staff of patients with features of delirium by assessing nursing documentation , showed poor documentation of delirium features . thus , a failure to recognize such patients will inevitably affect the efficiency surrounding the use of sitters . to help with identification of patients who would benefit from sitter use , and aid and improve sitter requests , an assessment tool , the patient attendant assessment tool ( paat ) was created . using the paat , an improvement was seen in completion of the requisition forms , hiring of sitters , and in actual sitter usage , which was accompanied by reduction in restraint use . the use of sitters as part of the multi - interventional approach for delirium has been well established in the hospital elderly life program ( help ) , initially described by inouye et al . this model used rigorously trained volunteers as part of an interdisciplinary approach to reduce the incidence and rates of delirium . the success of the program was based , in part , on the use of well - trained volunteers for delirium management . additional support for the use of sitters is provided from the results of the revive study , aimed at the prevention and reduction of delirium using a multi - interventional approach through the use of trained volunteers to provide patient interaction and re - orientation . the results revealed a reduction in the incidence and duration of delirium , together with a reduced frequency of falls , thus supporting the importance of this approach . in addition , it was associated with a reduced length of stay in hospital , providing indirectly evidence for the use of sitters for cost - saving purposes . this model is based on placing all patients who require constant or very close observation in close proximity to one another , either in a single room or unit in the presence of nursing staff , which provides for continuous or close monitoring for these patients . with the implementation of these safe units , the use of sitters was decreased significantly , in addition to a reduction in restraint use . one study that specifically focused on sitter costs stated median sitter costs as being $ 772 compared to $ 2397 for high sitter use . the interpretation of this information , however , is difficult due to the terminology used for average and high sitter usage for which there is a paucity of data . certain factors have been associated with higher rates of sitter usage and thus higher sitter costs , which include understaffing of units with registered nurses and certain patient characteristics . this same study proposed that increasing the number of hours worked by skilled nurses or by patient care assistants may reduce use of sitters and thus lead to lower sitter use and lower sitter costs . the cost - effectiveness of a patient sitter program was addressed using outcomes which included the frequency of patient falls and patient satisfaction . the results from this study showed only a marginal benefit was gained for both patient satisfaction and fall rates . in this study , the cost for a typical sitter shift was $ 160 , which , when adjusted for the ( slight ) benefit seen in fall rates and patient satisfaction , revealed a net cost expense of $ 156.24 . the help program , aimed at reducing the incidence and duration of delirium in hospitalized elderly patients , reduced hospital costs substantially , and has the potential to be replicated in alternative settings from that in which it was first described , possibly making it the most cost - effective way for employing sitters . however , a cochrane review looking at the cost - effectiveness of the implementation of similar programs has suggested that whilst this may reduce both incidence of delirium along with costs for intermediate risk patients , there was no significant benefit seen in the potential benefit offered by sitters is tremendous , especially considering the provision of constant observation on a busy acute medical ward . the one - to - one monitoring that can be given is invaluable , and provides a safety net for the nursing staff for high risk patients . this is supported by one study which showed the presence of sitters acted as a significant motivator and improved nurses confidence in ensuring the provision of adequate patient care . the results from the revive study provided evidence of the potential benefit that trained volunteers could offer with patient interaction , whilst providing additional benefits in terms of reduced numbers of nursing assistants required , as well as hospital cost savings . whilst the role of sitters for use with certain patient population has previously been established , evidence for the cost - effectiveness of sitter usage for patients with delirium compared to traditional management is conflicting . although the use of sitters may reduce restraint use , their impact on patient fall rates ( a major patient safety outcome measure ) is marginal at best , and not clinically significant . one study assessed the use of sitters for patients deemed at high risk of falling and patients in a psychiatric crisis . by identification of patient volume , current sitter usage , and an assessment of demand , the study authors concluded that the use of constant observation with sitters was an ineffective and costly way of attempting to improve patient care . the study further noted that it was more beneficial to identify high - risk patients on admission ( using assessment tools ) , and to train and employ the hospital s own health - care staff as sitters ( using a good quality sitter education program ) rather than use an external agency . from this review , it is evident that sitters are being employed in a variety of settings . sitter , a common underlying theme is apparent regarding their role as an observer or companion for the patient thus providing a potential definition for the sitter role as a person hired to provide constant observation or companionship. the question of which type of person would provide the most benefit in the sitter role is still not clear . studies have reported using a variety of people ranging from rn nurses and special care aides to ( untrained ) volunteers and patient relatives and family members . however , due to growing demands exerted on nursing duties , there has been an increasing trend towards using external hired agency staff as sitters , with a resulting rise in costs . family members have the potential to be effective sitters ; their familiarity with the patient , close relationship , and motivation to stay with the patient can provide essential patient interaction important for delirious patients . in addition , a family member s presence at the bedside can provide close observation , as well as familiarity , and thus enhance patient safety . their relationship with the patient , however , has the potential to negatively impact patient care if their goals are not the same as the health - care staff . volunteers may have the most potential in the capacity of the sitter , provided that they are adequately trained for the role and are freely available , which is evident from success of established program such as help model . this is supported by several studies that have justified the use of trained volunteers by showing them to be cost - effective . indeed , the use of sitters relieves the workload of overworked , stressed nursing staff who are often more than happy to have an additional pair of hands in the form of a volunteer sitter . as discussed earlier , sitters are currently employed across the country in several different settings with one of the most common being for delirious patients . precise details relating to the frequency of the use of sitters at any individual locations are difficult to establish due to a lack of documentation at the local , provincial , and federal levels . this highlights a potential role for the development of a national reporting scheme for recording their use . the absence of a standardized job description for the sitter role and lack of regulations regarding their use have led to the development of a certain degree of unease concerning their use , and accounts for the significant amount of variation and inconsistencies seen in the use of sitters . without a specific job description for the role of a sitter , health - care institutions are significantly restricted in this capacity , and hindered in benefiting from the use of sitters ( i.e. , provision of daily social interaction , cognitive stimulation , frequent reorientation therapy , etc . ) . this is important due to the significant clinical impact that can result following the implementation such interventions would have on delirium outcomes . however , without further clarification ( in the form of national guidance ) about their position , then these potential benefits offered by sitters can not be utilized . use of sitters for the provision of constant observation for high - risk psychiatric patients is well established . more creative and alternative roles for sitters have been successful , including their use as a although additional roles have used sitters in an attempt to reduce rates of wandering for high - risk patients and patient fall rates , these are theoretical advantages which are not supported by the current evidence available , and conflicts with the general consensus that sitters do not improve frequency of falling and may even increase fall rates ; however , the reasons for this are not clear . the process for identifying those patients who would benefit from the use of sitters has not been well established . the provision of pre - specified criteria or assessment tools to aid nursing staff in recognizing patients who would benefit from these costly resources is important for both reducing costs associated with using sitters and reducing rates of pharmacological therapy with its associated side effects . the importance of appropriate sitter usage is supported by evidence showing that whilst higher sitter usage was associated with decreased rates of soft restraint use , it was accompanied by an alarming increased rate of falls . if nursing staff are efficient in recognizing the presence of delirium , then this should naturally improve sitter use and cost - effectiveness . one prospective study looking at the accuracy of nursing documentation relating to their recognition of the signs and symptoms of delirium suggested possible poor recognition , with a possible exception being for severely ill patients . thus , the poor recognition of delirious patients will affect sitter usage within the hospital setting . although there are no currently no formal established assessments tools available to aid with this process , there is great potential in the form of a specified assessment tool the paat as discussed previously , which was created to help assist and thus improve the hiring and requisition of the use of sitters for high - risk patients . common sense states that the use of untrained sitters in potentially risky situations may have negative consequences . a lack of orientation to either the patient and/or to their role is distracting and problematic for both the patient and sitter and this , combined with the lack of familiarity between them , could create a potentially disastrous situation , as well as being an inefficient use of resources and a hindrance to patient care . in addition , inadequate training for the management of aggressive or agitated patients could put sitters , the patient , and staff at danger and has legal consequences . this is important , as many situations in which sitters are employed ( e.g. , delirious patients and palliative care ) are highly sensitive and emotionally charged settings in which the presence of a sitter can have a big impact on both the patient and relative . this further highlights the extreme importance of sitters receiving adequate training for this role . as a consequence of the lack of formal / recognized training or assessment , the competence of these sitters has been questioned several times , especially since it is often untrained volunteers that are commonly used in this capacity . the importance of this is evident from the dire consequences that can ensue due to the nature of settings in which these sitters are commonly employed . however , with adequate training , the effectiveness of fully trained volunteers in the sitter role is non - debatable , reconfirming the need for sufficient training for the position . the employment of sitters in the management of delirious patients may benefit the patient both in terms of delirium control ( through the application of consistent , frequent re - orientation therapy ) , as well as ( possibly ) reducing or preventing the use of pharmacological agents and/or restrictive devices . the help program has been shown to be successful in both reducing the rate and length of delirium episodes whilst reducing negative clinical outcomes associated with delirium . similar , smaller programs based on this concept have been set up , the findings of which have shown similar results . thus , the use of a well - established delirium program model for employing trained sitters ( i.e. , volunteers ) may offer the most cost - effective method . however , the translation of a large established program to implementation of smaller similar programs at the local level can be one of the hardest barriers to cross due to restrictions exerted in terms of costs , resistant attitudes from staff , and insufficient staffing levels ( to name a few ) . one article addressed this , and looked at the degree of implementation of similar help programs across the country and their success rates . this study showed that only 25% of those who initially expressed interest in developing the program actually succeeded in establishing an active , functioning help program in their local institution . certainly , in our local region , the use of sitters has been reduced substantially in the last couple of years due to financial restraints . this is hardly surprising considering the severe deficiency of good quality evidence for their use and lack of information supporting their role or even an accurate definition of their position . one study did produce a sitter guidance sheet , stating hints and tips which could offer useful information for current sitters . the impact of this on sitter activity and possible future training would be very interesting to assess . the same review suggested that management of patients who require sitters should avoid placing delirious patients in one single area due to a possible negative influence that may be exerted on one another . however , other studies do not support this concept , which conflicts with evidence from the aforementioned safe study . from the 37 articles included in the review , nine studies directly addressed the role for sitters in delirium , with sample sizes ranging from 244763 . addressed outcomes included markers of delirium ( incidence , severity , duration , number of episodes , and recurrence ) , patient outcomes ( cognitive change , functional change , sleep quality ) , nursing assistant use , and financial cost . the use of mostly trained volunteers in the sitter role provided the main limitations to these studies . one article addressed training for sitter role in delirium ( sample size : 1507 ) and six articles ( sample size : 375346 ) addressed cost - effectiveness for role of sitters in delirium . current evidence supports a role for the sitter as part of the management of patients with delirium . since the majority ( and strongest evidence ) is seen with the use of trained volunteers in the sitter role as part of an established delirium model ( such as help ) , then this may likely be the most cost - effective way for advocating their use , possibly in conjunction with use a recognized sitter assessment tool such as the paat . the specific type of training required for the sitter role is not clear and requires further research . certainly , when considering using a sitter , the health - care provider should carefully document the indications for sitter use , and monitor and reassess these indications alongside the frequency and use of the sitter in order to maximize cost - effectiveness . long term , the creation of a national set of regulations or guidelines would provide safeguards in the industry to ensure safe and effective patient care , in addition to the creation of a national reporting scheme for sitter usage .

purposethe concept behind constant observation is not new . whilst traditionally performed by nursing staff , it is now commonly performed by sitters . details surrounding the usage , job description , training , clinical and cost effectiveness of sitters are not known ; hence the reason for this review.methodsa literature search was performed in medline , cochrane database of systematic reviews , and pubmed from the years 1960 to october 2011 . the definition for sitter used in the articles was accepted for this review.resultsfrom this review , it is evident that sitters are being employed in a variety of settings . the question of which type of person would provide the most benefit in the sitter role is still not clear ; whilst sitters have typically included family and volunteers , it may be trained volunteers who may offer the most cost - effective solution . the paucity of information available regarding the training and assessments of sitters and the lack of formal guidelines regulating sitters use results in a lack of information available regarding these sitters , and current available evidence is conflicting regarding the benefits in terms of cost and clinical outcome . the only strong evidence relating to clinical benefit comes from the use of fully - trained sitters as part of a multi - interventional program ( i.e. , help)conclusionscurrent evidence supports a role for the sitter as part of the management of patients with delirium . the most cost - effective sitter role appears to be trained volunteers . further research is needed to determine the specific type of training required for the sitter role . the creation of a national set of regulations or guidelines would provide safeguards in the industry to ensure safe and effective patient care .

INTRODUCTION METHODS RESULTS The Sitter Role Current Sitter Usage Sitter Education and Training Employment of Sitters The Role of Sitters in Delirium Cost Benefit Benefits from Using Sitters Disadvantages of Sitter Use DISCUSSION CONCLUSION

a recognized component of this approach , both for the prevention and in the management of delirium , involves the employment of constant observation ( co ) ( also known as special observation ) . the concept of co has been around for some time , and associated definitions have ranged from an intervention in which continuous one - to - one monitoring is used to assure the safety and well - being of an individual patient or others , to direct observation of patients for the purpose of providing a safer environment for the patient. once mostly reserved for use within the psychiatric setting for patients at high risk of self - harm or suicide , the role of co has diversified and is now most commonly implemented for the management of delirious patients . , patient sitters , volunteers , client attendants , patient attendants , companions ) , for the purpose of this review they will be referred to as sitters . therefore , our primary objective for this literature review is to provide an overview of the current role of the sitter within the management of delirium in hospitalized patients by first addressing the current understanding of the role for sitters , the type of person employed in this role , indications for current usage , and the frequency they are used . secondary objectives include the identification of key training requirements required for the sitter role and establishment of the cost - effectiveness of their role within delirium management . a literature search was performed of medline , cochrane database of systematic reviews , and pubmed from the years 1960 to october 2011 . inclusion criteria required the articles ( and abstracts ) to be in the english language and contain relevant research involving at least one of the following : the roles of the sitter , people used for the sitter role , current usage of sitters , indications / assessment and/or tools / evaluation for sitter use , education / training requirements / pre - requisitions required for the role , costs associated with sitter use , sitter use / role within delirium setting including non - pharmalogical intervention models for delirium management . instead , one of the first reports documenting sitter use was in 1985 as part of a nursing intervention for post - operative orthopedic patients , in which a nurse visitor was employed to provide consistent contact with patient . whilst their presence may not have significantly influenced the rate or duration of delirium , it does provide evidence supporting the use of patient sitters in the management of delirium . currently , there is no guidance available regarding who should be used in the sitter role , and no evaluation has been performed to assess the impact that the type of person in this role has upon the patient s clinical outcome ; as a result the type of sitter used is determined by the local health region . the current literature describes a variety of people having been used in the sitter role . family members or other relatives are used quite often , inadvertently , as sitters by health - care staff , which is supported by a study that looked at family participation in patient care in the hospital setting and showed their involvement ranging from direct provision of patient care to companionship . although once used for observation of high - risk psychiatric patients , the use of sitters is now most commonly employed as part of the management for delirious patients . alternative uses for the sitter role have been seen as part of a preventative strategy for wandering patients and in fall prevention . this is when a family member or close friend stays in the patient s room , and has been shown to be effective for elderly orthopaedic patients , as described by one study on post - operative elderly orthopaedic patients to assess its clinical impact on delirium rates . this lack of standardized criteria regulating the use of sitters and the absence of formal guidelines ( provincial and federally ) has highlighted an important deficit in the system that requires addressing . one study based in the uk looked at sitters currently employed in the palliative care setting and identified the reasons why sitters got involved and showed ( indirectly ) that higher sitter turnover was linked with inadequate or insufficient training , which supports the need for adequate training for the role . a quality improvement project was done to assess the effectiveness of a one - hour educational program aimed at sitters ( and nurses ) which addressed key issues including policy and procedure , risk assessment , and symptom recognition , combined with self - learning . in addition to showing improvement through the comparisons of pre- and post - test results , it was also shown to be cost - effective , which supports the use of similar interventions in the provision of sufficient training for sitters . the use of sitters is dependent on the ability of nurses or health - care staff effectively recognizing patients requiring constant observation . however , one study , which addressed the recognition by nursing staff of patients with features of delirium by assessing nursing documentation , showed poor documentation of delirium features . thus , a failure to recognize such patients will inevitably affect the efficiency surrounding the use of sitters . using the paat , an improvement was seen in completion of the requisition forms , hiring of sitters , and in actual sitter usage , which was accompanied by reduction in restraint use . the use of sitters as part of the multi - interventional approach for delirium has been well established in the hospital elderly life program ( help ) , initially described by inouye et al . the success of the program was based , in part , on the use of well - trained volunteers for delirium management . additional support for the use of sitters is provided from the results of the revive study , aimed at the prevention and reduction of delirium using a multi - interventional approach through the use of trained volunteers to provide patient interaction and re - orientation . in addition , it was associated with a reduced length of stay in hospital , providing indirectly evidence for the use of sitters for cost - saving purposes . this model is based on placing all patients who require constant or very close observation in close proximity to one another , either in a single room or unit in the presence of nursing staff , which provides for continuous or close monitoring for these patients . with the implementation of these safe units , the use of sitters was decreased significantly , in addition to a reduction in restraint use . this same study proposed that increasing the number of hours worked by skilled nurses or by patient care assistants may reduce use of sitters and thus lead to lower sitter use and lower sitter costs . the cost - effectiveness of a patient sitter program was addressed using outcomes which included the frequency of patient falls and patient satisfaction . the help program , aimed at reducing the incidence and duration of delirium in hospitalized elderly patients , reduced hospital costs substantially , and has the potential to be replicated in alternative settings from that in which it was first described , possibly making it the most cost - effective way for employing sitters . however , a cochrane review looking at the cost - effectiveness of the implementation of similar programs has suggested that whilst this may reduce both incidence of delirium along with costs for intermediate risk patients , there was no significant benefit seen in the potential benefit offered by sitters is tremendous , especially considering the provision of constant observation on a busy acute medical ward . the one - to - one monitoring that can be given is invaluable , and provides a safety net for the nursing staff for high risk patients . the results from the revive study provided evidence of the potential benefit that trained volunteers could offer with patient interaction , whilst providing additional benefits in terms of reduced numbers of nursing assistants required , as well as hospital cost savings . whilst the role of sitters for use with certain patient population has previously been established , evidence for the cost - effectiveness of sitter usage for patients with delirium compared to traditional management is conflicting . although the use of sitters may reduce restraint use , their impact on patient fall rates ( a major patient safety outcome measure ) is marginal at best , and not clinically significant . one study assessed the use of sitters for patients deemed at high risk of falling and patients in a psychiatric crisis . by identification of patient volume , current sitter usage , and an assessment of demand , the study authors concluded that the use of constant observation with sitters was an ineffective and costly way of attempting to improve patient care . one of the first reports documenting sitter use was in 1985 as part of a nursing intervention for post - operative orthopedic patients , in which a nurse visitor was employed to provide consistent contact with patient . whilst their presence may not have significantly influenced the rate or duration of delirium , it does provide evidence supporting the use of patient sitters in the management of delirium . currently , there is no guidance available regarding who should be used in the sitter role , and no evaluation has been performed to assess the impact that the type of person in this role has upon the patient s clinical outcome ; as a result the type of sitter used is determined by the local health region . the current literature describes a variety of people having been used in the sitter role . although once used for observation of high - risk psychiatric patients , the use of sitters is now most commonly employed as part of the management for delirious patients . alternative uses for the sitter role have been seen as part of a preventative strategy for wandering patients and in fall prevention . an alternative role for the sitter has included their use for rooming with patients . this is when a family member or close friend stays in the patient s room , and has been shown to be effective for elderly orthopaedic patients , as described by one study on post - operative elderly orthopaedic patients to assess its clinical impact on delirium rates . although hospital length of stay and duration of delirium were not significantly reduced , it was shown to be feasible and may possibly be most beneficial for the acutely admitted elderly population . this lack of standardized criteria regulating the use of sitters and the absence of formal guidelines ( provincial and federally ) has highlighted an important deficit in the system that requires addressing . one study based in the uk looked at sitters currently employed in the palliative care setting and identified the reasons why sitters got involved and showed ( indirectly ) that higher sitter turnover was linked with inadequate or insufficient training , which supports the need for adequate training for the role . in addition to showing improvement through the comparisons of pre- and post - test results , it was also shown to be cost - effective , which supports the use of similar interventions in the provision of sufficient training for sitters . the use of sitters is dependent on the ability of nurses or health - care staff effectively recognizing patients requiring constant observation . however , one study , which addressed the recognition by nursing staff of patients with features of delirium by assessing nursing documentation , showed poor documentation of delirium features . thus , a failure to recognize such patients will inevitably affect the efficiency surrounding the use of sitters . using the paat , an improvement was seen in completion of the requisition forms , hiring of sitters , and in actual sitter usage , which was accompanied by reduction in restraint use . the use of sitters as part of the multi - interventional approach for delirium has been well established in the hospital elderly life program ( help ) , initially described by inouye et al . this model used rigorously trained volunteers as part of an interdisciplinary approach to reduce the incidence and rates of delirium . the success of the program was based , in part , on the use of well - trained volunteers for delirium management . additional support for the use of sitters is provided from the results of the revive study , aimed at the prevention and reduction of delirium using a multi - interventional approach through the use of trained volunteers to provide patient interaction and re - orientation . in addition , it was associated with a reduced length of stay in hospital , providing indirectly evidence for the use of sitters for cost - saving purposes . this model is based on placing all patients who require constant or very close observation in close proximity to one another , either in a single room or unit in the presence of nursing staff , which provides for continuous or close monitoring for these patients . with the implementation of these safe units , the use of sitters was decreased significantly , in addition to a reduction in restraint use . this same study proposed that increasing the number of hours worked by skilled nurses or by patient care assistants may reduce use of sitters and thus lead to lower sitter use and lower sitter costs . the help program , aimed at reducing the incidence and duration of delirium in hospitalized elderly patients , reduced hospital costs substantially , and has the potential to be replicated in alternative settings from that in which it was first described , possibly making it the most cost - effective way for employing sitters . however , a cochrane review looking at the cost - effectiveness of the implementation of similar programs has suggested that whilst this may reduce both incidence of delirium along with costs for intermediate risk patients , there was no significant benefit seen in the potential benefit offered by sitters is tremendous , especially considering the provision of constant observation on a busy acute medical ward . the results from the revive study provided evidence of the potential benefit that trained volunteers could offer with patient interaction , whilst providing additional benefits in terms of reduced numbers of nursing assistants required , as well as hospital cost savings . whilst the role of sitters for use with certain patient population has previously been established , evidence for the cost - effectiveness of sitter usage for patients with delirium compared to traditional management is conflicting . although the use of sitters may reduce restraint use , their impact on patient fall rates ( a major patient safety outcome measure ) is marginal at best , and not clinically significant . one study assessed the use of sitters for patients deemed at high risk of falling and patients in a psychiatric crisis . by identification of patient volume , current sitter usage , and an assessment of demand , the study authors concluded that the use of constant observation with sitters was an ineffective and costly way of attempting to improve patient care . from this review , it is evident that sitters are being employed in a variety of settings . sitter , a common underlying theme is apparent regarding their role as an observer or companion for the patient thus providing a potential definition for the sitter role as a person hired to provide constant observation or companionship. the question of which type of person would provide the most benefit in the sitter role is still not clear . volunteers may have the most potential in the capacity of the sitter , provided that they are adequately trained for the role and are freely available , which is evident from success of established program such as help model . this is supported by several studies that have justified the use of trained volunteers by showing them to be cost - effective . indeed , the use of sitters relieves the workload of overworked , stressed nursing staff who are often more than happy to have an additional pair of hands in the form of a volunteer sitter . precise details relating to the frequency of the use of sitters at any individual locations are difficult to establish due to a lack of documentation at the local , provincial , and federal levels . this highlights a potential role for the development of a national reporting scheme for recording their use . the absence of a standardized job description for the sitter role and lack of regulations regarding their use have led to the development of a certain degree of unease concerning their use , and accounts for the significant amount of variation and inconsistencies seen in the use of sitters . without a specific job description for the role of a sitter , health - care institutions are significantly restricted in this capacity , and hindered in benefiting from the use of sitters ( i.e. use of sitters for the provision of constant observation for high - risk psychiatric patients is well established . more creative and alternative roles for sitters have been successful , including their use as a although additional roles have used sitters in an attempt to reduce rates of wandering for high - risk patients and patient fall rates , these are theoretical advantages which are not supported by the current evidence available , and conflicts with the general consensus that sitters do not improve frequency of falling and may even increase fall rates ; however , the reasons for this are not clear . the process for identifying those patients who would benefit from the use of sitters has not been well established . if nursing staff are efficient in recognizing the presence of delirium , then this should naturally improve sitter use and cost - effectiveness . although there are no currently no formal established assessments tools available to aid with this process , there is great potential in the form of a specified assessment tool the paat as discussed previously , which was created to help assist and thus improve the hiring and requisition of the use of sitters for high - risk patients . a lack of orientation to either the patient and/or to their role is distracting and problematic for both the patient and sitter and this , combined with the lack of familiarity between them , could create a potentially disastrous situation , as well as being an inefficient use of resources and a hindrance to patient care . in addition , inadequate training for the management of aggressive or agitated patients could put sitters , the patient , and staff at danger and has legal consequences . as a consequence of the lack of formal / recognized training or assessment , the competence of these sitters has been questioned several times , especially since it is often untrained volunteers that are commonly used in this capacity . the importance of this is evident from the dire consequences that can ensue due to the nature of settings in which these sitters are commonly employed . however , with adequate training , the effectiveness of fully trained volunteers in the sitter role is non - debatable , reconfirming the need for sufficient training for the position . the employment of sitters in the management of delirious patients may benefit the patient both in terms of delirium control ( through the application of consistent , frequent re - orientation therapy ) , as well as ( possibly ) reducing or preventing the use of pharmacological agents and/or restrictive devices . thus , the use of a well - established delirium program model for employing trained sitters ( i.e. , volunteers ) may offer the most cost - effective method . however , the translation of a large established program to implementation of smaller similar programs at the local level can be one of the hardest barriers to cross due to restrictions exerted in terms of costs , resistant attitudes from staff , and insufficient staffing levels ( to name a few ) . certainly , in our local region , the use of sitters has been reduced substantially in the last couple of years due to financial restraints . the same review suggested that management of patients who require sitters should avoid placing delirious patients in one single area due to a possible negative influence that may be exerted on one another . from the 37 articles included in the review , nine studies directly addressed the role for sitters in delirium , with sample sizes ranging from 244763 . the use of mostly trained volunteers in the sitter role provided the main limitations to these studies . one article addressed training for sitter role in delirium ( sample size : 1507 ) and six articles ( sample size : 375346 ) addressed cost - effectiveness for role of sitters in delirium . current evidence supports a role for the sitter as part of the management of patients with delirium . since the majority ( and strongest evidence ) is seen with the use of trained volunteers in the sitter role as part of an established delirium model ( such as help ) , then this may likely be the most cost - effective way for advocating their use , possibly in conjunction with use a recognized sitter assessment tool such as the paat . the specific type of training required for the sitter role is not clear and requires further research . certainly , when considering using a sitter , the health - care provider should carefully document the indications for sitter use , and monitor and reassess these indications alongside the frequency and use of the sitter in order to maximize cost - effectiveness . long term , the creation of a national set of regulations or guidelines would provide safeguards in the industry to ensure safe and effective patient care , in addition to the creation of a national reporting scheme for sitter usage .

delirium is an extremely common problem occurring in over 50% of older , hospitalized patients , and is associated with both a decline in functional ability and increased morbidity and mortality . currently , the management for delirium is multifactorial and should include the treatment of reversible conditions and addressing specific patient and care factors which may be contributing to the delirium . a recognized component of this approach , both for the prevention and in the management of delirium , involves the employment of constant observation ( co ) ( also known as special observation ) . the concept of co has been around for some time , and associated definitions have ranged from an intervention in which continuous one - to - one monitoring is used to assure the safety and well - being of an individual patient or others , to direct observation of patients for the purpose of providing a safer environment for the patient. however , a more practical description for co uses co for the provision of one - to - one observation by a constant observer ( a dedicated person employed to sit with the patient ) . once mostly reserved for use within the psychiatric setting for patients at high risk of self - harm or suicide , the role of co has diversified and is now most commonly implemented for the management of delirious patients . therefore , our primary objective for this literature review is to provide an overview of the current role of the sitter within the management of delirium in hospitalized patients by first addressing the current understanding of the role for sitters , the type of person employed in this role , indications for current usage , and the frequency they are used . secondary objectives include the identification of key training requirements required for the sitter role and establishment of the cost - effectiveness of their role within delirium management . a literature search was performed of medline , cochrane database of systematic reviews , and pubmed from the years 1960 to october 2011 . the following search terms were used : sitter / patient sitter / companion / nurses aides / special care aides , confusion / delirium / acute confusional state , responsibilities / roles , constant observation / observation , outcomes , intervention , cost / cost analysis . inclusion criteria required the articles ( and abstracts ) to be in the english language and contain relevant research involving at least one of the following : the roles of the sitter , people used for the sitter role , current usage of sitters , indications / assessment and/or tools / evaluation for sitter use , education / training requirements / pre - requisitions required for the role , costs associated with sitter use , sitter use / role within delirium setting including non - pharmalogical intervention models for delirium management . instead , one of the first reports documenting sitter use was in 1985 as part of a nursing intervention for post - operative orthopedic patients , in which a nurse visitor was employed to provide consistent contact with patient . whilst their presence may not have significantly influenced the rate or duration of delirium , it does provide evidence supporting the use of patient sitters in the management of delirium . currently , there is no guidance available regarding who should be used in the sitter role , and no evaluation has been performed to assess the impact that the type of person in this role has upon the patient s clinical outcome ; as a result the type of sitter used is determined by the local health region . whilst the experience and training they could offer is vast , their use is significantly restricted by an associated high - cost expenditure combined with limited staffing levels . family members or other relatives are used quite often , inadvertently , as sitters by health - care staff , which is supported by a study that looked at family participation in patient care in the hospital setting and showed their involvement ranging from direct provision of patient care to companionship . one study based in australia used volunteers in the combined role of observers and companions for patients who had been highlighted as being at a high risk of falling . these volunteers remained with them in the room and were involved in patient interaction , in addition to alerting nursing staff any time patients were identified as being about to fall . whilst the results from this study showed significant reductions in patient fall rates , limited availability of these volunteers , combined with significant restrictions imposed from occupational health and safety regulations on their role , may limit their use in this capacity . although once used for observation of high - risk psychiatric patients , the use of sitters is now most commonly employed as part of the management for delirious patients . another established , albeit less frequently recognized role , is the provision of companionship for patients in palliative care . alternative uses for the sitter role have been seen as part of a preventative strategy for wandering patients and in fall prevention . this is when a family member or close friend stays in the patient s room , and has been shown to be effective for elderly orthopaedic patients , as described by one study on post - operative elderly orthopaedic patients to assess its clinical impact on delirium rates . although hospital length of stay and duration of delirium were not significantly reduced , it was shown to be feasible and may possibly be most beneficial for the acutely admitted elderly population . more recently , successful sitter use was shown in the role of a rehabilitation patient companion on an acute brain injury unit in an attempt to reduce costs and improve patient and nurse satisfaction . this lack of standardized criteria regulating the use of sitters and the absence of formal guidelines ( provincial and federally ) has highlighted an important deficit in the system that requires addressing . one study based in the uk looked at sitters currently employed in the palliative care setting and identified the reasons why sitters got involved and showed ( indirectly ) that higher sitter turnover was linked with inadequate or insufficient training , which supports the need for adequate training for the role . a quality improvement project was done to assess the effectiveness of a one - hour educational program aimed at sitters ( and nurses ) which addressed key issues including policy and procedure , risk assessment , and symptom recognition , combined with self - learning . in addition to showing improvement through the comparisons of pre- and post - test results , it was also shown to be cost - effective , which supports the use of similar interventions in the provision of sufficient training for sitters . however , one study , which addressed the recognition by nursing staff of patients with features of delirium by assessing nursing documentation , showed poor documentation of delirium features . to help with identification of patients who would benefit from sitter use , and aid and improve sitter requests , an assessment tool , the patient attendant assessment tool ( paat ) was created . using the paat , an improvement was seen in completion of the requisition forms , hiring of sitters , and in actual sitter usage , which was accompanied by reduction in restraint use . the use of sitters as part of the multi - interventional approach for delirium has been well established in the hospital elderly life program ( help ) , initially described by inouye et al . the success of the program was based , in part , on the use of well - trained volunteers for delirium management . additional support for the use of sitters is provided from the results of the revive study , aimed at the prevention and reduction of delirium using a multi - interventional approach through the use of trained volunteers to provide patient interaction and re - orientation . the results revealed a reduction in the incidence and duration of delirium , together with a reduced frequency of falls , thus supporting the importance of this approach . in addition , it was associated with a reduced length of stay in hospital , providing indirectly evidence for the use of sitters for cost - saving purposes . this model is based on placing all patients who require constant or very close observation in close proximity to one another , either in a single room or unit in the presence of nursing staff , which provides for continuous or close monitoring for these patients . with the implementation of these safe units , the use of sitters was decreased significantly , in addition to a reduction in restraint use . certain factors have been associated with higher rates of sitter usage and thus higher sitter costs , which include understaffing of units with registered nurses and certain patient characteristics . in this study , the cost for a typical sitter shift was $ 160 , which , when adjusted for the ( slight ) benefit seen in fall rates and patient satisfaction , revealed a net cost expense of $ 156.24 . the help program , aimed at reducing the incidence and duration of delirium in hospitalized elderly patients , reduced hospital costs substantially , and has the potential to be replicated in alternative settings from that in which it was first described , possibly making it the most cost - effective way for employing sitters . however , a cochrane review looking at the cost - effectiveness of the implementation of similar programs has suggested that whilst this may reduce both incidence of delirium along with costs for intermediate risk patients , there was no significant benefit seen in the potential benefit offered by sitters is tremendous , especially considering the provision of constant observation on a busy acute medical ward . the one - to - one monitoring that can be given is invaluable , and provides a safety net for the nursing staff for high risk patients . the results from the revive study provided evidence of the potential benefit that trained volunteers could offer with patient interaction , whilst providing additional benefits in terms of reduced numbers of nursing assistants required , as well as hospital cost savings . whilst the role of sitters for use with certain patient population has previously been established , evidence for the cost - effectiveness of sitter usage for patients with delirium compared to traditional management is conflicting . by identification of patient volume , current sitter usage , and an assessment of demand , the study authors concluded that the use of constant observation with sitters was an ineffective and costly way of attempting to improve patient care . the study further noted that it was more beneficial to identify high - risk patients on admission ( using assessment tools ) , and to train and employ the hospital s own health - care staff as sitters ( using a good quality sitter education program ) rather than use an external agency . one of the first reports documenting sitter use was in 1985 as part of a nursing intervention for post - operative orthopedic patients , in which a nurse visitor was employed to provide consistent contact with patient . whilst their presence may not have significantly influenced the rate or duration of delirium , it does provide evidence supporting the use of patient sitters in the management of delirium . currently , there is no guidance available regarding who should be used in the sitter role , and no evaluation has been performed to assess the impact that the type of person in this role has upon the patient s clinical outcome ; as a result the type of sitter used is determined by the local health region . family members or other relatives are used quite often , inadvertently , as sitters by health - care staff , which is supported by a study that looked at family participation in patient care in the hospital setting and showed their involvement ranging from direct provision of patient care to companionship . one study based in australia used volunteers in the combined role of observers and companions for patients who had been highlighted as being at a high risk of falling . these volunteers remained with them in the room and were involved in patient interaction , in addition to alerting nursing staff any time patients were identified as being about to fall . whilst the results from this study showed significant reductions in patient fall rates , limited availability of these volunteers , combined with significant restrictions imposed from occupational health and safety regulations on their role , may limit their use in this capacity . although once used for observation of high - risk psychiatric patients , the use of sitters is now most commonly employed as part of the management for delirious patients . this is when a family member or close friend stays in the patient s room , and has been shown to be effective for elderly orthopaedic patients , as described by one study on post - operative elderly orthopaedic patients to assess its clinical impact on delirium rates . one study based in the uk looked at sitters currently employed in the palliative care setting and identified the reasons why sitters got involved and showed ( indirectly ) that higher sitter turnover was linked with inadequate or insufficient training , which supports the need for adequate training for the role . a quality improvement project was done to assess the effectiveness of a one - hour educational program aimed at sitters ( and nurses ) which addressed key issues including policy and procedure , risk assessment , and symptom recognition , combined with self - learning . in addition to showing improvement through the comparisons of pre- and post - test results , it was also shown to be cost - effective , which supports the use of similar interventions in the provision of sufficient training for sitters . however , one study , which addressed the recognition by nursing staff of patients with features of delirium by assessing nursing documentation , showed poor documentation of delirium features . the use of sitters as part of the multi - interventional approach for delirium has been well established in the hospital elderly life program ( help ) , initially described by inouye et al . additional support for the use of sitters is provided from the results of the revive study , aimed at the prevention and reduction of delirium using a multi - interventional approach through the use of trained volunteers to provide patient interaction and re - orientation . the results revealed a reduction in the incidence and duration of delirium , together with a reduced frequency of falls , thus supporting the importance of this approach . in addition , it was associated with a reduced length of stay in hospital , providing indirectly evidence for the use of sitters for cost - saving purposes . certain factors have been associated with higher rates of sitter usage and thus higher sitter costs , which include understaffing of units with registered nurses and certain patient characteristics . in this study , the cost for a typical sitter shift was $ 160 , which , when adjusted for the ( slight ) benefit seen in fall rates and patient satisfaction , revealed a net cost expense of $ 156.24 . the help program , aimed at reducing the incidence and duration of delirium in hospitalized elderly patients , reduced hospital costs substantially , and has the potential to be replicated in alternative settings from that in which it was first described , possibly making it the most cost - effective way for employing sitters . however , a cochrane review looking at the cost - effectiveness of the implementation of similar programs has suggested that whilst this may reduce both incidence of delirium along with costs for intermediate risk patients , there was no significant benefit seen in the potential benefit offered by sitters is tremendous , especially considering the provision of constant observation on a busy acute medical ward . the one - to - one monitoring that can be given is invaluable , and provides a safety net for the nursing staff for high risk patients . the results from the revive study provided evidence of the potential benefit that trained volunteers could offer with patient interaction , whilst providing additional benefits in terms of reduced numbers of nursing assistants required , as well as hospital cost savings . whilst the role of sitters for use with certain patient population has previously been established , evidence for the cost - effectiveness of sitter usage for patients with delirium compared to traditional management is conflicting . by identification of patient volume , current sitter usage , and an assessment of demand , the study authors concluded that the use of constant observation with sitters was an ineffective and costly way of attempting to improve patient care . the study further noted that it was more beneficial to identify high - risk patients on admission ( using assessment tools ) , and to train and employ the hospital s own health - care staff as sitters ( using a good quality sitter education program ) rather than use an external agency . sitter , a common underlying theme is apparent regarding their role as an observer or companion for the patient thus providing a potential definition for the sitter role as a person hired to provide constant observation or companionship. family members have the potential to be effective sitters ; their familiarity with the patient , close relationship , and motivation to stay with the patient can provide essential patient interaction important for delirious patients . volunteers may have the most potential in the capacity of the sitter , provided that they are adequately trained for the role and are freely available , which is evident from success of established program such as help model . the absence of a standardized job description for the sitter role and lack of regulations regarding their use have led to the development of a certain degree of unease concerning their use , and accounts for the significant amount of variation and inconsistencies seen in the use of sitters . without a specific job description for the role of a sitter , health - care institutions are significantly restricted in this capacity , and hindered in benefiting from the use of sitters ( i.e. more creative and alternative roles for sitters have been successful , including their use as a although additional roles have used sitters in an attempt to reduce rates of wandering for high - risk patients and patient fall rates , these are theoretical advantages which are not supported by the current evidence available , and conflicts with the general consensus that sitters do not improve frequency of falling and may even increase fall rates ; however , the reasons for this are not clear . the provision of pre - specified criteria or assessment tools to aid nursing staff in recognizing patients who would benefit from these costly resources is important for both reducing costs associated with using sitters and reducing rates of pharmacological therapy with its associated side effects . one prospective study looking at the accuracy of nursing documentation relating to their recognition of the signs and symptoms of delirium suggested possible poor recognition , with a possible exception being for severely ill patients . although there are no currently no formal established assessments tools available to aid with this process , there is great potential in the form of a specified assessment tool the paat as discussed previously , which was created to help assist and thus improve the hiring and requisition of the use of sitters for high - risk patients . a lack of orientation to either the patient and/or to their role is distracting and problematic for both the patient and sitter and this , combined with the lack of familiarity between them , could create a potentially disastrous situation , as well as being an inefficient use of resources and a hindrance to patient care . however , with adequate training , the effectiveness of fully trained volunteers in the sitter role is non - debatable , reconfirming the need for sufficient training for the position . the employment of sitters in the management of delirious patients may benefit the patient both in terms of delirium control ( through the application of consistent , frequent re - orientation therapy ) , as well as ( possibly ) reducing or preventing the use of pharmacological agents and/or restrictive devices . however , the translation of a large established program to implementation of smaller similar programs at the local level can be one of the hardest barriers to cross due to restrictions exerted in terms of costs , resistant attitudes from staff , and insufficient staffing levels ( to name a few ) . from the 37 articles included in the review , nine studies directly addressed the role for sitters in delirium , with sample sizes ranging from 244763 . addressed outcomes included markers of delirium ( incidence , severity , duration , number of episodes , and recurrence ) , patient outcomes ( cognitive change , functional change , sleep quality ) , nursing assistant use , and financial cost . one article addressed training for sitter role in delirium ( sample size : 1507 ) and six articles ( sample size : 375346 ) addressed cost - effectiveness for role of sitters in delirium . since the majority ( and strongest evidence ) is seen with the use of trained volunteers in the sitter role as part of an established delirium model ( such as help ) , then this may likely be the most cost - effective way for advocating their use , possibly in conjunction with use a recognized sitter assessment tool such as the paat . certainly , when considering using a sitter , the health - care provider should carefully document the indications for sitter use , and monitor and reassess these indications alongside the frequency and use of the sitter in order to maximize cost - effectiveness .

in this review , we address the following questions : ( 1 ) what are the potential mechanisms linking type 1 diabetes with insulin resistance and how is this combination ( double diabetes ) associated with increased cardiovascular risk ? ( 2 ) might weight gain associated with intensive insulin regimens be associated with increased insulin resistance and what implications might this have for cardiovascular risk ? ( 3 ) what role does the liver play in double diabetes ? ( 4 ) might we need to consider changes in current clinical practice in the context of the issues raised in this review ? the term double diabetes was first coined in 1991 based on the observation that patients with type 1 diabetes who had a family history of type 2 diabetes were more likely to be overweight and rarely achieved adequate glycaemic control even with higher insulin doses . the more extensive , or stronger , the family history , the higher the dose the patient received . the authors suggested that this might indicate the presence of increased resistance to insulin - mediated glucose disposal in this subgroup of people with type 1 diabetes and asserted that , over a lifetime , some of these individuals would likely have been diagnosed with type 2 diabetes at some point , had they not first developed beta cell destruction by an independent pathological process ( i.e. type 1 diabetes ) . at this stage , it is important to differentiate this description of double diabetes , which considers autoimmune diabetes to be an independent process from obesity and insulin resistance , from the accelerator hypothesis , which describes triggering of autoimmune diabetes by factors including bmi and insulin resistance . other studies of people with type 1 diabetes and a family history of type 2 diabetes have supported the notion that this combination might promote both microvascular and macrovascular complications of type 1 diabetes . for example , in a prospective study of 3250 patients with type 1 diabetes recruited from 16 european countries ( eurodiab ) , it was demonstrated that women with a parental history of type 2 diabetes had a higher risk of developing albuminuria than those without a positive family history ( hr 1.36 , p = 0.04 ) . furthermore , in a cross - sectional study of 658 patients from the pittsburgh epidemiology of diabetes complications ( edc ) cohort , 112 of whom had a confirmed family history of type 2 diabetes , and 119 of whom had experienced a chd event , a positive family history was associated with a significant excess risk of chd ( hr 1.89 , 95% ci 1.27 , 2.84 ) . those with a greater number of affected family members had a greater risk ( p = 0.001 for trend ) , with one family member conferring an or of 1.62 and two family members increasing this to 5.13 . these data are consistent with either genetically determined insulin resistance / double diabetes or shared parental offspring lifestyle factors contributing to type 2 diabetes in parents and increasing the risk of complications . given the results of genome - wide association studies of type 2 diabetes published to date , the latter explanation currently appears more plausible . if insulin resistance , either inherited or acquired , is implicated in the pathogenesis of double diabetes , how is it likely to manifest and can it be identified ? the gold standard method of measuring insulin - mediated glucose uptake is the hyperinsulinaemic euglycaemic clamp technique . although this is a time - consuming and relatively invasive technique , several investigators have employed it to demonstrate increased insulin resistance in those with type 1 diabetes compared with age- , sex- and bmi - matched controls [ 616 ] . in one such study of 40 cases ( mean age 45 years , mean diabetes duration of type 1 diabetes 23 years ) and 47 age- and sex - matched control individuals , patients with type 1 diabetes not only had lower whole body insulin - mediated glucose uptake ( mean se : 6.19 0.72 vs 12.71 0.66 mg [ kg fat - free mass ] min , p < 0.0001 ) but also had lower insulin - mediated nefa suppression . both measurements were correlated ( p < 0.0001 within the type 1 diabetes group ) with coronary artery calcification ( cac ) measured by computed tomography . of note , hba1c did not correlate with either insulin resistance or cac . data from one clamp study , ( n = 24 ) were used to derive estimated glucose disposal rate ( egdr ) from a best - fit model:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mathrm{egdr}=24.31 - 12.22\left ( { \mathrm{whr } } \right)-3.29\left ( { \mathrm{hypertension}\ \mathrm{status } } \right)-0.57\left ( { \mathrm{hb}{{\mathrm{a}}_{1\mathrm{c } } } } \right ) $ $ \end{document}where hypertension status ( 1 or 0 ) is defined as bp > 140/90 mmhg or on anti - hypertensive drugs ; hba1c refers to the value in % . low egdr was proposed as a surrogate measure of insulin resistance for use either in a clinical setting or for epidemiological analyses . egdr was tested prospectively in 603 type 1 diabetes pittsburgh edc cohort participants ( mean baseline age 28 years , mean diabetes duration 19 years ) followed up over 10 years . independent predictors of cvd events ( n = 42 ) were disease duration , presence of nephropathy , non - hdl - cholesterol ( hdl - c ) , white cell count and egdr . however , in a subsequent analysis of the same cohort by the same group , egdr did not appear in chd risk prediction models ( although , importantly , these incorporated three of its individual components : whr , hdl - c and bp ) . in an independent examination of the dcct / epidemiology of diabetes interventions and complications ( edic ) data , kilpatrick et al estimated egdr retrospectively ( using bmi data instead of whr ) as a predictor of cvd events . in a cox regression model , high egdr as a surrogate for preserved insulin sensitivity was independently associated with a lower risk for cv events ( hr 0.70 ; 95% ci 0.56 , 0.88 ) . in contrast , total insulin dose requirement had no predictive value . intriguingly , high egdr was also associated with a lower risk for microvascular events , including retinopathy and nephropathy a finding also corroborated by data from the eurodiab study . given the mechanistic relevance of insulin resistance in the metabolic syndrome , a number of prospective studies have assessed the utility of various definitions of the metabolic syndrome in predicting risk in type 1 diabetes cohorts . however , as the glycaemia criterion of the who , national cholesterol education program ( ncep ) and international diabetes federation ( idf ) definitions are automatically fulfilled , there is a major caveat . for example , an individual with type 1 diabetes can meet the who definition by virtue of microalbuminuria alone , even if non - obese and with a normal lipid profile . in a prospective analysis of the pittsburgh edc cohort , the prevalence of the metabolic syndrome was 21% ( who definition ) , 12% ( ncep ) and 8% ( idf ) . using a composite endpoint of chd , renal failure and diabetes - related death , the metabolic syndrome conferred significantly elevated hrs ( ncep : 5.8 , 95% ci 3.9 , 8.6 ; who : 6.5 , 95% ci 4.5 , 9.4 ) ; however , these ratios were not higher than those for individual components of the syndrome ( for example , microalbuminuria : 6.3 , 95% ci 3.8 , 10.5 ; bp 4.5 , 95% ci 3.1 , 6.6 ; triacylglycerol : 4.4 , 95% ci 3.0 , 6.6 ) . in a similar analysis of the finndiane study , a high prevalence of the metabolic syndrome was reported ( 44% who definition ; 35% ncep ; 36% idf ) . the who definition predicted incident cv events after adjustment for traditional risk factors and nephropathy ( hr 2.05 , 95% ci 1.38 , 3.04 ) , even in participants with raised albumin excretion ( hr 1.44 , 95% ci 1.06 , 1.96 ) . however , the individual components of the metabolic syndrome were also significant predictors , with the exception of obesity . in the dcct / edic study , an adapted idf definition of the metabolic syndrome had a baseline prevalence of 22% , largely driven by low hdl - c , but did not predict either macro- or microvascular events over 17 years of follow - up . thus , in broad terms , the metabolic syndrome and its individual features are associated with more events but , as in the general population , dichotomous classification of individuals with type 1 diabetes according to the metabolic syndrome does not appear to add value for cv risk prediction in type 1 diabetes over and above its individual components . it is generally accepted that intensive insulin therapy reduces the incidence of cvd in type 1 diabetes . the most robust interventional clinical trial evidence for this is from the dcct in which 1,441 adolescents and young adults ( mean age 27 years , bmi 28 kg / m ) were randomised for 6.5 years to intensive or conventional glycaemic control and followed up post - randomisation in the edic study up to a total of 17 years . intensive glycaemic control was associated with a 57% reduction in rr ( 95% ci 12 , 79% , p = 0.02 ) in a composite endpoint of non - fatal mi , stroke or cv death ( approximate overall rate of six events per 1,000 patient - years ) . of course , one of the consequences of intensive therapy is weight gain ; the intensively treated group in the dcct study gained 4.6 kg more than the conventionally treated group over the 5 year study period . in this context , subgroup analyses of the dcct / edic study have raised concerns about participants in whom weight gain over the course of the trial was associated with the emergence of features associated with increased cv risk ( table 1 ) . those in the highest quartile for weight gain exhibited higher bp and ldl - cholesterol ( ldl - c ) values , a higher insulin dose requirement and whr and a more atherogenic lipid profile ( raised apolipoprotein b , high vldl , higher small , dense ldl , lower apolipoprotein a - i and lower hdl ) . in the intensive group , mean bmi increased from 24 to 31 kg / m . these differences remained significant after adjustment for hba1c . a similar pattern was observed among those allocated to conventional treatment ( albeit with a reduced effect size ) , which is consistent with the existence of individual predisposing factors to insulin - induced weight gain , an area that merits further study.table 1characteristics of the intensively treated group at follow - upquartiles of weight gain1234hba1c ( % ) [ mmol / mol]7.3 7.2 7.1 7.3 insulin dose ( units kg day)0.610.640.690.74achieved bmi ( kg / m)24252731systolic bp ( mmhg)113117115120triacylglycerol ( mmol / l)0.790.820.910.99ldl - cholesterol ( mmol / l)2.742.792.923.15hdl - cholesterol ( mmol / l)1.401.341.291.27baseline bmi was 24 kg / m for all quartilesadapted from with permission . all rights reserved characteristics of the intensively treated group at follow - up baseline bmi was 24 kg / m for all quartiles adapted from with permission . all rights reserved interestingly , in the dcct / edic study , a family history of type 2 diabetes was also a significant independent predictor of weight gain in both the conventional and intensive groups , consistent with an inherited tendency to store excess fat in response to insulin , a degree of inherited peripheral insulin resistance and/or a familial effect on environmental factors ( energy intake and physical activity ) . there is increasing contemporary evidence that the prevalence of obesity has risen substantially among the type 1 diabetic population , as it has in the general population . for example , in the pittsburgh edc cohort between 1987 and 2007 the prevalence of obesity rose sevenfold ( to 22.7% ) and overweight increased by 47% ( to 42% ) . while in the pre - dcct era baseline bmi in patients with type 1 diabetes was lower than population norms , probably owing to a combination of weight loss prior to diagnosis and suboptimal glycaemic control , current bmi patterns are similar . although much of the weight gain seen in contemporary patients with type 1 diabetes is likely to reflect cultural , societal and lifestyle changes , some of this increase may be due to use of more intensive insulin regimens . mechanisms for the latter may include reduced excretion of urinary glucose and altered feeding behaviour ( more frequent treatment of hypoglycaemic episodes , appetite stimulation ) , in addition to the known anabolic effects of insulin . adverse cv risk factors associated with weight gain in some recipients of intensive insulin therapy were also seen in the observational data from the eurodiab cohort , in which 1,800 type 1 diabetic patients ( mean age 33 years , duration of diabetes 14.8 years , hba1c 8.2% [ 66 mmol / mol ] ) were followed up for 7.3 years . those who gained more than 5 kg over this time had better glycaemic control ( mean hba1c difference of 0.2% ) than those with less or no weight gain but also had raised bp , ldl - c and triacylglycerol in association with lower hdl - c ( differences that remained significant after adjustment for glycaemic control ) . insulin resistance in type 1 insulin absorbed from subcutaneous depots results in relative peripheral hyperinsulinaemia and hepatic hypoinsulinaemia compared with normal physiology . chronic adaptation to this combination could reduce peripheral insulin - mediated glucose uptake and increase hepatic glucose production . in addition , it has been proposed that reduced hepatic insulin exposure results in a reduced level of circulating igf-1 , which , along with a parallel increase in growth hormone and igf - binding proteins , may also contribute to increased peripheral insulin resistance [ 30 , 31 ] . therefore , several factors could underlie the phenotype of double diabetes ( fig . 1 ) . first , the genetic and lifestyle factors that lead to type 2 diabetes may exist at similar frequency in those with type 1 diabetes this would be consistent with the robust data on family history of type 2 diabetes described above . third , exogenous insulin therapy might induce insulin resistance in patients with type 1 diabetes.fig . t1 dm , type 1 diabetes mechanisms potentially contributing to insulin resistance in type 1 diabetes patients with double diabetes . t1 dm , type 1 diabetes there are several important unanswered questions : are some people with type 1 diabetes ( including those with a family history of type 2 diabetes or of specific ethnicities , e.g. south asians ) more susceptible to weight gain during intensive insulin treatment , perhaps owing to differences in metabolism or substrate handling or can the observations regarding weight gain simply be explained by the fact that those who are more insulin resistant are treated with higher doses of insulin?what are the most important factors , e.g. inherited or familial factors , differences in feeding strategies for avoidance of hypoglycaemia , insulin resistance?to what extent do adverse cvd risk factors associated with higher weight gain attenuate or reverse the presumed earlier cv ( and other ) benefits of intensive glycaemic control in type 1 diabetes ? are some people with type 1 diabetes ( including those with a family history of type 2 diabetes or of specific ethnicities , e.g. south asians ) more susceptible to weight gain during intensive insulin treatment , perhaps owing to differences in metabolism or substrate handling or can the observations regarding weight gain simply be explained by the fact that those who are more insulin resistant are treated with higher doses of insulin ? what are the most important factors , e.g. inherited or familial factors , differences in feeding strategies for avoidance of hypoglycaemia , insulin resistance ? to what extent do adverse cvd risk factors associated with higher weight gain attenuate or reverse the presumed earlier cv ( and other ) benefits of intensive glycaemic control in type 1 diabetes ? insulin not only exerts effects on peripheral glucose uptake and suppression of hepatic glucose production , it also has profound effects on fat partitioning by promoting hepatic and peripheral lipogenesis ( fat storage ) as well as suppressing hepatic and peripheral lipolysis ( inhibition of fat oxidation ) . in this section , we compare lipid handling in type 1 diabetes with non - diabetic metabolism and with type 2 diabetes . the most obvious and striking difference between type 1 diabetes on the one hand and obesity / type 2 diabetes on the other , is low portal vein insulin concentration . this can be concluded by inference from the understanding of type 1 diabetes pathophysiology and anatomy ( i.e. if beta cells are not secreting insulin , then portal insulin levels will be low , only reflecting recirculating insulin from subcutaneous injection ) . further evidence for this is reviewed below and includes extrapolation from animal models . in type 1 diabetes , absent pancreatic insulin secretion is the opposite phenotype to the endogenous hyperinsulinaemia characteristic of most conditions characterised by insulin resistance . as type 1 diabetes is characterised by higher peripheral insulin concentrations and lower portal concentrations , it follows that contrasting hepatic and peripheral lipid handling might be predicted . in contrast to obesity and type 2 diabetes , in which the importance of non - alcoholic steatohepatitis ( fatty liver ) has been highlighted , there is some evidence that intra - hepatic fat content is reduced in type 1 diabetes . comparing 19 patients with type 1 diabetes ( mean age 35 , bmi 23 kg / m , hba1c 8.7% [ 72 mmol / mol ] ) and carefully matched non - diabetic controls using h magnetic resonance spectroscopy , perseghin et al demonstrated significantly reduced intra - hepatic fat content in those with type 1 diabetes ( 1.5 0.7% vs 2.2 1.0% , p < 0.03 ) . this was associated with increased fasting lipid oxidation ( 1.5 0.7 vs 0.8 0.4 mg / kg ) . the estimated hepatic insulin ( ehi ) level was lower and the glucagon : ehi ratio ( reported as an indicator of the balance between catabolism and anabolism ) was higher in the type 1 diabetes group ( both p < 0.05 ) . insulin stimulates sterol regulatory element - binding protein 1c ( srebp1c ) , which plays a crucial role in the regulation of triacylglycerol accumulation in the liver [ 3235 ] . moreover , two enzymes involved in de novo lipogenesis , namely , fatty acid synthase ( fas ) and acetyl - coa carboxylase , are activated by srebp1c . in parallel with this , hepatic fatty acid oxidation is inhibited and the balance shifts to lipid storage as triacylglycerol is incorporated into vldl [ 33 , 36 ] . it has been suggested that relative under - insulinisation of the liver with preserved glucagon secretion in type 1 diabetes favours a shift in metabolism from lipid storage to oxidation . fat partitioning ( the tissue distribution of fat storage ) reflects the balance of lipogenesis and fat oxidation in different tissues . in type 1 diabetes , this is likely to be influenced by the absence of endogenous insulin combined with therapeutic administration of subcutaneous insulin into the peripheral ( as opposed to portal ) circulation . this results in reduced inhibition of hepatic lipolysis with consequent increased levels of circulating nefas , which , in combination with peripheral hyperinsulinaemia , may promote relatively greater lipid storage in skeletal muscle [ 79 ] . this is supported by recent evidence from recipients of hepatic islet cell transplants , in whom ogtt - induced nefa suppresion was normalised . the subsequent pattern of increased ( ectopic ) intramyocellular fat storage is similar to that found in obesity / type 2 diabetes and is associated with impaired sensitivity of muscle to glucose uptake , i.e. peripheral insulin resistance . therefore , in type 1 diabetes , this may represent an indirect mechanism of insulin resistance . taking this idea further , anything that exaggerates these features ( i.e. increased fat mass , increased lipid / nefa flux , higher insulin dose requirements ) is likely to promote further insulin resistance . it follows that other sites of ectopic fat deposition might be influenced by abnormal fat partitioning . for example , epicardial and perivascular fat may play a role in modulating coronary function and pathophysiology , providing a potential mechanism to link double diabetes with cvd . in this regard , data have recently been published on epicardial fat thickness ( measured by echocardiography ) in 36 type 1 diabetic patients and 43 matched controls . not only was epicardial fat thicker in those with type 1 diabetes ( p < 0.0001 ) , but it also correlated significantly with both whr ( r = 0.67 , p = 0.003 ) and egdr ( r = 0.55 , p = 0.0004 ) within the patient group . hdl - c levels are normal or high in type 1 diabetes unless renal impairment develops , in which case hdl - c falls [ 7 , 4043 ] . moreover , long - term survivors of type 1 diabetes are characterised by unusually high hdl - c levels ( 1.84 mmol / l ) , and patients with chd events have been shown to have lower hdl - c levels ( along with higher triacylglycerol and ldl - c levels ) . exogenous insulin treatment usually drives higher mean hdl - c levels in type 1 diabetes , but even those individuals with poorer glycaemic control have higher levels than healthy controls . the most important of these appears to be lipoprotein lipase ( lpl ) , which is highly active in adipocytes and avidly hydrolyses triacylglycerol - rich particles , resulting in high hdl - c levels . peripheral hyperinsulinaemia associated with strict glycaemic control is associated with increased lpl activity and hdl - c , while poor glycaemic control results in lower lpl activity and hdl - c [ 46 , 47 ] . there may also be a smaller contribution from a reduction in the activity of hepatic lipase ( hl ) , an enzyme that hydrolyses triacylglycerol and phospholipids present in circulating plasma lipoproteins . low levels of portal vein insulin in type 1 diabetes are associated with reduced hl activity and increased hdl - c [ 4850 ] . increasing hepatic insulin exposure by changing insulin delivery from the subcutaneous to the intraperitoneal route reverses this pattern . in contrast , in type 2 diabetes , raised hl activity secondary to portal hyperinsulinaemia may contribute to low hdl - c levels . finally , phospholipid transfer protein activity is markedly elevated in patients with type 1 diabetes , and this activity is correlated with hdl - c levels . high hdl - c levels in patients with type 1 diabetes are likely to be atheroprotective , even though some compositional abnormalities may reduce the protective effect . however , if central obesity and insulin resistance then develop ( double diabetes ) , a more atherogenic lipid profile seems to emerge . this assertion is supported by data from a subgroup of 61 dcct participants , in whom increased hl activity accounted for most of the association between increased intra - abdominal fat stores and decreased hdl - c levels . as discussed above , the liver is under - insulinised in type 1 diabetes . this raises the hypothesis that type 1 diabetes may actually confer simultaneous protective and harmful cv effects . this is a difficult phenomenon to study , but interesting insights have been gained from the field of pancreas transplantation in type 1 diabetes . when an insulin - secreting graft is attached to the portal vein , an apparently more atherogenic lipid profile is later observed than when it is attached to a systemic vein . by contrast , this model has also been used to demonstrate that hepatic igf-1 secretion is increased and growth hormone secretion is reduced by increased portal insulin levels , with the resultant systemic effects on insulin resistance mentioned above . a number of other studies of patients on peritoneal dialysis ( i.e. receiving insulin by the portal vein route ) support these findings . in all cases , despite a more physiological method of delivering insulin , the lipid profile switched to an apparently more atherogenic pattern , with significant reductions in hdl - c and increases in ldl - c : hdl - c ratios [ 50 , 5355 ] . the pathophysiological significance of this is uncertain given mendelian randomisation studies showing that differences in hdl - c levels do not necessarily result in harm or benefit . whatever the net effect of portal insulinisation , it is of interest that new insulins have been specifically developed to have a relatively more hepatic than systemic mode of action ; thus , we will shortly have new tools to help answer the specific questions raised by this review . we hypothesise that these insulins will help avoid excess weight gain , peripheral insulin resistance and cardiac fat accumulation , but may lead to greater liver fat . the net effects of these insulins on cv outcomes will therefore be of major interest . a summary of our current knowledge of double diabetes and areas for future research is presented in the text box . while the risks of cvd in type 1 diabetes may be falling , the relative risk of cvd , chd , stroke and all - cause mortality continues to be unacceptably high for this patient population . our current understanding of double diabetes is insufficient to produce a rigid definition , but the literature we have summarised suggests that it has emerged as a relevant clinical concept in which there is : ( 1 ) marked weight gain over time ; ( 2 ) a high daily insulin requirement ; ( 3 ) a positive family history of type 2 diabetes , particularly when two or more relatives are affected ; and/ or ( 4 ) a low egdr . affected individuals may have high normal bp ( or hypertension ) and relatively low hdl - c . whether these effects associated with greater weight gain in at - risk individuals with double diabetes attenuate or reverse the vascular benefits of glycaemia reduction over time , remains to be fully established . in the meantime , avoidance of greater weight gain ( while retaining glycaemic benefits ) would have quality of life benefits for patients.summary of what is known and what remains unknown ( and thus requires further research ) in double diabetesknownunknown : areas for future researchindividuals with t1 dm and positive fht2 have increased risks of albuminuria and chd [ 3 , 4].is the effect of fht2 on complications in t1 dm mediated by genetic or familial factors ( or both)?peripheral insulin resistance is a consistent feature of t1 dm ( across the bmi range ) [ 616].is peripheral insulin resistance in t1 dm related to increased ectopic fat stores ( e.g. skeletal muscle ) as a result of abnormal fat partitioning owing to non - physiological subcutaneous insulin administration?clamp and derived measures of insulin resistance in t1 dm predict coronary disease [ 16 , 17 , 19].there are no prospective studies of baseline clamp insulin resistance in a t1 dm cohort with follow - up for incident cv events.defining metabolic syndrome in t1 dm does not add value for cv risk prediction [ 19 , 21 , 22].newer risk models in contemporary populations of t1 dm patients are urgently needed.dcct participants in the intensive insulin therapy group in the highest quartile for weight gain developed a higher bp and a more atherogenic lipid profile .numbers of cv events in the dcct / edic study are too small to demonstrate any excess risk in those patients who gained the most weight . it remains possible ( and clinically relevant ) that a beneficial cv effect of intensive glycaemic control might , in some patients , be outweighed by the adverse effects of weight gain.hepatic fat stores are reduced while intramuscular and epicardial fat stores are increased in lean patients with t1 dm compared with matched non - diabetic controls [ 9 , 39].what is the effect of weight gain on hepatic fat stores ( and ectopic fat stores ) in t1 dm ? any increase in perivascular or epicardial fat could have implications for cv risk.in t1 dm following pancreas transplant , portal vein drainage results in a more atherogenic lipid profile than systemic vein drainage . similar patterns are found in patients on peritoneal dialysis ( portal insulin delivery ) [ 5355].do newer insulins that target liver rather than systemic tissues lead to more liver fat but less weight gain overall and less cardiac and intramyocellular fat accumulation ? how do newer vs current insulins affect the function of hdl particles?fht2 , family history of type 2 diabetes ; t1 dm , type 1 diabetes when a patient with type 1 diabetes on an intensive insulin regimen is clearly gaining a significant amount of weight , early consideration should be given to adjusting that regimen in the context of diet and lifestyle in an effort to limit weight gain . it may also be useful to ascertain a more complete family history of type 2 diabetes in such patients . we suggest that the roles of structured educational approaches , continuous subcutaneous insulin infusion and closed - loop systems are potentially useful future strategies that should be assessed for the prevention and treatment of double diabetes . lower thresholds ( bp and lipid lowering ) for primary prevention of cvd should also be explored , as should the role of metformin as an insulin - sparing agent ( now being evaluated in the reducing with metformin vascular adverse lesions in type 1 diabetes [ removal ] trial ) . furthermore , it needs to be determined , whether newer insulins ( currently in early trials ) that target the liver more than the systemic tissues can lessen weight gain and , as a result , improve outcome in such patients . from a basic science perspective , further elucidation of the complex interplay of glucose and lipid factors driving cv risk in type 1 diabetes could help to better identify at - risk patients and improve primary prevention strategies .

in this review , we explore the concept of double diabetes , a combination of type 1 diabetes with features of insulin resistance and type 2 diabetes . after considering whether double diabetes is a useful concept , we discuss potential mechanisms of increased insulin resistance in type 1 diabetes before examining the extent to which double diabetes might increase the risk of cardiovascular disease ( cvd ) . we then go on to consider the proposal that weight gain from intensive insulin regimens may be associated with increased cv risk factors in some patients with type 1 diabetes , and explore the complex relationships between weight gain , insulin resistance , glycaemic control and cv outcome . important comparisons and contrasts between type 1 diabetes and type 2 diabetes are highlighted in terms of hepatic fat , fat partitioning and lipid profile , and how these may differ between type 1 diabetic patients with and without double diabetes . in so doing , we hope this work will stimulate much - needed research in this area and an improvement in clinical practice .

Introduction Double diabetes Measuring insulin resistance in type 1 diabetes and examining its association with complications Prediction of CVD using eGDR Other derived measures related to insulin resistance: does the metabolic syndrome identify CVD risk in type 1 diabetes? Intensive insulin therapy, weight/weight gain and their relationship to insulin resistance and CV risk in type 1 diabetes Lipid metabolism in type 1 diabetes Summary and conclusions

in this review , we address the following questions : ( 1 ) what are the potential mechanisms linking type 1 diabetes with insulin resistance and how is this combination ( double diabetes ) associated with increased cardiovascular risk ? ( 2 ) might weight gain associated with intensive insulin regimens be associated with increased insulin resistance and what implications might this have for cardiovascular risk ? ( 4 ) might we need to consider changes in current clinical practice in the context of the issues raised in this review ? the term double diabetes was first coined in 1991 based on the observation that patients with type 1 diabetes who had a family history of type 2 diabetes were more likely to be overweight and rarely achieved adequate glycaemic control even with higher insulin doses . the authors suggested that this might indicate the presence of increased resistance to insulin - mediated glucose disposal in this subgroup of people with type 1 diabetes and asserted that , over a lifetime , some of these individuals would likely have been diagnosed with type 2 diabetes at some point , had they not first developed beta cell destruction by an independent pathological process ( i.e. type 1 diabetes ) . at this stage , it is important to differentiate this description of double diabetes , which considers autoimmune diabetes to be an independent process from obesity and insulin resistance , from the accelerator hypothesis , which describes triggering of autoimmune diabetes by factors including bmi and insulin resistance . other studies of people with type 1 diabetes and a family history of type 2 diabetes have supported the notion that this combination might promote both microvascular and macrovascular complications of type 1 diabetes . for example , in a prospective study of 3250 patients with type 1 diabetes recruited from 16 european countries ( eurodiab ) , it was demonstrated that women with a parental history of type 2 diabetes had a higher risk of developing albuminuria than those without a positive family history ( hr 1.36 , p = 0.04 ) . furthermore , in a cross - sectional study of 658 patients from the pittsburgh epidemiology of diabetes complications ( edc ) cohort , 112 of whom had a confirmed family history of type 2 diabetes , and 119 of whom had experienced a chd event , a positive family history was associated with a significant excess risk of chd ( hr 1.89 , 95% ci 1.27 , 2.84 ) . these data are consistent with either genetically determined insulin resistance / double diabetes or shared parental offspring lifestyle factors contributing to type 2 diabetes in parents and increasing the risk of complications . given the results of genome - wide association studies of type 2 diabetes published to date , the latter explanation currently appears more plausible . if insulin resistance , either inherited or acquired , is implicated in the pathogenesis of double diabetes , how is it likely to manifest and can it be identified ? although this is a time - consuming and relatively invasive technique , several investigators have employed it to demonstrate increased insulin resistance in those with type 1 diabetes compared with age- , sex- and bmi - matched controls [ 616 ] . in one such study of 40 cases ( mean age 45 years , mean diabetes duration of type 1 diabetes 23 years ) and 47 age- and sex - matched control individuals , patients with type 1 diabetes not only had lower whole body insulin - mediated glucose uptake ( mean se : 6.19 0.72 vs 12.71 0.66 mg [ kg fat - free mass ] min , p < 0.0001 ) but also had lower insulin - mediated nefa suppression . both measurements were correlated ( p < 0.0001 within the type 1 diabetes group ) with coronary artery calcification ( cac ) measured by computed tomography . low egdr was proposed as a surrogate measure of insulin resistance for use either in a clinical setting or for epidemiological analyses . given the mechanistic relevance of insulin resistance in the metabolic syndrome , a number of prospective studies have assessed the utility of various definitions of the metabolic syndrome in predicting risk in type 1 diabetes cohorts . for example , an individual with type 1 diabetes can meet the who definition by virtue of microalbuminuria alone , even if non - obese and with a normal lipid profile . thus , in broad terms , the metabolic syndrome and its individual features are associated with more events but , as in the general population , dichotomous classification of individuals with type 1 diabetes according to the metabolic syndrome does not appear to add value for cv risk prediction in type 1 diabetes over and above its individual components . it is generally accepted that intensive insulin therapy reduces the incidence of cvd in type 1 diabetes . the most robust interventional clinical trial evidence for this is from the dcct in which 1,441 adolescents and young adults ( mean age 27 years , bmi 28 kg / m ) were randomised for 6.5 years to intensive or conventional glycaemic control and followed up post - randomisation in the edic study up to a total of 17 years . intensive glycaemic control was associated with a 57% reduction in rr ( 95% ci 12 , 79% , p = 0.02 ) in a composite endpoint of non - fatal mi , stroke or cv death ( approximate overall rate of six events per 1,000 patient - years ) . in this context , subgroup analyses of the dcct / edic study have raised concerns about participants in whom weight gain over the course of the trial was associated with the emergence of features associated with increased cv risk ( table 1 ) . those in the highest quartile for weight gain exhibited higher bp and ldl - cholesterol ( ldl - c ) values , a higher insulin dose requirement and whr and a more atherogenic lipid profile ( raised apolipoprotein b , high vldl , higher small , dense ldl , lower apolipoprotein a - i and lower hdl ) . a similar pattern was observed among those allocated to conventional treatment ( albeit with a reduced effect size ) , which is consistent with the existence of individual predisposing factors to insulin - induced weight gain , an area that merits further study.table 1characteristics of the intensively treated group at follow - upquartiles of weight gain1234hba1c ( % ) [ mmol / mol]7.3 7.2 7.1 7.3 insulin dose ( units kg day)0.610.640.690.74achieved bmi ( kg / m)24252731systolic bp ( mmhg)113117115120triacylglycerol ( mmol / l)0.790.820.910.99ldl - cholesterol ( mmol / l)2.742.792.923.15hdl - cholesterol ( mmol / l)1.401.341.291.27baseline bmi was 24 kg / m for all quartilesadapted from with permission . all rights reserved interestingly , in the dcct / edic study , a family history of type 2 diabetes was also a significant independent predictor of weight gain in both the conventional and intensive groups , consistent with an inherited tendency to store excess fat in response to insulin , a degree of inherited peripheral insulin resistance and/or a familial effect on environmental factors ( energy intake and physical activity ) . while in the pre - dcct era baseline bmi in patients with type 1 diabetes was lower than population norms , probably owing to a combination of weight loss prior to diagnosis and suboptimal glycaemic control , current bmi patterns are similar . although much of the weight gain seen in contemporary patients with type 1 diabetes is likely to reflect cultural , societal and lifestyle changes , some of this increase may be due to use of more intensive insulin regimens . adverse cv risk factors associated with weight gain in some recipients of intensive insulin therapy were also seen in the observational data from the eurodiab cohort , in which 1,800 type 1 diabetic patients ( mean age 33 years , duration of diabetes 14.8 years , hba1c 8.2% [ 66 mmol / mol ] ) were followed up for 7.3 years . those who gained more than 5 kg over this time had better glycaemic control ( mean hba1c difference of 0.2% ) than those with less or no weight gain but also had raised bp , ldl - c and triacylglycerol in association with lower hdl - c ( differences that remained significant after adjustment for glycaemic control ) . insulin resistance in type 1 insulin absorbed from subcutaneous depots results in relative peripheral hyperinsulinaemia and hepatic hypoinsulinaemia compared with normal physiology . first , the genetic and lifestyle factors that lead to type 2 diabetes may exist at similar frequency in those with type 1 diabetes this would be consistent with the robust data on family history of type 2 diabetes described above . third , exogenous insulin therapy might induce insulin resistance in patients with type 1 diabetes.fig . t1 dm , type 1 diabetes mechanisms potentially contributing to insulin resistance in type 1 diabetes patients with double diabetes . t1 dm , type 1 diabetes there are several important unanswered questions : are some people with type 1 diabetes ( including those with a family history of type 2 diabetes or of specific ethnicities , e.g. south asians ) more susceptible to weight gain during intensive insulin treatment , perhaps owing to differences in metabolism or substrate handling or can the observations regarding weight gain simply be explained by the fact that those who are more insulin resistant are treated with higher doses of insulin?what are the most important factors , e.g. inherited or familial factors , differences in feeding strategies for avoidance of hypoglycaemia , insulin resistance?to what extent do adverse cvd risk factors associated with higher weight gain attenuate or reverse the presumed earlier cv ( and other ) benefits of intensive glycaemic control in type 1 diabetes ? are some people with type 1 diabetes ( including those with a family history of type 2 diabetes or of specific ethnicities , e.g. south asians ) more susceptible to weight gain during intensive insulin treatment , perhaps owing to differences in metabolism or substrate handling or can the observations regarding weight gain simply be explained by the fact that those who are more insulin resistant are treated with higher doses of insulin ? inherited or familial factors , differences in feeding strategies for avoidance of hypoglycaemia , insulin resistance ? to what extent do adverse cvd risk factors associated with higher weight gain attenuate or reverse the presumed earlier cv ( and other ) benefits of intensive glycaemic control in type 1 diabetes ? in this section , we compare lipid handling in type 1 diabetes with non - diabetic metabolism and with type 2 diabetes . the most obvious and striking difference between type 1 diabetes on the one hand and obesity / type 2 diabetes on the other , is low portal vein insulin concentration . this can be concluded by inference from the understanding of type 1 diabetes pathophysiology and anatomy ( i.e. in type 1 diabetes , absent pancreatic insulin secretion is the opposite phenotype to the endogenous hyperinsulinaemia characteristic of most conditions characterised by insulin resistance . as type 1 diabetes is characterised by higher peripheral insulin concentrations and lower portal concentrations , it follows that contrasting hepatic and peripheral lipid handling might be predicted . in contrast to obesity and type 2 diabetes , in which the importance of non - alcoholic steatohepatitis ( fatty liver ) has been highlighted , there is some evidence that intra - hepatic fat content is reduced in type 1 diabetes . comparing 19 patients with type 1 diabetes ( mean age 35 , bmi 23 kg / m , hba1c 8.7% [ 72 mmol / mol ] ) and carefully matched non - diabetic controls using h magnetic resonance spectroscopy , perseghin et al demonstrated significantly reduced intra - hepatic fat content in those with type 1 diabetes ( 1.5 0.7% vs 2.2 1.0% , p < 0.03 ) . this was associated with increased fasting lipid oxidation ( 1.5 0.7 vs 0.8 0.4 mg / kg ) . it has been suggested that relative under - insulinisation of the liver with preserved glucagon secretion in type 1 diabetes favours a shift in metabolism from lipid storage to oxidation . in type 1 diabetes , this is likely to be influenced by the absence of endogenous insulin combined with therapeutic administration of subcutaneous insulin into the peripheral ( as opposed to portal ) circulation . the subsequent pattern of increased ( ectopic ) intramyocellular fat storage is similar to that found in obesity / type 2 diabetes and is associated with impaired sensitivity of muscle to glucose uptake , i.e. therefore , in type 1 diabetes , this may represent an indirect mechanism of insulin resistance . for example , epicardial and perivascular fat may play a role in modulating coronary function and pathophysiology , providing a potential mechanism to link double diabetes with cvd . in this regard , data have recently been published on epicardial fat thickness ( measured by echocardiography ) in 36 type 1 diabetic patients and 43 matched controls . not only was epicardial fat thicker in those with type 1 diabetes ( p < 0.0001 ) , but it also correlated significantly with both whr ( r = 0.67 , p = 0.003 ) and egdr ( r = 0.55 , p = 0.0004 ) within the patient group . hdl - c levels are normal or high in type 1 diabetes unless renal impairment develops , in which case hdl - c falls [ 7 , 4043 ] . moreover , long - term survivors of type 1 diabetes are characterised by unusually high hdl - c levels ( 1.84 mmol / l ) , and patients with chd events have been shown to have lower hdl - c levels ( along with higher triacylglycerol and ldl - c levels ) . exogenous insulin treatment usually drives higher mean hdl - c levels in type 1 diabetes , but even those individuals with poorer glycaemic control have higher levels than healthy controls . peripheral hyperinsulinaemia associated with strict glycaemic control is associated with increased lpl activity and hdl - c , while poor glycaemic control results in lower lpl activity and hdl - c [ 46 , 47 ] . low levels of portal vein insulin in type 1 diabetes are associated with reduced hl activity and increased hdl - c [ 4850 ] . in contrast , in type 2 diabetes , raised hl activity secondary to portal hyperinsulinaemia may contribute to low hdl - c levels . finally , phospholipid transfer protein activity is markedly elevated in patients with type 1 diabetes , and this activity is correlated with hdl - c levels . high hdl - c levels in patients with type 1 diabetes are likely to be atheroprotective , even though some compositional abnormalities may reduce the protective effect . however , if central obesity and insulin resistance then develop ( double diabetes ) , a more atherogenic lipid profile seems to emerge . as discussed above , the liver is under - insulinised in type 1 diabetes . this raises the hypothesis that type 1 diabetes may actually confer simultaneous protective and harmful cv effects . this is a difficult phenomenon to study , but interesting insights have been gained from the field of pancreas transplantation in type 1 diabetes . whatever the net effect of portal insulinisation , it is of interest that new insulins have been specifically developed to have a relatively more hepatic than systemic mode of action ; thus , we will shortly have new tools to help answer the specific questions raised by this review . we hypothesise that these insulins will help avoid excess weight gain , peripheral insulin resistance and cardiac fat accumulation , but may lead to greater liver fat . a summary of our current knowledge of double diabetes and areas for future research is presented in the text box . while the risks of cvd in type 1 diabetes may be falling , the relative risk of cvd , chd , stroke and all - cause mortality continues to be unacceptably high for this patient population . our current understanding of double diabetes is insufficient to produce a rigid definition , but the literature we have summarised suggests that it has emerged as a relevant clinical concept in which there is : ( 1 ) marked weight gain over time ; ( 2 ) a high daily insulin requirement ; ( 3 ) a positive family history of type 2 diabetes , particularly when two or more relatives are affected ; and/ or ( 4 ) a low egdr . whether these effects associated with greater weight gain in at - risk individuals with double diabetes attenuate or reverse the vascular benefits of glycaemia reduction over time , remains to be fully established . in the meantime , avoidance of greater weight gain ( while retaining glycaemic benefits ) would have quality of life benefits for patients.summary of what is known and what remains unknown ( and thus requires further research ) in double diabetesknownunknown : areas for future researchindividuals with t1 dm and positive fht2 have increased risks of albuminuria and chd [ 3 , 4].is the effect of fht2 on complications in t1 dm mediated by genetic or familial factors ( or both)?peripheral insulin resistance is a consistent feature of t1 dm ( across the bmi range ) [ 616].is peripheral insulin resistance in t1 dm related to increased ectopic fat stores ( e.g. skeletal muscle ) as a result of abnormal fat partitioning owing to non - physiological subcutaneous insulin administration?clamp and derived measures of insulin resistance in t1 dm predict coronary disease [ 16 , 17 , 19].there are no prospective studies of baseline clamp insulin resistance in a t1 dm cohort with follow - up for incident cv events.defining metabolic syndrome in t1 dm does not add value for cv risk prediction [ 19 , 21 , 22].newer risk models in contemporary populations of t1 dm patients are urgently needed.dcct participants in the intensive insulin therapy group in the highest quartile for weight gain developed a higher bp and a more atherogenic lipid profile .numbers of cv events in the dcct / edic study are too small to demonstrate any excess risk in those patients who gained the most weight . it remains possible ( and clinically relevant ) that a beneficial cv effect of intensive glycaemic control might , in some patients , be outweighed by the adverse effects of weight gain.hepatic fat stores are reduced while intramuscular and epicardial fat stores are increased in lean patients with t1 dm compared with matched non - diabetic controls [ 9 , 39].what is the effect of weight gain on hepatic fat stores ( and ectopic fat stores ) in t1 dm ? any increase in perivascular or epicardial fat could have implications for cv risk.in t1 dm following pancreas transplant , portal vein drainage results in a more atherogenic lipid profile than systemic vein drainage . how do newer vs current insulins affect the function of hdl particles?fht2 , family history of type 2 diabetes ; t1 dm , type 1 diabetes when a patient with type 1 diabetes on an intensive insulin regimen is clearly gaining a significant amount of weight , early consideration should be given to adjusting that regimen in the context of diet and lifestyle in an effort to limit weight gain . it may also be useful to ascertain a more complete family history of type 2 diabetes in such patients . we suggest that the roles of structured educational approaches , continuous subcutaneous insulin infusion and closed - loop systems are potentially useful future strategies that should be assessed for the prevention and treatment of double diabetes . lower thresholds ( bp and lipid lowering ) for primary prevention of cvd should also be explored , as should the role of metformin as an insulin - sparing agent ( now being evaluated in the reducing with metformin vascular adverse lesions in type 1 diabetes [ removal ] trial ) . from a basic science perspective , further elucidation of the complex interplay of glucose and lipid factors driving cv risk in type 1 diabetes could help to better identify at - risk patients and improve primary prevention strategies .

in this review , we address the following questions : ( 1 ) what are the potential mechanisms linking type 1 diabetes with insulin resistance and how is this combination ( double diabetes ) associated with increased cardiovascular risk ? the term double diabetes was first coined in 1991 based on the observation that patients with type 1 diabetes who had a family history of type 2 diabetes were more likely to be overweight and rarely achieved adequate glycaemic control even with higher insulin doses . the authors suggested that this might indicate the presence of increased resistance to insulin - mediated glucose disposal in this subgroup of people with type 1 diabetes and asserted that , over a lifetime , some of these individuals would likely have been diagnosed with type 2 diabetes at some point , had they not first developed beta cell destruction by an independent pathological process ( i.e. at this stage , it is important to differentiate this description of double diabetes , which considers autoimmune diabetes to be an independent process from obesity and insulin resistance , from the accelerator hypothesis , which describes triggering of autoimmune diabetes by factors including bmi and insulin resistance . other studies of people with type 1 diabetes and a family history of type 2 diabetes have supported the notion that this combination might promote both microvascular and macrovascular complications of type 1 diabetes . for example , in a prospective study of 3250 patients with type 1 diabetes recruited from 16 european countries ( eurodiab ) , it was demonstrated that women with a parental history of type 2 diabetes had a higher risk of developing albuminuria than those without a positive family history ( hr 1.36 , p = 0.04 ) . furthermore , in a cross - sectional study of 658 patients from the pittsburgh epidemiology of diabetes complications ( edc ) cohort , 112 of whom had a confirmed family history of type 2 diabetes , and 119 of whom had experienced a chd event , a positive family history was associated with a significant excess risk of chd ( hr 1.89 , 95% ci 1.27 , 2.84 ) . those with a greater number of affected family members had a greater risk ( p = 0.001 for trend ) , with one family member conferring an or of 1.62 and two family members increasing this to 5.13 . although this is a time - consuming and relatively invasive technique , several investigators have employed it to demonstrate increased insulin resistance in those with type 1 diabetes compared with age- , sex- and bmi - matched controls [ 616 ] . in one such study of 40 cases ( mean age 45 years , mean diabetes duration of type 1 diabetes 23 years ) and 47 age- and sex - matched control individuals , patients with type 1 diabetes not only had lower whole body insulin - mediated glucose uptake ( mean se : 6.19 0.72 vs 12.71 0.66 mg [ kg fat - free mass ] min , p < 0.0001 ) but also had lower insulin - mediated nefa suppression . data from one clamp study , ( n = 24 ) were used to derive estimated glucose disposal rate ( egdr ) from a best - fit model:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mathrm{egdr}=24.31 - 12.22\left ( { \mathrm{whr } } \right)-3.29\left ( { \mathrm{hypertension}\ \mathrm{status } } \right)-0.57\left ( { \mathrm{hb}{{\mathrm{a}}_{1\mathrm{c } } } } \right ) $ $ \end{document}where hypertension status ( 1 or 0 ) is defined as bp > 140/90 mmhg or on anti - hypertensive drugs ; hba1c refers to the value in % . independent predictors of cvd events ( n = 42 ) were disease duration , presence of nephropathy , non - hdl - cholesterol ( hdl - c ) , white cell count and egdr . however , in a subsequent analysis of the same cohort by the same group , egdr did not appear in chd risk prediction models ( although , importantly , these incorporated three of its individual components : whr , hdl - c and bp ) . in an independent examination of the dcct / epidemiology of diabetes interventions and complications ( edic ) data , kilpatrick et al estimated egdr retrospectively ( using bmi data instead of whr ) as a predictor of cvd events . in a cox regression model , high egdr as a surrogate for preserved insulin sensitivity was independently associated with a lower risk for cv events ( hr 0.70 ; 95% ci 0.56 , 0.88 ) . given the mechanistic relevance of insulin resistance in the metabolic syndrome , a number of prospective studies have assessed the utility of various definitions of the metabolic syndrome in predicting risk in type 1 diabetes cohorts . in a prospective analysis of the pittsburgh edc cohort , the prevalence of the metabolic syndrome was 21% ( who definition ) , 12% ( ncep ) and 8% ( idf ) . using a composite endpoint of chd , renal failure and diabetes - related death , the metabolic syndrome conferred significantly elevated hrs ( ncep : 5.8 , 95% ci 3.9 , 8.6 ; who : 6.5 , 95% ci 4.5 , 9.4 ) ; however , these ratios were not higher than those for individual components of the syndrome ( for example , microalbuminuria : 6.3 , 95% ci 3.8 , 10.5 ; bp 4.5 , 95% ci 3.1 , 6.6 ; triacylglycerol : 4.4 , 95% ci 3.0 , 6.6 ) . in a similar analysis of the finndiane study , a high prevalence of the metabolic syndrome was reported ( 44% who definition ; 35% ncep ; 36% idf ) . the who definition predicted incident cv events after adjustment for traditional risk factors and nephropathy ( hr 2.05 , 95% ci 1.38 , 3.04 ) , even in participants with raised albumin excretion ( hr 1.44 , 95% ci 1.06 , 1.96 ) . in the dcct / edic study , an adapted idf definition of the metabolic syndrome had a baseline prevalence of 22% , largely driven by low hdl - c , but did not predict either macro- or microvascular events over 17 years of follow - up . thus , in broad terms , the metabolic syndrome and its individual features are associated with more events but , as in the general population , dichotomous classification of individuals with type 1 diabetes according to the metabolic syndrome does not appear to add value for cv risk prediction in type 1 diabetes over and above its individual components . the most robust interventional clinical trial evidence for this is from the dcct in which 1,441 adolescents and young adults ( mean age 27 years , bmi 28 kg / m ) were randomised for 6.5 years to intensive or conventional glycaemic control and followed up post - randomisation in the edic study up to a total of 17 years . intensive glycaemic control was associated with a 57% reduction in rr ( 95% ci 12 , 79% , p = 0.02 ) in a composite endpoint of non - fatal mi , stroke or cv death ( approximate overall rate of six events per 1,000 patient - years ) . of course , one of the consequences of intensive therapy is weight gain ; the intensively treated group in the dcct study gained 4.6 kg more than the conventionally treated group over the 5 year study period . in this context , subgroup analyses of the dcct / edic study have raised concerns about participants in whom weight gain over the course of the trial was associated with the emergence of features associated with increased cv risk ( table 1 ) . those in the highest quartile for weight gain exhibited higher bp and ldl - cholesterol ( ldl - c ) values , a higher insulin dose requirement and whr and a more atherogenic lipid profile ( raised apolipoprotein b , high vldl , higher small , dense ldl , lower apolipoprotein a - i and lower hdl ) . a similar pattern was observed among those allocated to conventional treatment ( albeit with a reduced effect size ) , which is consistent with the existence of individual predisposing factors to insulin - induced weight gain , an area that merits further study.table 1characteristics of the intensively treated group at follow - upquartiles of weight gain1234hba1c ( % ) [ mmol / mol]7.3 7.2 7.1 7.3 insulin dose ( units kg day)0.610.640.690.74achieved bmi ( kg / m)24252731systolic bp ( mmhg)113117115120triacylglycerol ( mmol / l)0.790.820.910.99ldl - cholesterol ( mmol / l)2.742.792.923.15hdl - cholesterol ( mmol / l)1.401.341.291.27baseline bmi was 24 kg / m for all quartilesadapted from with permission . all rights reserved interestingly , in the dcct / edic study , a family history of type 2 diabetes was also a significant independent predictor of weight gain in both the conventional and intensive groups , consistent with an inherited tendency to store excess fat in response to insulin , a degree of inherited peripheral insulin resistance and/or a familial effect on environmental factors ( energy intake and physical activity ) . while in the pre - dcct era baseline bmi in patients with type 1 diabetes was lower than population norms , probably owing to a combination of weight loss prior to diagnosis and suboptimal glycaemic control , current bmi patterns are similar . although much of the weight gain seen in contemporary patients with type 1 diabetes is likely to reflect cultural , societal and lifestyle changes , some of this increase may be due to use of more intensive insulin regimens . adverse cv risk factors associated with weight gain in some recipients of intensive insulin therapy were also seen in the observational data from the eurodiab cohort , in which 1,800 type 1 diabetic patients ( mean age 33 years , duration of diabetes 14.8 years , hba1c 8.2% [ 66 mmol / mol ] ) were followed up for 7.3 years . those who gained more than 5 kg over this time had better glycaemic control ( mean hba1c difference of 0.2% ) than those with less or no weight gain but also had raised bp , ldl - c and triacylglycerol in association with lower hdl - c ( differences that remained significant after adjustment for glycaemic control ) . in addition , it has been proposed that reduced hepatic insulin exposure results in a reduced level of circulating igf-1 , which , along with a parallel increase in growth hormone and igf - binding proteins , may also contribute to increased peripheral insulin resistance [ 30 , 31 ] . first , the genetic and lifestyle factors that lead to type 2 diabetes may exist at similar frequency in those with type 1 diabetes this would be consistent with the robust data on family history of type 2 diabetes described above . t1 dm , type 1 diabetes there are several important unanswered questions : are some people with type 1 diabetes ( including those with a family history of type 2 diabetes or of specific ethnicities , e.g. south asians ) more susceptible to weight gain during intensive insulin treatment , perhaps owing to differences in metabolism or substrate handling or can the observations regarding weight gain simply be explained by the fact that those who are more insulin resistant are treated with higher doses of insulin?what are the most important factors , e.g. inherited or familial factors , differences in feeding strategies for avoidance of hypoglycaemia , insulin resistance?to what extent do adverse cvd risk factors associated with higher weight gain attenuate or reverse the presumed earlier cv ( and other ) benefits of intensive glycaemic control in type 1 diabetes ? insulin not only exerts effects on peripheral glucose uptake and suppression of hepatic glucose production , it also has profound effects on fat partitioning by promoting hepatic and peripheral lipogenesis ( fat storage ) as well as suppressing hepatic and peripheral lipolysis ( inhibition of fat oxidation ) . in contrast to obesity and type 2 diabetes , in which the importance of non - alcoholic steatohepatitis ( fatty liver ) has been highlighted , there is some evidence that intra - hepatic fat content is reduced in type 1 diabetes . comparing 19 patients with type 1 diabetes ( mean age 35 , bmi 23 kg / m , hba1c 8.7% [ 72 mmol / mol ] ) and carefully matched non - diabetic controls using h magnetic resonance spectroscopy , perseghin et al demonstrated significantly reduced intra - hepatic fat content in those with type 1 diabetes ( 1.5 0.7% vs 2.2 1.0% , p < 0.03 ) . the estimated hepatic insulin ( ehi ) level was lower and the glucagon : ehi ratio ( reported as an indicator of the balance between catabolism and anabolism ) was higher in the type 1 diabetes group ( both p < 0.05 ) . in type 1 diabetes , this is likely to be influenced by the absence of endogenous insulin combined with therapeutic administration of subcutaneous insulin into the peripheral ( as opposed to portal ) circulation . this results in reduced inhibition of hepatic lipolysis with consequent increased levels of circulating nefas , which , in combination with peripheral hyperinsulinaemia , may promote relatively greater lipid storage in skeletal muscle [ 79 ] . not only was epicardial fat thicker in those with type 1 diabetes ( p < 0.0001 ) , but it also correlated significantly with both whr ( r = 0.67 , p = 0.003 ) and egdr ( r = 0.55 , p = 0.0004 ) within the patient group . moreover , long - term survivors of type 1 diabetes are characterised by unusually high hdl - c levels ( 1.84 mmol / l ) , and patients with chd events have been shown to have lower hdl - c levels ( along with higher triacylglycerol and ldl - c levels ) . peripheral hyperinsulinaemia associated with strict glycaemic control is associated with increased lpl activity and hdl - c , while poor glycaemic control results in lower lpl activity and hdl - c [ 46 , 47 ] . in all cases , despite a more physiological method of delivering insulin , the lipid profile switched to an apparently more atherogenic pattern , with significant reductions in hdl - c and increases in ldl - c : hdl - c ratios [ 50 , 5355 ] . whatever the net effect of portal insulinisation , it is of interest that new insulins have been specifically developed to have a relatively more hepatic than systemic mode of action ; thus , we will shortly have new tools to help answer the specific questions raised by this review . while the risks of cvd in type 1 diabetes may be falling , the relative risk of cvd , chd , stroke and all - cause mortality continues to be unacceptably high for this patient population . our current understanding of double diabetes is insufficient to produce a rigid definition , but the literature we have summarised suggests that it has emerged as a relevant clinical concept in which there is : ( 1 ) marked weight gain over time ; ( 2 ) a high daily insulin requirement ; ( 3 ) a positive family history of type 2 diabetes , particularly when two or more relatives are affected ; and/ or ( 4 ) a low egdr . in the meantime , avoidance of greater weight gain ( while retaining glycaemic benefits ) would have quality of life benefits for patients.summary of what is known and what remains unknown ( and thus requires further research ) in double diabetesknownunknown : areas for future researchindividuals with t1 dm and positive fht2 have increased risks of albuminuria and chd [ 3 , 4].is the effect of fht2 on complications in t1 dm mediated by genetic or familial factors ( or both)?peripheral insulin resistance is a consistent feature of t1 dm ( across the bmi range ) [ 616].is peripheral insulin resistance in t1 dm related to increased ectopic fat stores ( e.g. skeletal muscle ) as a result of abnormal fat partitioning owing to non - physiological subcutaneous insulin administration?clamp and derived measures of insulin resistance in t1 dm predict coronary disease [ 16 , 17 , 19].there are no prospective studies of baseline clamp insulin resistance in a t1 dm cohort with follow - up for incident cv events.defining metabolic syndrome in t1 dm does not add value for cv risk prediction [ 19 , 21 , 22].newer risk models in contemporary populations of t1 dm patients are urgently needed.dcct participants in the intensive insulin therapy group in the highest quartile for weight gain developed a higher bp and a more atherogenic lipid profile .numbers of cv events in the dcct / edic study are too small to demonstrate any excess risk in those patients who gained the most weight . it remains possible ( and clinically relevant ) that a beneficial cv effect of intensive glycaemic control might , in some patients , be outweighed by the adverse effects of weight gain.hepatic fat stores are reduced while intramuscular and epicardial fat stores are increased in lean patients with t1 dm compared with matched non - diabetic controls [ 9 , 39].what is the effect of weight gain on hepatic fat stores ( and ectopic fat stores ) in t1 dm ? how do newer vs current insulins affect the function of hdl particles?fht2 , family history of type 2 diabetes ; t1 dm , type 1 diabetes when a patient with type 1 diabetes on an intensive insulin regimen is clearly gaining a significant amount of weight , early consideration should be given to adjusting that regimen in the context of diet and lifestyle in an effort to limit weight gain . we suggest that the roles of structured educational approaches , continuous subcutaneous insulin infusion and closed - loop systems are potentially useful future strategies that should be assessed for the prevention and treatment of double diabetes . lower thresholds ( bp and lipid lowering ) for primary prevention of cvd should also be explored , as should the role of metformin as an insulin - sparing agent ( now being evaluated in the reducing with metformin vascular adverse lesions in type 1 diabetes [ removal ] trial ) . from a basic science perspective , further elucidation of the complex interplay of glucose and lipid factors driving cv risk in type 1 diabetes could help to better identify at - risk patients and improve primary prevention strategies .

elucidation of metabolic regulation in plants has been an academic pursuit spanning many decades . indeed the question of how metabolism is regulated was first raised in the scientific era in which metabolism was first truly defined ( buchner , 1907 ; krebs and henseleit , 1932 ; plaxton , 1996 ; kornberg , 2000 ) . whilst the ability to regulate the rates of metabolic processes in response to cellular circumstance is a common feature of all organisms , it is particularly acute in sessile organism such as plants ( plaxton , 1996 ) . from a textbook perspective , metabolic regulation is classically divided into coarse and fine levels of control ( dennis et al . , 1997 ; fell , 1997 ) . coarse control refers to long - term mechanisms that are energetically expensive and lead to changes in the total cellular population of a protein . by contrast , fine control describes generally fast ( and therefore energetically inexpensive ) regulatory devices that modulate the activity of pre - existing enzyme molecules . whilst this arbitrary division can be useful for descriptive purposes , recent reports suggest that several regulatory mechanisms can not be so easily defined and hence such classification is commonly regarded as outdated . however , unfortunately , despite the massive amount of data available regarding steady - state rna levels afforded by microarrays ( see for example the data stored in genevestigator ; http://www.genevestigator.ethz.ch ) and more recently by next generation sequence analyses ( see for example gonzalez - ballester et al . that said , important recent advances have been made both regarding protein synthesis ( mustroph et al . , 2009 ; piques et al . , 2009 ) and degradation ( arajo et al . , 2010 , 2011 ; our understanding of regulation of central ( primary ) plant metabolism has been largely defined by the discovery of such features within the last 5060 years , whereas understanding of specialized ( secondary ) metabolism has made similar strides within the last 30 years ( for reviews see pichersky and gang , 2000 ; dauria and gershenzon , 2005 ; gachon et al . , 2005 ; yonekura - sakakibara and saito , 2009 ) . in brief , such mechanisms include ( i ) alteration in substrate or co - substrate concentration , ( ii ) variation in ph , ( iii ) allosteric effectors . the first , of these is essentially the most simple and certainly the most rapid to affect metabolic systems with the rate of an enzyme - catalyzed reaction proceeding more rapidly upon an increase in sub - saturating substrate a case that is common in vivo all enzyme reactions are , to a greater or lesser extent regulated in this manner . however , the situation is complicated by the fact that not all reactions display simple michaelis menton - like kinetics and by the fact that many co - substrates are shared by multiple reactions . these factors alone render understanding the systemic response to prevailing fluctuations in substrate conditions unpredictable . for example , regulation of enzymes of the calvin cycle is well documented to be ph regulated ; stromal ph is 8.0 in that the light and 7.0 in the dark ( see dennis et al . , thirdly , allosteric effectors are immensely important in the regulation of plant metabolic networks , be they activators or inhibitors . within the major pathways of carbohydrate metabolism , several examples of the importance of such metabolites exist , including the 3 phosphoglycerate ( 3pga)/inorganic phosphate ( pi ) ratio in activating adp glucose pyrophosphorylase ( agpase ; preiss , 1982 ; tiessen et al . , 2002 ) , the fructose 2,6-bisphosphatase ( fru 2,6p2ase ) system ( stitt , 1990 ; fernie et al . , 2001 ) and the effect of pyruvate on the alternative oxidase of mitochondrial respiration ( millar et al . , 1993 understanding the function of a given enzyme within a biological process has until recently largely followed a set protocol by which novel genes associated with a specific process are identified by means of similar patterns of expression across a wide range of experiments and subsequently their function tested . this is initially carried out by analyzing the metabolite profiles of genotypes deficient in the expression of the gene . confirmation of kinetic properties of the enzyme either in planta ( in the case that the gene encodes the only isoform of an enzyme ) or following expression of the gene in a heterologous system lacking the activity is subsequently required ( see for details tohge and fernie , 2010 ) . whilst this approach has been tremendously successful ( hirai et al . , 2005 , 2007 ; tohge et al . , 2005 ; okazaki et al . , 2009 ) in terms of annotating the precise biochemical function of individual genes , it does not enable elucidation of the exact physiological function in vivo . in the last 25 years the roles of specific metabolic enzymes have been addressed via the use of transgenic plants ( stitt and sonnewald , 1995 ; lytovchenko et al . , 2007 ) . such studies have greatly advanced our understanding of metabolic regulation . however , such directed approaches sometimes fail to uncover complex inter - pathway interactions and pathway regulators . given that the chemical constituents of any life form determine the development and functioning of the organism ( sumner et al . , 2003 ; fernie et al . , 2004a ; dauria and gershenzon , 2005 ) , strategies to characterize the chemical complement of the cell are becoming increasingly important . the diversity of metabolites is controlled by a complex interaction involving many structural and regulatory genes as well as environmental influences ( harrigan et al . , 2007a ) . although chemical profiles differ between and even within species , thousands of diverse metabolites are usually found in a single plant ( de luca and st pierre , 2000 ; sumner et al . these range from small and simple structures such as vitamins and amino acids to more complex compounds such as polycyclic antioxidants and protease inhibitors . other compounds function as energy carriers that can store or release energy upon formation or degradation respectively ( fernie et al . , 2004b ) . for example , glucose synthesized during gluconeogenesis can be polymerized to form starch or be broken down during glycolysis . this regulated interconversion of compounds is perhaps the most important hallmark of plant metabolism , enabling the organism to respond to specific demands over the course of its life and on a minute - by - minute basis ( keuentjes and fernie , 2011 ) . metabolites are often classified as being either primary or secondary , although no strict discrimination can be made and interactions between the two classes are manifold . primary metabolism includes essential metabolites such as those in central carbohydrate metabolism ( koch , 1996 ; rontein et al . , 2002 ) whereas secondary metabolism is often connected to interactions with environmental cues , including cell signaling , interspecies communication , and responses to biotic and abiotic stress ( see for example wink , 1988 ; mitchell - olds and pedersen , 1998 ; lehmann et al . , 2009 ; rubin et al . , 2009 ) . although primary metabolic pathways are strongly conserved between species , quantitative variation is often observed , possibly related to the different growth characteristics of various species ( mitchell - olds and pedersen , 1998 ) . qualitative and quantitative variation in secondary metabolism is , however , much more extensive and it is widely accepted that secondary metabolism determines to a great extent the success of plant adaptation ( herms and mattson , 1992 ; pichersky and gang , 2000 ) . as stated above , thousands of different metabolites can be found in a single plant species ( de luca and st pierre , 2000 ; sumner et al . , 2003 ; dauria and gershenzon , 2005 ; fernie , 2007 ) . that said , we are only starting to explore the composition of the metabolome , let alone unravel all of the biosynthetic pathways leading to this diversity of chemical structures . in the current article we concentrate on results from broad screening of the natural genetic diversity of metabolism in arabidopsis rosettes and tomato fruit . following intensive statistical analysis , clear patterns of metabolic regulation can be demarcated via these approaches and a sub - set of these patterns can be resolved at the genetic level . we conclude that the screening of diverse genetic populations by metabolic profiling significantly adds to our understand metabolic regulation . in recent years much research has exploited natural variance in the pre - eminent model species arabidopsis thaliana ( kliebenstein et al . , 2001 ; koornneef et al . , 2004 ; weigel and nordborg , 2005 ; borevitz et al . study of metabolic traits in arabidopsis largely focuses on understanding the principles underlying metabolic regulation and the influence of metabolism on growth and development ( kliebenstein et al . , 2001 ; keurentjes et al . , 2006 ; meyer et al . , 2007 ; , 2008 , 2009 ; rowe et al . , 2008 ; sulpice et al . , establishment of the genetic basis of quantitative traits commonly referred to as quantitative trait loci ( qtl ) , has often been hampered due to their complex multigenic inheritance and strong interactions with the environment . the principle of qtl mapping in segregating populations is based on genotyping of progeny derived from a cross between distinct genotypes for the trait under study . phenotypic values for the trait are then compared with molecular markers in the progeny to search for particular genomic regions showing statistically significant associations with the trait variation ( broman , 2001 ; slate , 2005 ) . over the past few decades , the ease with which such markers can be developed has facilitated qtl mapping studies of even the most complex traits ( borevitz and nordborg , 2003 ) . quantitative trait loci analysis makes use of the natural variation present within species ( alonso - blanco and koornneef , 2000 ; maloof , 2003 ; fernie et al . , 2006 ) and has been successfully applied to various types of segregating populations . in plants , the use of immortal mapping populations consisting of homozygous individuals that , at least theoretically , can be propagated indefinitely is preferred because it permits replication and multiple analyses of the same population . homozygous populations can be obtained by repeated selfing , as is the case for recombinant inbred lines ( rils ) , but also by induced chromosomal doubling of haploids , such as for doubled haploids ( dhs ; han et al . , 1997 ; rae et al . , 1999 ; von korff et al . , 2004 ) . rils are likely advantageous over dhs since they are characterized by a higher frequency of recombination within the population , resulting from multiple meiotic events occurred during repeated selfing ( jansen , 2003 ; keurentjes and fernie , 2011 ) . another type of immortal population consists of introgression lines ( ils ; eshed and zamir , 1994 ) , which are obtained through repeated backcrossing and extensive genotyping . these are also referred to as near isogenic lines ( nils ; monforte and tanksley , 2000 ) , or backcross inbred lines ( bils ; jeuken and lindhout , 2004 ; blanco et al . , 2006 ) . these lines contain a single or a small number of genomic introgression fragments from a donor parent into an otherwise homogeneous genetic background . in plants , rils and nils are the most common types of experimental populations used for the analysis of quantitative traits ( for an illustration of these populations see figure 1 ) . in both cases the accuracy of qtl localization , referred to as mapping resolution , depends on population size . for rils , the position of the recombination event is fixed and can therefore only be increased within the population by adding more lines ( i.e. , more independent recombination events ) . alternatively , recombination frequency can be increased by intercrossing lines before fixation as homozygous lines by inbreeding ( zou et al . , 2005 ; balasubramanian et al . , 2009 ) . in nil populations resolution can be improved by minimizing the introgression size of each nil . consequently , to maintain genome - wide coverage either a larger number of lines or a high proportion of overlapping regions , or both , are needed . despite the similarities between these two types of mapping populations , large differences exist in the genetic makeup of the respective individuals and the resulting mapping approach . in general , recombination frequency in ril populations each ril contains several introgressed fragments and , on average , each genomic region is represented by an equal number of both parental genotypes in the population . therefore , replication of individual lines is often not necessary because the effect of each genomic region on phenotypic traits is independently tested multiple times by comparing the two genotypic ril classes . in addition , the multiple introgressions per ril can potentially reveal epistasis between loci . however , this may negatively bias the power to detect qtl . furthermore , the wide variation of morphological and developmental traits among individuals within most ril populations may hamper analysis of traits requiring the same growth and developmental stage of the individual lines . when many traits segregate simultaneously , this often affects the expression of other traits due to genetic interactions . by contrast , nils preferably contain only a single introgressed segment per line , increasing the power to detect small - effect qtl . however , the presence of a single introgressed segment limits testing for genetic interactions and thereby the detection of epistatic qtl . since most of the genetic background is identical for all lines , nils show more limited developmental and growth variation , increasing the homogeneity of growth stage within experiments . introgression lines are created by backcrossing an f1 of a cross between two parental lines to a recurrent parent for several times . recombinant inbred lines are generated by selfing an f1 for at least eight generations when full homozygosity is reached . recombinant inbred lines allow for the testing of epistasis . also because of the higher recombination frequency they often offer higher resolution than introgression lines . introgression lines , however , often display greater statistical power in the detection of small - effect qtl . in arabidopsis , the ease of generating fertile ril populations with complete genome coverage has led to their extensive use in qtl mapping ( oneill et al . , nils have also been developed to confirm and fine map qtl previously identified in rils ( alonso - blanco et al . , 1998 , 2003 ; swarup et al . , 1999 ; bentsink et al . , 2003 ; edwards et al . , 2005 ; juenger et al . , 2005 ; teng et al . , 2005 ) . genome - wide sets of nils and rils , descending from identical intercrosses , that allow mapping to chromosomal sections have been described in arabidopsis and empirical comparative studies have been performed between the two population types ( keurentjes et al . these studies illustrate the complementary benefits of both resources , facilitating the genetic dissection of various quantitative traits in arabidopsis . ril populations allow mapping at higher resolution , whilst nils have the advantage of detecting small - effect qtl ( keurentjes and fernie , 2011 ) . having extensively described the approaches , we now detail the biological significance of results obtained to date in arabidopsis . although many simple metabolic traits affecting protein , oil , and starch content have been studied using targeted approaches ( see moose and mumm , 2008 ; fernie and schauer , 2009 ) , the adoption of methods able to detect the levels of multiple metabolites at once greatly expanded our ability to pose questions about regulation at the pathway and network level ( sweetlove et al . , 2008 ; stitt et al . , 2010 ) . studies , largely reliant on gas chromatography mass spectrometry ( gc ms ) or liquid chromatography mass spectrometry ( lc ms ) have been carried out in arabidopsis by three different research groups . the first publication of note focused on a ril population derived from a cross between the landsberg erecta ( ler ) and cape verde islands ( cvi ) accessions and evaluated the variance of some 2000 mass peaks across this population ( keurentjes et al . , 2006 ) . interestingly , almost one - third of these peaks were not detected in either parent , implying a vast potential for manipulation of chemotypes via classical breeding . another intriguing finding of this study was the fact that colocation of qtl coincided with clusters of highly correlated mass peaks . comparison of these data with those of previous targeted work showed that these co - locations were at positions of known regulators of glucosinolate metabolism to which peptide methionine sulfoxide reductase ( mam ) and qtl for production of alkenyl or hydroxyalkyl glucosinolates ( aop ) mapped ( kliebenstein et al . further work from this group focused on parallel analysis of 15 enzyme activities in tandem with their corresponding transcript levels and a set of relevant metabolites . the results revealed that traits affecting primary metabolism are often correlated and many activity qtl co - localize with expression qtl ( although a fair number do not , suggesting that such multilevel approaches will be highly useful for distinguishing between transcriptional and metabolic control ( keurentjes et al . , 2008 ) . as an extension of this work they next performed a multiplexed transcriptome , proteome , and metabolome study on the same material , combining these data with publically available data ( fu et al . , 2009 ) . the surprising finding of this study was that following mapping of over 40000 molecular and 100 phenotypic traits , there were only six qtl hotspots . the authors concluded that there are thus only six breakpoints in a system otherwise buffered against many of the half - million single nucleotide polymorphisms ( snps ) between the parental lines ( fu et al . , 2009 ) . following a similar approach to that mentioned above , groups at the max - planck - institute in golm focused on primary metabolism . two different strategies were employed ; the study of rils and nils resulting from a cross between col-0 and c24 ( meyer et al . , 2007 ; 2011 ) and analysis of the natural variance of metabolite accumulation inherent in ecotypes ( cross et al . , metabolites were not treated in isolation but rather evaluated with respect to the influence they exerted toward plant growth with a metabolic signature for high growth being defined from results obtained in the col-0/c24 rils ( meyer et al . , 2007 ) . a more detailed study examined both rils and nils derived from the same parents ( lisec et al . , 2008 ) , revealing a couple of hotspots in which yield qtl overlapped with a large number of metabolite accumulation qtl . in this study , metabolic pathway - derived candidate genes were found for 2467% of all tested metabolite qtl in the database aracyc 3.5 , demonstrating the power of this approach to identify possible sites of metabolic regulation . it is , however , important to note that this is only the first step and considerable further experimentation is required to confirm the existence and physiological relevance of such regulations . a recent paper describes the identification of a cytosolic isoform of fumarase as the causal gene underlying traits of decreased fumarate and increased malate ( brotman et al . , 2011 ) . this result represents an elegant proof - of - concept study and beautifully fits the cross - over theorem , which states that if an equilibrium reaction of a linear pathway is inhibited , a build - up of substrate and a depletion of product of that reaction will occur ( rolleston , 1972 ) . an illustration of the power of this theorem is shown in its application to starch synthesis in potato , which prompted a successful search for a novel post - translational modifier of the agpase enzyme ( stitt et al . bearing this example in mind , analyses of metabolite ratios in advanced genetic populations will likely prove to be an important route to identify previously uncharacterized mechanisms of metabolic regulation in the future . the rapidly increasing availability of genome information for multiple arabidopsis ecotypes ( weigel and mott , 2009 ; schneeberger and weigel , 2011 ) , alongside the increase in the number of groups performing metabolomic analyses should accelerate gene discovery . a second approach to identification of metabolic regulators is comparative analysis of various arabidopsis ecotypes . this approach was initiated to assess the natural variance in enzyme activities ; the first study examined the activities of seven enzymes across 24 arabidopsis ecotypes ( cross et al . , 2006 ) whilst the second evaluated the activity of the most abundant protein in photosynthetic tissues , ribulose-1,5-bisphosphate - carboxylase/-oxygenase ( rubisco ) , across the abovementioned col-0/c24 ril population ( sulpice et al . , 2007 ) in the former study it was observed that enzyme activities largely vary on mass , but are not well correlated to the levels of the metabolites measured in the same sample ( cross et al . , 2006 ) . this observation is consistent with the concept that metabolism is highly regulated at multiple levels . the latter study described application of a novel rubisco assay to describe the characteristics of this enzyme in 118 arabidopsis accessions , defining two loci for rubisco activity and two for rubisco activation state ( sulpice et al . these analyses were subsequently massively expanded to encompass either 94 or 112 accessions as well as quantification of select metabolites in an attempt to establish the major integrator of growth within the species ( sulpice et al . , 2009 , 2010 ) . the results of these analyses spotlighted starch and protein biosynthesis as the major controlling factors with respect to total biomass ( sulpice et al . transcript profiling in 21 accessions further revealed coordinated changes in expression of more than 70 carbon - regulated genes , identifying two ( myo - inositol-1-phosphate synthase and a kelch - domain protein ) whose transcripts correlate with biomass . the impact of allelic variation at these two loci was shown by association mapping , identifying them as candidate genes to increase biomass production . kliebenstein and co - workers have also taken both association mapping and qtl - based approaches in their studies , profiling both primary and secondary metabolites alongside studies of gene expression ( kliebenstein et al . , 2006 ; rowe et al . , 2008 ; kliebenstein , 2009 ; chan et al . , they have expended considerable effort to understand whole genome expression qtl , revealing a high incidence of both cis- and trans - acting qtl , including non - additive variation such as epistasis and transgressive segregation as well as genetic variation affecting entire transcriptional networks ( kliebenstein et al . , 2006 ; kliebenstein , 2009 ) . they additionally linked this variation to phenotypic alterations in secondary metabolite content ( wentzell et al . , 2007 ; hansen et al . , 2008 ) and more recently profiled primary metabolites ( rowe et al . , 2008 ) in a 210 member ril population . statistical analysis of the resultant dataset suggested that epistatic interactions control a majority of the variation in the network of plant primary metabolism . they also identified 11 metabolite qtl hotspots , two of which overlapped the aop and mam loci previously characterized as qtl for glucosinolate accumulation . taking this analysis one step further , the authors constructed two biochemical networks de novo ; however , it is too early to judge the accuracy of such an approach . the group subsequently profiled some 327 metabolites against greater than 200 000 snps ( chan et al . , 2010 ) . however , comparison of the resultant data from this study and that described above on the ril population revealed that the higher level of genetic variation in the accession population was not reflected by a higher variation in the metabolome . clearly such studies must be conducted in a range of environmental conditions since not only do individual metabolites change with the environment but the entire network behavior changes as well . while arabidopsis is the best characterized plant species with respect to natural variation in the metabolome , an increasing number of studies are being carried out in crop species . unfortunately , a limited number of these studies have addressed environmental influences across independent harvests and/or locations . nonetheless , considerable information has been gleaned from single - harvest studies ( see for example fraser et al . , 2007 ; kusano et al . , 2007 ) . despite these limitations , studies on rice , the staple food crop of almost half of the world s population ( hall et al . , 2008 ) , are particularly pertinent from an applied perspective . ( 2007 ) recently profiled a total of 70 rice cultivars using a combination of two - dimensional gc similar smaller scale studies have also been carried out in sesame , broccoli , and mustard ( magrath et al . , 1993 ; laurentin et al . , 2008 ; rochfort et al . , adoption of maldi / tof - ms to individual mutagenized plants and solanum pennellii ils has been used for screening of fruit containing high levels of nutraceutical compounds such as carotenoids ( fraser et al . , 2007 ) . these studies all reveal large diversity within populations at the genetic and metabolic levels and hint that such information is likely of high value for breeding programs ( fernie and schauer , 2009 ) . as we will discuss in the following paragraphs it is also highly interesting material in which to study metabolic regulation . to date the majority of these have been focused on tomato and these studies are the focus of this section . however , interesting examples in maize ( harrigan et al . , 2007a , b ; zhang et al . , 2010 ; lisec et al . , 2011 ) , lolium perenne ( koulman et al . , 2009 ) , and wheat ( hazehzarghani et al . , 2008 ) will also be discussed . in maize a range of compositional traits including protein , oil , fatty acid , amino acid , and organic acid content was carried out in two independent maize hybrids grown at three separate locations ( harrigan et al . this important proof - of - concept study demonstrated the high non - genetic variability in crop composition , illustrating the need for replicated trials . more recently a comparative analysis of the root metabolome of six parental maize inbred lines and their corresponding 14 hybrids was performed ( lisec et al . , 2011 ) . the metabolic profile of each hybrid when compared to its parents is distinct and even reciprocal hybrids are easily distinguished . reconstructed metabolic networks display a higher network density in most hybrids as compared to the corresponding inbred lines , suggesting that metabolite levels are subject to tighter control . on a broader scope , a maize diversity panel was screened for 10 key enzyme activities and heritable variation was found in each one ( zhang et al . , 2010 ) . association mapping subsequently identified a novel amino acid substitution associated with a variation in isocitrate dehydrogenase activity , demonstrating that this approach can identify putative functional sites . a later study of the same 10 enzymes across a maize intermated b73 mo17 mapping population provided almost a four - fold increase in genetic map distance compared with conventional mapping populations ( zhang et al . , 2011 ) . in total , 73 significant qtl that influence the activity of these 10 enzymes as well as 8 qtl that influence biomass were identified . while some qtl were shared by different enzymes or biomass , all enzyme activity qtl were in trans to the known genomic locations of structural genes ( i.e. , genes that encode enzymes operating within the pathways ) , except for single cis - qtl for nitrate reductase , glutamate dehydrogenase , and shikimate dehydrogenase ; the low frequency and low additive magnitude compared with trans - qtl indicates that , at least in this population , cis - regulation is relatively unimportant versus trans - regulation . returning to metabolite content qtl , large datasets have been obtained for l. perenne populations ( rasmussen et al . , 2008 ) , as well as wheat infected with fusarium head blight ( hazehzarghani et al . , 2008 ) . by far the best characterized crop system , however , is tomato ( klee , 2010 ; keurentjes and fernie , 2011 ) . in this species , a broad profiling of fruit volatiles , which are extremely important flavor components , in a population consisting of 74 solanum lycopersicum s. pennellii ils yielded 100 qtl that were conserved across harvests ( tieman et al . , 2006b ) . metabolic and flux profiling of one of these qtl was instrumental in defining the pathway for synthesis of important phenylalanine - derived aromatic compounds in the fruit ( tieman et al . thirty additional qtl that affect the volatile emissions of red - ripe fruit were identified in a second population ofils derived from a cross between s. lycopersicum and s. habrochaites grown in multiple seasons and locations ( mathieu et al . , 2010 ) . the same population has also recently been characterized for qtl for ripening - associated ethylene release ( dal cin et al . , 2009 ) and used to define a novel pathway for sesquiterpene biosynthesis from z , z - farnesyl pyrophosphate ( sallaud et al . in other studies of note , the volatile metabolite composition of some 300 compounds was determined across a population of 94 elite cultivars of tomato ( tikunov et al . , 2005 ) , whilst the primary metabolite composition of five wild species of tomato was assessed in comparison to the cultivated tomato ( schauer et al . that said , it will be some time before our knowledge is sufficiently advanced that we can facilely use such information for predictive breeding ( keurentjes and fernie , 2011 ) . similar , albeit not quite so extensive , studies were performed on intraspecific crosses of s. lycopersicum cultivars ( causse et al . , 2002 ) , and have subsequently been validated in replicated experiments ( zanor et al . , 2009 ) . ms method in replicated harvests resulting in identification of 889 qtl covering 74 metabolites including important primary metabolites such as sugars and organic acids as well as essential amino acids , intermediate metabolites and vitamins ( schauer et al . , 2006 ) . it is important to note that despite the fact that in many cases metabolite content was elevated , the vast majority of these qtl were associated with a yield penalty . in a subsequent study the heritability of these traits were established ( schauer et al . , 2008 ) . for this purpose , the s. penellii ils were grown alongside lines heterozygous for the introgression ( ilhs ) allowing evaluation of both heritability and the mode of inheritance . these studies revealed that mean heritability of the metabolite qtl was generally relatively low ( as in arabidopsis ; rowe et al . , 2008 ) . however , a handful of the traits were nevertheless highly conserved and displayed reasonable heritability . comparative study of the ils and ilhs revealed that most of the metabolic qtl were dominant with a considerable number displaying additive or recessive mode of action and only a negligible number displaying overdominant phenotypes . interestingly , the mode of inheritance was quantitatively different between diverse compound classes and several metabolite pairs displayed a similar mode of inheritance at the same chromosomal loci , suggesting that the variation is likely to be mediated by enzymes involved in their interconversion ( schauer et al . , 2008 ) . the s. pennellii ils have also been characterized for trichome specialized metabolites including terpenes , flavonoids , and acyl sugars ( schilmiller et al . , 2010 ) . metabolite profiling led to the discovery of ils producing different acyl chain substitutions on acyl sugar metabolites as well as two regions quantitatively influencing acyl sugar content . these results illustrate the power of qtl mapping for identification of novel enzymatic functions and pathways . following a similar approach tieman et al . ( 2006b ) used ils and reverse genetics in combination with volatile and isotope tracer analysis to establish the route of 2-phenylethanol and phenylacetaldehyde biosynthesis in tomato . a second flavor volatile qtl responsible for synthesis of methylsalicylate was shown to be the consequence of altered expression qtl ( eqtl ) of the gene encoding the biosynthetic enzyme , salicylic acid methyltransferase ( tieman et al . , 2010 ) . another important agronomic property in tomato , total soluble solids content , has been defined at the genetic level . an early study utilizing the s. pennellii ils mapped the moderate qtl brix 9 - 2 - 5 to a 484-bp region of the cell wall invertase gene lin5 ( fridman et al . , 2000 ) , although there was no difference in expression or protein content of lin5 in the il harboring this qtl ( fridman et al . qtl analysis of five different tomato species delimited the functional polymorphism of brix 9 - 2 - 5 to an amino acid at the fructosyl binding site near the catalytic site of the invertase crystal with enzyme kinetic analysis of recombinant protein demonstrating that the s. pennellii allele was more efficient in degrading sucrose ( fridman et al . , subsequent experiments involving rna interference of this isoform resulted in reduced brix and sink strength , thus confirming the results obtained in the heterologous system ( zanor et al . , currently , optimized assays ( steinhauser et al . , 2010 ) for a set of key enzymes of central metabolism are being used to profile the s. pennellii ils . once finished it will be highly informative to integrate these analyses with those of the metabolites . further proof of the value of metabolic qtl analysis is illustrated by studies on branched chain amino acid metabolism for which four co - ordinate qtl were identified ( schauer et al . , 2006 ) . as a first approach we focused on branched chain aminotransferases ( bcats ) , mapping all six members of the family . bcat1 is an eqtl whereas bcat4 is a protein quality qtl ( i.e. , a qtl that does not affect protein abundance but rather its relative efficiency within a given biological process ; maloney et al . , 2010 ) . we next mapped a further 22 putative gene functions associated with branched chain amino acid metabolism . mapping the chromosomal locations of these enzymes , it was possible to define the map positions of 24 genes ( with two of the putative gene functions being encoded by two independent genes ) . eight co - localized with bcaa qtl including those encoding ketol - acid reductoisomerase ( kari ) , dihydroxy - acid dehydratase ( dhad ) , and isopropylmalate dehydratase ( ipmd ; kochevenko and fernie , 2011 ) . quantitative evaluation of the expression of these genes revealed that the s. pennellii allele exhibited altered expression of ipmd , whereas expression of kari and dhad were invariant across the genotypes . whilst the antisense inhibition of ipmd resulted in increased bcaa , the antisense inhibition of kari or dhad had no effect on fruit bcaa contents ( kochevenko and fernie , 2011 ) . the above examples in both arabidopsis and crop species illustrate how forward genetic approaches based on natural variation can produce high resolution information concerning metabolic regulation . this is the case at the levels of the individual enzyme , whole pathways , and even metabolic networks . they furthermore , demonstrate how natural variance is an undermined resource of biochemical diversity and one that will certainly be an important resource for breeding approaches toward metabolic engineering ( fernie et al . , 2006 ) . given the ever increasing number of crop species for which full - genome sequences are becoming available it is highly likely that such studies will be greatly aided by the development of translational approaches both at the molecular ( mutwil et al . , 2011 ) and phenotyping levels ( tohge and fernie , 2010 ) . as yet one drawback of this approach that said , the development of a wide array of genetic materials including those resulting from tilling ( till et al . , 2006 ) and the adoption of every more sophisticated rapid screening technologies such as those afforded by viral induced gene silencing ( ruiz et al . , 1998 ; regardless , the forward genetic approach has at least two advantages ( i ) by taking a top - down approach it may uncover different levels of metabolic regulation within an experiment and ( ii ) given that the whole genome is considered , this approach is not restricted to evaluation of previously known enzymes or regulators thereof . while the forward genetic approach is in its infancy we are convinced that it will prove a highly powerful tool for the identification of novel mechanisms of metabolic regulation . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

the study of metabolic regulation has traditionally focused on analysis of specific enzymes , emphasizing kinetic properties , and the influence of protein interactions and post - translational modifications . more recently , reverse genetic approaches permit researchers to directly determine the effects of a deficiency or a surplus of a given enzyme on the biochemistry and physiology of a plant . furthermore , in many model species , gene expression atlases that give important spatial information concerning the quantitative expression level of metabolism - associated genes are being produced . in parallel , top - down approaches to understand metabolic regulation have recently been instigated whereby broad genetic diversity is screened for metabolic traits and the genetic basis of this diversity is defined thereafter . in this article we will review recent examples of this latter approach both in the model species arabidopsis thaliana and the crop species tomato ( solanum lycopersicum ) . in addition to highlighting examples in which this genetic diversity approach has proven promising , we will discuss the challenges associated with this approach and provide a perspective for its future utility .

Introduction Metabolic Variance in Metabolic Variance in Crop Species Concluding Remarks Conflict of Interest Statement

elucidation of metabolic regulation in plants has been an academic pursuit spanning many decades . indeed the question of how metabolism is regulated was first raised in the scientific era in which metabolism was first truly defined ( buchner , 1907 ; krebs and henseleit , 1932 ; plaxton , 1996 ; kornberg , 2000 ) . whilst the ability to regulate the rates of metabolic processes in response to cellular circumstance is a common feature of all organisms , it is particularly acute in sessile organism such as plants ( plaxton , 1996 ) . from a textbook perspective , metabolic regulation is classically divided into coarse and fine levels of control ( dennis et al . coarse control refers to long - term mechanisms that are energetically expensive and lead to changes in the total cellular population of a protein . however , unfortunately , despite the massive amount of data available regarding steady - state rna levels afforded by microarrays ( see for example the data stored in genevestigator ; http://www.genevestigator.ethz.ch ) and more recently by next generation sequence analyses ( see for example gonzalez - ballester et al . the first , of these is essentially the most simple and certainly the most rapid to affect metabolic systems with the rate of an enzyme - catalyzed reaction proceeding more rapidly upon an increase in sub - saturating substrate a case that is common in vivo all enzyme reactions are , to a greater or lesser extent regulated in this manner . for example , regulation of enzymes of the calvin cycle is well documented to be ph regulated ; stromal ph is 8.0 in that the light and 7.0 in the dark ( see dennis et al . , thirdly , allosteric effectors are immensely important in the regulation of plant metabolic networks , be they activators or inhibitors . within the major pathways of carbohydrate metabolism , several examples of the importance of such metabolites exist , including the 3 phosphoglycerate ( 3pga)/inorganic phosphate ( pi ) ratio in activating adp glucose pyrophosphorylase ( agpase ; preiss , 1982 ; tiessen et al . , 2001 ) and the effect of pyruvate on the alternative oxidase of mitochondrial respiration ( millar et al . , 1993 understanding the function of a given enzyme within a biological process has until recently largely followed a set protocol by which novel genes associated with a specific process are identified by means of similar patterns of expression across a wide range of experiments and subsequently their function tested . this is initially carried out by analyzing the metabolite profiles of genotypes deficient in the expression of the gene . confirmation of kinetic properties of the enzyme either in planta ( in the case that the gene encodes the only isoform of an enzyme ) or following expression of the gene in a heterologous system lacking the activity is subsequently required ( see for details tohge and fernie , 2010 ) . whilst this approach has been tremendously successful ( hirai et al . , 2009 ) in terms of annotating the precise biochemical function of individual genes , it does not enable elucidation of the exact physiological function in vivo . in the last 25 years the roles of specific metabolic enzymes have been addressed via the use of transgenic plants ( stitt and sonnewald , 1995 ; lytovchenko et al . such studies have greatly advanced our understanding of metabolic regulation . however , such directed approaches sometimes fail to uncover complex inter - pathway interactions and pathway regulators . given that the chemical constituents of any life form determine the development and functioning of the organism ( sumner et al . although chemical profiles differ between and even within species , thousands of diverse metabolites are usually found in a single plant ( de luca and st pierre , 2000 ; sumner et al . , 2002 ) whereas secondary metabolism is often connected to interactions with environmental cues , including cell signaling , interspecies communication , and responses to biotic and abiotic stress ( see for example wink , 1988 ; mitchell - olds and pedersen , 1998 ; lehmann et al . although primary metabolic pathways are strongly conserved between species , quantitative variation is often observed , possibly related to the different growth characteristics of various species ( mitchell - olds and pedersen , 1998 ) . that said , we are only starting to explore the composition of the metabolome , let alone unravel all of the biosynthetic pathways leading to this diversity of chemical structures . in the current article we concentrate on results from broad screening of the natural genetic diversity of metabolism in arabidopsis rosettes and tomato fruit . following intensive statistical analysis , clear patterns of metabolic regulation can be demarcated via these approaches and a sub - set of these patterns can be resolved at the genetic level . we conclude that the screening of diverse genetic populations by metabolic profiling significantly adds to our understand metabolic regulation . in recent years much research has exploited natural variance in the pre - eminent model species arabidopsis thaliana ( kliebenstein et al . study of metabolic traits in arabidopsis largely focuses on understanding the principles underlying metabolic regulation and the influence of metabolism on growth and development ( kliebenstein et al . , establishment of the genetic basis of quantitative traits commonly referred to as quantitative trait loci ( qtl ) , has often been hampered due to their complex multigenic inheritance and strong interactions with the environment . phenotypic values for the trait are then compared with molecular markers in the progeny to search for particular genomic regions showing statistically significant associations with the trait variation ( broman , 2001 ; slate , 2005 ) . these lines contain a single or a small number of genomic introgression fragments from a donor parent into an otherwise homogeneous genetic background . in plants , rils and nils are the most common types of experimental populations used for the analysis of quantitative traits ( for an illustration of these populations see figure 1 ) . consequently , to maintain genome - wide coverage either a larger number of lines or a high proportion of overlapping regions , or both , are needed . despite the similarities between these two types of mapping populations , large differences exist in the genetic makeup of the respective individuals and the resulting mapping approach . in general , recombination frequency in ril populations each ril contains several introgressed fragments and , on average , each genomic region is represented by an equal number of both parental genotypes in the population . in addition , the multiple introgressions per ril can potentially reveal epistasis between loci . furthermore , the wide variation of morphological and developmental traits among individuals within most ril populations may hamper analysis of traits requiring the same growth and developmental stage of the individual lines . however , the presence of a single introgressed segment limits testing for genetic interactions and thereby the detection of epistatic qtl . since most of the genetic background is identical for all lines , nils show more limited developmental and growth variation , increasing the homogeneity of growth stage within experiments . introgression lines are created by backcrossing an f1 of a cross between two parental lines to a recurrent parent for several times . introgression lines , however , often display greater statistical power in the detection of small - effect qtl . these studies illustrate the complementary benefits of both resources , facilitating the genetic dissection of various quantitative traits in arabidopsis . having extensively described the approaches , we now detail the biological significance of results obtained to date in arabidopsis . although many simple metabolic traits affecting protein , oil , and starch content have been studied using targeted approaches ( see moose and mumm , 2008 ; fernie and schauer , 2009 ) , the adoption of methods able to detect the levels of multiple metabolites at once greatly expanded our ability to pose questions about regulation at the pathway and network level ( sweetlove et al . the first publication of note focused on a ril population derived from a cross between the landsberg erecta ( ler ) and cape verde islands ( cvi ) accessions and evaluated the variance of some 2000 mass peaks across this population ( keurentjes et al . another intriguing finding of this study was the fact that colocation of qtl coincided with clusters of highly correlated mass peaks . further work from this group focused on parallel analysis of 15 enzyme activities in tandem with their corresponding transcript levels and a set of relevant metabolites . as an extension of this work they next performed a multiplexed transcriptome , proteome , and metabolome study on the same material , combining these data with publically available data ( fu et al . the surprising finding of this study was that following mapping of over 40000 molecular and 100 phenotypic traits , there were only six qtl hotspots . following a similar approach to that mentioned above , groups at the max - planck - institute in golm focused on primary metabolism . two different strategies were employed ; the study of rils and nils resulting from a cross between col-0 and c24 ( meyer et al . , 2007 ; 2011 ) and analysis of the natural variance of metabolite accumulation inherent in ecotypes ( cross et al . , metabolites were not treated in isolation but rather evaluated with respect to the influence they exerted toward plant growth with a metabolic signature for high growth being defined from results obtained in the col-0/c24 rils ( meyer et al . , 2008 ) , revealing a couple of hotspots in which yield qtl overlapped with a large number of metabolite accumulation qtl . in this study , metabolic pathway - derived candidate genes were found for 2467% of all tested metabolite qtl in the database aracyc 3.5 , demonstrating the power of this approach to identify possible sites of metabolic regulation . this result represents an elegant proof - of - concept study and beautifully fits the cross - over theorem , which states that if an equilibrium reaction of a linear pathway is inhibited , a build - up of substrate and a depletion of product of that reaction will occur ( rolleston , 1972 ) . an illustration of the power of this theorem is shown in its application to starch synthesis in potato , which prompted a successful search for a novel post - translational modifier of the agpase enzyme ( stitt et al . bearing this example in mind , analyses of metabolite ratios in advanced genetic populations will likely prove to be an important route to identify previously uncharacterized mechanisms of metabolic regulation in the future . the rapidly increasing availability of genome information for multiple arabidopsis ecotypes ( weigel and mott , 2009 ; schneeberger and weigel , 2011 ) , alongside the increase in the number of groups performing metabolomic analyses should accelerate gene discovery . a second approach to identification of metabolic regulators is comparative analysis of various arabidopsis ecotypes . this approach was initiated to assess the natural variance in enzyme activities ; the first study examined the activities of seven enzymes across 24 arabidopsis ecotypes ( cross et al . , 2007 ) in the former study it was observed that enzyme activities largely vary on mass , but are not well correlated to the levels of the metabolites measured in the same sample ( cross et al . the latter study described application of a novel rubisco assay to describe the characteristics of this enzyme in 118 arabidopsis accessions , defining two loci for rubisco activity and two for rubisco activation state ( sulpice et al . kliebenstein and co - workers have also taken both association mapping and qtl - based approaches in their studies , profiling both primary and secondary metabolites alongside studies of gene expression ( kliebenstein et al . , they have expended considerable effort to understand whole genome expression qtl , revealing a high incidence of both cis- and trans - acting qtl , including non - additive variation such as epistasis and transgressive segregation as well as genetic variation affecting entire transcriptional networks ( kliebenstein et al . , 2008 ) and more recently profiled primary metabolites ( rowe et al . , 2008 ) in a 210 member ril population . statistical analysis of the resultant dataset suggested that epistatic interactions control a majority of the variation in the network of plant primary metabolism . however , comparison of the resultant data from this study and that described above on the ril population revealed that the higher level of genetic variation in the accession population was not reflected by a higher variation in the metabolome . while arabidopsis is the best characterized plant species with respect to natural variation in the metabolome , an increasing number of studies are being carried out in crop species . ( 2007 ) recently profiled a total of 70 rice cultivars using a combination of two - dimensional gc similar smaller scale studies have also been carried out in sesame , broccoli , and mustard ( magrath et al . these studies all reveal large diversity within populations at the genetic and metabolic levels and hint that such information is likely of high value for breeding programs ( fernie and schauer , 2009 ) . as we will discuss in the following paragraphs it is also highly interesting material in which to study metabolic regulation . to date the majority of these have been focused on tomato and these studies are the focus of this section . however , interesting examples in maize ( harrigan et al . , 2009 ) , and wheat ( hazehzarghani et al . in maize a range of compositional traits including protein , oil , fatty acid , amino acid , and organic acid content was carried out in two independent maize hybrids grown at three separate locations ( harrigan et al . more recently a comparative analysis of the root metabolome of six parental maize inbred lines and their corresponding 14 hybrids was performed ( lisec et al . on a broader scope , a maize diversity panel was screened for 10 key enzyme activities and heritable variation was found in each one ( zhang et al . association mapping subsequently identified a novel amino acid substitution associated with a variation in isocitrate dehydrogenase activity , demonstrating that this approach can identify putative functional sites . a later study of the same 10 enzymes across a maize intermated b73 mo17 mapping population provided almost a four - fold increase in genetic map distance compared with conventional mapping populations ( zhang et al . , genes that encode enzymes operating within the pathways ) , except for single cis - qtl for nitrate reductase , glutamate dehydrogenase , and shikimate dehydrogenase ; the low frequency and low additive magnitude compared with trans - qtl indicates that , at least in this population , cis - regulation is relatively unimportant versus trans - regulation . by far the best characterized crop system , however , is tomato ( klee , 2010 ; keurentjes and fernie , 2011 ) . in this species , a broad profiling of fruit volatiles , which are extremely important flavor components , in a population consisting of 74 solanum lycopersicum s. pennellii ils yielded 100 qtl that were conserved across harvests ( tieman et al . metabolic and flux profiling of one of these qtl was instrumental in defining the pathway for synthesis of important phenylalanine - derived aromatic compounds in the fruit ( tieman et al . the same population has also recently been characterized for qtl for ripening - associated ethylene release ( dal cin et al . in other studies of note , the volatile metabolite composition of some 300 compounds was determined across a population of 94 elite cultivars of tomato ( tikunov et al . , 2005 ) , whilst the primary metabolite composition of five wild species of tomato was assessed in comparison to the cultivated tomato ( schauer et al . , 2002 ) , and have subsequently been validated in replicated experiments ( zanor et al . it is important to note that despite the fact that in many cases metabolite content was elevated , the vast majority of these qtl were associated with a yield penalty . for this purpose , the s. penellii ils were grown alongside lines heterozygous for the introgression ( ilhs ) allowing evaluation of both heritability and the mode of inheritance . comparative study of the ils and ilhs revealed that most of the metabolic qtl were dominant with a considerable number displaying additive or recessive mode of action and only a negligible number displaying overdominant phenotypes . the s. pennellii ils have also been characterized for trichome specialized metabolites including terpenes , flavonoids , and acyl sugars ( schilmiller et al . another important agronomic property in tomato , total soluble solids content , has been defined at the genetic level . , 2000 ) , although there was no difference in expression or protein content of lin5 in the il harboring this qtl ( fridman et al . qtl analysis of five different tomato species delimited the functional polymorphism of brix 9 - 2 - 5 to an amino acid at the fructosyl binding site near the catalytic site of the invertase crystal with enzyme kinetic analysis of recombinant protein demonstrating that the s. pennellii allele was more efficient in degrading sucrose ( fridman et al . , subsequent experiments involving rna interference of this isoform resulted in reduced brix and sink strength , thus confirming the results obtained in the heterologous system ( zanor et al . , 2010 ) for a set of key enzymes of central metabolism are being used to profile the s. pennellii ils . further proof of the value of metabolic qtl analysis is illustrated by studies on branched chain amino acid metabolism for which four co - ordinate qtl were identified ( schauer et al . as a first approach we focused on branched chain aminotransferases ( bcats ) , mapping all six members of the family . , a qtl that does not affect protein abundance but rather its relative efficiency within a given biological process ; maloney et al . we next mapped a further 22 putative gene functions associated with branched chain amino acid metabolism . mapping the chromosomal locations of these enzymes , it was possible to define the map positions of 24 genes ( with two of the putative gene functions being encoded by two independent genes ) . eight co - localized with bcaa qtl including those encoding ketol - acid reductoisomerase ( kari ) , dihydroxy - acid dehydratase ( dhad ) , and isopropylmalate dehydratase ( ipmd ; kochevenko and fernie , 2011 ) . the above examples in both arabidopsis and crop species illustrate how forward genetic approaches based on natural variation can produce high resolution information concerning metabolic regulation . this is the case at the levels of the individual enzyme , whole pathways , and even metabolic networks . they furthermore , demonstrate how natural variance is an undermined resource of biochemical diversity and one that will certainly be an important resource for breeding approaches toward metabolic engineering ( fernie et al . given the ever increasing number of crop species for which full - genome sequences are becoming available it is highly likely that such studies will be greatly aided by the development of translational approaches both at the molecular ( mutwil et al . as yet one drawback of this approach that said , the development of a wide array of genetic materials including those resulting from tilling ( till et al . , 2006 ) and the adoption of every more sophisticated rapid screening technologies such as those afforded by viral induced gene silencing ( ruiz et al . , 1998 ; regardless , the forward genetic approach has at least two advantages ( i ) by taking a top - down approach it may uncover different levels of metabolic regulation within an experiment and ( ii ) given that the whole genome is considered , this approach is not restricted to evaluation of previously known enzymes or regulators thereof . while the forward genetic approach is in its infancy we are convinced that it will prove a highly powerful tool for the identification of novel mechanisms of metabolic regulation . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

indeed the question of how metabolism is regulated was first raised in the scientific era in which metabolism was first truly defined ( buchner , 1907 ; krebs and henseleit , 1932 ; plaxton , 1996 ; kornberg , 2000 ) . whilst the ability to regulate the rates of metabolic processes in response to cellular circumstance is a common feature of all organisms , it is particularly acute in sessile organism such as plants ( plaxton , 1996 ) . from a textbook perspective , metabolic regulation is classically divided into coarse and fine levels of control ( dennis et al . coarse control refers to long - term mechanisms that are energetically expensive and lead to changes in the total cellular population of a protein . by contrast , fine control describes generally fast ( and therefore energetically inexpensive ) regulatory devices that modulate the activity of pre - existing enzyme molecules . whilst this arbitrary division can be useful for descriptive purposes , recent reports suggest that several regulatory mechanisms can not be so easily defined and hence such classification is commonly regarded as outdated . however , unfortunately , despite the massive amount of data available regarding steady - state rna levels afforded by microarrays ( see for example the data stored in genevestigator ; http://www.genevestigator.ethz.ch ) and more recently by next generation sequence analyses ( see for example gonzalez - ballester et al . , 2010 , 2011 ; our understanding of regulation of central ( primary ) plant metabolism has been largely defined by the discovery of such features within the last 5060 years , whereas understanding of specialized ( secondary ) metabolism has made similar strides within the last 30 years ( for reviews see pichersky and gang , 2000 ; dauria and gershenzon , 2005 ; gachon et al . in brief , such mechanisms include ( i ) alteration in substrate or co - substrate concentration , ( ii ) variation in ph , ( iii ) allosteric effectors . the first , of these is essentially the most simple and certainly the most rapid to affect metabolic systems with the rate of an enzyme - catalyzed reaction proceeding more rapidly upon an increase in sub - saturating substrate a case that is common in vivo all enzyme reactions are , to a greater or lesser extent regulated in this manner . however , the situation is complicated by the fact that not all reactions display simple michaelis menton - like kinetics and by the fact that many co - substrates are shared by multiple reactions . for example , regulation of enzymes of the calvin cycle is well documented to be ph regulated ; stromal ph is 8.0 in that the light and 7.0 in the dark ( see dennis et al . , thirdly , allosteric effectors are immensely important in the regulation of plant metabolic networks , be they activators or inhibitors . within the major pathways of carbohydrate metabolism , several examples of the importance of such metabolites exist , including the 3 phosphoglycerate ( 3pga)/inorganic phosphate ( pi ) ratio in activating adp glucose pyrophosphorylase ( agpase ; preiss , 1982 ; tiessen et al . , 1993 understanding the function of a given enzyme within a biological process has until recently largely followed a set protocol by which novel genes associated with a specific process are identified by means of similar patterns of expression across a wide range of experiments and subsequently their function tested . this is initially carried out by analyzing the metabolite profiles of genotypes deficient in the expression of the gene . confirmation of kinetic properties of the enzyme either in planta ( in the case that the gene encodes the only isoform of an enzyme ) or following expression of the gene in a heterologous system lacking the activity is subsequently required ( see for details tohge and fernie , 2010 ) . , 2009 ) in terms of annotating the precise biochemical function of individual genes , it does not enable elucidation of the exact physiological function in vivo . in the last 25 years the roles of specific metabolic enzymes have been addressed via the use of transgenic plants ( stitt and sonnewald , 1995 ; lytovchenko et al . given that the chemical constituents of any life form determine the development and functioning of the organism ( sumner et al . , 2004a ; dauria and gershenzon , 2005 ) , strategies to characterize the chemical complement of the cell are becoming increasingly important . this regulated interconversion of compounds is perhaps the most important hallmark of plant metabolism , enabling the organism to respond to specific demands over the course of its life and on a minute - by - minute basis ( keuentjes and fernie , 2011 ) . , 2002 ) whereas secondary metabolism is often connected to interactions with environmental cues , including cell signaling , interspecies communication , and responses to biotic and abiotic stress ( see for example wink , 1988 ; mitchell - olds and pedersen , 1998 ; lehmann et al . although primary metabolic pathways are strongly conserved between species , quantitative variation is often observed , possibly related to the different growth characteristics of various species ( mitchell - olds and pedersen , 1998 ) . qualitative and quantitative variation in secondary metabolism is , however , much more extensive and it is widely accepted that secondary metabolism determines to a great extent the success of plant adaptation ( herms and mattson , 1992 ; pichersky and gang , 2000 ) . that said , we are only starting to explore the composition of the metabolome , let alone unravel all of the biosynthetic pathways leading to this diversity of chemical structures . in the current article we concentrate on results from broad screening of the natural genetic diversity of metabolism in arabidopsis rosettes and tomato fruit . following intensive statistical analysis , clear patterns of metabolic regulation can be demarcated via these approaches and a sub - set of these patterns can be resolved at the genetic level . we conclude that the screening of diverse genetic populations by metabolic profiling significantly adds to our understand metabolic regulation . study of metabolic traits in arabidopsis largely focuses on understanding the principles underlying metabolic regulation and the influence of metabolism on growth and development ( kliebenstein et al . , establishment of the genetic basis of quantitative traits commonly referred to as quantitative trait loci ( qtl ) , has often been hampered due to their complex multigenic inheritance and strong interactions with the environment . the principle of qtl mapping in segregating populations is based on genotyping of progeny derived from a cross between distinct genotypes for the trait under study . phenotypic values for the trait are then compared with molecular markers in the progeny to search for particular genomic regions showing statistically significant associations with the trait variation ( broman , 2001 ; slate , 2005 ) . over the past few decades , the ease with which such markers can be developed has facilitated qtl mapping studies of even the most complex traits ( borevitz and nordborg , 2003 ) . in plants , the use of immortal mapping populations consisting of homozygous individuals that , at least theoretically , can be propagated indefinitely is preferred because it permits replication and multiple analyses of the same population . homozygous populations can be obtained by repeated selfing , as is the case for recombinant inbred lines ( rils ) , but also by induced chromosomal doubling of haploids , such as for doubled haploids ( dhs ; han et al . in plants , rils and nils are the most common types of experimental populations used for the analysis of quantitative traits ( for an illustration of these populations see figure 1 ) . despite the similarities between these two types of mapping populations , large differences exist in the genetic makeup of the respective individuals and the resulting mapping approach . furthermore , the wide variation of morphological and developmental traits among individuals within most ril populations may hamper analysis of traits requiring the same growth and developmental stage of the individual lines . however , the presence of a single introgressed segment limits testing for genetic interactions and thereby the detection of epistatic qtl . since most of the genetic background is identical for all lines , nils show more limited developmental and growth variation , increasing the homogeneity of growth stage within experiments . genome - wide sets of nils and rils , descending from identical intercrosses , that allow mapping to chromosomal sections have been described in arabidopsis and empirical comparative studies have been performed between the two population types ( keurentjes et al . these studies illustrate the complementary benefits of both resources , facilitating the genetic dissection of various quantitative traits in arabidopsis . having extensively described the approaches , we now detail the biological significance of results obtained to date in arabidopsis . although many simple metabolic traits affecting protein , oil , and starch content have been studied using targeted approaches ( see moose and mumm , 2008 ; fernie and schauer , 2009 ) , the adoption of methods able to detect the levels of multiple metabolites at once greatly expanded our ability to pose questions about regulation at the pathway and network level ( sweetlove et al . the first publication of note focused on a ril population derived from a cross between the landsberg erecta ( ler ) and cape verde islands ( cvi ) accessions and evaluated the variance of some 2000 mass peaks across this population ( keurentjes et al . comparison of these data with those of previous targeted work showed that these co - locations were at positions of known regulators of glucosinolate metabolism to which peptide methionine sulfoxide reductase ( mam ) and qtl for production of alkenyl or hydroxyalkyl glucosinolates ( aop ) mapped ( kliebenstein et al . further work from this group focused on parallel analysis of 15 enzyme activities in tandem with their corresponding transcript levels and a set of relevant metabolites . the results revealed that traits affecting primary metabolism are often correlated and many activity qtl co - localize with expression qtl ( although a fair number do not , suggesting that such multilevel approaches will be highly useful for distinguishing between transcriptional and metabolic control ( keurentjes et al . as an extension of this work they next performed a multiplexed transcriptome , proteome , and metabolome study on the same material , combining these data with publically available data ( fu et al . the surprising finding of this study was that following mapping of over 40000 molecular and 100 phenotypic traits , there were only six qtl hotspots . , metabolites were not treated in isolation but rather evaluated with respect to the influence they exerted toward plant growth with a metabolic signature for high growth being defined from results obtained in the col-0/c24 rils ( meyer et al . in this study , metabolic pathway - derived candidate genes were found for 2467% of all tested metabolite qtl in the database aracyc 3.5 , demonstrating the power of this approach to identify possible sites of metabolic regulation . a recent paper describes the identification of a cytosolic isoform of fumarase as the causal gene underlying traits of decreased fumarate and increased malate ( brotman et al . this result represents an elegant proof - of - concept study and beautifully fits the cross - over theorem , which states that if an equilibrium reaction of a linear pathway is inhibited , a build - up of substrate and a depletion of product of that reaction will occur ( rolleston , 1972 ) . an illustration of the power of this theorem is shown in its application to starch synthesis in potato , which prompted a successful search for a novel post - translational modifier of the agpase enzyme ( stitt et al . bearing this example in mind , analyses of metabolite ratios in advanced genetic populations will likely prove to be an important route to identify previously uncharacterized mechanisms of metabolic regulation in the future . the rapidly increasing availability of genome information for multiple arabidopsis ecotypes ( weigel and mott , 2009 ; schneeberger and weigel , 2011 ) , alongside the increase in the number of groups performing metabolomic analyses should accelerate gene discovery . this approach was initiated to assess the natural variance in enzyme activities ; the first study examined the activities of seven enzymes across 24 arabidopsis ecotypes ( cross et al . , 2006 ) whilst the second evaluated the activity of the most abundant protein in photosynthetic tissues , ribulose-1,5-bisphosphate - carboxylase/-oxygenase ( rubisco ) , across the abovementioned col-0/c24 ril population ( sulpice et al . , 2007 ) in the former study it was observed that enzyme activities largely vary on mass , but are not well correlated to the levels of the metabolites measured in the same sample ( cross et al . the latter study described application of a novel rubisco assay to describe the characteristics of this enzyme in 118 arabidopsis accessions , defining two loci for rubisco activity and two for rubisco activation state ( sulpice et al . transcript profiling in 21 accessions further revealed coordinated changes in expression of more than 70 carbon - regulated genes , identifying two ( myo - inositol-1-phosphate synthase and a kelch - domain protein ) whose transcripts correlate with biomass . kliebenstein and co - workers have also taken both association mapping and qtl - based approaches in their studies , profiling both primary and secondary metabolites alongside studies of gene expression ( kliebenstein et al . , they have expended considerable effort to understand whole genome expression qtl , revealing a high incidence of both cis- and trans - acting qtl , including non - additive variation such as epistasis and transgressive segregation as well as genetic variation affecting entire transcriptional networks ( kliebenstein et al . however , comparison of the resultant data from this study and that described above on the ril population revealed that the higher level of genetic variation in the accession population was not reflected by a higher variation in the metabolome . while arabidopsis is the best characterized plant species with respect to natural variation in the metabolome , an increasing number of studies are being carried out in crop species . , adoption of maldi / tof - ms to individual mutagenized plants and solanum pennellii ils has been used for screening of fruit containing high levels of nutraceutical compounds such as carotenoids ( fraser et al . these studies all reveal large diversity within populations at the genetic and metabolic levels and hint that such information is likely of high value for breeding programs ( fernie and schauer , 2009 ) . as we will discuss in the following paragraphs it is also highly interesting material in which to study metabolic regulation . in maize a range of compositional traits including protein , oil , fatty acid , amino acid , and organic acid content was carried out in two independent maize hybrids grown at three separate locations ( harrigan et al . this important proof - of - concept study demonstrated the high non - genetic variability in crop composition , illustrating the need for replicated trials . reconstructed metabolic networks display a higher network density in most hybrids as compared to the corresponding inbred lines , suggesting that metabolite levels are subject to tighter control . association mapping subsequently identified a novel amino acid substitution associated with a variation in isocitrate dehydrogenase activity , demonstrating that this approach can identify putative functional sites . in total , 73 significant qtl that influence the activity of these 10 enzymes as well as 8 qtl that influence biomass were identified . , genes that encode enzymes operating within the pathways ) , except for single cis - qtl for nitrate reductase , glutamate dehydrogenase , and shikimate dehydrogenase ; the low frequency and low additive magnitude compared with trans - qtl indicates that , at least in this population , cis - regulation is relatively unimportant versus trans - regulation . in this species , a broad profiling of fruit volatiles , which are extremely important flavor components , in a population consisting of 74 solanum lycopersicum s. pennellii ils yielded 100 qtl that were conserved across harvests ( tieman et al . metabolic and flux profiling of one of these qtl was instrumental in defining the pathway for synthesis of important phenylalanine - derived aromatic compounds in the fruit ( tieman et al . thirty additional qtl that affect the volatile emissions of red - ripe fruit were identified in a second population ofils derived from a cross between s. lycopersicum and s. habrochaites grown in multiple seasons and locations ( mathieu et al . , 2005 ) , whilst the primary metabolite composition of five wild species of tomato was assessed in comparison to the cultivated tomato ( schauer et al . for this purpose , the s. penellii ils were grown alongside lines heterozygous for the introgression ( ilhs ) allowing evaluation of both heritability and the mode of inheritance . comparative study of the ils and ilhs revealed that most of the metabolic qtl were dominant with a considerable number displaying additive or recessive mode of action and only a negligible number displaying overdominant phenotypes . interestingly , the mode of inheritance was quantitatively different between diverse compound classes and several metabolite pairs displayed a similar mode of inheritance at the same chromosomal loci , suggesting that the variation is likely to be mediated by enzymes involved in their interconversion ( schauer et al . metabolite profiling led to the discovery of ils producing different acyl chain substitutions on acyl sugar metabolites as well as two regions quantitatively influencing acyl sugar content . a second flavor volatile qtl responsible for synthesis of methylsalicylate was shown to be the consequence of altered expression qtl ( eqtl ) of the gene encoding the biosynthetic enzyme , salicylic acid methyltransferase ( tieman et al . an early study utilizing the s. pennellii ils mapped the moderate qtl brix 9 - 2 - 5 to a 484-bp region of the cell wall invertase gene lin5 ( fridman et al . qtl analysis of five different tomato species delimited the functional polymorphism of brix 9 - 2 - 5 to an amino acid at the fructosyl binding site near the catalytic site of the invertase crystal with enzyme kinetic analysis of recombinant protein demonstrating that the s. pennellii allele was more efficient in degrading sucrose ( fridman et al . mapping the chromosomal locations of these enzymes , it was possible to define the map positions of 24 genes ( with two of the putative gene functions being encoded by two independent genes ) . eight co - localized with bcaa qtl including those encoding ketol - acid reductoisomerase ( kari ) , dihydroxy - acid dehydratase ( dhad ) , and isopropylmalate dehydratase ( ipmd ; kochevenko and fernie , 2011 ) . quantitative evaluation of the expression of these genes revealed that the s. pennellii allele exhibited altered expression of ipmd , whereas expression of kari and dhad were invariant across the genotypes . whilst the antisense inhibition of ipmd resulted in increased bcaa , the antisense inhibition of kari or dhad had no effect on fruit bcaa contents ( kochevenko and fernie , 2011 ) . given the ever increasing number of crop species for which full - genome sequences are becoming available it is highly likely that such studies will be greatly aided by the development of translational approaches both at the molecular ( mutwil et al . , 1998 ; regardless , the forward genetic approach has at least two advantages ( i ) by taking a top - down approach it may uncover different levels of metabolic regulation within an experiment and ( ii ) given that the whole genome is considered , this approach is not restricted to evaluation of previously known enzymes or regulators thereof . while the forward genetic approach is in its infancy we are convinced that it will prove a highly powerful tool for the identification of novel mechanisms of metabolic regulation .

alzheimer disease ( ad ) is a progressive central nervous system degeneration , which is characterized by a gradual decline in cognitive abilities and activities of daily living . the most common symptoms of ad are an expanding variety of cognitive disorders , which usually starts with dysfunction of memory , and , as the ad progresses , involves language , orientation , praxis , attention , and perception functions . the proteinopathy , cholinergic deficit , and neurodegeneration in early ad involves brain areas responsible for noncognitive functions , including structures and pathways of vision [ 48 ] . visual changes occur early in the course of ad and have a potential role as noninvasive biomarkers of ad - related neurodegeneration . ad is associated with a wide variety of pathological changes in the retina and brain visual system . some of the disorders of visual function may become noninvasive markers for the diagnosis of ad , and also may provide a better understanding of the mechanisms of visual perception per se [ 917 ] . one of the most promising and potentially insightful provisional biomarker is color vision [ 1018 ] . some researchers have reported dysfunctional color vision and color memory in ad patients [ 1820 ] . some research reports indicate deficits of color perception at specific color axes according to the rgb ( red - green - blue ) system , corresponding to the maximum sensitivity to different light wavelengths of 3 types of retinal cone photoreceptors , while others did not identify selective color vision deficits . most research reports have found abnormalities in the tritan axis in ad patients [ 14,1820 ] , while reports from other research groups suggest that the deutan axis may be dysfunctional . cholinergic neurons of the nucleus basalis of meynert have a number of important functions in modulation of visual perception . in ad patients , the nucleus basalis is one of the brain structures involved , degenerating at the earliest stages of the disease and leading to the cholinergic deficit in the cerebral cortex . in addition , according to the 2-streams ( dorsal and ventral ) hypothesis of the neural processing of visual perception , the ventral stream is associated with form and color representation ; it goes to areas of the inferior temporal lobe and has strong connections to the medial temporal lobe , which encodes long - term memories . this indicates that research on disorders of color perception may be a promising strategy in search of functional markers of early ad . among the provisional visual markers of ad , one very interesting and still poorly understood phenomenon fechner illusory colors has received no attention in ad research . our search of databases of published research articles found no scientific reports dealing with the investigation of fechner illusory colors in ad . fechner illusory colors are also called subjective colors , fechner - behnam subjective colors , and pattern - induced flicker colors ( pifc ) . fechner colors are the illusory colors that are perceived from the repeated flashing of black and white patterns . benham s disk ( also known as benham s top ) is a spinning disk with an alternating black and white pattern developed to create the experience of pifc . the fechner illusory colors are experienced when the flicker rate of black and white stimuli is neither too slow nor too fast . usually pfics are seen when the speed of rotation ( frequency ) is going from 120 revolutions per minute ( rpm ) or 2 hz up to 360 rpm or 6 hz . the most common method for measuring fechner illusory colors is some form of color matching to determine the color experienced by the participant . while pfics have some individual variation , there is a remarkable degree of agreement across observers . initially , the most popular explanation of how the monochromatic or neutral flickering stimuli produce the perception of color was based on the difference in information processing speed by different types of retinal cones , especially by the relative slowness of the blue or short - wavelength cone ( s cone ) compared to the speed of the other 2 cones the green or medium - wavelength cone ( m cone ) and the red or long - wavelength cone ( l - cone ) . differences in responsivity spectra of s , m , and l type cone cells could not be an explanation , because the stimulus was monochromatic ( neutral ) black and white and despite that , they produced several concentric bands of different colors on the spinning disk . also , there were no differing compositions of red , green , and blue components , which are able to imitate the real physical colors ( which is the case for computer monitors and tv screens ) . if illusory colors are experienced due to the slowness of the s cones , then the most probable neuroanatomical substrate of pfics are the retinal ganglion cells , responsible for the blue - yellow opponent process . despite the seeming plausibility of this explanation , there are some facts that can not be explained by the difference of information processing speed in different types of cones . some more recent fmri investigations showed that fechner illusory colors may be a cortical phenomenon associated with a ventral stream of visual information ( ie , with the interaction between visual areas v1 , v2 , and v4 ) , which anatomically is ongoing at the inferior temporal cortex . the inferior temporal cortex has numerous connections with the medial temporal lobe and is affected by the neurodegeneration early in the course of ad . the purpose of the study was to explore and compare perception of fechner illusory colors in ad patients and normal controls . the present prospective , cross - sectional , case - control study was carried out at the alzheimer disease and dementia subunit of the department of neurology , vuh santariskiu clinics . we enrolled 40 ad patients ( the ad group ) and 40 control participants ( the control group , cg ) , who were matched by age , sex , and education . informed consent forms ( icf ) , which were approved by the vu hospital santariskiu clinics biomedical ethics commission , were signed by all participants . the research protocol and icf were approved by the vu hospital santariskiu clinics biomedical ethics commission ( approval no . inclusion criteria for ad group participants were : 1 ) ad diagnosis established based on nincds - adrda criteria ; 2 ) mild or moderate dementia based on mmse score 14 or more ; 3 ) at least 65 years old ; 4 ) education at least 8 years ; 5 ) hachinski index equal to or less than 4 ; 6 ) yesavage gds depression score equal to or less than 19 ; 7 ) no significant vascular signs or other neurodegenerative pathology results of the head - on ct or mri . exclusion criteria for ad group participants were : 1 ) diagnosis or any signs of any other than ad neurodegenerative disease ; 2 ) diagnosis of any significant psychiatric disorder , such as schizophrenia , delirium , psychosis , or other ; 3 ) diagnosis or any symptoms of significant cardiovascular , hepatic , or metabolic disorders ; 4 ) history or evidence of past or current drug or alcohol abuse . inclusion criteria for control group participants were : 1 ) normal cognition based on mmse score 27 or more ; 2 ) at least 65 years old ; 3 ) hachinski index equal to or less than 4 ; 4 ) yesavage gds depression score equal to or less than 19 ; 5 ) education at least 8 years . for the cg group , mini - mental state examination ( mmse ) score was used as a measure of overall cognitive status . we used a benham s disk attached to a device of original construction to control the speed and direction of rotation . the device could rotate the benham disk at speeds of 90 rpm , 120 rpm , 150 rpm , 180 rpm , and 210 rpm clockwise and counter - clockwise . the device was constructed and calibrated at the gediminas technical university , vilnius , lithuania . the rotation speed of 210 rpm was chosen for the main study after initial proof - of - concept investigation because it had less flicker and greater stability and saturation of observed colors . we used a variant of the benham disk with one half of the disk colored solid black and the other half containing 4 perfectly concentric arcs consisting of 4 black lines . the observation angle was 11.5. letters from a to d were assigned to the bands of stripes for the clockwise rotating disk and letters e to h were assigned for the bands of the counter - clockwise rotating disk for the purposes of further analysis ( figure 1 ) . we used a 210 rpm ( revolutions per minute ) speed of rotation , or 3.5 hz , for the reasons mentioned above . the benham disk was covered and not visible to the participants until the disk achieved a stable angular speed . the pc monitor used to the evaluate the color template was switched off before the participant provided answers regarding the colors of the bands of stripes on the rotating disk , and only after that the monitor was switched on and the participant was asked to indicate the most resembling color on the screen . the names of the colors were not used for further analysis due to heavy influence of the color categorization in the language and a wide variety of color vocabulary used to describe the illusory fechner colors , but the naming was needed to be sure that the participants are actually seeing some kind of color . rgb primary , secondary , and tertiary colors with the addition of 4 neutral colors were used to standardize the participants responses . the participants were asked to choose the color from the template on the monitor that most resembles the illusory color they had seen . the template for color matching was constructed from vertical bands of rgb primary , secondary , and tertiary colors with the addition of 4 neutral colors . a forced - choice paradigm was used , which was necessary to standardize the responses of participants . codes and order of color bands used in the template for matching fechner illusory colors with rgb colors are provided in table 1 . numbers of colors were used for the purposes of further evaluation , but were not visible on the screen . we used the shapiro - wilk test for the verification of normality of data distribution . comparisons between groups were performed using parametric analysis . for comparison of the groups on the basis of categorical variables , the present prospective , cross - sectional , case - control study was carried out at the alzheimer disease and dementia subunit of the department of neurology , vuh santariskiu clinics . we enrolled 40 ad patients ( the ad group ) and 40 control participants ( the control group , cg ) , who were matched by age , sex , and education . informed consent forms ( icf ) , which were approved by the vu hospital santariskiu clinics biomedical ethics commission , were signed by all participants . the research protocol and icf were approved by the vu hospital santariskiu clinics biomedical ethics commission ( approval no . inclusion criteria for ad group participants were : 1 ) ad diagnosis established based on nincds - adrda criteria ; 2 ) mild or moderate dementia based on mmse score 14 or more ; 3 ) at least 65 years old ; 4 ) education at least 8 years ; 5 ) hachinski index equal to or less than 4 ; 6 ) yesavage gds depression score equal to or less than 19 ; 7 ) no significant vascular signs or other neurodegenerative pathology results of the head - on ct or mri . exclusion criteria for ad group participants were : 1 ) diagnosis or any signs of any other than ad neurodegenerative disease ; 2 ) diagnosis of any significant psychiatric disorder , such as schizophrenia , delirium , psychosis , or other ; 3 ) diagnosis or any symptoms of significant cardiovascular , hepatic , or metabolic disorders ; 4 ) history or evidence of past or current drug or alcohol abuse . inclusion criteria for control group participants were : 1 ) normal cognition based on mmse score 27 or more ; 2 ) at least 65 years old ; 3 ) hachinski index equal to or less than 4 ; 4 ) yesavage gds depression score equal to or less than 19 ; 5 ) education at least 8 years . for the cg group , mini - mental state examination ( mmse ) score was used as a measure of overall cognitive status . we used a benham s disk attached to a device of original construction to control the speed and direction of rotation . the device could rotate the benham disk at speeds of 90 rpm , 120 rpm , 150 rpm , 180 rpm , and 210 rpm clockwise and counter - clockwise . the device was constructed and calibrated at the gediminas technical university , vilnius , lithuania . the rotation speed of 210 rpm was chosen for the main study after initial proof - of - concept investigation because it had less flicker and greater stability and saturation of observed colors . we used a variant of the benham disk with one half of the disk colored solid black and the other half containing 4 perfectly concentric arcs consisting of 4 black lines . the observation angle was 11.5. letters from a to d were assigned to the bands of stripes for the clockwise rotating disk and letters e to h were assigned for the bands of the counter - clockwise rotating disk for the purposes of further analysis ( figure 1 ) . we used a 210 rpm ( revolutions per minute ) speed of rotation , or 3.5 hz , for the reasons mentioned above . the benham disk was covered and not visible to the participants until the disk achieved a stable angular speed . the pc monitor used to the evaluate the color template was switched off before the participant provided answers regarding the colors of the bands of stripes on the rotating disk , and only after that the monitor was switched on and the participant was asked to indicate the most resembling color on the screen . the names of the colors were not used for further analysis due to heavy influence of the color categorization in the language and a wide variety of color vocabulary used to describe the illusory fechner colors , but the naming was needed to be sure that the participants are actually seeing some kind of color . rgb primary , secondary , and tertiary colors with the addition of 4 neutral colors were used to standardize the participants responses . the participants were asked to choose the color from the template on the monitor that most resembles the illusory color they had seen . the template for color matching was constructed from vertical bands of rgb primary , secondary , and tertiary colors with the addition of 4 neutral colors . a forced - choice paradigm was used , which was necessary to standardize the responses of participants . codes and order of color bands used in the template for matching fechner illusory colors with rgb colors are provided in table 1 . numbers of colors were used for the purposes of further evaluation , but were not visible on the screen . we used the shapiro - wilk test for the verification of normality of data distribution . comparisons between groups were performed using parametric analysis . for comparison of the groups on the basis of categorical variables , the age ( p=0.91 ) , education ( p=0.21 ) , and sex ( p=0.82 ) of ad and cg groups did not differ . demographic characteristics and mmse results in ad and cg groups are presented in table 2 . the number of ad and cg participants reporting the perception of fechner illusory color on each arc when the benham s disk is rotating clockwise and counter - clockwise are provided in table 3 , based on the matching template color indicated by the participant . reversing of the colors when the direction of rotation reverses is evident in table 3 . this reversal of color order , which is typical for healthy controls , remains intact in the ad group . the numbers of ad and cg participants perceiving spectral versus neutral color on each arc of lines when the benham s disk is rotating clockwise ( arcs a to d ) and counter - clockwise ( arcs e to h ) is provided in table 4 . for further analysis , the grouping of fechner illusory colors according to blue - yellow opponent process theory was performed . based on data provided in table 1 , pure blue color without any constituent parts of red or green ( hexadecimal html code # 0000ff ) was assigned to group 1 . the mixed colors containing both blue and yellow ( red plus green , only red , or only green by rgb system ) components were assigned to group 2 . the colors lacking any amount of blue constituent and composed only from components of the yellow part of the blue - yellow opponent axis ( ie , colors containing red plus green , or only red , or only green based on rgb system ) were assigned to group 3 . the scheme of assigning the matching template colors to the groups based on the blue - yellow opponent process theory is provided in table 5 . the differences in perception of fechner illusory colors were grouped according to the blue - yellow opponent process constituent parts in ad and cg ( figure 2 ) . the age ( p=0.91 ) , education ( p=0.21 ) , and sex ( p=0.82 ) of ad and cg groups did not differ . demographic characteristics and mmse results in ad and cg groups are presented in table 2 . the number of ad and cg participants reporting the perception of fechner illusory color on each arc when the benham s disk is rotating clockwise and counter - clockwise are provided in table 3 , based on the matching template color indicated by the participant . reversing of the colors when the direction of rotation reverses is evident in table 3 . this reversal of color order , which is typical for healthy controls , remains intact in the ad group . the numbers of ad and cg participants perceiving spectral versus neutral color on each arc of lines when the benham s disk is rotating clockwise ( arcs a to d ) and counter - clockwise ( arcs e to h ) is provided in table 4 . for further analysis , the grouping of fechner illusory colors according to blue - yellow opponent process theory was performed . based on data provided in table 1 , pure blue color without any constituent parts of red or green ( hexadecimal html code # 0000ff ) was assigned to group 1 . the mixed colors containing both blue and yellow ( red plus green , only red , or only green by rgb system ) components were assigned to group 2 . the colors lacking any amount of blue constituent and composed only from components of the yellow part of the blue - yellow opponent axis ( ie , colors containing red plus green , or only red , or only green based on rgb system ) were assigned to group 3 . the scheme of assigning the matching template colors to the groups based on the blue - yellow opponent process theory is provided in table 5 . the differences in perception of fechner illusory colors were grouped according to the blue - yellow opponent process constituent parts in ad and cg ( figure 2 ) . ad patients retain the ability to see fechner illusory colors , but they significantly more frequently report neutral colors ( e.g. , shades of gray , black , white ) than participants from the control group ( table 4 ) . at 210 rpm , clockwise , the outermost ( 1 ) arc appears dark saturated blue , the second set of lines pale magenta , the third arc of lines green , and the innermost ( 4 ) arc of lines looks clear or luminescent yellow , orange , or red . when the direction of benham disk rotation is changed to counter - clockwise , the colors reverse order . this reversal of color order when changing the direction of disk rotation remains intact in ad subjects . a possible explanation of why the study participants see colors when looking at the rotating achromatic stimulus disk may be that the cone receptors in the human retina respond at different rates to red , green , and blue . even more importantly , the latencies at the center and the surroundings differ for the different types of color - specific retinal ganglion cells . substantial evidence exists that pifcs appear due to neural activity in a wide range of nervous system structures , from the retina to the visual cortex , and there are some indications that the blue - yellow opponent process may explain subjects experience of all the different fechner colors . the color opponent process is a color theory that indicates that the human visual system interprets information about color by processing signals from cones and rods in an antagonistic manner . the 3 types of cones ( l for long , m for medium , and s for short wavelengths of light ) have significant overlap in the wavelengths of light to which they respond ; therefore , it is more efficient for the human visual system to record differences between the responses of cone cells , rather than to analyze and compare individual responses from each type of cone . the opponent process theory posits the existence of 3 different opponent channels : red vs. green , blue vs. yellow , and black vs. white ( achromatic ) . the opponent process theory accounts for neural mechanisms that receive information from the 3 types of cones and process it in a more efficient way , finally producing the various phenomena of color experience . according to the opponent process theory , 2 other types of cells are involved in response to color stimuli : bipolar cells and ganglion cells . the parvocellular layer cells process the color - related information and may be assigned to 2 groups : one of these groups processes the information about the difference of activity of l and m cones , while the other group processes the difference between s cones and a combined activity of both l and m cones . the first type of cell is responsible for red - green opponent process axis and the second type is responsible for blue - yellow opponent process axis ; specifically , the latencies of processes at the center and the surrounding differ among the different types of color - specific ganglion cells . our results show that a significant difference in reporting various colors between ad and cg groups . very few ad patients reported pure blue color ( # 0000ff ) when viewing the outermost arc of lines on a benham disk rotating clockwise and the innermost arc of lines on a disk rotating counter - clockwise ; whereas control group participants most frequently reported clear and saturated pure blue in these arcs on the disk rotated in the same direction ( table 3 ) . when colors seen by the study participants moved to the yellow part of the blue - yellow opponent axis , the difference between colors reported by ad and cg groups gradually vanished . importantly , the 4 colors visible on the disk by the healthy control group participants do not follow the rgb - derived sequence , usually provided in the published literature as blue green yellow red . magenta ( or rose ) green yellow ( or orange , or red ) . this clearly is not the sequence expected if a unidimensional wavelength - dependent process is underlying the illusory fechner colors . the sequence of colors reported by participants in our study is explained much more reasonably by the opponent process theory . even more unexpected is that if all phenomenal properties of fechner illusory colors are explained exclusively by the blue - yellow opponent axis , as is contended by schramme et al . , the arc next to the one that appears as blue should be seen as greenish . in our study , the second arc after blue most frequently was seen as magenta or rose by the participants of both groups , and only the third one was green . when the disk was rotating clockwise , there was much more scattered reporting of colors by ad group subjects , clearly skewed to the red but not to the green ( table 3 ) . it may be hypothesized that the colors observed on the outer and inner arcs of lines , and which are at the greatest distance from each other , are reasonably explained by the blue - yellow opponent process . the 2 middle arcs ( the 2 and 3 clockwise and counter - clockwise ) exhibit signs of intrusion of the red - green opponent process , which significantly affects visible colors only in a small range located inside of a much wider interval , which is under the influence of the blue - yellow opponent process . if this explanation of 2 opponent processes involved is feasible , there still remains the difference between blue - yellow opponent process , responsible for coloring of distant arcs and red - green opponent process , which is responsible for colors of the 2 middle arcs , located near each other : when the disk is rotated clockwise , colors of the outermost and innermost arcs follow the direction from shorter ( blue ) to longer ( yellow , or a sum of red and green ) wavelengths ; in contrast to this , the colors of 2 middle arcs follow the direction from longer ( red ) to shorter ( green ) wavelengths . furthermore , red and green seem unequal in this double - opponent process explanation because the ad group tended to shift reported colors from green to red in both extremities of the wide blue - yellow opponent process activity range ( table 3 ) . this can not be explained just by the action of the 2 opponent processes at different ranges . consequently , there is good reason to suppose that the processes ongoing at the retinal ganglion cells level are not sufficient to explain the illusory color sequence in its entirety . thus , the phenomenon of fechner illusory colors originates from neural activity in the retina and the more central structures of the visual system . the fmri research findings reported by tanabe et al suggest that the visual cortex , which processes pattern recognition , may be involved in the generation of illusory colors . the results from this fmri study indicate that modulation from v4 to v2 to v1 plays a significant role in the perception of fechner illusory colors . the utilization of pifcs in medical diagnosis and the biological interpretation of the neural network involved would also benefit from research on fechner illusory colors in ad . the decline in blue opponent perception , if properly standardized , may be used as a cheap and noninvasive diagnostic marker in early diagnosis of ad as an addition to other cognitive , electrophysiologic , behavioral , and emotional regulation methods [ 2831 ] . on the other hand , the pattern of decline in perception of fechner illusory colors in people with ad can be helpful for further research in order to understand the nature of visual perception of color , and may have even broader implications for the fundamental mind - body relationship . this kind of empirical research may lead to reconsideration of some basic premises of such renowned thought experiment as mary s room or mary the super - scientist proposed by frank jackson and intended to argue for the existence of qualia and against physicalism . bearing in mind the results of research using fechner illusory colors , the initial basic premise of this thought experiment about the possibility of existence of an entirely black and white room , where mary the super - scientist is working without any possibility to see spectral colors , seems quite problematic due to the established fact that movement of black and white patterns may produce the experience of illusory colors , indistinguishable from real physical spectral colors . this means that isolation of mary the super - scientist in the black and white room does not provide a possibility to deprive her of subjectively experiencing colors . the reversal of color order when changing the direction of disk rotation remains intact in ad patients . the visibility of spectral illusory colors is significantly less frequent in ad patients compared with normal controls . the perception of pure blue fechner color was markedly lower in the ad group than in the control group . the illusory colors reported by ad patients when observing the blue - producing arcs of lines are much more scattered around the pure blue color than in the control group . while a clear shift to the yellow opponent is observed among ad patients , this shift is asymmetric for red and green constituent parts of the yellow opponent . a larger shift to the red constituent is found instead of green , which makes it harder to explain all the phenomena of fechner illusory colors solely on the basis of blue - yellow opponent process , and definitely does not correspond to the sequence of physical spectral colors . the decreased perception of blue opponent was very large , but we did not observe any impairment in perception of yellow opponent colors that do not contain blue subcomponent according to the rgb system . there is impaired perception of intermediary colors containing the blue subcomponent , but less pronounced than for pure blue . this impaired pattern of visibility of illusory colors in ad patients could be explained by the prevailing disorder in the blue - yellow opponent axis , with some noticeable effect of disorder in the red - green opponent axis only in the middle part of the much broader blue - yellow axis , which may indicate the cortical , but not the retinal , origin of the disorder in perception of fechner illusory colors in people with ad .

backgroundalzheimer disease ( ad ) primarily affects cognition . a variety of visual disorders was established in ad . fechner illusory colors are produced by a rotating disk with a black and white pattern . the purpose of our research was to explore the perception of illusory colors in ad.material/methodsw recruited 40 ad patients ( mmse 14 ) and 40 normal controls ( cg group ) matched by age , education , gender in this prospective , cross - sectional , case - control study . an achromatic benham s disk attached to a device to control the speed and direction of rotation was used to produce illusory colors . primary , secondary , and tertiary rgb system colors were used for matching of illusory and physical colors.resultssubjects in the ad group perceived less illusory colors in 5 arcs ( p<0.05 ) of the 8 arcs assessed . the biggest difference was found between ad and cg groups for pure blue ( 2=26.87 , p<0.001 clockwise , 2=22.75 , p<0.001 counter - clockwise ) . groups did not differ in perception of pure yellow opponent colors ( p>0.05 ) . mixed colors of the blue - yellow axis were perceived less often in ad , but more frequently than pure blue ( # 0000ff ) . the sequence of colors on benham s disk followed a complex pattern , different from the order of physical spectral colors and opponent processes - based colors.conclusionsad patients retained reduced perception of illusory colors . the perception of pure blue illusory color is almost absent in ad . the asymmetrical shift to the yellow opponent is observed in ad with red prevailing over green constituent . this may indicate cortical rather than retinal impairment .

Background Material and Methods Study participants Inclusion\exclusion criteria Investigation tools Statistical analysis Results Demographic characteristics and MMSE scores Discussion Conclusions

alzheimer disease ( ad ) is a progressive central nervous system degeneration , which is characterized by a gradual decline in cognitive abilities and activities of daily living . the most common symptoms of ad are an expanding variety of cognitive disorders , which usually starts with dysfunction of memory , and , as the ad progresses , involves language , orientation , praxis , attention , and perception functions . visual changes occur early in the course of ad and have a potential role as noninvasive biomarkers of ad - related neurodegeneration . ad is associated with a wide variety of pathological changes in the retina and brain visual system . some of the disorders of visual function may become noninvasive markers for the diagnosis of ad , and also may provide a better understanding of the mechanisms of visual perception per se [ 917 ] . some research reports indicate deficits of color perception at specific color axes according to the rgb ( red - green - blue ) system , corresponding to the maximum sensitivity to different light wavelengths of 3 types of retinal cone photoreceptors , while others did not identify selective color vision deficits . most research reports have found abnormalities in the tritan axis in ad patients [ 14,1820 ] , while reports from other research groups suggest that the deutan axis may be dysfunctional . in ad patients , the nucleus basalis is one of the brain structures involved , degenerating at the earliest stages of the disease and leading to the cholinergic deficit in the cerebral cortex . in addition , according to the 2-streams ( dorsal and ventral ) hypothesis of the neural processing of visual perception , the ventral stream is associated with form and color representation ; it goes to areas of the inferior temporal lobe and has strong connections to the medial temporal lobe , which encodes long - term memories . among the provisional visual markers of ad , one very interesting and still poorly understood phenomenon fechner illusory colors has received no attention in ad research . our search of databases of published research articles found no scientific reports dealing with the investigation of fechner illusory colors in ad . fechner illusory colors are also called subjective colors , fechner - behnam subjective colors , and pattern - induced flicker colors ( pifc ) . fechner colors are the illusory colors that are perceived from the repeated flashing of black and white patterns . benham s disk ( also known as benham s top ) is a spinning disk with an alternating black and white pattern developed to create the experience of pifc . the fechner illusory colors are experienced when the flicker rate of black and white stimuli is neither too slow nor too fast . initially , the most popular explanation of how the monochromatic or neutral flickering stimuli produce the perception of color was based on the difference in information processing speed by different types of retinal cones , especially by the relative slowness of the blue or short - wavelength cone ( s cone ) compared to the speed of the other 2 cones the green or medium - wavelength cone ( m cone ) and the red or long - wavelength cone ( l - cone ) . differences in responsivity spectra of s , m , and l type cone cells could not be an explanation , because the stimulus was monochromatic ( neutral ) black and white and despite that , they produced several concentric bands of different colors on the spinning disk . also , there were no differing compositions of red , green , and blue components , which are able to imitate the real physical colors ( which is the case for computer monitors and tv screens ) . if illusory colors are experienced due to the slowness of the s cones , then the most probable neuroanatomical substrate of pfics are the retinal ganglion cells , responsible for the blue - yellow opponent process . some more recent fmri investigations showed that fechner illusory colors may be a cortical phenomenon associated with a ventral stream of visual information ( ie , with the interaction between visual areas v1 , v2 , and v4 ) , which anatomically is ongoing at the inferior temporal cortex . the purpose of the study was to explore and compare perception of fechner illusory colors in ad patients and normal controls . the present prospective , cross - sectional , case - control study was carried out at the alzheimer disease and dementia subunit of the department of neurology , vuh santariskiu clinics . we enrolled 40 ad patients ( the ad group ) and 40 control participants ( the control group , cg ) , who were matched by age , sex , and education . inclusion criteria for ad group participants were : 1 ) ad diagnosis established based on nincds - adrda criteria ; 2 ) mild or moderate dementia based on mmse score 14 or more ; 3 ) at least 65 years old ; 4 ) education at least 8 years ; 5 ) hachinski index equal to or less than 4 ; 6 ) yesavage gds depression score equal to or less than 19 ; 7 ) no significant vascular signs or other neurodegenerative pathology results of the head - on ct or mri . for the cg group , mini - mental state examination ( mmse ) score was used as a measure of overall cognitive status . we used a benham s disk attached to a device of original construction to control the speed and direction of rotation . the device could rotate the benham disk at speeds of 90 rpm , 120 rpm , 150 rpm , 180 rpm , and 210 rpm clockwise and counter - clockwise . we used a variant of the benham disk with one half of the disk colored solid black and the other half containing 4 perfectly concentric arcs consisting of 4 black lines . the observation angle was 11.5. letters from a to d were assigned to the bands of stripes for the clockwise rotating disk and letters e to h were assigned for the bands of the counter - clockwise rotating disk for the purposes of further analysis ( figure 1 ) . the pc monitor used to the evaluate the color template was switched off before the participant provided answers regarding the colors of the bands of stripes on the rotating disk , and only after that the monitor was switched on and the participant was asked to indicate the most resembling color on the screen . the names of the colors were not used for further analysis due to heavy influence of the color categorization in the language and a wide variety of color vocabulary used to describe the illusory fechner colors , but the naming was needed to be sure that the participants are actually seeing some kind of color . rgb primary , secondary , and tertiary colors with the addition of 4 neutral colors were used to standardize the participants responses . the template for color matching was constructed from vertical bands of rgb primary , secondary , and tertiary colors with the addition of 4 neutral colors . codes and order of color bands used in the template for matching fechner illusory colors with rgb colors are provided in table 1 . numbers of colors were used for the purposes of further evaluation , but were not visible on the screen . for comparison of the groups on the basis of categorical variables , the present prospective , cross - sectional , case - control study was carried out at the alzheimer disease and dementia subunit of the department of neurology , vuh santariskiu clinics . we enrolled 40 ad patients ( the ad group ) and 40 control participants ( the control group , cg ) , who were matched by age , sex , and education . for the cg group , mini - mental state examination ( mmse ) score was used as a measure of overall cognitive status . we used a benham s disk attached to a device of original construction to control the speed and direction of rotation . the device could rotate the benham disk at speeds of 90 rpm , 120 rpm , 150 rpm , 180 rpm , and 210 rpm clockwise and counter - clockwise . we used a variant of the benham disk with one half of the disk colored solid black and the other half containing 4 perfectly concentric arcs consisting of 4 black lines . the observation angle was 11.5. letters from a to d were assigned to the bands of stripes for the clockwise rotating disk and letters e to h were assigned for the bands of the counter - clockwise rotating disk for the purposes of further analysis ( figure 1 ) . the pc monitor used to the evaluate the color template was switched off before the participant provided answers regarding the colors of the bands of stripes on the rotating disk , and only after that the monitor was switched on and the participant was asked to indicate the most resembling color on the screen . the names of the colors were not used for further analysis due to heavy influence of the color categorization in the language and a wide variety of color vocabulary used to describe the illusory fechner colors , but the naming was needed to be sure that the participants are actually seeing some kind of color . rgb primary , secondary , and tertiary colors with the addition of 4 neutral colors were used to standardize the participants responses . the template for color matching was constructed from vertical bands of rgb primary , secondary , and tertiary colors with the addition of 4 neutral colors . codes and order of color bands used in the template for matching fechner illusory colors with rgb colors are provided in table 1 . numbers of colors were used for the purposes of further evaluation , but were not visible on the screen . for comparison of the groups on the basis of categorical variables , the age ( p=0.91 ) , education ( p=0.21 ) , and sex ( p=0.82 ) of ad and cg groups did not differ . demographic characteristics and mmse results in ad and cg groups are presented in table 2 . the number of ad and cg participants reporting the perception of fechner illusory color on each arc when the benham s disk is rotating clockwise and counter - clockwise are provided in table 3 , based on the matching template color indicated by the participant . reversing of the colors when the direction of rotation reverses is evident in table 3 . this reversal of color order , which is typical for healthy controls , remains intact in the ad group . the numbers of ad and cg participants perceiving spectral versus neutral color on each arc of lines when the benham s disk is rotating clockwise ( arcs a to d ) and counter - clockwise ( arcs e to h ) is provided in table 4 . for further analysis , the grouping of fechner illusory colors according to blue - yellow opponent process theory was performed . based on data provided in table 1 , pure blue color without any constituent parts of red or green ( hexadecimal html code # 0000ff ) was assigned to group 1 . the colors lacking any amount of blue constituent and composed only from components of the yellow part of the blue - yellow opponent axis ( ie , colors containing red plus green , or only red , or only green based on rgb system ) were assigned to group 3 . the scheme of assigning the matching template colors to the groups based on the blue - yellow opponent process theory is provided in table 5 . the differences in perception of fechner illusory colors were grouped according to the blue - yellow opponent process constituent parts in ad and cg ( figure 2 ) . the age ( p=0.91 ) , education ( p=0.21 ) , and sex ( p=0.82 ) of ad and cg groups did not differ . demographic characteristics and mmse results in ad and cg groups are presented in table 2 . the number of ad and cg participants reporting the perception of fechner illusory color on each arc when the benham s disk is rotating clockwise and counter - clockwise are provided in table 3 , based on the matching template color indicated by the participant . reversing of the colors when the direction of rotation reverses is evident in table 3 . this reversal of color order , which is typical for healthy controls , remains intact in the ad group . the numbers of ad and cg participants perceiving spectral versus neutral color on each arc of lines when the benham s disk is rotating clockwise ( arcs a to d ) and counter - clockwise ( arcs e to h ) is provided in table 4 . for further analysis , the grouping of fechner illusory colors according to blue - yellow opponent process theory was performed . based on data provided in table 1 , pure blue color without any constituent parts of red or green ( hexadecimal html code # 0000ff ) was assigned to group 1 . the colors lacking any amount of blue constituent and composed only from components of the yellow part of the blue - yellow opponent axis ( ie , colors containing red plus green , or only red , or only green based on rgb system ) were assigned to group 3 . the scheme of assigning the matching template colors to the groups based on the blue - yellow opponent process theory is provided in table 5 . the differences in perception of fechner illusory colors were grouped according to the blue - yellow opponent process constituent parts in ad and cg ( figure 2 ) . ad patients retain the ability to see fechner illusory colors , but they significantly more frequently report neutral colors ( e.g. at 210 rpm , clockwise , the outermost ( 1 ) arc appears dark saturated blue , the second set of lines pale magenta , the third arc of lines green , and the innermost ( 4 ) arc of lines looks clear or luminescent yellow , orange , or red . when the direction of benham disk rotation is changed to counter - clockwise , the colors reverse order . substantial evidence exists that pifcs appear due to neural activity in a wide range of nervous system structures , from the retina to the visual cortex , and there are some indications that the blue - yellow opponent process may explain subjects experience of all the different fechner colors . the 3 types of cones ( l for long , m for medium , and s for short wavelengths of light ) have significant overlap in the wavelengths of light to which they respond ; therefore , it is more efficient for the human visual system to record differences between the responses of cone cells , rather than to analyze and compare individual responses from each type of cone . the first type of cell is responsible for red - green opponent process axis and the second type is responsible for blue - yellow opponent process axis ; specifically , the latencies of processes at the center and the surrounding differ among the different types of color - specific ganglion cells . our results show that a significant difference in reporting various colors between ad and cg groups . very few ad patients reported pure blue color ( # 0000ff ) when viewing the outermost arc of lines on a benham disk rotating clockwise and the innermost arc of lines on a disk rotating counter - clockwise ; whereas control group participants most frequently reported clear and saturated pure blue in these arcs on the disk rotated in the same direction ( table 3 ) . when colors seen by the study participants moved to the yellow part of the blue - yellow opponent axis , the difference between colors reported by ad and cg groups gradually vanished . the sequence of colors reported by participants in our study is explained much more reasonably by the opponent process theory . even more unexpected is that if all phenomenal properties of fechner illusory colors are explained exclusively by the blue - yellow opponent axis , as is contended by schramme et al . when the disk was rotating clockwise , there was much more scattered reporting of colors by ad group subjects , clearly skewed to the red but not to the green ( table 3 ) . it may be hypothesized that the colors observed on the outer and inner arcs of lines , and which are at the greatest distance from each other , are reasonably explained by the blue - yellow opponent process . the 2 middle arcs ( the 2 and 3 clockwise and counter - clockwise ) exhibit signs of intrusion of the red - green opponent process , which significantly affects visible colors only in a small range located inside of a much wider interval , which is under the influence of the blue - yellow opponent process . if this explanation of 2 opponent processes involved is feasible , there still remains the difference between blue - yellow opponent process , responsible for coloring of distant arcs and red - green opponent process , which is responsible for colors of the 2 middle arcs , located near each other : when the disk is rotated clockwise , colors of the outermost and innermost arcs follow the direction from shorter ( blue ) to longer ( yellow , or a sum of red and green ) wavelengths ; in contrast to this , the colors of 2 middle arcs follow the direction from longer ( red ) to shorter ( green ) wavelengths . furthermore , red and green seem unequal in this double - opponent process explanation because the ad group tended to shift reported colors from green to red in both extremities of the wide blue - yellow opponent process activity range ( table 3 ) . thus , the phenomenon of fechner illusory colors originates from neural activity in the retina and the more central structures of the visual system . the fmri research findings reported by tanabe et al suggest that the visual cortex , which processes pattern recognition , may be involved in the generation of illusory colors . the results from this fmri study indicate that modulation from v4 to v2 to v1 plays a significant role in the perception of fechner illusory colors . the utilization of pifcs in medical diagnosis and the biological interpretation of the neural network involved would also benefit from research on fechner illusory colors in ad . on the other hand , the pattern of decline in perception of fechner illusory colors in people with ad can be helpful for further research in order to understand the nature of visual perception of color , and may have even broader implications for the fundamental mind - body relationship . bearing in mind the results of research using fechner illusory colors , the initial basic premise of this thought experiment about the possibility of existence of an entirely black and white room , where mary the super - scientist is working without any possibility to see spectral colors , seems quite problematic due to the established fact that movement of black and white patterns may produce the experience of illusory colors , indistinguishable from real physical spectral colors . this means that isolation of mary the super - scientist in the black and white room does not provide a possibility to deprive her of subjectively experiencing colors . the reversal of color order when changing the direction of disk rotation remains intact in ad patients . the visibility of spectral illusory colors is significantly less frequent in ad patients compared with normal controls . the perception of pure blue fechner color was markedly lower in the ad group than in the control group . the illusory colors reported by ad patients when observing the blue - producing arcs of lines are much more scattered around the pure blue color than in the control group . while a clear shift to the yellow opponent is observed among ad patients , this shift is asymmetric for red and green constituent parts of the yellow opponent . a larger shift to the red constituent is found instead of green , which makes it harder to explain all the phenomena of fechner illusory colors solely on the basis of blue - yellow opponent process , and definitely does not correspond to the sequence of physical spectral colors . the decreased perception of blue opponent was very large , but we did not observe any impairment in perception of yellow opponent colors that do not contain blue subcomponent according to the rgb system . there is impaired perception of intermediary colors containing the blue subcomponent , but less pronounced than for pure blue . this impaired pattern of visibility of illusory colors in ad patients could be explained by the prevailing disorder in the blue - yellow opponent axis , with some noticeable effect of disorder in the red - green opponent axis only in the middle part of the much broader blue - yellow axis , which may indicate the cortical , but not the retinal , origin of the disorder in perception of fechner illusory colors in people with ad .

alzheimer disease ( ad ) is a progressive central nervous system degeneration , which is characterized by a gradual decline in cognitive abilities and activities of daily living . the most common symptoms of ad are an expanding variety of cognitive disorders , which usually starts with dysfunction of memory , and , as the ad progresses , involves language , orientation , praxis , attention , and perception functions . the proteinopathy , cholinergic deficit , and neurodegeneration in early ad involves brain areas responsible for noncognitive functions , including structures and pathways of vision [ 48 ] . visual changes occur early in the course of ad and have a potential role as noninvasive biomarkers of ad - related neurodegeneration . ad is associated with a wide variety of pathological changes in the retina and brain visual system . some of the disorders of visual function may become noninvasive markers for the diagnosis of ad , and also may provide a better understanding of the mechanisms of visual perception per se [ 917 ] . one of the most promising and potentially insightful provisional biomarker is color vision [ 1018 ] . some research reports indicate deficits of color perception at specific color axes according to the rgb ( red - green - blue ) system , corresponding to the maximum sensitivity to different light wavelengths of 3 types of retinal cone photoreceptors , while others did not identify selective color vision deficits . most research reports have found abnormalities in the tritan axis in ad patients [ 14,1820 ] , while reports from other research groups suggest that the deutan axis may be dysfunctional . cholinergic neurons of the nucleus basalis of meynert have a number of important functions in modulation of visual perception . in ad patients , the nucleus basalis is one of the brain structures involved , degenerating at the earliest stages of the disease and leading to the cholinergic deficit in the cerebral cortex . in addition , according to the 2-streams ( dorsal and ventral ) hypothesis of the neural processing of visual perception , the ventral stream is associated with form and color representation ; it goes to areas of the inferior temporal lobe and has strong connections to the medial temporal lobe , which encodes long - term memories . this indicates that research on disorders of color perception may be a promising strategy in search of functional markers of early ad . among the provisional visual markers of ad , one very interesting and still poorly understood phenomenon fechner illusory colors has received no attention in ad research . our search of databases of published research articles found no scientific reports dealing with the investigation of fechner illusory colors in ad . initially , the most popular explanation of how the monochromatic or neutral flickering stimuli produce the perception of color was based on the difference in information processing speed by different types of retinal cones , especially by the relative slowness of the blue or short - wavelength cone ( s cone ) compared to the speed of the other 2 cones the green or medium - wavelength cone ( m cone ) and the red or long - wavelength cone ( l - cone ) . differences in responsivity spectra of s , m , and l type cone cells could not be an explanation , because the stimulus was monochromatic ( neutral ) black and white and despite that , they produced several concentric bands of different colors on the spinning disk . also , there were no differing compositions of red , green , and blue components , which are able to imitate the real physical colors ( which is the case for computer monitors and tv screens ) . if illusory colors are experienced due to the slowness of the s cones , then the most probable neuroanatomical substrate of pfics are the retinal ganglion cells , responsible for the blue - yellow opponent process . despite the seeming plausibility of this explanation , there are some facts that can not be explained by the difference of information processing speed in different types of cones . some more recent fmri investigations showed that fechner illusory colors may be a cortical phenomenon associated with a ventral stream of visual information ( ie , with the interaction between visual areas v1 , v2 , and v4 ) , which anatomically is ongoing at the inferior temporal cortex . the inferior temporal cortex has numerous connections with the medial temporal lobe and is affected by the neurodegeneration early in the course of ad . the purpose of the study was to explore and compare perception of fechner illusory colors in ad patients and normal controls . the present prospective , cross - sectional , case - control study was carried out at the alzheimer disease and dementia subunit of the department of neurology , vuh santariskiu clinics . we enrolled 40 ad patients ( the ad group ) and 40 control participants ( the control group , cg ) , who were matched by age , sex , and education . inclusion criteria for ad group participants were : 1 ) ad diagnosis established based on nincds - adrda criteria ; 2 ) mild or moderate dementia based on mmse score 14 or more ; 3 ) at least 65 years old ; 4 ) education at least 8 years ; 5 ) hachinski index equal to or less than 4 ; 6 ) yesavage gds depression score equal to or less than 19 ; 7 ) no significant vascular signs or other neurodegenerative pathology results of the head - on ct or mri . exclusion criteria for ad group participants were : 1 ) diagnosis or any signs of any other than ad neurodegenerative disease ; 2 ) diagnosis of any significant psychiatric disorder , such as schizophrenia , delirium , psychosis , or other ; 3 ) diagnosis or any symptoms of significant cardiovascular , hepatic , or metabolic disorders ; 4 ) history or evidence of past or current drug or alcohol abuse . inclusion criteria for control group participants were : 1 ) normal cognition based on mmse score 27 or more ; 2 ) at least 65 years old ; 3 ) hachinski index equal to or less than 4 ; 4 ) yesavage gds depression score equal to or less than 19 ; 5 ) education at least 8 years . for the cg group , mini - mental state examination ( mmse ) score was used as a measure of overall cognitive status . the rotation speed of 210 rpm was chosen for the main study after initial proof - of - concept investigation because it had less flicker and greater stability and saturation of observed colors . we used a variant of the benham disk with one half of the disk colored solid black and the other half containing 4 perfectly concentric arcs consisting of 4 black lines . the observation angle was 11.5. letters from a to d were assigned to the bands of stripes for the clockwise rotating disk and letters e to h were assigned for the bands of the counter - clockwise rotating disk for the purposes of further analysis ( figure 1 ) . the pc monitor used to the evaluate the color template was switched off before the participant provided answers regarding the colors of the bands of stripes on the rotating disk , and only after that the monitor was switched on and the participant was asked to indicate the most resembling color on the screen . the names of the colors were not used for further analysis due to heavy influence of the color categorization in the language and a wide variety of color vocabulary used to describe the illusory fechner colors , but the naming was needed to be sure that the participants are actually seeing some kind of color . rgb primary , secondary , and tertiary colors with the addition of 4 neutral colors were used to standardize the participants responses . the template for color matching was constructed from vertical bands of rgb primary , secondary , and tertiary colors with the addition of 4 neutral colors . numbers of colors were used for the purposes of further evaluation , but were not visible on the screen . we used the shapiro - wilk test for the verification of normality of data distribution . for comparison of the groups on the basis of categorical variables , the present prospective , cross - sectional , case - control study was carried out at the alzheimer disease and dementia subunit of the department of neurology , vuh santariskiu clinics . we enrolled 40 ad patients ( the ad group ) and 40 control participants ( the control group , cg ) , who were matched by age , sex , and education . inclusion criteria for ad group participants were : 1 ) ad diagnosis established based on nincds - adrda criteria ; 2 ) mild or moderate dementia based on mmse score 14 or more ; 3 ) at least 65 years old ; 4 ) education at least 8 years ; 5 ) hachinski index equal to or less than 4 ; 6 ) yesavage gds depression score equal to or less than 19 ; 7 ) no significant vascular signs or other neurodegenerative pathology results of the head - on ct or mri . exclusion criteria for ad group participants were : 1 ) diagnosis or any signs of any other than ad neurodegenerative disease ; 2 ) diagnosis of any significant psychiatric disorder , such as schizophrenia , delirium , psychosis , or other ; 3 ) diagnosis or any symptoms of significant cardiovascular , hepatic , or metabolic disorders ; 4 ) history or evidence of past or current drug or alcohol abuse . for the cg group , mini - mental state examination ( mmse ) score was used as a measure of overall cognitive status . the rotation speed of 210 rpm was chosen for the main study after initial proof - of - concept investigation because it had less flicker and greater stability and saturation of observed colors . we used a variant of the benham disk with one half of the disk colored solid black and the other half containing 4 perfectly concentric arcs consisting of 4 black lines . the observation angle was 11.5. letters from a to d were assigned to the bands of stripes for the clockwise rotating disk and letters e to h were assigned for the bands of the counter - clockwise rotating disk for the purposes of further analysis ( figure 1 ) . the pc monitor used to the evaluate the color template was switched off before the participant provided answers regarding the colors of the bands of stripes on the rotating disk , and only after that the monitor was switched on and the participant was asked to indicate the most resembling color on the screen . the names of the colors were not used for further analysis due to heavy influence of the color categorization in the language and a wide variety of color vocabulary used to describe the illusory fechner colors , but the naming was needed to be sure that the participants are actually seeing some kind of color . rgb primary , secondary , and tertiary colors with the addition of 4 neutral colors were used to standardize the participants responses . the template for color matching was constructed from vertical bands of rgb primary , secondary , and tertiary colors with the addition of 4 neutral colors . we used the shapiro - wilk test for the verification of normality of data distribution . for comparison of the groups on the basis of categorical variables , the age ( p=0.91 ) , education ( p=0.21 ) , and sex ( p=0.82 ) of ad and cg groups did not differ . the number of ad and cg participants reporting the perception of fechner illusory color on each arc when the benham s disk is rotating clockwise and counter - clockwise are provided in table 3 , based on the matching template color indicated by the participant . the numbers of ad and cg participants perceiving spectral versus neutral color on each arc of lines when the benham s disk is rotating clockwise ( arcs a to d ) and counter - clockwise ( arcs e to h ) is provided in table 4 . the colors lacking any amount of blue constituent and composed only from components of the yellow part of the blue - yellow opponent axis ( ie , colors containing red plus green , or only red , or only green based on rgb system ) were assigned to group 3 . the scheme of assigning the matching template colors to the groups based on the blue - yellow opponent process theory is provided in table 5 . the differences in perception of fechner illusory colors were grouped according to the blue - yellow opponent process constituent parts in ad and cg ( figure 2 ) . the age ( p=0.91 ) , education ( p=0.21 ) , and sex ( p=0.82 ) of ad and cg groups did not differ . the number of ad and cg participants reporting the perception of fechner illusory color on each arc when the benham s disk is rotating clockwise and counter - clockwise are provided in table 3 , based on the matching template color indicated by the participant . the numbers of ad and cg participants perceiving spectral versus neutral color on each arc of lines when the benham s disk is rotating clockwise ( arcs a to d ) and counter - clockwise ( arcs e to h ) is provided in table 4 . the colors lacking any amount of blue constituent and composed only from components of the yellow part of the blue - yellow opponent axis ( ie , colors containing red plus green , or only red , or only green based on rgb system ) were assigned to group 3 . the scheme of assigning the matching template colors to the groups based on the blue - yellow opponent process theory is provided in table 5 . the differences in perception of fechner illusory colors were grouped according to the blue - yellow opponent process constituent parts in ad and cg ( figure 2 ) . at 210 rpm , clockwise , the outermost ( 1 ) arc appears dark saturated blue , the second set of lines pale magenta , the third arc of lines green , and the innermost ( 4 ) arc of lines looks clear or luminescent yellow , orange , or red . a possible explanation of why the study participants see colors when looking at the rotating achromatic stimulus disk may be that the cone receptors in the human retina respond at different rates to red , green , and blue . even more importantly , the latencies at the center and the surroundings differ for the different types of color - specific retinal ganglion cells . substantial evidence exists that pifcs appear due to neural activity in a wide range of nervous system structures , from the retina to the visual cortex , and there are some indications that the blue - yellow opponent process may explain subjects experience of all the different fechner colors . the 3 types of cones ( l for long , m for medium , and s for short wavelengths of light ) have significant overlap in the wavelengths of light to which they respond ; therefore , it is more efficient for the human visual system to record differences between the responses of cone cells , rather than to analyze and compare individual responses from each type of cone . the opponent process theory posits the existence of 3 different opponent channels : red vs. green , blue vs. yellow , and black vs. white ( achromatic ) . the opponent process theory accounts for neural mechanisms that receive information from the 3 types of cones and process it in a more efficient way , finally producing the various phenomena of color experience . according to the opponent process theory , 2 other types of cells are involved in response to color stimuli : bipolar cells and ganglion cells . the parvocellular layer cells process the color - related information and may be assigned to 2 groups : one of these groups processes the information about the difference of activity of l and m cones , while the other group processes the difference between s cones and a combined activity of both l and m cones . the first type of cell is responsible for red - green opponent process axis and the second type is responsible for blue - yellow opponent process axis ; specifically , the latencies of processes at the center and the surrounding differ among the different types of color - specific ganglion cells . our results show that a significant difference in reporting various colors between ad and cg groups . very few ad patients reported pure blue color ( # 0000ff ) when viewing the outermost arc of lines on a benham disk rotating clockwise and the innermost arc of lines on a disk rotating counter - clockwise ; whereas control group participants most frequently reported clear and saturated pure blue in these arcs on the disk rotated in the same direction ( table 3 ) . when colors seen by the study participants moved to the yellow part of the blue - yellow opponent axis , the difference between colors reported by ad and cg groups gradually vanished . importantly , the 4 colors visible on the disk by the healthy control group participants do not follow the rgb - derived sequence , usually provided in the published literature as blue green yellow red . it may be hypothesized that the colors observed on the outer and inner arcs of lines , and which are at the greatest distance from each other , are reasonably explained by the blue - yellow opponent process . the 2 middle arcs ( the 2 and 3 clockwise and counter - clockwise ) exhibit signs of intrusion of the red - green opponent process , which significantly affects visible colors only in a small range located inside of a much wider interval , which is under the influence of the blue - yellow opponent process . if this explanation of 2 opponent processes involved is feasible , there still remains the difference between blue - yellow opponent process , responsible for coloring of distant arcs and red - green opponent process , which is responsible for colors of the 2 middle arcs , located near each other : when the disk is rotated clockwise , colors of the outermost and innermost arcs follow the direction from shorter ( blue ) to longer ( yellow , or a sum of red and green ) wavelengths ; in contrast to this , the colors of 2 middle arcs follow the direction from longer ( red ) to shorter ( green ) wavelengths . furthermore , red and green seem unequal in this double - opponent process explanation because the ad group tended to shift reported colors from green to red in both extremities of the wide blue - yellow opponent process activity range ( table 3 ) . consequently , there is good reason to suppose that the processes ongoing at the retinal ganglion cells level are not sufficient to explain the illusory color sequence in its entirety . thus , the phenomenon of fechner illusory colors originates from neural activity in the retina and the more central structures of the visual system . the results from this fmri study indicate that modulation from v4 to v2 to v1 plays a significant role in the perception of fechner illusory colors . the utilization of pifcs in medical diagnosis and the biological interpretation of the neural network involved would also benefit from research on fechner illusory colors in ad . the decline in blue opponent perception , if properly standardized , may be used as a cheap and noninvasive diagnostic marker in early diagnosis of ad as an addition to other cognitive , electrophysiologic , behavioral , and emotional regulation methods [ 2831 ] . on the other hand , the pattern of decline in perception of fechner illusory colors in people with ad can be helpful for further research in order to understand the nature of visual perception of color , and may have even broader implications for the fundamental mind - body relationship . bearing in mind the results of research using fechner illusory colors , the initial basic premise of this thought experiment about the possibility of existence of an entirely black and white room , where mary the super - scientist is working without any possibility to see spectral colors , seems quite problematic due to the established fact that movement of black and white patterns may produce the experience of illusory colors , indistinguishable from real physical spectral colors . this means that isolation of mary the super - scientist in the black and white room does not provide a possibility to deprive her of subjectively experiencing colors . a larger shift to the red constituent is found instead of green , which makes it harder to explain all the phenomena of fechner illusory colors solely on the basis of blue - yellow opponent process , and definitely does not correspond to the sequence of physical spectral colors . this impaired pattern of visibility of illusory colors in ad patients could be explained by the prevailing disorder in the blue - yellow opponent axis , with some noticeable effect of disorder in the red - green opponent axis only in the middle part of the much broader blue - yellow axis , which may indicate the cortical , but not the retinal , origin of the disorder in perception of fechner illusory colors in people with ad .

type 2 diabetes is associated with an approximately twofold greater risk of dementia among older adults ( 13 ) , but it is not clear whether risk of dementia for people with diabetes varies across racial / ethnic groups . in the general population , the prevalence of dementia has been reported to be higher among african americans and caribbean hispanics , lower among japanese americans , and similar among native americans and mexican americans compared with non - hispanic whites ( 4,5 ) . a study of community - dwelling older adults in new york city reported that the higher prevalence of type 2 diabetes in african americans and hispanics compared with whites partially contributes to the higher observed incidence of dementia and cognitive impairment in these populations ( 6 ) . to our knowledge , however , racial / ethnic differences in dementia risk have not been investigated among people with type 2 diabetes . ( 7 ) , and the prevalence is highest in some racial / ethnic minorities , including latinos , african americans , native americans , and some asian american groups ( 7,8 ) . because type 2 diabetes increases the risk of dementia , it is important to understand the risk of dementia among older adults with type 2 diabetes of diverse racial / ethnic backgrounds . with the aging ( 9 ) and growing diversity ( 10 ) of the u.s . population , the increasing prevalence of type 2 diabetes ( 11,12 ) , and the national goal of eliminating health disparities ( 13 ) , understanding racial / ethnic differences in diabetes - related complications , including dementia , among older adults is increasingly important . the primary goal of this study was to examine whether there are racial / ethnic differences in the 10-year incidence of dementia among patients with type 2 diabetes . secondarily , we explored whether these differences are explained by diabetes duration , markers of clinical control , and vascular diabetes - related complications in a multiethnic population of older type 2 diabetic patients . we evaluated patients who were members of the kaiser permanente northern california ( kpnc ) diabetes registry , a well - characterized population of diabetic patients that has been the basis of a wide range of epidemiologic research ( 1417 ) . the registry was formed in 1993 and is updated annually to include all kpnc health plan members who are identified as having diabetes on the basis of laboratory results , pharmacy records , and outpatient diagnoses ( 14 ) . kpnc is a large , integrated health care delivery system that provides comprehensive medical care to > 3.3 million members ( 30% of the geographic region ) . the kpnc population is generally representative of the overall population of the region , with the exception that individuals at the extreme tails of the income distribution are underrepresented ( 18,19 ) . between 1994 and 1996 , all noninstitutionalized kpnc diabetes registry members 19 years of age were sent a self - administered survey ( mailed or online ) or contacted for a computer - assisted telephone interview on sociodemographic factors and health behaviors . the written and web surveys were in english only , but the computer - assisted telephone interview was available in english , spanish , cantonese , mandarin , and tagalog through certified translations of an english script and was intended to maximize accessibility to those with language barriers or limited english literacy or fluency . a detailed description of the kpnc diabetes registry and survey has been published ( 14,17 ) . the present study was approved by the kaiser foundation research institute and university of chicago institutional review boards . we included kpnc diabetes registry members in this analysis if they completed the 19941996 survey , had type 2 diabetes , were 60 years of age as of 1 january 1998 ( baseline ) , had no prior diagnosis of dementia , and had no gaps in membership > 3 months in the 2 years before baseline ( n = 29,956 ) . we excluded patients who reported mixed race or other ( n = 139 ) and pacific islanders ( n = 183 ) because of insufficient sample size to support statistical comparisons . we also excluded 7,162 patients missing information on the following key covariates : level of education ( n = 1,842 ) , glycosylated hemoglobin ( a1c ) ( n = 2,976 ) , duration of diabetes ( n = 1,745 ) , or more than one of these variables ( n = 599 ) . the proportion of patients who were excluded because of missing key covariates was similar across racial / ethnic groups and ranged from 23.0% in non - hispanic whites to 28.2% in latinos . dementia cases were identified from clinical diagnoses in electronic medical records from inpatient and outpatient encounters between 1 january 1998 and 31 december 2007 based on icd-9-cm diagnostic codes for alzheimer disease ( 331.0 ) ; senile dementia , uncomplicated ( 290.0 ) ; presenile dementia ( 290.1x ) ; senile dementia with delusional or depressive features ( 290.2x ) ; senile dementia with delirium ( 290.3 ) ; and vascular dementia ( 290.4x ) . this method of dementia ascertainment has been used successfully in kpnc health plan members in other publications ( 15,16,20 ) . race / ethnicity ( non - hispanic white , african american , latino , asian , pacific islander , native american , or mixed race / other ) and educational attainment were ascertained by self - report from the survey . neighborhood deprivation index , based on a validated methodology , was calculated from each patient s 1996 address and 1990 census information ( 21 ) . duration of diabetes was estimated from electronic health records according to initial date of diagnosis . acute metabolic events ( severe hyperglycemia or hypoglycemia requiring hospitalization ) were identified from electronic hospitalization and emergency department records during the period of 1 january 1996 to 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . a1c levels were abstracted from kpnc electronic laboratory databases , using the most recent measurement during the period of 1 january 1996 to 31 december 1997 . the median time from the a1c measurement to baseline ( 1 january 1998 ) was 0.34 years ( interquartile range 0.160.73 ) . diabetes pharmacotherapy was defined by a 30-day supply of glycemia - lowering medication dispensed at a kpnc pharmacy during the 6 months before baseline ( 1 january 1998 ) . prevalent diabetes - related microvascular and macrovascular complications were identified from electronic medical records from inpatient and outpatient visits that took place between 1 january 1996 and 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . microvascular complications included end - stage renal disease , diabetic retinal disease , and lower - extremity complications ( lower - limb ulcer , gangrene , and lower - extremity amputation ) . macrovascular complications included cardiovascular disease ( congestive heart failure , peripheral vascular disease , and myocardial infarction ) and cerebrovascular disease . hypertension was identified from electronic medical records ( 1 january 1996 to 31 december 1997 ) . as a measure of health care utilization , the average number of clinical visits per year during follow - up for each individual was calculated , resulting in a medical utilization rate . baseline characteristics were compared across racial / ethnic groups with anova tests for continuous variables and tests for categorical variables . age - adjusted dementia incidence densities ( incidence rates ) by racial / ethnic group were estimated after directly standardizing to the 2000 census population . we specified cox proportional hazards models to estimate the association between race / ethnicity and risk of dementia . to facilitate comparisons among racial / ethnic groups included in the study , we present hazard ratios for each model , with each racial / ethnic group as the reference group . because of the strong association between age and dementia incidence , we used age as the time scale in all proportional hazard models to best control for the influence of age on dementia risk . participants were followed from age as of 1 january 1998 until age at diagnosis of dementia , death , a gap in membership > 3 months , or 31 december 2007 ( end of study period ) . we estimated racial / ethnic differences in dementia risk after adjustment for age ( as the time scale ) , sex , and level of education . we evaluated whether the association between race / ethnicity and risk of dementia was explained by racial / ethnic differences in diabetes duration and clinical control ( pharmacotherapy , a1c level , acute metabolic events ) , microvascular complications ( end - stage renal disease , diabetic retinopathy , lower - extremity complications ) , macrovascular complications ( cardiovascular disease , cerebrovascular disease ) , neighborhood deprivation index , bmi , and hypertension . analyses were performed with sas versions 9.1 and 9.3 statistical software ( sas institute inc . , cary , nc ) . we evaluated patients who were members of the kaiser permanente northern california ( kpnc ) diabetes registry , a well - characterized population of diabetic patients that has been the basis of a wide range of epidemiologic research ( 1417 ) . the registry was formed in 1993 and is updated annually to include all kpnc health plan members who are identified as having diabetes on the basis of laboratory results , pharmacy records , and outpatient diagnoses ( 14 ) . kpnc is a large , integrated health care delivery system that provides comprehensive medical care to > 3.3 million members ( 30% of the geographic region ) . the kpnc population is generally representative of the overall population of the region , with the exception that individuals at the extreme tails of the income distribution are underrepresented ( 18,19 ) . between 1994 and 1996 , all noninstitutionalized kpnc diabetes registry members 19 years of age were sent a self - administered survey ( mailed or online ) or contacted for a computer - assisted telephone interview on sociodemographic factors and health behaviors . the written and web surveys were in english only , but the computer - assisted telephone interview was available in english , spanish , cantonese , mandarin , and tagalog through certified translations of an english script and was intended to maximize accessibility to those with language barriers or limited english literacy or fluency . a detailed description of the kpnc diabetes registry and survey has been published ( 14,17 ) . the present study was approved by the kaiser foundation research institute and university of chicago institutional review boards . we included kpnc diabetes registry members in this analysis if they completed the 19941996 survey , had type 2 diabetes , were 60 years of age as of 1 january 1998 ( baseline ) , had no prior diagnosis of dementia , and had no gaps in membership > 3 months in the 2 years before baseline ( n = 29,956 ) . we excluded patients who reported mixed race or other ( n = 139 ) and pacific islanders ( n = 183 ) because of insufficient sample size to support statistical comparisons . we also excluded 7,162 patients missing information on the following key covariates : level of education ( n = 1,842 ) , glycosylated hemoglobin ( a1c ) ( n = 2,976 ) , duration of diabetes ( n = 1,745 ) , or more than one of these variables ( n = 599 ) . the proportion of patients who were excluded because of missing key covariates was similar across racial / ethnic groups and ranged from 23.0% in non - hispanic whites to 28.2% in latinos . dementia cases were identified from clinical diagnoses in electronic medical records from inpatient and outpatient encounters between 1 january 1998 and 31 december 2007 based on icd-9-cm diagnostic codes for alzheimer disease ( 331.0 ) ; senile dementia , uncomplicated ( 290.0 ) ; presenile dementia ( 290.1x ) ; senile dementia with delusional or depressive features ( 290.2x ) ; senile dementia with delirium ( 290.3 ) ; and vascular dementia ( 290.4x ) . this method of dementia ascertainment has been used successfully in kpnc health plan members in other publications ( 15,16,20 ) . race / ethnicity ( non - hispanic white , african american , latino , asian , pacific islander , native american , or mixed race / other ) and educational attainment were ascertained by self - report from the survey . neighborhood deprivation index , based on a validated methodology , was calculated from each patient s 1996 address and 1990 census information ( 21 ) . duration of diabetes was estimated from electronic health records according to initial date of diagnosis . acute metabolic events ( severe hyperglycemia or hypoglycemia requiring hospitalization ) were identified from electronic hospitalization and emergency department records during the period of 1 january 1996 to 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . a1c levels were abstracted from kpnc electronic laboratory databases , using the most recent measurement during the period of 1 january 1996 to 31 december 1997 . the median time from the a1c measurement to baseline ( 1 january 1998 ) was 0.34 years ( interquartile range 0.160.73 ) . diabetes pharmacotherapy was defined by a 30-day supply of glycemia - lowering medication dispensed at a kpnc pharmacy during the 6 months before baseline ( 1 january 1998 ) . prevalent diabetes - related microvascular and macrovascular complications were identified from electronic medical records from inpatient and outpatient visits that took place between 1 january 1996 and 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . microvascular complications included end - stage renal disease , diabetic retinal disease , and lower - extremity complications ( lower - limb ulcer , gangrene , and lower - extremity amputation ) . macrovascular complications included cardiovascular disease ( congestive heart failure , peripheral vascular disease , and myocardial infarction ) and cerebrovascular disease . hypertension was identified from electronic medical records ( 1 january 1996 to 31 december 1997 ) . as a measure of health care utilization , the average number of clinical visits per year during follow - up for each individual was calculated , resulting in a medical utilization rate . race / ethnicity ( non - hispanic white , african american , latino , asian , pacific islander , native american , or mixed race / other ) and educational attainment were ascertained by self - report from the survey . neighborhood deprivation index , based on a validated methodology , was calculated from each patient s 1996 address and 1990 census information ( 21 ) . duration of diabetes was estimated from electronic health records according to initial date of diagnosis . acute metabolic events ( severe hyperglycemia or hypoglycemia requiring hospitalization ) were identified from electronic hospitalization and emergency department records during the period of 1 january 1996 to 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . a1c levels were abstracted from kpnc electronic laboratory databases , using the most recent measurement during the period of 1 january 1996 to 31 december 1997 . the median time from the a1c measurement to baseline ( 1 january 1998 ) was 0.34 years ( interquartile range 0.160.73 ) . diabetes pharmacotherapy was defined by a 30-day supply of glycemia - lowering medication dispensed at a kpnc pharmacy during the 6 months before baseline ( 1 january 1998 ) . prevalent diabetes - related microvascular and macrovascular complications were identified from electronic medical records from inpatient and outpatient visits that took place between 1 january 1996 and 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . microvascular complications included end - stage renal disease , diabetic retinal disease , and lower - extremity complications ( lower - limb ulcer , gangrene , and lower - extremity amputation ) . macrovascular complications included cardiovascular disease ( congestive heart failure , peripheral vascular disease , and myocardial infarction ) and cerebrovascular disease . hypertension was identified from electronic medical records ( 1 january 1996 to 31 december 1997 ) . as a measure of health care utilization , the average number of clinical visits per year during follow - up for each individual was calculated , resulting in a medical utilization rate . baseline characteristics were compared across racial / ethnic groups with anova tests for continuous variables and tests for categorical variables . age - adjusted dementia incidence densities ( incidence rates ) by racial / ethnic group were estimated after directly standardizing to the 2000 census population . we specified cox proportional hazards models to estimate the association between race / ethnicity and risk of dementia . to facilitate comparisons among racial / ethnic groups included in the study , we present hazard ratios for each model , with each racial / ethnic group as the reference group . because of the strong association between age and dementia incidence , we used age as the time scale in all proportional hazard models to best control for the influence of age on dementia risk . participants were followed from age as of 1 january 1998 until age at diagnosis of dementia , death , a gap in membership > 3 months , or 31 december 2007 ( end of study period ) . we estimated racial / ethnic differences in dementia risk after adjustment for age ( as the time scale ) , sex , and level of education . we evaluated whether the association between race / ethnicity and risk of dementia was explained by racial / ethnic differences in diabetes duration and clinical control ( pharmacotherapy , a1c level , acute metabolic events ) , microvascular complications ( end - stage renal disease , diabetic retinopathy , lower - extremity complications ) , macrovascular complications ( cardiovascular disease , cerebrovascular disease ) , neighborhood deprivation index , bmi , and hypertension . analyses were performed with sas versions 9.1 and 9.3 statistical software ( sas institute inc . , cary , nc ) . approximately 66% were non - hispanic white , 11% african american , 11% latino , 10% asian , and 2% native american ( table 1 ) . education levels were lowest among latinos and highest among asians , and higher proportions of african americans and latinos lived in deprived neighborhoods . overall , the mean duration of diabetes at baseline was 6 years , and 82.2% of participants were receiving diabetes pharmacotherapy . a1c levels tended to be highest among african americans , latinos , and asians , and acute metabolic events were more common among african americans and native americans . individual diabetes - related complications varied by race / ethnicity , but there was no consistent pattern of any one racial / ethnic group consistently having a higher prevalence of diabetes - related complications ( microvascular or macrovascular ) overall . among microvascular complications , end - stage renal disease was rare in all groups , the prevalence of lower - extremity complications was lowest among asians , and diabetic retinopathy was most prevalent among african americans and latinos . among macrovascular complications , peripheral vascular disease was least common among asians , myocardial infarction was more prevalent among native americans and non - hispanic whites , congestive heart failure was least common among asians and latinos , and the prevalence of cerebrovascular disease was highest among non - hispanic whites and native americans . baseline characteristics of the sample by race / ethnicity ( 19961997 ) the average length of follow - up was 6.7 years , and the average age at dementia diagnosis was 79.2 years . the median number of clinical visits per year was similar across race groups but was lowest among native americans ( median 9.0 ) and highest among latinos ( median 10.5 ) . during follow - up , there were 10,525 deaths ( 47% of the cohort ) . over 10 years of follow - up , 3,796 ( 17.1% ) participants were diagnosed with dementia . among patients with dementia , 676 ( 17.8% ) were diagnosed with alzheimer disease , 474 ( 12.5% ) with vascular dementia , and 128 ( 3.4% ) with both alzheimer disease and vascular dementia . the remaining 2,518 ( 66.3% ) dementia cases were unspecified dementia diagnoses made in primary care . because the majority of dementia diagnoses were for unspecified dementia , we did not evaluate racial / ethnic differences for specific dementia subtypes . age - adjusted dementia cumulative incidence and incidence densities were highest among native americans and african americans and lowest among asians ( table 2 ) . adjustment for age , sex , and level of education in the cox proportional hazards models showed consistent patterns ( table 3 , model 1 ) . the measures of diabetes control and microvascular and macrovascular complications were significant risk factors for incident dementia in bivariate cox proportional hazards models ( data not shown ) . however , adjustment for these measures ( table 3 , models 24 ) imparted no substantive change in the association between race / ethnicity and dementia incidence . in all four models , dementia risk was significantly higher among native americans and african americans than among the other racial / ethnic groups , whereas dementia risk was significantly lower among asians than among all other racial / ethnic groups . dementia risk was similar for non - hispanic whites and latinos , whose risk of dementia was lower than that of native americans and african americans and higher than that of asians . native americans had a 60% increased risk of dementia in models that accounted for sociodemographic characteristics , diabetes duration , markers of clinical control , and microvascular and macrovascular complications . we also adjusted for bmi , hypertension , and neighborhood deprivation index , but these variables did not change the magnitude of the association between race / ethnicity and risk of dementia ( data not shown ) . the observed trends persisted when we included individuals with missing information on education , a1c , and/or duration of diabetes , when we accounted for the competing risk of death with competing risk regression models ( 22 ) , and when we stratified by sex ( data not shown ) . dementia incidence densities by race / ethnicity ( 19982007 ) hazard ratios for race / ethnicity and dementia risk ( 19982007 ) among older type 2 diabetic patients , 10-year dementia incidence was highest among native americans and african americans , lowest among asians , and intermediate for non - hispanic whites and latinos . the observed differences persisted after accounting for sociodemographic characteristics , diabetes duration , markers of clinical control , and a host of diabetes - related microvascular or macrovascular complications . to our knowledge , this study is the first to examine racial / ethnic differences in dementia incidence among people with diabetes , a group at high - risk for dementia . cerebral microvascular and macrocerebrovascular disease are recognized as important risk factors for dementia ( 23 ) and are potential pathways that link diabetes and dementia ( 24,25 ) . although microvascular complications , macrovascular complications , and markers of clinical control were associated with increased dementia incidence , the observed racial / ethnic differences in dementia risk persisted after adjustment for these factors . although the prevalence of microvascular and macrovascular complications varied by race / ethnicity in the current study , there was no general pattern of a greater burden of complications in certain racial / ethnic groups . previous population - based studies reported that among people with diabetes , the rates of microvascular complications , such as end - stage renal disease , tend to be higher and the rates of macrovascular complications lower among racial / ethnic minorities compared with non - hispanic whites ( 14,26,27 ) . however , the current finding of no general pattern with respect to race / ethnicity and diabetes - related complications is consistent with earlier research on diabetes - related complications in this population ( 14 ) . unmeasured behavioral or environmental factors years of education varied across racial / ethnic groups and tended to be lowest among latinos and highest among asians . we adjusted for years of education , but this may not account for differences in quality of education , which may partially explain the observed racial / ethnic differences in dementia incidence . we adjusted for neighborhood deprivation index , which may have negative effects on health by influencing health behaviors and stress ( 21 ) . a greater proportion of african american and latino patients lived in deprived neighborhoods compared with other groups , but adjusting for this variable did not alter observed associations and was not included in final models . the findings on racial / ethnic difference in dementia risk among older diabetic patients are in accord with previous population - based studies of older adults with and without diabetes , which found that dementia rates were higher among african americans relative to non - hispanic whites ( 4 ) and similar among mexican americans ( 5 ) compared with non - hispanic whites . the current finding that dementia rates are lower among asian americans than among non - hispanic whites is consistent with previous reports among japanese americans ( 4 ) . although the incidence of dementia was higher among native americans than among other racial / ethnic groups in the current study , one small canadian cross - sectional study reported a similar prevalence of dementia among native americans living on two reservations in manitoba relative to non - hispanic whites living in winnipeg ( 4 ) . the results from the current study may differ from this previous study because the previous study examined dementia prevalence and the current study examined incidence . the previous study comprised native americans living on reservations in canada , and the current study examined native americans living in northern california . to our knowledge , this study is the first to evaluate racial / ethnic differences in dementia risk among older adults with diabetes , a population particularly vulnerable to dementia . previous studies evaluated racial / ethnic differences in incidence of traditional diabetes - related complications ( 14,26,27 ) but provided much fewer data on diabetes - related comorbidities specific to older patients , including dementia . it is important to evaluate possible racial / ethnic differences in dementia incidence as part of the public health effort to eliminate health disparities . the kpnc diabetes registry is a large , well - characterized , diverse cohort of diabetic patients with detailed information on clinical control and diabetes - related complications . the kpnc diabetes registry is an excellent setting in which to examine racial / ethnic differences because health plan members have uniform access to care , so observed racial / ethnic differences in dementia risk should not be attributable to differences in access to care . the long duration of follow - up and generalizability to diabetic patients with access to medical care are also strengths of this study . although study patients had uniform access to care , differences in quality of care may still be present . dementia diagnosis was based on clinical diagnosis from electronic medical records rather than on standardized assessments administered regularly to all cohort members . it is possible that patients of some racial / ethnic groups are more or less likely to receive a dementia diagnosis from their health care provider . although our models included adjustment for a1c levels as a measure of glycemic control , evidence from previous studies suggest that a1c levels might not accurately capture average glycemic control across all racial / ethnic groups ( 28,29 ) . in conclusion , among older type 2 diabetic patients , african american and native american patients are at higher risk of dementia than other racial / ethnic groups , and asian patients are at lower risk of dementia . somewhat surprising are the observed differences not explained by sociodemographic characteristics or differences in diabetes duration , markers of clinical control , or microvascular or macrovascular complications of diabetes . given the epidemic of type 2 diabetes and its association with increased risk of dementia , more work is needed to identify factors that may explain these differences in dementia incidence and ways to eliminate them .

objectivealthough patients with type 2 diabetes have double the risk of dementia , potential racial / ethnic differences in dementia risk have not been explored in this population . we evaluated racial / ethnic differences in dementia and potential explanatory factors among older diabetic patients.research design and methodswe identified 22,171 diabetic patients without preexisting dementia aged 60 years ( 14,546 non - hispanic whites , 2,484 african americans , 2,363 latinos , 2,262 asians , 516 native americans ) from the kaiser permanente northern california diabetes registry . we abstracted prevalent medical history ( 1 january 1996 to 31 december 1997 ) and dementia incidence ( 1 january 1998 to 31 december 2007 ) from medical records and calculated age - adjusted incidence densities . we fit cox proportional hazards models adjusted for age , sex , education , diabetes duration , and markers of clinical control.resultsdementia was diagnosed in 3,796 ( 17.1% ) patients . age - adjusted dementia incidence densities were highest among native americans ( 34/1,000 person - years ) and african americans ( 27/1,000 person - years ) and lowest among asians ( 19/1,000 person - years ) . in the fully adjusted model , hazard ratios ( 95% cis ) ( relative to asians ) were 1.64 ( 1.302.06 ) for native americans , 1.44 ( 1.241.67 ) for african americans , 1.30 ( 1.151.47 ) for non - hispanic whites , and 1.19 ( 1.021.40 ) for latinos . adjustment for diabetes - related complications and neighborhood deprivation index did not change the results.conclusionsamong type 2 diabetic patients followed for 10 years , african americans and native americans had a 4060% greater risk of dementia compared with asians , and risk was intermediate for non - hispanic whites and latinos . adjustment for sociodemographics , diabetes - related complications , and markers of clinical control did not explain observed differences . future studies should investigate why these differences exist and ways to reduce them .

Introduction Research Design and Methods Study Population Dementia Diagnosis Other Measures Sociodemographic Characteristics. Diabetes Duration and Clinical Control (Baseline). Diabetes-Related Complications (Baseline). Other Covariates (Baseline). Statistical Analyses Results Conclusions Supplementary Material

type 2 diabetes is associated with an approximately twofold greater risk of dementia among older adults ( 13 ) , but it is not clear whether risk of dementia for people with diabetes varies across racial / ethnic groups . in the general population , the prevalence of dementia has been reported to be higher among african americans and caribbean hispanics , lower among japanese americans , and similar among native americans and mexican americans compared with non - hispanic whites ( 4,5 ) . a study of community - dwelling older adults in new york city reported that the higher prevalence of type 2 diabetes in african americans and hispanics compared with whites partially contributes to the higher observed incidence of dementia and cognitive impairment in these populations ( 6 ) . to our knowledge , however , racial / ethnic differences in dementia risk have not been investigated among people with type 2 diabetes . ( 7 ) , and the prevalence is highest in some racial / ethnic minorities , including latinos , african americans , native americans , and some asian american groups ( 7,8 ) . because type 2 diabetes increases the risk of dementia , it is important to understand the risk of dementia among older adults with type 2 diabetes of diverse racial / ethnic backgrounds . population , the increasing prevalence of type 2 diabetes ( 11,12 ) , and the national goal of eliminating health disparities ( 13 ) , understanding racial / ethnic differences in diabetes - related complications , including dementia , among older adults is increasingly important . the primary goal of this study was to examine whether there are racial / ethnic differences in the 10-year incidence of dementia among patients with type 2 diabetes . secondarily , we explored whether these differences are explained by diabetes duration , markers of clinical control , and vascular diabetes - related complications in a multiethnic population of older type 2 diabetic patients . we evaluated patients who were members of the kaiser permanente northern california ( kpnc ) diabetes registry , a well - characterized population of diabetic patients that has been the basis of a wide range of epidemiologic research ( 1417 ) . we included kpnc diabetes registry members in this analysis if they completed the 19941996 survey , had type 2 diabetes , were 60 years of age as of 1 january 1998 ( baseline ) , had no prior diagnosis of dementia , and had no gaps in membership > 3 months in the 2 years before baseline ( n = 29,956 ) . the proportion of patients who were excluded because of missing key covariates was similar across racial / ethnic groups and ranged from 23.0% in non - hispanic whites to 28.2% in latinos . dementia cases were identified from clinical diagnoses in electronic medical records from inpatient and outpatient encounters between 1 january 1998 and 31 december 2007 based on icd-9-cm diagnostic codes for alzheimer disease ( 331.0 ) ; senile dementia , uncomplicated ( 290.0 ) ; presenile dementia ( 290.1x ) ; senile dementia with delusional or depressive features ( 290.2x ) ; senile dementia with delirium ( 290.3 ) ; and vascular dementia ( 290.4x ) . race / ethnicity ( non - hispanic white , african american , latino , asian , pacific islander , native american , or mixed race / other ) and educational attainment were ascertained by self - report from the survey . acute metabolic events ( severe hyperglycemia or hypoglycemia requiring hospitalization ) were identified from electronic hospitalization and emergency department records during the period of 1 january 1996 to 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . a1c levels were abstracted from kpnc electronic laboratory databases , using the most recent measurement during the period of 1 january 1996 to 31 december 1997 . the median time from the a1c measurement to baseline ( 1 january 1998 ) was 0.34 years ( interquartile range 0.160.73 ) . diabetes pharmacotherapy was defined by a 30-day supply of glycemia - lowering medication dispensed at a kpnc pharmacy during the 6 months before baseline ( 1 january 1998 ) . prevalent diabetes - related microvascular and macrovascular complications were identified from electronic medical records from inpatient and outpatient visits that took place between 1 january 1996 and 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . hypertension was identified from electronic medical records ( 1 january 1996 to 31 december 1997 ) . age - adjusted dementia incidence densities ( incidence rates ) by racial / ethnic group were estimated after directly standardizing to the 2000 census population . we specified cox proportional hazards models to estimate the association between race / ethnicity and risk of dementia . to facilitate comparisons among racial / ethnic groups included in the study , we present hazard ratios for each model , with each racial / ethnic group as the reference group . because of the strong association between age and dementia incidence , we used age as the time scale in all proportional hazard models to best control for the influence of age on dementia risk . participants were followed from age as of 1 january 1998 until age at diagnosis of dementia , death , a gap in membership > 3 months , or 31 december 2007 ( end of study period ) . we estimated racial / ethnic differences in dementia risk after adjustment for age ( as the time scale ) , sex , and level of education . we evaluated whether the association between race / ethnicity and risk of dementia was explained by racial / ethnic differences in diabetes duration and clinical control ( pharmacotherapy , a1c level , acute metabolic events ) , microvascular complications ( end - stage renal disease , diabetic retinopathy , lower - extremity complications ) , macrovascular complications ( cardiovascular disease , cerebrovascular disease ) , neighborhood deprivation index , bmi , and hypertension . we evaluated patients who were members of the kaiser permanente northern california ( kpnc ) diabetes registry , a well - characterized population of diabetic patients that has been the basis of a wide range of epidemiologic research ( 1417 ) . we included kpnc diabetes registry members in this analysis if they completed the 19941996 survey , had type 2 diabetes , were 60 years of age as of 1 january 1998 ( baseline ) , had no prior diagnosis of dementia , and had no gaps in membership > 3 months in the 2 years before baseline ( n = 29,956 ) . the proportion of patients who were excluded because of missing key covariates was similar across racial / ethnic groups and ranged from 23.0% in non - hispanic whites to 28.2% in latinos . dementia cases were identified from clinical diagnoses in electronic medical records from inpatient and outpatient encounters between 1 january 1998 and 31 december 2007 based on icd-9-cm diagnostic codes for alzheimer disease ( 331.0 ) ; senile dementia , uncomplicated ( 290.0 ) ; presenile dementia ( 290.1x ) ; senile dementia with delusional or depressive features ( 290.2x ) ; senile dementia with delirium ( 290.3 ) ; and vascular dementia ( 290.4x ) . race / ethnicity ( non - hispanic white , african american , latino , asian , pacific islander , native american , or mixed race / other ) and educational attainment were ascertained by self - report from the survey . acute metabolic events ( severe hyperglycemia or hypoglycemia requiring hospitalization ) were identified from electronic hospitalization and emergency department records during the period of 1 january 1996 to 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . a1c levels were abstracted from kpnc electronic laboratory databases , using the most recent measurement during the period of 1 january 1996 to 31 december 1997 . the median time from the a1c measurement to baseline ( 1 january 1998 ) was 0.34 years ( interquartile range 0.160.73 ) . diabetes pharmacotherapy was defined by a 30-day supply of glycemia - lowering medication dispensed at a kpnc pharmacy during the 6 months before baseline ( 1 january 1998 ) . prevalent diabetes - related microvascular and macrovascular complications were identified from electronic medical records from inpatient and outpatient visits that took place between 1 january 1996 and 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . hypertension was identified from electronic medical records ( 1 january 1996 to 31 december 1997 ) . race / ethnicity ( non - hispanic white , african american , latino , asian , pacific islander , native american , or mixed race / other ) and educational attainment were ascertained by self - report from the survey . acute metabolic events ( severe hyperglycemia or hypoglycemia requiring hospitalization ) were identified from electronic hospitalization and emergency department records during the period of 1 january 1996 to 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . a1c levels were abstracted from kpnc electronic laboratory databases , using the most recent measurement during the period of 1 january 1996 to 31 december 1997 . the median time from the a1c measurement to baseline ( 1 january 1998 ) was 0.34 years ( interquartile range 0.160.73 ) . diabetes pharmacotherapy was defined by a 30-day supply of glycemia - lowering medication dispensed at a kpnc pharmacy during the 6 months before baseline ( 1 january 1998 ) . prevalent diabetes - related microvascular and macrovascular complications were identified from electronic medical records from inpatient and outpatient visits that took place between 1 january 1996 and 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . macrovascular complications included cardiovascular disease ( congestive heart failure , peripheral vascular disease , and myocardial infarction ) and cerebrovascular disease . hypertension was identified from electronic medical records ( 1 january 1996 to 31 december 1997 ) . age - adjusted dementia incidence densities ( incidence rates ) by racial / ethnic group were estimated after directly standardizing to the 2000 census population . we specified cox proportional hazards models to estimate the association between race / ethnicity and risk of dementia . to facilitate comparisons among racial / ethnic groups included in the study , we present hazard ratios for each model , with each racial / ethnic group as the reference group . because of the strong association between age and dementia incidence , we used age as the time scale in all proportional hazard models to best control for the influence of age on dementia risk . participants were followed from age as of 1 january 1998 until age at diagnosis of dementia , death , a gap in membership > 3 months , or 31 december 2007 ( end of study period ) . we estimated racial / ethnic differences in dementia risk after adjustment for age ( as the time scale ) , sex , and level of education . we evaluated whether the association between race / ethnicity and risk of dementia was explained by racial / ethnic differences in diabetes duration and clinical control ( pharmacotherapy , a1c level , acute metabolic events ) , microvascular complications ( end - stage renal disease , diabetic retinopathy , lower - extremity complications ) , macrovascular complications ( cardiovascular disease , cerebrovascular disease ) , neighborhood deprivation index , bmi , and hypertension . approximately 66% were non - hispanic white , 11% african american , 11% latino , 10% asian , and 2% native american ( table 1 ) . education levels were lowest among latinos and highest among asians , and higher proportions of african americans and latinos lived in deprived neighborhoods . a1c levels tended to be highest among african americans , latinos , and asians , and acute metabolic events were more common among african americans and native americans . individual diabetes - related complications varied by race / ethnicity , but there was no consistent pattern of any one racial / ethnic group consistently having a higher prevalence of diabetes - related complications ( microvascular or macrovascular ) overall . among microvascular complications , end - stage renal disease was rare in all groups , the prevalence of lower - extremity complications was lowest among asians , and diabetic retinopathy was most prevalent among african americans and latinos . among macrovascular complications , peripheral vascular disease was least common among asians , myocardial infarction was more prevalent among native americans and non - hispanic whites , congestive heart failure was least common among asians and latinos , and the prevalence of cerebrovascular disease was highest among non - hispanic whites and native americans . the median number of clinical visits per year was similar across race groups but was lowest among native americans ( median 9.0 ) and highest among latinos ( median 10.5 ) . over 10 years of follow - up , 3,796 ( 17.1% ) participants were diagnosed with dementia . among patients with dementia , 676 ( 17.8% ) were diagnosed with alzheimer disease , 474 ( 12.5% ) with vascular dementia , and 128 ( 3.4% ) with both alzheimer disease and vascular dementia . because the majority of dementia diagnoses were for unspecified dementia , we did not evaluate racial / ethnic differences for specific dementia subtypes . age - adjusted dementia cumulative incidence and incidence densities were highest among native americans and african americans and lowest among asians ( table 2 ) . adjustment for age , sex , and level of education in the cox proportional hazards models showed consistent patterns ( table 3 , model 1 ) . the measures of diabetes control and microvascular and macrovascular complications were significant risk factors for incident dementia in bivariate cox proportional hazards models ( data not shown ) . however , adjustment for these measures ( table 3 , models 24 ) imparted no substantive change in the association between race / ethnicity and dementia incidence . in all four models , dementia risk was significantly higher among native americans and african americans than among the other racial / ethnic groups , whereas dementia risk was significantly lower among asians than among all other racial / ethnic groups . dementia risk was similar for non - hispanic whites and latinos , whose risk of dementia was lower than that of native americans and african americans and higher than that of asians . native americans had a 60% increased risk of dementia in models that accounted for sociodemographic characteristics , diabetes duration , markers of clinical control , and microvascular and macrovascular complications . we also adjusted for bmi , hypertension , and neighborhood deprivation index , but these variables did not change the magnitude of the association between race / ethnicity and risk of dementia ( data not shown ) . the observed trends persisted when we included individuals with missing information on education , a1c , and/or duration of diabetes , when we accounted for the competing risk of death with competing risk regression models ( 22 ) , and when we stratified by sex ( data not shown ) . dementia incidence densities by race / ethnicity ( 19982007 ) hazard ratios for race / ethnicity and dementia risk ( 19982007 ) among older type 2 diabetic patients , 10-year dementia incidence was highest among native americans and african americans , lowest among asians , and intermediate for non - hispanic whites and latinos . the observed differences persisted after accounting for sociodemographic characteristics , diabetes duration , markers of clinical control , and a host of diabetes - related microvascular or macrovascular complications . to our knowledge , this study is the first to examine racial / ethnic differences in dementia incidence among people with diabetes , a group at high - risk for dementia . although microvascular complications , macrovascular complications , and markers of clinical control were associated with increased dementia incidence , the observed racial / ethnic differences in dementia risk persisted after adjustment for these factors . although the prevalence of microvascular and macrovascular complications varied by race / ethnicity in the current study , there was no general pattern of a greater burden of complications in certain racial / ethnic groups . previous population - based studies reported that among people with diabetes , the rates of microvascular complications , such as end - stage renal disease , tend to be higher and the rates of macrovascular complications lower among racial / ethnic minorities compared with non - hispanic whites ( 14,26,27 ) . however , the current finding of no general pattern with respect to race / ethnicity and diabetes - related complications is consistent with earlier research on diabetes - related complications in this population ( 14 ) . unmeasured behavioral or environmental factors years of education varied across racial / ethnic groups and tended to be lowest among latinos and highest among asians . we adjusted for years of education , but this may not account for differences in quality of education , which may partially explain the observed racial / ethnic differences in dementia incidence . we adjusted for neighborhood deprivation index , which may have negative effects on health by influencing health behaviors and stress ( 21 ) . the findings on racial / ethnic difference in dementia risk among older diabetic patients are in accord with previous population - based studies of older adults with and without diabetes , which found that dementia rates were higher among african americans relative to non - hispanic whites ( 4 ) and similar among mexican americans ( 5 ) compared with non - hispanic whites . the current finding that dementia rates are lower among asian americans than among non - hispanic whites is consistent with previous reports among japanese americans ( 4 ) . although the incidence of dementia was higher among native americans than among other racial / ethnic groups in the current study , one small canadian cross - sectional study reported a similar prevalence of dementia among native americans living on two reservations in manitoba relative to non - hispanic whites living in winnipeg ( 4 ) . the previous study comprised native americans living on reservations in canada , and the current study examined native americans living in northern california . to our knowledge , this study is the first to evaluate racial / ethnic differences in dementia risk among older adults with diabetes , a population particularly vulnerable to dementia . previous studies evaluated racial / ethnic differences in incidence of traditional diabetes - related complications ( 14,26,27 ) but provided much fewer data on diabetes - related comorbidities specific to older patients , including dementia . it is important to evaluate possible racial / ethnic differences in dementia incidence as part of the public health effort to eliminate health disparities . the kpnc diabetes registry is a large , well - characterized , diverse cohort of diabetic patients with detailed information on clinical control and diabetes - related complications . the kpnc diabetes registry is an excellent setting in which to examine racial / ethnic differences because health plan members have uniform access to care , so observed racial / ethnic differences in dementia risk should not be attributable to differences in access to care . the long duration of follow - up and generalizability to diabetic patients with access to medical care are also strengths of this study . although our models included adjustment for a1c levels as a measure of glycemic control , evidence from previous studies suggest that a1c levels might not accurately capture average glycemic control across all racial / ethnic groups ( 28,29 ) . in conclusion , among older type 2 diabetic patients , african american and native american patients are at higher risk of dementia than other racial / ethnic groups , and asian patients are at lower risk of dementia . somewhat surprising are the observed differences not explained by sociodemographic characteristics or differences in diabetes duration , markers of clinical control , or microvascular or macrovascular complications of diabetes . given the epidemic of type 2 diabetes and its association with increased risk of dementia , more work is needed to identify factors that may explain these differences in dementia incidence and ways to eliminate them .

type 2 diabetes is associated with an approximately twofold greater risk of dementia among older adults ( 13 ) , but it is not clear whether risk of dementia for people with diabetes varies across racial / ethnic groups . in the general population , the prevalence of dementia has been reported to be higher among african americans and caribbean hispanics , lower among japanese americans , and similar among native americans and mexican americans compared with non - hispanic whites ( 4,5 ) . a study of community - dwelling older adults in new york city reported that the higher prevalence of type 2 diabetes in african americans and hispanics compared with whites partially contributes to the higher observed incidence of dementia and cognitive impairment in these populations ( 6 ) . to our knowledge , however , racial / ethnic differences in dementia risk have not been investigated among people with type 2 diabetes . ( 7 ) , and the prevalence is highest in some racial / ethnic minorities , including latinos , african americans , native americans , and some asian american groups ( 7,8 ) . because type 2 diabetes increases the risk of dementia , it is important to understand the risk of dementia among older adults with type 2 diabetes of diverse racial / ethnic backgrounds . population , the increasing prevalence of type 2 diabetes ( 11,12 ) , and the national goal of eliminating health disparities ( 13 ) , understanding racial / ethnic differences in diabetes - related complications , including dementia , among older adults is increasingly important . the primary goal of this study was to examine whether there are racial / ethnic differences in the 10-year incidence of dementia among patients with type 2 diabetes . secondarily , we explored whether these differences are explained by diabetes duration , markers of clinical control , and vascular diabetes - related complications in a multiethnic population of older type 2 diabetic patients . we evaluated patients who were members of the kaiser permanente northern california ( kpnc ) diabetes registry , a well - characterized population of diabetic patients that has been the basis of a wide range of epidemiologic research ( 1417 ) . the registry was formed in 1993 and is updated annually to include all kpnc health plan members who are identified as having diabetes on the basis of laboratory results , pharmacy records , and outpatient diagnoses ( 14 ) . kpnc is a large , integrated health care delivery system that provides comprehensive medical care to > 3.3 million members ( 30% of the geographic region ) . the kpnc population is generally representative of the overall population of the region , with the exception that individuals at the extreme tails of the income distribution are underrepresented ( 18,19 ) . between 1994 and 1996 , all noninstitutionalized kpnc diabetes registry members 19 years of age were sent a self - administered survey ( mailed or online ) or contacted for a computer - assisted telephone interview on sociodemographic factors and health behaviors . the written and web surveys were in english only , but the computer - assisted telephone interview was available in english , spanish , cantonese , mandarin , and tagalog through certified translations of an english script and was intended to maximize accessibility to those with language barriers or limited english literacy or fluency . we included kpnc diabetes registry members in this analysis if they completed the 19941996 survey , had type 2 diabetes , were 60 years of age as of 1 january 1998 ( baseline ) , had no prior diagnosis of dementia , and had no gaps in membership > 3 months in the 2 years before baseline ( n = 29,956 ) . we also excluded 7,162 patients missing information on the following key covariates : level of education ( n = 1,842 ) , glycosylated hemoglobin ( a1c ) ( n = 2,976 ) , duration of diabetes ( n = 1,745 ) , or more than one of these variables ( n = 599 ) . dementia cases were identified from clinical diagnoses in electronic medical records from inpatient and outpatient encounters between 1 january 1998 and 31 december 2007 based on icd-9-cm diagnostic codes for alzheimer disease ( 331.0 ) ; senile dementia , uncomplicated ( 290.0 ) ; presenile dementia ( 290.1x ) ; senile dementia with delusional or depressive features ( 290.2x ) ; senile dementia with delirium ( 290.3 ) ; and vascular dementia ( 290.4x ) . race / ethnicity ( non - hispanic white , african american , latino , asian , pacific islander , native american , or mixed race / other ) and educational attainment were ascertained by self - report from the survey . acute metabolic events ( severe hyperglycemia or hypoglycemia requiring hospitalization ) were identified from electronic hospitalization and emergency department records during the period of 1 january 1996 to 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . prevalent diabetes - related microvascular and macrovascular complications were identified from electronic medical records from inpatient and outpatient visits that took place between 1 january 1996 and 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . microvascular complications included end - stage renal disease , diabetic retinal disease , and lower - extremity complications ( lower - limb ulcer , gangrene , and lower - extremity amputation ) . as a measure of health care utilization , the average number of clinical visits per year during follow - up for each individual was calculated , resulting in a medical utilization rate . age - adjusted dementia incidence densities ( incidence rates ) by racial / ethnic group were estimated after directly standardizing to the 2000 census population . we specified cox proportional hazards models to estimate the association between race / ethnicity and risk of dementia . to facilitate comparisons among racial / ethnic groups included in the study , we present hazard ratios for each model , with each racial / ethnic group as the reference group . because of the strong association between age and dementia incidence , we used age as the time scale in all proportional hazard models to best control for the influence of age on dementia risk . participants were followed from age as of 1 january 1998 until age at diagnosis of dementia , death , a gap in membership > 3 months , or 31 december 2007 ( end of study period ) . we estimated racial / ethnic differences in dementia risk after adjustment for age ( as the time scale ) , sex , and level of education . we evaluated whether the association between race / ethnicity and risk of dementia was explained by racial / ethnic differences in diabetes duration and clinical control ( pharmacotherapy , a1c level , acute metabolic events ) , microvascular complications ( end - stage renal disease , diabetic retinopathy , lower - extremity complications ) , macrovascular complications ( cardiovascular disease , cerebrovascular disease ) , neighborhood deprivation index , bmi , and hypertension . we evaluated patients who were members of the kaiser permanente northern california ( kpnc ) diabetes registry , a well - characterized population of diabetic patients that has been the basis of a wide range of epidemiologic research ( 1417 ) . the registry was formed in 1993 and is updated annually to include all kpnc health plan members who are identified as having diabetes on the basis of laboratory results , pharmacy records , and outpatient diagnoses ( 14 ) . kpnc is a large , integrated health care delivery system that provides comprehensive medical care to > 3.3 million members ( 30% of the geographic region ) . the kpnc population is generally representative of the overall population of the region , with the exception that individuals at the extreme tails of the income distribution are underrepresented ( 18,19 ) . between 1994 and 1996 , all noninstitutionalized kpnc diabetes registry members 19 years of age were sent a self - administered survey ( mailed or online ) or contacted for a computer - assisted telephone interview on sociodemographic factors and health behaviors . the written and web surveys were in english only , but the computer - assisted telephone interview was available in english , spanish , cantonese , mandarin , and tagalog through certified translations of an english script and was intended to maximize accessibility to those with language barriers or limited english literacy or fluency . we included kpnc diabetes registry members in this analysis if they completed the 19941996 survey , had type 2 diabetes , were 60 years of age as of 1 january 1998 ( baseline ) , had no prior diagnosis of dementia , and had no gaps in membership > 3 months in the 2 years before baseline ( n = 29,956 ) . we also excluded 7,162 patients missing information on the following key covariates : level of education ( n = 1,842 ) , glycosylated hemoglobin ( a1c ) ( n = 2,976 ) , duration of diabetes ( n = 1,745 ) , or more than one of these variables ( n = 599 ) . dementia cases were identified from clinical diagnoses in electronic medical records from inpatient and outpatient encounters between 1 january 1998 and 31 december 2007 based on icd-9-cm diagnostic codes for alzheimer disease ( 331.0 ) ; senile dementia , uncomplicated ( 290.0 ) ; presenile dementia ( 290.1x ) ; senile dementia with delusional or depressive features ( 290.2x ) ; senile dementia with delirium ( 290.3 ) ; and vascular dementia ( 290.4x ) . race / ethnicity ( non - hispanic white , african american , latino , asian , pacific islander , native american , or mixed race / other ) and educational attainment were ascertained by self - report from the survey . acute metabolic events ( severe hyperglycemia or hypoglycemia requiring hospitalization ) were identified from electronic hospitalization and emergency department records during the period of 1 january 1996 to 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . prevalent diabetes - related microvascular and macrovascular complications were identified from electronic medical records from inpatient and outpatient visits that took place between 1 january 1996 and 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . microvascular complications included end - stage renal disease , diabetic retinal disease , and lower - extremity complications ( lower - limb ulcer , gangrene , and lower - extremity amputation ) . as a measure of health care utilization , the average number of clinical visits per year during follow - up for each individual was calculated , resulting in a medical utilization rate . race / ethnicity ( non - hispanic white , african american , latino , asian , pacific islander , native american , or mixed race / other ) and educational attainment were ascertained by self - report from the survey . acute metabolic events ( severe hyperglycemia or hypoglycemia requiring hospitalization ) were identified from electronic hospitalization and emergency department records during the period of 1 january 1996 to 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . prevalent diabetes - related microvascular and macrovascular complications were identified from electronic medical records from inpatient and outpatient visits that took place between 1 january 1996 and 31 december 1997 , using primary icd-9-cm diagnostic codes ( supplementary data ) . microvascular complications included end - stage renal disease , diabetic retinal disease , and lower - extremity complications ( lower - limb ulcer , gangrene , and lower - extremity amputation ) . as a measure of health care utilization , the average number of clinical visits per year during follow - up for each individual was calculated , resulting in a medical utilization rate . to facilitate comparisons among racial / ethnic groups included in the study , we present hazard ratios for each model , with each racial / ethnic group as the reference group . because of the strong association between age and dementia incidence , we used age as the time scale in all proportional hazard models to best control for the influence of age on dementia risk . participants were followed from age as of 1 january 1998 until age at diagnosis of dementia , death , a gap in membership > 3 months , or 31 december 2007 ( end of study period ) . we estimated racial / ethnic differences in dementia risk after adjustment for age ( as the time scale ) , sex , and level of education . we evaluated whether the association between race / ethnicity and risk of dementia was explained by racial / ethnic differences in diabetes duration and clinical control ( pharmacotherapy , a1c level , acute metabolic events ) , microvascular complications ( end - stage renal disease , diabetic retinopathy , lower - extremity complications ) , macrovascular complications ( cardiovascular disease , cerebrovascular disease ) , neighborhood deprivation index , bmi , and hypertension . overall , the mean duration of diabetes at baseline was 6 years , and 82.2% of participants were receiving diabetes pharmacotherapy . a1c levels tended to be highest among african americans , latinos , and asians , and acute metabolic events were more common among african americans and native americans . individual diabetes - related complications varied by race / ethnicity , but there was no consistent pattern of any one racial / ethnic group consistently having a higher prevalence of diabetes - related complications ( microvascular or macrovascular ) overall . among microvascular complications , end - stage renal disease was rare in all groups , the prevalence of lower - extremity complications was lowest among asians , and diabetic retinopathy was most prevalent among african americans and latinos . among macrovascular complications , peripheral vascular disease was least common among asians , myocardial infarction was more prevalent among native americans and non - hispanic whites , congestive heart failure was least common among asians and latinos , and the prevalence of cerebrovascular disease was highest among non - hispanic whites and native americans . baseline characteristics of the sample by race / ethnicity ( 19961997 ) the average length of follow - up was 6.7 years , and the average age at dementia diagnosis was 79.2 years . the median number of clinical visits per year was similar across race groups but was lowest among native americans ( median 9.0 ) and highest among latinos ( median 10.5 ) . among patients with dementia , 676 ( 17.8% ) were diagnosed with alzheimer disease , 474 ( 12.5% ) with vascular dementia , and 128 ( 3.4% ) with both alzheimer disease and vascular dementia . age - adjusted dementia cumulative incidence and incidence densities were highest among native americans and african americans and lowest among asians ( table 2 ) . adjustment for age , sex , and level of education in the cox proportional hazards models showed consistent patterns ( table 3 , model 1 ) . the measures of diabetes control and microvascular and macrovascular complications were significant risk factors for incident dementia in bivariate cox proportional hazards models ( data not shown ) . in all four models , dementia risk was significantly higher among native americans and african americans than among the other racial / ethnic groups , whereas dementia risk was significantly lower among asians than among all other racial / ethnic groups . dementia risk was similar for non - hispanic whites and latinos , whose risk of dementia was lower than that of native americans and african americans and higher than that of asians . native americans had a 60% increased risk of dementia in models that accounted for sociodemographic characteristics , diabetes duration , markers of clinical control , and microvascular and macrovascular complications . we also adjusted for bmi , hypertension , and neighborhood deprivation index , but these variables did not change the magnitude of the association between race / ethnicity and risk of dementia ( data not shown ) . the observed trends persisted when we included individuals with missing information on education , a1c , and/or duration of diabetes , when we accounted for the competing risk of death with competing risk regression models ( 22 ) , and when we stratified by sex ( data not shown ) . dementia incidence densities by race / ethnicity ( 19982007 ) hazard ratios for race / ethnicity and dementia risk ( 19982007 ) among older type 2 diabetic patients , 10-year dementia incidence was highest among native americans and african americans , lowest among asians , and intermediate for non - hispanic whites and latinos . the observed differences persisted after accounting for sociodemographic characteristics , diabetes duration , markers of clinical control , and a host of diabetes - related microvascular or macrovascular complications . to our knowledge , this study is the first to examine racial / ethnic differences in dementia incidence among people with diabetes , a group at high - risk for dementia . although microvascular complications , macrovascular complications , and markers of clinical control were associated with increased dementia incidence , the observed racial / ethnic differences in dementia risk persisted after adjustment for these factors . although the prevalence of microvascular and macrovascular complications varied by race / ethnicity in the current study , there was no general pattern of a greater burden of complications in certain racial / ethnic groups . previous population - based studies reported that among people with diabetes , the rates of microvascular complications , such as end - stage renal disease , tend to be higher and the rates of macrovascular complications lower among racial / ethnic minorities compared with non - hispanic whites ( 14,26,27 ) . however , the current finding of no general pattern with respect to race / ethnicity and diabetes - related complications is consistent with earlier research on diabetes - related complications in this population ( 14 ) . a greater proportion of african american and latino patients lived in deprived neighborhoods compared with other groups , but adjusting for this variable did not alter observed associations and was not included in final models . the findings on racial / ethnic difference in dementia risk among older diabetic patients are in accord with previous population - based studies of older adults with and without diabetes , which found that dementia rates were higher among african americans relative to non - hispanic whites ( 4 ) and similar among mexican americans ( 5 ) compared with non - hispanic whites . although the incidence of dementia was higher among native americans than among other racial / ethnic groups in the current study , one small canadian cross - sectional study reported a similar prevalence of dementia among native americans living on two reservations in manitoba relative to non - hispanic whites living in winnipeg ( 4 ) . to our knowledge , this study is the first to evaluate racial / ethnic differences in dementia risk among older adults with diabetes , a population particularly vulnerable to dementia . previous studies evaluated racial / ethnic differences in incidence of traditional diabetes - related complications ( 14,26,27 ) but provided much fewer data on diabetes - related comorbidities specific to older patients , including dementia . the kpnc diabetes registry is a large , well - characterized , diverse cohort of diabetic patients with detailed information on clinical control and diabetes - related complications . the kpnc diabetes registry is an excellent setting in which to examine racial / ethnic differences because health plan members have uniform access to care , so observed racial / ethnic differences in dementia risk should not be attributable to differences in access to care . although our models included adjustment for a1c levels as a measure of glycemic control , evidence from previous studies suggest that a1c levels might not accurately capture average glycemic control across all racial / ethnic groups ( 28,29 ) . in conclusion , among older type 2 diabetic patients , african american and native american patients are at higher risk of dementia than other racial / ethnic groups , and asian patients are at lower risk of dementia . somewhat surprising are the observed differences not explained by sociodemographic characteristics or differences in diabetes duration , markers of clinical control , or microvascular or macrovascular complications of diabetes . given the epidemic of type 2 diabetes and its association with increased risk of dementia , more work is needed to identify factors that may explain these differences in dementia incidence and ways to eliminate them .

within the last few years , multistate collaboratives or networks have become a more prevalent mode for implementing health care quality improvement initiatives . there are many benefits to states for participating in such collaboratives , but at the center is that they give states the opportunity to learn from each other , as well as from experts , about successful practices and policies . further , the sharing of information can take place rapidly and without the significant time lag needed for published research to become available . our experience from participating in the medicaid medical directors learning network ( mmdln ) whose motto is to steal shamelessly and share senselessly , has borne out the importance of such collaboratives . as the colorado medicaid medical director ( jz ) , mmdln manager ( kg ) , and medicaid medical director ( mmd)-led study principle investigators ( gf , tt ) , our leadership roles have given us a unique perspective on the benefits and importance of such collaboratives to states and the field at large . in this paper , we share our experiences and lessons about the value of participating in this learning network . the mmdln is focused on the development and use of evidence - based medicine , measurement , and improvement of health care quality ; and on the redesign of health care delivery systems through expert presentations and peer - to - peer learning . the mmdln is one of several state - level collaboratives or networks , all of which share the ultimate goal of improving health care quality . while these collaboratives may have slightly different purposes or populations of focus , they have in common the ultimate purpose of providing actionable evidence to aid in states decision - making . for example , the health resources and services administration s ( hrsa ) collaborative improvement & innovation network ( coiin ) is facilitating learning and adoption of common , proven quality improvement practices across all 50 states to reduce infant mortality and improve birth outcomes,1 the centers medicare & medicaid services funded national improvement partnership network ( nipn ) is a network of over 20 states developed to advance quality and transform health care for children and their families,2 and the medicaid evidence - based decisions ( med ) project was established as a self - governing , state - funded collaboration of state medicaid agencies to provide policymakers with the tools and resources they need to make evidence - based decisions including producing independent and objective evaluations of clinical evidence and convening states around clinical topics of interest.3 a major value of collaboratives like the mmdln and these others is that they allow for rapid learning among state members . evidence provided to the states through these collaboratives comes in varying forms including data analysis results , literature reviews , expert presentations , and even anecdotal references from peers . for the mmds , evidence to date has also come from administrative claims data from participating states , aggregated and assembled to show patterns at the national level . a companion paper describes methods for using this distributed claims data resource.4 along with the value of rapid learning in these collaboratives is the fact that knowledge builds on prior experiences as practices are shared . for example , when a new quality improvement practice is implemented by one state based on information shared through collaboratives , it is often picked up and further improved upon by other states . thus , states , and ultimately their constituents , benefit in a timely manner from the continuous cycle of sharing , implementation , assessment , refinement , and more sharing . however , there are also challenges associated with participating in claims - based data aggregation projects and maintaining a learning network that are also discussed in the companion paper . despite these challenges , this commentary examines these benefits and illustrates the importance of quality improvement collaborations to decision - making in state medicaid programs . department of health and human services agency for healthcare research and quality ( ahrq ) in 2005 under contract to academyhealth . while that contract ended in 2013 , the work of the network is continuing under the national association of medicaid directors as a clinical arm of the national association of state medicaid programs . the network , as an integrated national resource , seeks to advance the health of medicaid patients in the states and across the nation while best stewarding available resources . network participation is open to mmds and those in similar clinical leadership positions who advise the medicaid director for one or more components of a medicaid program administered by a state , territory , or the district of columbia . for the last eight years , network activities have focused on regular in - person and virtual learning sessions addressing topics of high relevance and importance for the medicaid population . in addition , we have a regularly maintained website and use a web forum to share documents and pose questions to other members of the network . these activities have allowed mmds the opportunity to interact with our peers and learn from each other s experiences . in 2007 , through the mmdln , mmds also began conducting quality improvement studies using their own state - level administrative data to better understand the major clinical issues facing the medicaid populations and to work together on policies to improve outcomes . mmds saw an opportunity as clinical leaders in the states to assess clinical outcomes , to better understand an issue , and to make quality improvement decisions . the first project dealt with antipsychotic medication use in children and adolescents;5 the second examined hospital admissions and readmissions , while the third focused on early elective deliveries . the companion paper describes in detail the methods used to conduct them ; the present commentary focuses on the benefits of such studies and their importance to quality improvement in medicaid . findings in this commentary , as well as the companion paper , are based on review of documents ; personal experiences ; and discussions with mmds leaders , researchers involved in these studies , and state analysts . all problems selected by mmds for quality improvement monitoring involved an important national problem one that involved both morbidity and cost for the medicaid population and were amenable to policy solutions . the importance of understanding and improving care and cost in medicaid can hardly be overstated . with 74 million beneficiaries,6 cost from excess utilization can be enormous , and conversely , even small improvements can result in cost savings . in the selected areas , a the first study examined the use of antipsychotic medication in children , and was selected in response to the high level of concern among the mmds about this issue . the results of this multistate study that antipsychotic medication use was prevalent , even among young children and especially among children in foster care highlighted a problem that had previously received little attention nationally and galvanized action to address it . states also shared policy tools to address antipsychotic medication use in children and the evidence that these policies worked . department of health & human services ( hhs ) centers for medicare & medicaid services ( cms ) in letters to medicaid directors when discussing the seriousness of the issue , and was the topic of a summit in the fall of 2012 sponsored by the hhs administration for children , youth , and families . further , in recognition of the importance of this issue , there has been subsequent funding to states to improve monitoring of prescribing in foster care children . this first study made clear the need for such efforts in medicaid and the benefit to states . the mmds selected this topic as the second area of focus due to the prevalence , expense and presumed preventability of hospital use ( at least for some diagnoses).78 national data showed that about 1 in 12 adults discharged from a hospital was readmitted within 30 days , adding an additional $ 16 billion to health care costs in the united states.9 additionally , the mmds took note of the fact that medicare had targeted hospital readmissions as an important area for both cost savings and care improvement and crafted nonpayment policies for some readmissions.1011 many state officials expected that similar policies might be applied to the medicaid population . it was thus deemed critically important to understand the extent and nature of hospital readmissions in medicaid , which had not been studied , in order to create effective state - level policies . this study found that readmissions were costly for the states and were more prevalent in those with certain diagnoses such as mental health . further , readmission rates varied considerably among the 19 participating states , which allowed states with higher rates to learn from those with lower rates.12 the third multistate study , which is currently under way , is examining early elective deliveries ( i.e. , labor induction or cesarean deliveries occurring before 39 weeks ) . first , there was a large body of evidence showing that these early deliveries led to poorer neonatal outcomes and long - term problems , as well as excess cost.1318 second , there was a clear policy statement from the american college of obstetricians and gynecologists ( acog ) calling for the restriction of these early elective deliveries.1920 third , an mmd - led effort in partnership with public health colleagues , could exert a multiplier effect by building on and complementing other efforts including the hrsa coiin and cms strong start for mothers and newborns initiative to reduce preterm births and improve birth outcomes.21 finally , since medicaid pays for up to 48 percent of the births nationally ( 1.8 million births),2223 the program has a clear vested interest in developing policies to reduce complications and costs associated with these early elective deliveries . this study initially found that , of the medicaid singleton deliveries in 22 states , early elective deliveries are prevalent and that states can improve their reported rates by implementing policies to reduce them and improving the reporting on the birth certificates of medical indications for an early labor induction or cesarean delivery . the first study examined the use of antipsychotic medication in children , and was selected in response to the high level of concern among the mmds about this issue . the results of this multistate study that antipsychotic medication use was prevalent , even among young children and especially among children in foster care highlighted a problem that had previously received little attention nationally and galvanized action to address it . states also shared policy tools to address antipsychotic medication use in children and the evidence that these policies worked . department of health & human services ( hhs ) centers for medicare & medicaid services ( cms ) in letters to medicaid directors when discussing the seriousness of the issue , and was the topic of a summit in the fall of 2012 sponsored by the hhs administration for children , youth , and families . further , in recognition of the importance of this issue , there has been subsequent funding to states to improve monitoring of prescribing in foster care children . this first study made clear the need for such efforts in medicaid and the benefit to states . the mmds selected this topic as the second area of focus due to the prevalence , expense and presumed preventability of hospital use ( at least for some diagnoses).78 national data showed that about 1 in 12 adults discharged from a hospital was readmitted within 30 days , adding an additional $ 16 billion to health care costs in the united states.9 additionally , the mmds took note of the fact that medicare had targeted hospital readmissions as an important area for both cost savings and care improvement and crafted nonpayment policies for some readmissions.1011 many state officials expected that similar policies might be applied to the medicaid population . it was thus deemed critically important to understand the extent and nature of hospital readmissions in medicaid , which had not been studied , in order to create effective state - level policies . this study found that readmissions were costly for the states and were more prevalent in those with certain diagnoses such as mental health . further , readmission rates varied considerably among the 19 participating states , which allowed states with higher rates to learn from those with lower rates.12 the third multistate study , which is currently under way , is examining early elective deliveries ( i.e. , labor induction or cesarean deliveries occurring before 39 weeks ) . first , there was a large body of evidence showing that these early deliveries led to poorer neonatal outcomes and long - term problems , as well as excess cost.1318 second , there was a clear policy statement from the american college of obstetricians and gynecologists ( acog ) calling for the restriction of these early elective deliveries.1920 third , an mmd - led effort in partnership with public health colleagues , could exert a multiplier effect by building on and complementing other efforts including the hrsa coiin and cms strong start for mothers and newborns initiative to reduce preterm births and improve birth outcomes.21 finally , since medicaid pays for up to 48 percent of the births nationally ( 1.8 million births),2223 the program has a clear vested interest in developing policies to reduce complications and costs associated with these early elective deliveries . this study initially found that , of the medicaid singleton deliveries in 22 states , early elective deliveries are prevalent and that states can improve their reported rates by implementing policies to reduce them and improving the reporting on the birth certificates of medical indications for an early labor induction or cesarean delivery . there are a number of reasons why these studies are important and useful to state policy making and practices for the medicaid program . one of the most important reasons is that these studies respond to crucially important national health and policy concerns for the medicaid population . medicaid is currently at the vortex of policy making for the nation s lowest - income , most vulnerable , and sickest residents , and as medicaid expands under the affordable care act ( aca ) the size and importance of this vulnerable population will increase . this will bring new demands to the program and heighten the need for these collaborative , rapid learning projects on areas of concern to multiple states . the multistate clinical quality projects conducted by the mmds offer a key mechanism ( possibly the best mechanism ) to help medicaid monitor program outcomes across the country and continuously measure efforts to deliver value - driven , high - quality , cost - effective health care in an efficient manner . quality improvement studies often start discussions about improvement goals and drive action in the states . for example , the studies can lead to important state - level actions to implement effective policies that then are built upon by other states reassessment . as one mmd said , instead of this is our strategy and that is yours now it will be , what can we do together to get there faster ? that is a very exciting conversation to have . some mmds believe that these multistate studies also enable states to share and implement policies and practices used by others . another mmd said that the outcomes were not just [ about ] the data , but several states picked up on other states practices and implemented them similar to the effect of sharing and understanding data on reducing practice variation , work must be done between states to accelerate policy changes . the mmd s quality improvement projects provide participating states with data to inform policies internally . such data is important because state legislatures and policymakers are often more persuaded by local state data than by studies from another state or at the national level . only aggregated data from all states are shared publicly , but each state receives information on its own progress . specifically , individual states receive information comparing its outcomes with the average across all states and to the range of outcomes . this information allows the medicaid agency to highlight specific areas for policy or payment change . these studies are especially important in that there is a relative dearth of published research relying on medicaid administrative data . even though the reports are not the first to examine given issues , they are important because state policymakers often need to see their own data in comparison to the aggregate multistate results to make a strong case for improvements . and indeed , states have used the findings from the reports in a variety of ways , including the following : to identify and correct problems such as disparities , to set policies , to plan within the state , and to continue to monitor the data on their own . an example of identifying and correcting problems includes some mmds taking the data on readmissions to hospitals and nursing homes that were in the top for readmissions . as an example , one children s hospital did not believe that they had a problem with readmissions , but the results convinced them otherwise , and convinced them to take action by implementing an internal quality improvement program . many states have used the results of the studies to set policy , for example , one state used the antipsychotic medications study learnings to require prior review and authorization for use . one mmd stated the report on antipsychotic drug use , has been really impactful in that it has pushed the policy conversation that is taking place especially in the kids in foster care . you see antipsychotics in those kids and what we do around that and it really pushes the physical and behavioral health integration issues . other states incorporated readmissions into pay - for - performance hospital policies and used the read - missions results to inform decisions on incentive payments to hospitals . in addition , one state used the antipsychotic medication data for planning purposes , as a basis for a grant to further address the problem of inappropriate use . finally other states use the project materials , such as the data dictionaries , to continue to monitor the issue . one state is continuing to track antipsychotic use ( using the code developed in this project ) , and another state mentioned that tracking readmissions and the readmission report pushed the discussions of the problems and solutions in the state . in addition , lessons learned from these studies will be valuable in the future as the sources of data to support new evidence changes . for mmds , evidence to date has primarily come from administrative claims since few studies are done on the medicaid population . these administrative claims have sometimes been linked to other state data sources , such as birth certificates , but to date the distributed claims databases in various states have formed the backbone of the available evidence . in the future , the mmds hope to use clinical data from electronic health records , as interoperability methods for extracting , storing , and analyzing these centrally become more robust . including additional clinical data will permit monitoring of clinical outcomes across the states , in addition to utilization and cost information from claims . it will create a more complete picture of the issue , for example , more specific diagnoses contributing to readmissions . one of the most important reasons is that these studies respond to crucially important national health and policy concerns for the medicaid population . medicaid is currently at the vortex of policy making for the nation s lowest - income , most vulnerable , and sickest residents , and as medicaid expands under the affordable care act ( aca ) the size and importance of this vulnerable population will increase . this will bring new demands to the program and heighten the need for these collaborative , rapid learning projects on areas of concern to multiple states . the multistate clinical quality projects conducted by the mmds offer a key mechanism ( possibly the best mechanism ) to help medicaid monitor program outcomes across the country and continuously measure efforts to deliver value - driven , high - quality , cost - effective health care in an efficient manner . quality improvement studies often start discussions about improvement goals and drive action in the states . for example , the studies can lead to important state - level actions to implement effective policies that then are built upon by other states reassessment . as one mmd said , instead of this is our strategy and that is yours now it will be , what can we do together to get there faster ? that is a very exciting conversation to have . some mmds believe that these multistate studies also enable states to share and implement policies and practices used by others . another mmd said that the outcomes were not just [ about ] the data , but several states picked up on other states practices and implemented them similar to the effect of sharing and understanding data on reducing practice variation , work must be done between states to accelerate policy changes . the mmd s quality improvement projects provide participating states with data to inform policies internally . such data is important because state legislatures and policymakers are often more persuaded by local state data than by studies from another state or at the national level . only aggregated data from all states are shared publicly , but each state receives information on its own progress . specifically , individual states receive information comparing its outcomes with the average across all states and to the range of outcomes . this information allows the medicaid agency to highlight specific areas for policy or payment change . these studies are especially important in that there is a relative dearth of published research relying on medicaid administrative data . even though the reports are not the first to examine given issues , they are important because state policymakers often need to see their own data in comparison to the aggregate multistate results to make a strong case for improvements . and indeed , states have used the findings from the reports in a variety of ways , including the following : to identify and correct problems such as disparities , to set policies , to plan within the state , and to continue to monitor the data on their own . an example of identifying and correcting problems includes some mmds taking the data on readmissions to hospitals and nursing homes that were in the top for readmissions . as an example , one children s hospital did not believe that they had a problem with readmissions , but the results convinced them otherwise , and convinced them to take action by implementing an internal quality improvement program . many states have used the results of the studies to set policy , for example , one state used the antipsychotic medications study learnings to require prior review and authorization for use . one mmd stated the report on antipsychotic drug use , has been really impactful in that it has pushed the policy conversation that is taking place especially in the kids in foster care . you see antipsychotics in those kids and what we do around that and it really pushes the physical and behavioral health integration issues . other states incorporated readmissions into pay - for - performance hospital policies and used the read - missions results to inform decisions on incentive payments to hospitals . in addition , one state used the antipsychotic medication data for planning purposes , as a basis for a grant to further address the problem of inappropriate use . finally other states use the project materials , such as the data dictionaries , to continue to monitor the issue . one state is continuing to track antipsychotic use ( using the code developed in this project ) , and another state mentioned that tracking readmissions and the readmission report pushed the discussions of the problems and solutions in the state . an example of identifying and correcting problems includes some mmds taking the data on readmissions to hospitals and nursing homes that were in the top for readmissions . as an example , one children s hospital did not believe that they had a problem with readmissions , but the results convinced them otherwise , and convinced them to take action by implementing an internal quality improvement program . many states have used the results of the studies to set policy , for example , one state used the antipsychotic medications study learnings to require prior review and authorization for use . one mmd stated the report on antipsychotic drug use , has been really impactful in that it has pushed the policy conversation that is taking place especially in the kids in foster care . you see antipsychotics in those kids and what we do around that and it really pushes the physical and behavioral health integration issues . other states incorporated readmissions into pay - for - performance hospital policies and used the read - missions results to inform decisions on incentive payments to hospitals . in addition , one state used the antipsychotic medication data for planning purposes , as a basis for a grant to further address the problem of inappropriate use . finally other states use the project materials , such as the data dictionaries , to continue to monitor the issue . one state is continuing to track antipsychotic use ( using the code developed in this project ) , and another state mentioned that tracking readmissions and the readmission report pushed the discussions of the problems and solutions in the state . in addition , lessons learned from these studies will be valuable in the future as the sources of data to support new evidence changes . for mmds , evidence to date has primarily come from administrative claims since few studies are done on the medicaid population . these administrative claims have sometimes been linked to other state data sources , such as birth certificates , but to date the distributed claims databases in various states have formed the backbone of the available evidence . in the future , the mmds hope to use clinical data from electronic health records , as interoperability methods for extracting , storing , and analyzing these centrally become more robust . including additional clinical data will permit monitoring of clinical outcomes across the states , in addition to utilization and cost information from claims . it will create a more complete picture of the issue , for example , more specific diagnoses contributing to readmissions . the mmdln is an important vehicle for improving quality and reducing costs in the medicaid program . in order for the quality of health care to improve , the system needs to be structured as a learning health care system : one that is always accessing evidence , implementing a variation of it ( i.e. , with new data sources or tools such as electronic clinical data ) , assessing effectiveness , and sharing results for others to repeat the cycle . its ability to function as a learning health care system will grow as electronic health data become more available and . this will give the mmds information on both quality outcomes for the population as well as cost . the importance of a learning network in medicaid will grow rapidly as medicaid expands under the aca to take on more of the nation s most vulnerable and costly individuals .

introduction : there are many benefits of multistate collaboratives or networks to states , but at the center is that they allow for the opportunity to learn from other states and experts about the practices and policies states have implemented without the significant time lag of published research . this commentary examines these benefits and illustrates the importance of quality improvement collaborations to decision - making in state medicaid programs.background:in 2007 , the medicaid medical directors learning network ( mmdln ) began conducting quality improvement studies using their own state - level administrative data to better understand the major clinical issues facing the medicaid populations and to work together on policies to improve outcomes.rationale and results : the three issues selected by mmds for quality improvement monitoring to date involved an important national problem including both morbidity and cost and were amenable to policy solutions . the studies examined the use of antipsychotic medication in children , hospital admissions and readmissions , and early elective deliveries ( i.e. , elective deliveries occurring before 39 weeks).importance and utility : the multistate clinical quality projects conducted offer a key mechanism for achieving the goal of helping the medicaid program deliver value - driven , high - quality , cost - effective health care in an efficient manner . these projects also provide the participating states with data to inform policies internally.conclusions:in order for the quality of health care to improve , the system needs to be structured as a learning health care system ; one that is always accessing evidence , implementing a variation of it ( i.e. , with new data sources or tools such as electronic clinical data ) , assessing effectiveness , and sharing results for others to repeat the cycle .

Introduction Background Rationale and Results of Medicaid Medical Directors Collaborative Studies Antipsychotic Medication Use in Children and Adolescents Hospital Admission and Readmission Early Elective Deliveries Importance and Utility of Multistate Collaborative Projects Learning about Medicaid to Improve Population Health Quality Improvement Projects Can Spur Action Benefits to State-Level Quality Improvement Projects Identifying and correcting problems Using results to set policy Using data for planning and continued monitoring Lessons Can Be Used for Future Studies with New Data Sources Conclusions

within the last few years , multistate collaboratives or networks have become a more prevalent mode for implementing health care quality improvement initiatives . there are many benefits to states for participating in such collaboratives , but at the center is that they give states the opportunity to learn from each other , as well as from experts , about successful practices and policies . further , the sharing of information can take place rapidly and without the significant time lag needed for published research to become available . our experience from participating in the medicaid medical directors learning network ( mmdln ) whose motto is to steal shamelessly and share senselessly , has borne out the importance of such collaboratives . as the colorado medicaid medical director ( jz ) , mmdln manager ( kg ) , and medicaid medical director ( mmd)-led study principle investigators ( gf , tt ) , our leadership roles have given us a unique perspective on the benefits and importance of such collaboratives to states and the field at large . in this paper , we share our experiences and lessons about the value of participating in this learning network . the mmdln is focused on the development and use of evidence - based medicine , measurement , and improvement of health care quality ; and on the redesign of health care delivery systems through expert presentations and peer - to - peer learning . the mmdln is one of several state - level collaboratives or networks , all of which share the ultimate goal of improving health care quality . while these collaboratives may have slightly different purposes or populations of focus , they have in common the ultimate purpose of providing actionable evidence to aid in states decision - making . for example , the health resources and services administration s ( hrsa ) collaborative improvement & innovation network ( coiin ) is facilitating learning and adoption of common , proven quality improvement practices across all 50 states to reduce infant mortality and improve birth outcomes,1 the centers medicare & medicaid services funded national improvement partnership network ( nipn ) is a network of over 20 states developed to advance quality and transform health care for children and their families,2 and the medicaid evidence - based decisions ( med ) project was established as a self - governing , state - funded collaboration of state medicaid agencies to provide policymakers with the tools and resources they need to make evidence - based decisions including producing independent and objective evaluations of clinical evidence and convening states around clinical topics of interest.3 a major value of collaboratives like the mmdln and these others is that they allow for rapid learning among state members . for the mmds , evidence to date has also come from administrative claims data from participating states , aggregated and assembled to show patterns at the national level . for example , when a new quality improvement practice is implemented by one state based on information shared through collaboratives , it is often picked up and further improved upon by other states . thus , states , and ultimately their constituents , benefit in a timely manner from the continuous cycle of sharing , implementation , assessment , refinement , and more sharing . however , there are also challenges associated with participating in claims - based data aggregation projects and maintaining a learning network that are also discussed in the companion paper . despite these challenges , this commentary examines these benefits and illustrates the importance of quality improvement collaborations to decision - making in state medicaid programs . while that contract ended in 2013 , the work of the network is continuing under the national association of medicaid directors as a clinical arm of the national association of state medicaid programs . network participation is open to mmds and those in similar clinical leadership positions who advise the medicaid director for one or more components of a medicaid program administered by a state , territory , or the district of columbia . for the last eight years , network activities have focused on regular in - person and virtual learning sessions addressing topics of high relevance and importance for the medicaid population . these activities have allowed mmds the opportunity to interact with our peers and learn from each other s experiences . in 2007 , through the mmdln , mmds also began conducting quality improvement studies using their own state - level administrative data to better understand the major clinical issues facing the medicaid populations and to work together on policies to improve outcomes . mmds saw an opportunity as clinical leaders in the states to assess clinical outcomes , to better understand an issue , and to make quality improvement decisions . the first project dealt with antipsychotic medication use in children and adolescents;5 the second examined hospital admissions and readmissions , while the third focused on early elective deliveries . the companion paper describes in detail the methods used to conduct them ; the present commentary focuses on the benefits of such studies and their importance to quality improvement in medicaid . findings in this commentary , as well as the companion paper , are based on review of documents ; personal experiences ; and discussions with mmds leaders , researchers involved in these studies , and state analysts . all problems selected by mmds for quality improvement monitoring involved an important national problem one that involved both morbidity and cost for the medicaid population and were amenable to policy solutions . the importance of understanding and improving care and cost in medicaid can hardly be overstated . in the selected areas , a the first study examined the use of antipsychotic medication in children , and was selected in response to the high level of concern among the mmds about this issue . states also shared policy tools to address antipsychotic medication use in children and the evidence that these policies worked . department of health & human services ( hhs ) centers for medicare & medicaid services ( cms ) in letters to medicaid directors when discussing the seriousness of the issue , and was the topic of a summit in the fall of 2012 sponsored by the hhs administration for children , youth , and families . further , in recognition of the importance of this issue , there has been subsequent funding to states to improve monitoring of prescribing in foster care children . the mmds selected this topic as the second area of focus due to the prevalence , expense and presumed preventability of hospital use ( at least for some diagnoses).78 national data showed that about 1 in 12 adults discharged from a hospital was readmitted within 30 days , adding an additional $ 16 billion to health care costs in the united states.9 additionally , the mmds took note of the fact that medicare had targeted hospital readmissions as an important area for both cost savings and care improvement and crafted nonpayment policies for some readmissions.1011 many state officials expected that similar policies might be applied to the medicaid population . it was thus deemed critically important to understand the extent and nature of hospital readmissions in medicaid , which had not been studied , in order to create effective state - level policies . this study found that readmissions were costly for the states and were more prevalent in those with certain diagnoses such as mental health . further , readmission rates varied considerably among the 19 participating states , which allowed states with higher rates to learn from those with lower rates.12 the third multistate study , which is currently under way , is examining early elective deliveries ( i.e. , labor induction or cesarean deliveries occurring before 39 weeks ) . first , there was a large body of evidence showing that these early deliveries led to poorer neonatal outcomes and long - term problems , as well as excess cost.1318 second , there was a clear policy statement from the american college of obstetricians and gynecologists ( acog ) calling for the restriction of these early elective deliveries.1920 third , an mmd - led effort in partnership with public health colleagues , could exert a multiplier effect by building on and complementing other efforts including the hrsa coiin and cms strong start for mothers and newborns initiative to reduce preterm births and improve birth outcomes.21 finally , since medicaid pays for up to 48 percent of the births nationally ( 1.8 million births),2223 the program has a clear vested interest in developing policies to reduce complications and costs associated with these early elective deliveries . this study initially found that , of the medicaid singleton deliveries in 22 states , early elective deliveries are prevalent and that states can improve their reported rates by implementing policies to reduce them and improving the reporting on the birth certificates of medical indications for an early labor induction or cesarean delivery . the first study examined the use of antipsychotic medication in children , and was selected in response to the high level of concern among the mmds about this issue . states also shared policy tools to address antipsychotic medication use in children and the evidence that these policies worked . department of health & human services ( hhs ) centers for medicare & medicaid services ( cms ) in letters to medicaid directors when discussing the seriousness of the issue , and was the topic of a summit in the fall of 2012 sponsored by the hhs administration for children , youth , and families . further , in recognition of the importance of this issue , there has been subsequent funding to states to improve monitoring of prescribing in foster care children . this first study made clear the need for such efforts in medicaid and the benefit to states . the mmds selected this topic as the second area of focus due to the prevalence , expense and presumed preventability of hospital use ( at least for some diagnoses).78 national data showed that about 1 in 12 adults discharged from a hospital was readmitted within 30 days , adding an additional $ 16 billion to health care costs in the united states.9 additionally , the mmds took note of the fact that medicare had targeted hospital readmissions as an important area for both cost savings and care improvement and crafted nonpayment policies for some readmissions.1011 many state officials expected that similar policies might be applied to the medicaid population . it was thus deemed critically important to understand the extent and nature of hospital readmissions in medicaid , which had not been studied , in order to create effective state - level policies . this study found that readmissions were costly for the states and were more prevalent in those with certain diagnoses such as mental health . further , readmission rates varied considerably among the 19 participating states , which allowed states with higher rates to learn from those with lower rates.12 the third multistate study , which is currently under way , is examining early elective deliveries ( i.e. , labor induction or cesarean deliveries occurring before 39 weeks ) . first , there was a large body of evidence showing that these early deliveries led to poorer neonatal outcomes and long - term problems , as well as excess cost.1318 second , there was a clear policy statement from the american college of obstetricians and gynecologists ( acog ) calling for the restriction of these early elective deliveries.1920 third , an mmd - led effort in partnership with public health colleagues , could exert a multiplier effect by building on and complementing other efforts including the hrsa coiin and cms strong start for mothers and newborns initiative to reduce preterm births and improve birth outcomes.21 finally , since medicaid pays for up to 48 percent of the births nationally ( 1.8 million births),2223 the program has a clear vested interest in developing policies to reduce complications and costs associated with these early elective deliveries . this study initially found that , of the medicaid singleton deliveries in 22 states , early elective deliveries are prevalent and that states can improve their reported rates by implementing policies to reduce them and improving the reporting on the birth certificates of medical indications for an early labor induction or cesarean delivery . there are a number of reasons why these studies are important and useful to state policy making and practices for the medicaid program . one of the most important reasons is that these studies respond to crucially important national health and policy concerns for the medicaid population . medicaid is currently at the vortex of policy making for the nation s lowest - income , most vulnerable , and sickest residents , and as medicaid expands under the affordable care act ( aca ) the size and importance of this vulnerable population will increase . the multistate clinical quality projects conducted by the mmds offer a key mechanism ( possibly the best mechanism ) to help medicaid monitor program outcomes across the country and continuously measure efforts to deliver value - driven , high - quality , cost - effective health care in an efficient manner . quality improvement studies often start discussions about improvement goals and drive action in the states . for example , the studies can lead to important state - level actions to implement effective policies that then are built upon by other states reassessment . another mmd said that the outcomes were not just [ about ] the data , but several states picked up on other states practices and implemented them similar to the effect of sharing and understanding data on reducing practice variation , work must be done between states to accelerate policy changes . the mmd s quality improvement projects provide participating states with data to inform policies internally . specifically , individual states receive information comparing its outcomes with the average across all states and to the range of outcomes . these studies are especially important in that there is a relative dearth of published research relying on medicaid administrative data . and indeed , states have used the findings from the reports in a variety of ways , including the following : to identify and correct problems such as disparities , to set policies , to plan within the state , and to continue to monitor the data on their own . as an example , one children s hospital did not believe that they had a problem with readmissions , but the results convinced them otherwise , and convinced them to take action by implementing an internal quality improvement program . many states have used the results of the studies to set policy , for example , one state used the antipsychotic medications study learnings to require prior review and authorization for use . other states incorporated readmissions into pay - for - performance hospital policies and used the read - missions results to inform decisions on incentive payments to hospitals . in addition , one state used the antipsychotic medication data for planning purposes , as a basis for a grant to further address the problem of inappropriate use . finally other states use the project materials , such as the data dictionaries , to continue to monitor the issue . one state is continuing to track antipsychotic use ( using the code developed in this project ) , and another state mentioned that tracking readmissions and the readmission report pushed the discussions of the problems and solutions in the state . for mmds , evidence to date has primarily come from administrative claims since few studies are done on the medicaid population . these administrative claims have sometimes been linked to other state data sources , such as birth certificates , but to date the distributed claims databases in various states have formed the backbone of the available evidence . in the future , the mmds hope to use clinical data from electronic health records , as interoperability methods for extracting , storing , and analyzing these centrally become more robust . including additional clinical data will permit monitoring of clinical outcomes across the states , in addition to utilization and cost information from claims . one of the most important reasons is that these studies respond to crucially important national health and policy concerns for the medicaid population . medicaid is currently at the vortex of policy making for the nation s lowest - income , most vulnerable , and sickest residents , and as medicaid expands under the affordable care act ( aca ) the size and importance of this vulnerable population will increase . the multistate clinical quality projects conducted by the mmds offer a key mechanism ( possibly the best mechanism ) to help medicaid monitor program outcomes across the country and continuously measure efforts to deliver value - driven , high - quality , cost - effective health care in an efficient manner . quality improvement studies often start discussions about improvement goals and drive action in the states . for example , the studies can lead to important state - level actions to implement effective policies that then are built upon by other states reassessment . another mmd said that the outcomes were not just [ about ] the data , but several states picked up on other states practices and implemented them similar to the effect of sharing and understanding data on reducing practice variation , work must be done between states to accelerate policy changes . the mmd s quality improvement projects provide participating states with data to inform policies internally . specifically , individual states receive information comparing its outcomes with the average across all states and to the range of outcomes . these studies are especially important in that there is a relative dearth of published research relying on medicaid administrative data . even though the reports are not the first to examine given issues , they are important because state policymakers often need to see their own data in comparison to the aggregate multistate results to make a strong case for improvements . and indeed , states have used the findings from the reports in a variety of ways , including the following : to identify and correct problems such as disparities , to set policies , to plan within the state , and to continue to monitor the data on their own . as an example , one children s hospital did not believe that they had a problem with readmissions , but the results convinced them otherwise , and convinced them to take action by implementing an internal quality improvement program . many states have used the results of the studies to set policy , for example , one state used the antipsychotic medications study learnings to require prior review and authorization for use . other states incorporated readmissions into pay - for - performance hospital policies and used the read - missions results to inform decisions on incentive payments to hospitals . in addition , one state used the antipsychotic medication data for planning purposes , as a basis for a grant to further address the problem of inappropriate use . finally other states use the project materials , such as the data dictionaries , to continue to monitor the issue . one state is continuing to track antipsychotic use ( using the code developed in this project ) , and another state mentioned that tracking readmissions and the readmission report pushed the discussions of the problems and solutions in the state . as an example , one children s hospital did not believe that they had a problem with readmissions , but the results convinced them otherwise , and convinced them to take action by implementing an internal quality improvement program . many states have used the results of the studies to set policy , for example , one state used the antipsychotic medications study learnings to require prior review and authorization for use . other states incorporated readmissions into pay - for - performance hospital policies and used the read - missions results to inform decisions on incentive payments to hospitals . in addition , one state used the antipsychotic medication data for planning purposes , as a basis for a grant to further address the problem of inappropriate use . finally other states use the project materials , such as the data dictionaries , to continue to monitor the issue . one state is continuing to track antipsychotic use ( using the code developed in this project ) , and another state mentioned that tracking readmissions and the readmission report pushed the discussions of the problems and solutions in the state . for mmds , evidence to date has primarily come from administrative claims since few studies are done on the medicaid population . these administrative claims have sometimes been linked to other state data sources , such as birth certificates , but to date the distributed claims databases in various states have formed the backbone of the available evidence . in the future , the mmds hope to use clinical data from electronic health records , as interoperability methods for extracting , storing , and analyzing these centrally become more robust . including additional clinical data will permit monitoring of clinical outcomes across the states , in addition to utilization and cost information from claims . the mmdln is an important vehicle for improving quality and reducing costs in the medicaid program . in order for the quality of health care to improve , the system needs to be structured as a learning health care system : one that is always accessing evidence , implementing a variation of it ( i.e. , with new data sources or tools such as electronic clinical data ) , assessing effectiveness , and sharing results for others to repeat the cycle . its ability to function as a learning health care system will grow as electronic health data become more available and . the importance of a learning network in medicaid will grow rapidly as medicaid expands under the aca to take on more of the nation s most vulnerable and costly individuals .

within the last few years , multistate collaboratives or networks have become a more prevalent mode for implementing health care quality improvement initiatives . there are many benefits to states for participating in such collaboratives , but at the center is that they give states the opportunity to learn from each other , as well as from experts , about successful practices and policies . further , the sharing of information can take place rapidly and without the significant time lag needed for published research to become available . our experience from participating in the medicaid medical directors learning network ( mmdln ) whose motto is to steal shamelessly and share senselessly , has borne out the importance of such collaboratives . as the colorado medicaid medical director ( jz ) , mmdln manager ( kg ) , and medicaid medical director ( mmd)-led study principle investigators ( gf , tt ) , our leadership roles have given us a unique perspective on the benefits and importance of such collaboratives to states and the field at large . in this paper , we share our experiences and lessons about the value of participating in this learning network . the mmdln is focused on the development and use of evidence - based medicine , measurement , and improvement of health care quality ; and on the redesign of health care delivery systems through expert presentations and peer - to - peer learning . the mmdln is one of several state - level collaboratives or networks , all of which share the ultimate goal of improving health care quality . while these collaboratives may have slightly different purposes or populations of focus , they have in common the ultimate purpose of providing actionable evidence to aid in states decision - making . for example , the health resources and services administration s ( hrsa ) collaborative improvement & innovation network ( coiin ) is facilitating learning and adoption of common , proven quality improvement practices across all 50 states to reduce infant mortality and improve birth outcomes,1 the centers medicare & medicaid services funded national improvement partnership network ( nipn ) is a network of over 20 states developed to advance quality and transform health care for children and their families,2 and the medicaid evidence - based decisions ( med ) project was established as a self - governing , state - funded collaboration of state medicaid agencies to provide policymakers with the tools and resources they need to make evidence - based decisions including producing independent and objective evaluations of clinical evidence and convening states around clinical topics of interest.3 a major value of collaboratives like the mmdln and these others is that they allow for rapid learning among state members . evidence provided to the states through these collaboratives comes in varying forms including data analysis results , literature reviews , expert presentations , and even anecdotal references from peers . a companion paper describes methods for using this distributed claims data resource.4 along with the value of rapid learning in these collaboratives is the fact that knowledge builds on prior experiences as practices are shared . thus , states , and ultimately their constituents , benefit in a timely manner from the continuous cycle of sharing , implementation , assessment , refinement , and more sharing . however , there are also challenges associated with participating in claims - based data aggregation projects and maintaining a learning network that are also discussed in the companion paper . despite these challenges , this commentary examines these benefits and illustrates the importance of quality improvement collaborations to decision - making in state medicaid programs . department of health and human services agency for healthcare research and quality ( ahrq ) in 2005 under contract to academyhealth . while that contract ended in 2013 , the work of the network is continuing under the national association of medicaid directors as a clinical arm of the national association of state medicaid programs . the network , as an integrated national resource , seeks to advance the health of medicaid patients in the states and across the nation while best stewarding available resources . network participation is open to mmds and those in similar clinical leadership positions who advise the medicaid director for one or more components of a medicaid program administered by a state , territory , or the district of columbia . for the last eight years , network activities have focused on regular in - person and virtual learning sessions addressing topics of high relevance and importance for the medicaid population . in addition , we have a regularly maintained website and use a web forum to share documents and pose questions to other members of the network . in 2007 , through the mmdln , mmds also began conducting quality improvement studies using their own state - level administrative data to better understand the major clinical issues facing the medicaid populations and to work together on policies to improve outcomes . mmds saw an opportunity as clinical leaders in the states to assess clinical outcomes , to better understand an issue , and to make quality improvement decisions . the first project dealt with antipsychotic medication use in children and adolescents;5 the second examined hospital admissions and readmissions , while the third focused on early elective deliveries . the companion paper describes in detail the methods used to conduct them ; the present commentary focuses on the benefits of such studies and their importance to quality improvement in medicaid . findings in this commentary , as well as the companion paper , are based on review of documents ; personal experiences ; and discussions with mmds leaders , researchers involved in these studies , and state analysts . all problems selected by mmds for quality improvement monitoring involved an important national problem one that involved both morbidity and cost for the medicaid population and were amenable to policy solutions . the importance of understanding and improving care and cost in medicaid can hardly be overstated . with 74 million beneficiaries,6 cost from excess utilization can be enormous , and conversely , even small improvements can result in cost savings . in the selected areas , a the first study examined the use of antipsychotic medication in children , and was selected in response to the high level of concern among the mmds about this issue . the results of this multistate study that antipsychotic medication use was prevalent , even among young children and especially among children in foster care highlighted a problem that had previously received little attention nationally and galvanized action to address it . states also shared policy tools to address antipsychotic medication use in children and the evidence that these policies worked . department of health & human services ( hhs ) centers for medicare & medicaid services ( cms ) in letters to medicaid directors when discussing the seriousness of the issue , and was the topic of a summit in the fall of 2012 sponsored by the hhs administration for children , youth , and families . further , in recognition of the importance of this issue , there has been subsequent funding to states to improve monitoring of prescribing in foster care children . this first study made clear the need for such efforts in medicaid and the benefit to states . the mmds selected this topic as the second area of focus due to the prevalence , expense and presumed preventability of hospital use ( at least for some diagnoses).78 national data showed that about 1 in 12 adults discharged from a hospital was readmitted within 30 days , adding an additional $ 16 billion to health care costs in the united states.9 additionally , the mmds took note of the fact that medicare had targeted hospital readmissions as an important area for both cost savings and care improvement and crafted nonpayment policies for some readmissions.1011 many state officials expected that similar policies might be applied to the medicaid population . it was thus deemed critically important to understand the extent and nature of hospital readmissions in medicaid , which had not been studied , in order to create effective state - level policies . this study found that readmissions were costly for the states and were more prevalent in those with certain diagnoses such as mental health . further , readmission rates varied considerably among the 19 participating states , which allowed states with higher rates to learn from those with lower rates.12 the third multistate study , which is currently under way , is examining early elective deliveries ( i.e. first , there was a large body of evidence showing that these early deliveries led to poorer neonatal outcomes and long - term problems , as well as excess cost.1318 second , there was a clear policy statement from the american college of obstetricians and gynecologists ( acog ) calling for the restriction of these early elective deliveries.1920 third , an mmd - led effort in partnership with public health colleagues , could exert a multiplier effect by building on and complementing other efforts including the hrsa coiin and cms strong start for mothers and newborns initiative to reduce preterm births and improve birth outcomes.21 finally , since medicaid pays for up to 48 percent of the births nationally ( 1.8 million births),2223 the program has a clear vested interest in developing policies to reduce complications and costs associated with these early elective deliveries . this study initially found that , of the medicaid singleton deliveries in 22 states , early elective deliveries are prevalent and that states can improve their reported rates by implementing policies to reduce them and improving the reporting on the birth certificates of medical indications for an early labor induction or cesarean delivery . the first study examined the use of antipsychotic medication in children , and was selected in response to the high level of concern among the mmds about this issue . the results of this multistate study that antipsychotic medication use was prevalent , even among young children and especially among children in foster care highlighted a problem that had previously received little attention nationally and galvanized action to address it . department of health & human services ( hhs ) centers for medicare & medicaid services ( cms ) in letters to medicaid directors when discussing the seriousness of the issue , and was the topic of a summit in the fall of 2012 sponsored by the hhs administration for children , youth , and families . further , in recognition of the importance of this issue , there has been subsequent funding to states to improve monitoring of prescribing in foster care children . this first study made clear the need for such efforts in medicaid and the benefit to states . the mmds selected this topic as the second area of focus due to the prevalence , expense and presumed preventability of hospital use ( at least for some diagnoses).78 national data showed that about 1 in 12 adults discharged from a hospital was readmitted within 30 days , adding an additional $ 16 billion to health care costs in the united states.9 additionally , the mmds took note of the fact that medicare had targeted hospital readmissions as an important area for both cost savings and care improvement and crafted nonpayment policies for some readmissions.1011 many state officials expected that similar policies might be applied to the medicaid population . it was thus deemed critically important to understand the extent and nature of hospital readmissions in medicaid , which had not been studied , in order to create effective state - level policies . this study found that readmissions were costly for the states and were more prevalent in those with certain diagnoses such as mental health . further , readmission rates varied considerably among the 19 participating states , which allowed states with higher rates to learn from those with lower rates.12 the third multistate study , which is currently under way , is examining early elective deliveries ( i.e. first , there was a large body of evidence showing that these early deliveries led to poorer neonatal outcomes and long - term problems , as well as excess cost.1318 second , there was a clear policy statement from the american college of obstetricians and gynecologists ( acog ) calling for the restriction of these early elective deliveries.1920 third , an mmd - led effort in partnership with public health colleagues , could exert a multiplier effect by building on and complementing other efforts including the hrsa coiin and cms strong start for mothers and newborns initiative to reduce preterm births and improve birth outcomes.21 finally , since medicaid pays for up to 48 percent of the births nationally ( 1.8 million births),2223 the program has a clear vested interest in developing policies to reduce complications and costs associated with these early elective deliveries . this study initially found that , of the medicaid singleton deliveries in 22 states , early elective deliveries are prevalent and that states can improve their reported rates by implementing policies to reduce them and improving the reporting on the birth certificates of medical indications for an early labor induction or cesarean delivery . there are a number of reasons why these studies are important and useful to state policy making and practices for the medicaid program . one of the most important reasons is that these studies respond to crucially important national health and policy concerns for the medicaid population . medicaid is currently at the vortex of policy making for the nation s lowest - income , most vulnerable , and sickest residents , and as medicaid expands under the affordable care act ( aca ) the size and importance of this vulnerable population will increase . this will bring new demands to the program and heighten the need for these collaborative , rapid learning projects on areas of concern to multiple states . the multistate clinical quality projects conducted by the mmds offer a key mechanism ( possibly the best mechanism ) to help medicaid monitor program outcomes across the country and continuously measure efforts to deliver value - driven , high - quality , cost - effective health care in an efficient manner . as one mmd said , instead of this is our strategy and that is yours now it will be , what can we do together to get there faster ? another mmd said that the outcomes were not just [ about ] the data , but several states picked up on other states practices and implemented them similar to the effect of sharing and understanding data on reducing practice variation , work must be done between states to accelerate policy changes . specifically , individual states receive information comparing its outcomes with the average across all states and to the range of outcomes . these studies are especially important in that there is a relative dearth of published research relying on medicaid administrative data . even though the reports are not the first to examine given issues , they are important because state policymakers often need to see their own data in comparison to the aggregate multistate results to make a strong case for improvements . and indeed , states have used the findings from the reports in a variety of ways , including the following : to identify and correct problems such as disparities , to set policies , to plan within the state , and to continue to monitor the data on their own . an example of identifying and correcting problems includes some mmds taking the data on readmissions to hospitals and nursing homes that were in the top for readmissions . many states have used the results of the studies to set policy , for example , one state used the antipsychotic medications study learnings to require prior review and authorization for use . one mmd stated the report on antipsychotic drug use , has been really impactful in that it has pushed the policy conversation that is taking place especially in the kids in foster care . other states incorporated readmissions into pay - for - performance hospital policies and used the read - missions results to inform decisions on incentive payments to hospitals . in addition , one state used the antipsychotic medication data for planning purposes , as a basis for a grant to further address the problem of inappropriate use . one state is continuing to track antipsychotic use ( using the code developed in this project ) , and another state mentioned that tracking readmissions and the readmission report pushed the discussions of the problems and solutions in the state . in addition , lessons learned from these studies will be valuable in the future as the sources of data to support new evidence changes . these administrative claims have sometimes been linked to other state data sources , such as birth certificates , but to date the distributed claims databases in various states have formed the backbone of the available evidence . in the future , the mmds hope to use clinical data from electronic health records , as interoperability methods for extracting , storing , and analyzing these centrally become more robust . including additional clinical data will permit monitoring of clinical outcomes across the states , in addition to utilization and cost information from claims . one of the most important reasons is that these studies respond to crucially important national health and policy concerns for the medicaid population . medicaid is currently at the vortex of policy making for the nation s lowest - income , most vulnerable , and sickest residents , and as medicaid expands under the affordable care act ( aca ) the size and importance of this vulnerable population will increase . this will bring new demands to the program and heighten the need for these collaborative , rapid learning projects on areas of concern to multiple states . the multistate clinical quality projects conducted by the mmds offer a key mechanism ( possibly the best mechanism ) to help medicaid monitor program outcomes across the country and continuously measure efforts to deliver value - driven , high - quality , cost - effective health care in an efficient manner . another mmd said that the outcomes were not just [ about ] the data , but several states picked up on other states practices and implemented them similar to the effect of sharing and understanding data on reducing practice variation , work must be done between states to accelerate policy changes . specifically , individual states receive information comparing its outcomes with the average across all states and to the range of outcomes . even though the reports are not the first to examine given issues , they are important because state policymakers often need to see their own data in comparison to the aggregate multistate results to make a strong case for improvements . and indeed , states have used the findings from the reports in a variety of ways , including the following : to identify and correct problems such as disparities , to set policies , to plan within the state , and to continue to monitor the data on their own . an example of identifying and correcting problems includes some mmds taking the data on readmissions to hospitals and nursing homes that were in the top for readmissions . many states have used the results of the studies to set policy , for example , one state used the antipsychotic medications study learnings to require prior review and authorization for use . one mmd stated the report on antipsychotic drug use , has been really impactful in that it has pushed the policy conversation that is taking place especially in the kids in foster care . other states incorporated readmissions into pay - for - performance hospital policies and used the read - missions results to inform decisions on incentive payments to hospitals . one state is continuing to track antipsychotic use ( using the code developed in this project ) , and another state mentioned that tracking readmissions and the readmission report pushed the discussions of the problems and solutions in the state . many states have used the results of the studies to set policy , for example , one state used the antipsychotic medications study learnings to require prior review and authorization for use . one mmd stated the report on antipsychotic drug use , has been really impactful in that it has pushed the policy conversation that is taking place especially in the kids in foster care . other states incorporated readmissions into pay - for - performance hospital policies and used the read - missions results to inform decisions on incentive payments to hospitals . one state is continuing to track antipsychotic use ( using the code developed in this project ) , and another state mentioned that tracking readmissions and the readmission report pushed the discussions of the problems and solutions in the state . in addition , lessons learned from these studies will be valuable in the future as the sources of data to support new evidence changes . these administrative claims have sometimes been linked to other state data sources , such as birth certificates , but to date the distributed claims databases in various states have formed the backbone of the available evidence . including additional clinical data will permit monitoring of clinical outcomes across the states , in addition to utilization and cost information from claims . in order for the quality of health care to improve , the system needs to be structured as a learning health care system : one that is always accessing evidence , implementing a variation of it ( i.e. the importance of a learning network in medicaid will grow rapidly as medicaid expands under the aca to take on more of the nation s most vulnerable and costly individuals .

detection of acute conditions of neglected infectious diseases ( nids ) is hindered by low primary health care utilization as well as low priority and quality of health care services . chagas disease ( cd ) is an infection caused by the parasite trypanosoma cruzi and is transmitted to humans , among other ways , by an insect vector , the triatomine bug . recent estimates suggest that more than 5.7 million people are infected with cd , mostly socially and economically vulnerable populations . it produces 14,000 annual deaths in latin america and is a cause of morbidity ( who , 2002 ) , work disability , and increased health care costs . a notable clinical consequence of t. cruzi is chagasic cardiomyopathy which may lead to heart failure and sudden death . cd acute phase takes place two to four weeks after infection but is symptomatic in only 1% to 2% of cases . typically , the acute phase develops in children under 10 years , with a 28% mortality rate . diagnosis is complicated due to the nonspecific nature of the signs and symptoms and often leads to misdiagnosis . according to health regulations , all confirmed cd cases require notification to the epidemiological office . despite the success in parasite and vector control , argentina regularly reports acute cases of cd , mostly young individuals . between 1996 and 2014 the number of reported acute cases ranged from a maximum of twenty - nine to a minimum of one [ 9 , 10 ] . as has been underlined by official sources , most cd infection cases go unnoticed . underreporting cd figures might be explained by several factors hindering diagnosis : the largely asymptomatic nature of disease , organizational and human resources factors at the health system level , and social and cultural factors at the patient level . although there are estimates on patterns of health care system utilization by cd patients developed by experts , there is a void of knowledge of health care seeking behavior in subjects experiencing a cd acute condition . in this article , through a retrospective lens we explore the latter through the health care seeking paths followed by acute cases which have been successfully diagnosed and specifically prescribed for cd treatment . by doing this , we want to better understand the factors that surround people 's experience with acute cd in their quest for a diagnosis . the understanding of health care seeking behaviors is critical to determine how it affects the diagnosis and treatment of conditions . parsons described the sick role which discusses the social role that is implied in health behavior . the ill is excused to fulfill normal obligations and is not blamed for their condition . therefore , they are not expected to heal without assistance , yet the individual holds the responsibility of seeking care and complying with medical directions . however , in the context of mainly poor rural populations that face several social and economic constraints , it is crucial to trace back the path that allows them to fulfill the main requirement of the sick role . broadly , the literature discusses health care seeking behaviors at the level of households and health systems . at the level of the household , health may be dependent on the availability of time , resources , priority allocated to health , cultural norms , and access to cash and transport . at the level of the health system , issues like quality of health care delivery , that is , human resources , and organizational arrangements and environment influence care seekers ' decisions [ 21 , 22 ] . several studies demonstrate that the cost of care or transportation to care facilities is a major barrier influencing care seeking behaviors [ 23 , 24 ] . in a study done in colombia , the most frequent path was to initially seek care at the most basic level of care which was influenced by insurance coverage and resources . in the united states the absence of insurance restricted care for cd . when mothers of sick children in uganda perceived the distance to health services to be further , seeking care was delayed compared to those who perceived a shorter distance . poverty plays a role in the time it takes to seek care as well as the type of care being sought . cost relating to care is a significant contributor to delaying care which affects diagnoses and treatment efficacy . the health care seeking behaviors of mothers with low socioeconomic status in uganda delayed care seeking compared to those with high socioeconomic status . a study on maternal health care seeking behaviors in rural ecuador supported the hypotheses that families with lower socioeconomic status were more likely not to seek care or to treat their children 's respiratory infection symptoms with home remedies . also , poverty has symbolic and moral implications . in several countries poor and rural populations are stigmatized and mistreated for being infected by cd . these barriers further limit access to care and delay diagnosis in those who carry most of the disease burden . health system perception is an important factor that drives health care seeking behaviors . in a study done in bolivia , rural women who followed their family 's tradition of giving birth at home felt a sense of exclusion and believed the hospital would increase health complications . others felt they would be mistreated and not informed by the staff about the procedures being done to them . latino immigrant women in the united states recognized that traditional remedies were not always the most effective but were the first line of treatment due to its lower cost . this caused a further delay in seeking care when traditional remedies were ineffective since women waited for symptoms to disappear before seeking mainstream health care . seeking care is marked by cultural and traditional elements influencing the perception of symptoms and conditions . in mexico , bites by triatomines were considered as normal daily events and only sometimes thought to transmit disease , while others thought they had developed a resistance . for other participants , patients with schistosomiasis - related symptoms were more likely to seek care only when symptoms were more severe . the lack of information about symptoms , diseases , treatments , and services plays an important , yet detrimental role in health care seeking behaviors . in bolivia , similarly , one of the main barriers for latino immigrants in europe to access chagas - related care was the lack of awareness of services provided to them . moreover , the fear related to cd is linked to the lack of knowledge of the disease and the treatment process . not recognizing the severe implications of symptoms was also a significant element in delaying or not seeking health care [ 15 , 16 , 30 , 31 ] . the review shows the different factors contributing to an array of health care seeking behaviors . our research approaches patterns to health care from the patients ' perspective , exploring the way in which families respond to the symptoms , how they address contextual limitations , and , specifically , their interactions with the health care system . an ethnographic approach of reported acute cases was applied . at the time of the study , the province of santiago del estero was a high risk , cd endemic area with reported acute cases , a household infestation index > 5% , no active surveillance , and sero - prevalence above 5% among children less than 5 years of age . the purpose was to collect data regarding the experience of the acute case 's family from the time of the outbreak to the sero - positive laboratory result and to find care seeking patterns in these processes . twenty - five acute cases out of forty reported in 2003 , 2004 , and 2005 [ 32 , 33 ] were interviewed , 14 females and 11 males , that were registered by the health provincial system ( table 1 ) . as minors , most of the acute cases were represented by a care taker , who was the respondent in the interview . as twenty - three out of the twenty - five subjects were under 15 years old , the retrospective information collected during the interview was provided by close relatives , usually the mother . both long and short term retrospective recall can provide an accurate report of occurrence of health related events . also , family members playing a caring role , particularly mothers , are in position to provide accurate proxy reports of disease and symptoms . distance from the provincial capital city , santiago del estero , to acute cases ' houses ranged from 12 to 300 km , in villages distributed across 11 departamentos , a subprovincial administrative level ( figures 1 and 2 ) . most of the acute cases were located in rural areas of particularly difficult access : unpaved and dirt roads , narrow and fainting trails , and with no means of public transportation . the average time between the outbreak and the interview was 11 months , ranging from 3 to 22 months . mostly , the study population was from poor rural families with minimal access to water and electricity . most homes were made out of mud , with cracks and crevices that were ideal to accommodate t. infestans ( known in argentina as vinchuca ) . homes that had a bathroom , access to electricity , and easy access to water were considered as having standard living conditions ( table 1 ) . those who had health posts in their area mostly had irregular or minimal attendance of health practitioners or did not have the resources to deal with the health problems presented by the families . the most common symptom of the acute cases was a swollen eye ( 17 out of 25 ) which is a clinical symptom specific to cd . at times , the main condition was accompanied by other symptoms including fever , red eyes , or body pain . some families were unaware that these symptoms could be a result of cd and these nonspecific symptoms led them to think they acquired other conditions , which influenced their method of care . others were not aware of the existence of cd and how it was caused ; there were few families who were knowledgeable about the transmission of the disease . direct parasitological methods , that is , micromethod in children and strout method in adults , were used to diagnose trypanosoma cruzi infection at the acute phase . there were five different health care seeking behavior pathways the families took in order to finally get the subject diagnosed with acute cd . in 2000 , 51 hospitals and 254 primary health care units ( phcus ) organized with different levels of complexity were part of the provincial health care system . at the bottom , the phcus which were mostly located in rural areas offered outpatient services . although some of them were staffed with physicians , many were looked after by auxiliary nurses with basic training . at the top of the system , in the capital city it had a specialized center for cd diagnosis and treatment as well as a laboratory equipped for specific studies . in an intermediate position , smaller and lower complexity hospitals , with permanent on - call doctors , delivered basic health services . at the onset of symptoms , subjects searched for care at different levels of the system . in this endeavor , they followed a variety of paths ending with a sero - positivity diagnosis . one individual directly attended the hospital due to the absence of a primary health care unit ( phcu ) , four dismissed the local phcu as the first line of care , and one was taken to gh by health personnel working in the field where they were diagnosed . there are many reasons for which subjects dismissed the phcu as the lack of consistency in the schedule , e : but it 's nothing but a tale because sometimes the nurse is there and sometimes he is n't ( ) because in this zone we are disadvantaged ( subject 11 ) ; even with a stable schedule , patients had no choice but to seek care at the hospital when doctors attended the post infrequently , e : yes there is a health post here ( does n't the doctor come ? ) ( and do you go for consultations there?) e : if it is n't in the morning , if you go in the afternoon you need to go to santiago . ( subject 10 ) . some health posts only had nurses , and local residents could not reach a doctor unless they went to the hospital . in some cases , it had been so long since a doctor had attended the health post they did not expect one to be present , ( do you have a health post ? ) e : yes nothing but a nurse . e : no , sometimes from medelln every two months . ( subject 3 ) . those who had access to a primary health care unit were not always able to be cared for due to policies regarding the need for a doctor 's reference : ( you do n't go to the health post ? e : yes , sometimes but you need a doctor 's referral , otherwise they will not attend to you ) in addition to the limitations mentioned in these four cases , the health care seeking behaviors were driven by the proximity to the hospital . the family of the fifth case was an exception as they chose to be transferred to the capital city ( 135 km ) as a relative lived there . obtaining a diagnosis was not free of complications ; an 11-year - old boy was diagnosed with an eye infection . immediately , upon his father 's insistence , a second test was carried out in the same hospital where cd was diagnosed . as the first line of care , twelve individuals attended a hospital , one visited a phcu , and one consulted a healer . all these cases ended their pathway in gh , where they were diagnosed and/or treated . from the total of the 12 individuals that made it to different hospitals for their first consult , 5 were diagnosed with cd and were transferred to gh for treatment , six cases were transferred directly to gh without diagnosis , with the presumption of cd , and one attended the gh not by transfer but guided by the informal advice of a nurse . of the 12 cases , 10 travelled between 3 and 25 km and attended hospitals located outside their area of residence , while two attended a hospital in their area of residence . among those who travelled outside of their locality , three did not have a phcu in their area of residence and went to nearby hospitals . in the rest of the seven cases , the families had a phcu in their locality ; however these were dismissed , as their first consult . they preferred to attend the hospital , even when this choice implied heavier costs in terms of time and money . five of the cases had a negative view on the care provided by the phcu in regard to the lack of supplies and the lack of consistency in care yes , ( ) the post is for decoration because we do n't have medications , nothing and now the nurse is going on leave and we no longer have one ( a post ) ( ) until january . although satisfied with the closest phcu in another village because it was a source of milk and vaccination , and doctors attended regularly , decided to attend a hospital . the only subject that chose the phcu as first line of care lived in a small area with difficult access , 13 km away . faced with the symptoms , the mother attended the post where the acute case was transferred to gh , 127 km away , with the presumption of cd . distrusting the health system , this subject sought care from a healer who had cured one of the adults in the family from cd and had also treated symptoms of parasitic infections in children . the healer suggested transferring the girl to gh : e : he is a healer in the countryside that heals then i took her there , and he tells me ( ) this is a vinchuca bite . he tells me you have to take her to gh ( ) and then i ( ) took her . it 's already advanced , he tells me , i will not be able to cure her for you . ( subject 17 ) . of the 14 acute cases , the four that received a misdiagnosis attended diverse hospitals . these wrongfully diagnosed cases returned to their homes and with the persistence of the acute symptoms went back for another consult . the fourth case , unsatisfied with a second diagnosis , attended gh by own decision , without referral . the first step for two of them was to attend the phcu while the remaining was unidentifiable . in the second line of care , the cases attended different hospitals , until they ended their pathway in gh . in this search , they were all misdiagnosed . the choice of the health post was driven by its proximity . in one of the cases , the urgency to respond to the symptoms of the acute case prevailed over the negative view of the care offered by the phcu . the closest hospital was located at 12 km : e : no , because here they do n't want to medicate , they do n't want to examine . ( ) if you go ask for a remedy they say there is not any , they do n't want to give it to you , so you have to travel by force ( subject 7 ) . the other case that attended the phcu belonged to a small rural area without any services for this family , the closest phcu was located at 19 km and offered unsatisfactory care . the three subjects received false diagnoses in their first consult and returned home : e : she became worse , she did n't sleep and at night she would cry and cry , on top of that she had fever from there , we started to get worried and from there i took her to ( hospital ) , but they gave her drops , but for the eye . ( subject 7 ) . similarly to other misdiagnosis , the persistence and the worsening of symptoms was a source of worry for the family , where the acute cases felt an impulse to search for other answers : e : and she started to ( ) have inflammation in the eye , and since i did n't know what chagas was ( ) , and i took her to the doctor and they gave me a lot of drops to give her . e : he said that it was conjunctivitis , and from there a nurse told me : no , i do n't think it 's conjunctivitis because it needs to be in both eyes . he was transferred twice , always without a diagnosis despite having done tests , until reaching gh . at first , the mother attributed the inflammation of the eye erroneously to an accident involving a nail . she treated him with edible oil , but once the symptoms worsened she searched for help : e : well , there was the health train that came from buenos aires , ( ) here to the village ( ) i took him ( ) and there they did nothing but transfer me urgently to f ( zonal hospital ) , because they thought that since i had told them it was from a nail from f they transferred me urgently to santiago , to hospital from the zonal hospital located 52 km from their place of residence , the acute case was transferred to the hospital the mother , due to past family history , was knowledgeable about cd . during her interactions with health personnel she communicated her suspicion that grew stronger as the days passed and the symptoms persisted : e : i told her , look doctor , please i ask you to do an analysis of chagas . because i had told her that i had vinchucas ( ) and i tell her there has already been a case doctor , a chagas problem there too and the girl was like him , like this . since the boy did not get better , the mother requested the director of the hospital to transfer him to gh : ( ) finally , i had to get angry ; i had no other choice ( ) . i was going to sign and i was going to take him because i saw no improvement , i was going to take him somewhere else , look for other doctors , my intentions were to take him to gh . the director authorized the transfer to gh in an ambulance accompanied by a nurse and the doctor who had treated him . upon arriving to gh , and based on his clinical manifestations , cd was diagnosed . this individual went through three different hospitals and a healer and ended up being diagnosed with cd in gh . the acute symptom initially appeared as general inflammation of the body , discomfort , crying , and sleeping problems . the parents dismissed the phcu in their zone where they had not been treated well . the suspicion of child physical abuse was the reason this individual had to pass through five steps of seeking diagnosis . each hospital believed that the symptoms were caused by burns and did not believe the parents . ( ) they gave him some drops so it does n't burn , you see . e2 : they said the same thing there at ( other hospital ) , that it was a burn . , the boy received a diagnosis for a throat condition , for which he was prescribed an antibiotic treatment : e : he started to swell up ( ) and he cried all night , we took him and the doctors did n't give him anything . from there i took him to ( other hospital ) again and the doctors gave me medication because they said it 's a sore throat . ( subject 18 ) . faced with persistent symptoms , the parents consulted a healer , who determined that the boy suffered from a scare , which he had treated successfully before , and thereafter , the discomfort and crying disappeared . however , within a few days , the boy presented with a lesion in the arm , which prompted a new consult at the hospital confirmed that it was a burn , although the parents insisted that the boy had not gotten burned . finally , he was transferred to gh where he received diagnosis for cd and treatment . the trajectories of the 25 subjects bring to light active health care seeking behaviors , of which there are five identifiable pathways . these pathways include phcus , different levels of hospitals , the referral hospital ( gh ) , and natural healers . as the number of steps increased , so did the cost of the transfers that came with the search and the disruption that took place in the everyday lives of the families . however , this did not prevent seeking care , in contrast to oxaal & cook 's findings . attending health centers was not delayed either , despite the distances that had to be travelled , as was the case in rutebemberwa et al . . various studies have shown that payments for medical services cause a differentiation in health care seeking [ 15 , 23 ] . in argentina , this restriction is not relevant considering that access to public health care services is free . the socioeconomic condition of the study population is mainly homogenous . although there are three families with more stable incomes , the majority have precarious financial means with incomes consisting of governmental cash transfers , remittances from family members , and occasional paid work . the insecurity of income explains the predominance of substandard dwellings and with that a favorable habitat for t. infestans . in contrast to what is noted in other studies , the relative socioeconomic conditions of the acute cases studied did not influence the number of steps the households took to seek care . the difficulty to diagnose the disease during the acute phase is shown in various studies and countries due to the heterogeneity of symptoms that tend to overlap with other diseases . in accordance with the guidelines for patients ' care , the weak surveillance systems hindered the capacity of catching new cases . according to a study conducted in venezuela the acute - phase patient is only detected by chance or by extending sero - parasitologic diagnosis to people living in the vicinity of an identified acute case . coincidently , in the study population , only one case was detected as a result of a blood extraction campaign at the population level . as the first symptoms appeared , in many cases seeking care was oriented by the expectation of an effective answer from the system . in 12 cases , the first decision consisted of not attending the phcu due to a negative view on the care they offer , which is consistent with other studies . despite a negative opinion , 's study on the latino immigrant population in the united states reports a widespread use of home remedies . in the study population , only two individuals used home remedies as the first response to symptoms , which delayed seeking care in the health system . the role of healers was also very small , for those who resorted to one as the first line of care . similar to the findings reported in mexico , the study subjects were familiar with triatomine bugs but were not able to connect it with the disease , nor did they have the necessary information for its prevention . only in few cases where relatives or neighbors had experienced the disease were they aware of cd . the reported acute cases ' pathways show the effort and the perseverance of families to overcome obstacles presented by vulnerable social and economic conditions . most individuals with acute cd reached diagnosis after going through several health services . from the patient perspective , that process was costly in material and symbolic terms . poor information on cd and its symptoms was a factor that conditioned the way families sought care for their health concerns . on the system 's side , health care delivery was inefficient . this study stresses the health system 's weakness for the detection of acute cases and consequently its epidemiological registry . more effort is needed to strengthen medical training to diagnose cd conditions in endemic areas . the effects of a better diagnostic capacity will be reflected in more accurate cd statistics .

chagas disease ( cd ) is a tropical parasitic disease largely underdiagnosed and mostly asymptomatic affecting marginalized rural populations . argentina regularly reports acute cases of cd , mostly young individuals under 14 years old . there is a void of knowledge of health care seeking behavior in subjects experiencing a cd acute condition . early treatment of the acute case is crucial to limit subsequent development of disease . the article explores how the health outcome of persons with acute cd may be conditioned by their health care seeking behavior . the study , with a qualitative approach , was carried out in rural areas of santiago del estero province , a high risk endemic region for vector transmission of cd . narratives of 25 in - depth interviews carried out in 2005 and 2006 are analyzed identifying patterns of health care seeking behavior followed by acute cases . through the retrospective recall of paths for diagnoses , weaknesses of disease information , knowledge at the household level , and underperformance at the provincial health care system level are detected . the misdiagnoses were a major factor in delaying a health care response . the study results expose lost opportunities for the health care system to effectively record cd acute cases .

1. Introduction 2. Health Seeking Behavior 3. Methodology 4. Findings 5. Discussion 6. Conclusions

detection of acute conditions of neglected infectious diseases ( nids ) is hindered by low primary health care utilization as well as low priority and quality of health care services . chagas disease ( cd ) is an infection caused by the parasite trypanosoma cruzi and is transmitted to humans , among other ways , by an insect vector , the triatomine bug . recent estimates suggest that more than 5.7 million people are infected with cd , mostly socially and economically vulnerable populations . it produces 14,000 annual deaths in latin america and is a cause of morbidity ( who , 2002 ) , work disability , and increased health care costs . cd acute phase takes place two to four weeks after infection but is symptomatic in only 1% to 2% of cases . typically , the acute phase develops in children under 10 years , with a 28% mortality rate . diagnosis is complicated due to the nonspecific nature of the signs and symptoms and often leads to misdiagnosis . despite the success in parasite and vector control , argentina regularly reports acute cases of cd , mostly young individuals . between 1996 and 2014 the number of reported acute cases ranged from a maximum of twenty - nine to a minimum of one [ 9 , 10 ] . underreporting cd figures might be explained by several factors hindering diagnosis : the largely asymptomatic nature of disease , organizational and human resources factors at the health system level , and social and cultural factors at the patient level . although there are estimates on patterns of health care system utilization by cd patients developed by experts , there is a void of knowledge of health care seeking behavior in subjects experiencing a cd acute condition . in this article , through a retrospective lens we explore the latter through the health care seeking paths followed by acute cases which have been successfully diagnosed and specifically prescribed for cd treatment . by doing this , we want to better understand the factors that surround people 's experience with acute cd in their quest for a diagnosis . the understanding of health care seeking behaviors is critical to determine how it affects the diagnosis and treatment of conditions . however , in the context of mainly poor rural populations that face several social and economic constraints , it is crucial to trace back the path that allows them to fulfill the main requirement of the sick role . broadly , the literature discusses health care seeking behaviors at the level of households and health systems . at the level of the household , health may be dependent on the availability of time , resources , priority allocated to health , cultural norms , and access to cash and transport . at the level of the health system , issues like quality of health care delivery , that is , human resources , and organizational arrangements and environment influence care seekers ' decisions [ 21 , 22 ] . several studies demonstrate that the cost of care or transportation to care facilities is a major barrier influencing care seeking behaviors [ 23 , 24 ] . in a study done in colombia , the most frequent path was to initially seek care at the most basic level of care which was influenced by insurance coverage and resources . poverty plays a role in the time it takes to seek care as well as the type of care being sought . cost relating to care is a significant contributor to delaying care which affects diagnoses and treatment efficacy . the health care seeking behaviors of mothers with low socioeconomic status in uganda delayed care seeking compared to those with high socioeconomic status . a study on maternal health care seeking behaviors in rural ecuador supported the hypotheses that families with lower socioeconomic status were more likely not to seek care or to treat their children 's respiratory infection symptoms with home remedies . in several countries poor and rural populations are stigmatized and mistreated for being infected by cd . these barriers further limit access to care and delay diagnosis in those who carry most of the disease burden . health system perception is an important factor that drives health care seeking behaviors . in a study done in bolivia , rural women who followed their family 's tradition of giving birth at home felt a sense of exclusion and believed the hospital would increase health complications . others felt they would be mistreated and not informed by the staff about the procedures being done to them . this caused a further delay in seeking care when traditional remedies were ineffective since women waited for symptoms to disappear before seeking mainstream health care . in mexico , bites by triatomines were considered as normal daily events and only sometimes thought to transmit disease , while others thought they had developed a resistance . for other participants , patients with schistosomiasis - related symptoms were more likely to seek care only when symptoms were more severe . the lack of information about symptoms , diseases , treatments , and services plays an important , yet detrimental role in health care seeking behaviors . in bolivia , similarly , one of the main barriers for latino immigrants in europe to access chagas - related care was the lack of awareness of services provided to them . moreover , the fear related to cd is linked to the lack of knowledge of the disease and the treatment process . not recognizing the severe implications of symptoms was also a significant element in delaying or not seeking health care [ 15 , 16 , 30 , 31 ] . the review shows the different factors contributing to an array of health care seeking behaviors . our research approaches patterns to health care from the patients ' perspective , exploring the way in which families respond to the symptoms , how they address contextual limitations , and , specifically , their interactions with the health care system . an ethnographic approach of reported acute cases was applied . at the time of the study , the province of santiago del estero was a high risk , cd endemic area with reported acute cases , a household infestation index > 5% , no active surveillance , and sero - prevalence above 5% among children less than 5 years of age . the purpose was to collect data regarding the experience of the acute case 's family from the time of the outbreak to the sero - positive laboratory result and to find care seeking patterns in these processes . twenty - five acute cases out of forty reported in 2003 , 2004 , and 2005 [ 32 , 33 ] were interviewed , 14 females and 11 males , that were registered by the health provincial system ( table 1 ) . as minors , most of the acute cases were represented by a care taker , who was the respondent in the interview . as twenty - three out of the twenty - five subjects were under 15 years old , the retrospective information collected during the interview was provided by close relatives , usually the mother . both long and short term retrospective recall can provide an accurate report of occurrence of health related events . also , family members playing a caring role , particularly mothers , are in position to provide accurate proxy reports of disease and symptoms . distance from the provincial capital city , santiago del estero , to acute cases ' houses ranged from 12 to 300 km , in villages distributed across 11 departamentos , a subprovincial administrative level ( figures 1 and 2 ) . most of the acute cases were located in rural areas of particularly difficult access : unpaved and dirt roads , narrow and fainting trails , and with no means of public transportation . the average time between the outbreak and the interview was 11 months , ranging from 3 to 22 months . mostly , the study population was from poor rural families with minimal access to water and electricity . most homes were made out of mud , with cracks and crevices that were ideal to accommodate t. infestans ( known in argentina as vinchuca ) . homes that had a bathroom , access to electricity , and easy access to water were considered as having standard living conditions ( table 1 ) . those who had health posts in their area mostly had irregular or minimal attendance of health practitioners or did not have the resources to deal with the health problems presented by the families . the most common symptom of the acute cases was a swollen eye ( 17 out of 25 ) which is a clinical symptom specific to cd . some families were unaware that these symptoms could be a result of cd and these nonspecific symptoms led them to think they acquired other conditions , which influenced their method of care . others were not aware of the existence of cd and how it was caused ; there were few families who were knowledgeable about the transmission of the disease . direct parasitological methods , that is , micromethod in children and strout method in adults , were used to diagnose trypanosoma cruzi infection at the acute phase . there were five different health care seeking behavior pathways the families took in order to finally get the subject diagnosed with acute cd . in 2000 , 51 hospitals and 254 primary health care units ( phcus ) organized with different levels of complexity were part of the provincial health care system . at the bottom , the phcus which were mostly located in rural areas offered outpatient services . at the top of the system , in the capital city it had a specialized center for cd diagnosis and treatment as well as a laboratory equipped for specific studies . in an intermediate position , smaller and lower complexity hospitals , with permanent on - call doctors , delivered basic health services . at the onset of symptoms , subjects searched for care at different levels of the system . in this endeavor , they followed a variety of paths ending with a sero - positivity diagnosis . one individual directly attended the hospital due to the absence of a primary health care unit ( phcu ) , four dismissed the local phcu as the first line of care , and one was taken to gh by health personnel working in the field where they were diagnosed . there are many reasons for which subjects dismissed the phcu as the lack of consistency in the schedule , e : but it 's nothing but a tale because sometimes the nurse is there and sometimes he is n't ( ) because in this zone we are disadvantaged ( subject 11 ) ; even with a stable schedule , patients had no choice but to seek care at the hospital when doctors attended the post infrequently , e : yes there is a health post here ( does n't the doctor come ? ) some health posts only had nurses , and local residents could not reach a doctor unless they went to the hospital . in some cases , it had been so long since a doctor had attended the health post they did not expect one to be present , ( do you have a health post ? ) those who had access to a primary health care unit were not always able to be cared for due to policies regarding the need for a doctor 's reference : ( you do n't go to the health post ? e : yes , sometimes but you need a doctor 's referral , otherwise they will not attend to you ) in addition to the limitations mentioned in these four cases , the health care seeking behaviors were driven by the proximity to the hospital . the family of the fifth case was an exception as they chose to be transferred to the capital city ( 135 km ) as a relative lived there . immediately , upon his father 's insistence , a second test was carried out in the same hospital where cd was diagnosed . as the first line of care , twelve individuals attended a hospital , one visited a phcu , and one consulted a healer . from the total of the 12 individuals that made it to different hospitals for their first consult , 5 were diagnosed with cd and were transferred to gh for treatment , six cases were transferred directly to gh without diagnosis , with the presumption of cd , and one attended the gh not by transfer but guided by the informal advice of a nurse . of the 12 cases , 10 travelled between 3 and 25 km and attended hospitals located outside their area of residence , while two attended a hospital in their area of residence . in the rest of the seven cases , the families had a phcu in their locality ; however these were dismissed , as their first consult . they preferred to attend the hospital , even when this choice implied heavier costs in terms of time and money . five of the cases had a negative view on the care provided by the phcu in regard to the lack of supplies and the lack of consistency in care yes , ( ) the post is for decoration because we do n't have medications , nothing and now the nurse is going on leave and we no longer have one ( a post ) ( ) until january . although satisfied with the closest phcu in another village because it was a source of milk and vaccination , and doctors attended regularly , decided to attend a hospital . the only subject that chose the phcu as first line of care lived in a small area with difficult access , 13 km away . faced with the symptoms , the mother attended the post where the acute case was transferred to gh , 127 km away , with the presumption of cd . distrusting the health system , this subject sought care from a healer who had cured one of the adults in the family from cd and had also treated symptoms of parasitic infections in children . the healer suggested transferring the girl to gh : e : he is a healer in the countryside that heals then i took her there , and he tells me ( ) this is a vinchuca bite . ( subject 17 ) . of the 14 acute cases , the four that received a misdiagnosis attended diverse hospitals . these wrongfully diagnosed cases returned to their homes and with the persistence of the acute symptoms went back for another consult . the fourth case , unsatisfied with a second diagnosis , attended gh by own decision , without referral . in the second line of care , the cases attended different hospitals , until they ended their pathway in gh . the choice of the health post was driven by its proximity . in one of the cases , the urgency to respond to the symptoms of the acute case prevailed over the negative view of the care offered by the phcu . ( ) if you go ask for a remedy they say there is not any , they do n't want to give it to you , so you have to travel by force ( subject 7 ) . the other case that attended the phcu belonged to a small rural area without any services for this family , the closest phcu was located at 19 km and offered unsatisfactory care . the three subjects received false diagnoses in their first consult and returned home : e : she became worse , she did n't sleep and at night she would cry and cry , on top of that she had fever from there , we started to get worried and from there i took her to ( hospital ) , but they gave her drops , but for the eye . ( subject 7 ) . similarly to other misdiagnosis , the persistence and the worsening of symptoms was a source of worry for the family , where the acute cases felt an impulse to search for other answers : e : and she started to ( ) have inflammation in the eye , and since i did n't know what chagas was ( ) , and i took her to the doctor and they gave me a lot of drops to give her . e : he said that it was conjunctivitis , and from there a nurse told me : no , i do n't think it 's conjunctivitis because it needs to be in both eyes . at first , the mother attributed the inflammation of the eye erroneously to an accident involving a nail . she treated him with edible oil , but once the symptoms worsened she searched for help : e : well , there was the health train that came from buenos aires , ( ) here to the village ( ) i took him ( ) and there they did nothing but transfer me urgently to f ( zonal hospital ) , because they thought that since i had told them it was from a nail from f they transferred me urgently to santiago , to hospital from the zonal hospital located 52 km from their place of residence , the acute case was transferred to the hospital the mother , due to past family history , was knowledgeable about cd . because i had told her that i had vinchucas ( ) and i tell her there has already been a case doctor , a chagas problem there too and the girl was like him , like this . since the boy did not get better , the mother requested the director of the hospital to transfer him to gh : ( ) finally , i had to get angry ; i had no other choice ( ) . upon arriving to gh , and based on his clinical manifestations , cd was diagnosed . the acute symptom initially appeared as general inflammation of the body , discomfort , crying , and sleeping problems . each hospital believed that the symptoms were caused by burns and did not believe the parents . ( ) they gave him some drops so it does n't burn , you see . e2 : they said the same thing there at ( other hospital ) , that it was a burn . , the boy received a diagnosis for a throat condition , for which he was prescribed an antibiotic treatment : e : he started to swell up ( ) and he cried all night , we took him and the doctors did n't give him anything . faced with persistent symptoms , the parents consulted a healer , who determined that the boy suffered from a scare , which he had treated successfully before , and thereafter , the discomfort and crying disappeared . however , within a few days , the boy presented with a lesion in the arm , which prompted a new consult at the hospital confirmed that it was a burn , although the parents insisted that the boy had not gotten burned . the trajectories of the 25 subjects bring to light active health care seeking behaviors , of which there are five identifiable pathways . these pathways include phcus , different levels of hospitals , the referral hospital ( gh ) , and natural healers . as the number of steps increased , so did the cost of the transfers that came with the search and the disruption that took place in the everyday lives of the families . various studies have shown that payments for medical services cause a differentiation in health care seeking [ 15 , 23 ] . in argentina , this restriction is not relevant considering that access to public health care services is free . the socioeconomic condition of the study population is mainly homogenous . although there are three families with more stable incomes , the majority have precarious financial means with incomes consisting of governmental cash transfers , remittances from family members , and occasional paid work . the insecurity of income explains the predominance of substandard dwellings and with that a favorable habitat for t. infestans . in contrast to what is noted in other studies , the relative socioeconomic conditions of the acute cases studied did not influence the number of steps the households took to seek care . the difficulty to diagnose the disease during the acute phase is shown in various studies and countries due to the heterogeneity of symptoms that tend to overlap with other diseases . in accordance with the guidelines for patients ' care , the weak surveillance systems hindered the capacity of catching new cases . according to a study conducted in venezuela the acute - phase patient is only detected by chance or by extending sero - parasitologic diagnosis to people living in the vicinity of an identified acute case . coincidently , in the study population , only one case was detected as a result of a blood extraction campaign at the population level . in the study population , only two individuals used home remedies as the first response to symptoms , which delayed seeking care in the health system . similar to the findings reported in mexico , the study subjects were familiar with triatomine bugs but were not able to connect it with the disease , nor did they have the necessary information for its prevention . only in few cases where relatives or neighbors had experienced the disease were they aware of cd . the reported acute cases ' pathways show the effort and the perseverance of families to overcome obstacles presented by vulnerable social and economic conditions . most individuals with acute cd reached diagnosis after going through several health services . poor information on cd and its symptoms was a factor that conditioned the way families sought care for their health concerns . on the system 's side , health care delivery was inefficient . this study stresses the health system 's weakness for the detection of acute cases and consequently its epidemiological registry .

detection of acute conditions of neglected infectious diseases ( nids ) is hindered by low primary health care utilization as well as low priority and quality of health care services . chagas disease ( cd ) is an infection caused by the parasite trypanosoma cruzi and is transmitted to humans , among other ways , by an insect vector , the triatomine bug . recent estimates suggest that more than 5.7 million people are infected with cd , mostly socially and economically vulnerable populations . it produces 14,000 annual deaths in latin america and is a cause of morbidity ( who , 2002 ) , work disability , and increased health care costs . a notable clinical consequence of t. cruzi is chagasic cardiomyopathy which may lead to heart failure and sudden death . cd acute phase takes place two to four weeks after infection but is symptomatic in only 1% to 2% of cases . typically , the acute phase develops in children under 10 years , with a 28% mortality rate . diagnosis is complicated due to the nonspecific nature of the signs and symptoms and often leads to misdiagnosis . despite the success in parasite and vector control , argentina regularly reports acute cases of cd , mostly young individuals . between 1996 and 2014 the number of reported acute cases ranged from a maximum of twenty - nine to a minimum of one [ 9 , 10 ] . underreporting cd figures might be explained by several factors hindering diagnosis : the largely asymptomatic nature of disease , organizational and human resources factors at the health system level , and social and cultural factors at the patient level . although there are estimates on patterns of health care system utilization by cd patients developed by experts , there is a void of knowledge of health care seeking behavior in subjects experiencing a cd acute condition . in this article , through a retrospective lens we explore the latter through the health care seeking paths followed by acute cases which have been successfully diagnosed and specifically prescribed for cd treatment . by doing this , we want to better understand the factors that surround people 's experience with acute cd in their quest for a diagnosis . the understanding of health care seeking behaviors is critical to determine how it affects the diagnosis and treatment of conditions . parsons described the sick role which discusses the social role that is implied in health behavior . therefore , they are not expected to heal without assistance , yet the individual holds the responsibility of seeking care and complying with medical directions . however , in the context of mainly poor rural populations that face several social and economic constraints , it is crucial to trace back the path that allows them to fulfill the main requirement of the sick role . broadly , the literature discusses health care seeking behaviors at the level of households and health systems . at the level of the household , health may be dependent on the availability of time , resources , priority allocated to health , cultural norms , and access to cash and transport . at the level of the health system , issues like quality of health care delivery , that is , human resources , and organizational arrangements and environment influence care seekers ' decisions [ 21 , 22 ] . several studies demonstrate that the cost of care or transportation to care facilities is a major barrier influencing care seeking behaviors [ 23 , 24 ] . in a study done in colombia , the most frequent path was to initially seek care at the most basic level of care which was influenced by insurance coverage and resources . in the united states the absence of insurance restricted care for cd . when mothers of sick children in uganda perceived the distance to health services to be further , seeking care was delayed compared to those who perceived a shorter distance . poverty plays a role in the time it takes to seek care as well as the type of care being sought . cost relating to care is a significant contributor to delaying care which affects diagnoses and treatment efficacy . the health care seeking behaviors of mothers with low socioeconomic status in uganda delayed care seeking compared to those with high socioeconomic status . a study on maternal health care seeking behaviors in rural ecuador supported the hypotheses that families with lower socioeconomic status were more likely not to seek care or to treat their children 's respiratory infection symptoms with home remedies . in several countries poor and rural populations are stigmatized and mistreated for being infected by cd . these barriers further limit access to care and delay diagnosis in those who carry most of the disease burden . health system perception is an important factor that drives health care seeking behaviors . in a study done in bolivia , rural women who followed their family 's tradition of giving birth at home felt a sense of exclusion and believed the hospital would increase health complications . others felt they would be mistreated and not informed by the staff about the procedures being done to them . latino immigrant women in the united states recognized that traditional remedies were not always the most effective but were the first line of treatment due to its lower cost . this caused a further delay in seeking care when traditional remedies were ineffective since women waited for symptoms to disappear before seeking mainstream health care . seeking care is marked by cultural and traditional elements influencing the perception of symptoms and conditions . for other participants , patients with schistosomiasis - related symptoms were more likely to seek care only when symptoms were more severe . the lack of information about symptoms , diseases , treatments , and services plays an important , yet detrimental role in health care seeking behaviors . in bolivia , similarly , one of the main barriers for latino immigrants in europe to access chagas - related care was the lack of awareness of services provided to them . moreover , the fear related to cd is linked to the lack of knowledge of the disease and the treatment process . not recognizing the severe implications of symptoms was also a significant element in delaying or not seeking health care [ 15 , 16 , 30 , 31 ] . the review shows the different factors contributing to an array of health care seeking behaviors . our research approaches patterns to health care from the patients ' perspective , exploring the way in which families respond to the symptoms , how they address contextual limitations , and , specifically , their interactions with the health care system . at the time of the study , the province of santiago del estero was a high risk , cd endemic area with reported acute cases , a household infestation index > 5% , no active surveillance , and sero - prevalence above 5% among children less than 5 years of age . the purpose was to collect data regarding the experience of the acute case 's family from the time of the outbreak to the sero - positive laboratory result and to find care seeking patterns in these processes . twenty - five acute cases out of forty reported in 2003 , 2004 , and 2005 [ 32 , 33 ] were interviewed , 14 females and 11 males , that were registered by the health provincial system ( table 1 ) . as minors , most of the acute cases were represented by a care taker , who was the respondent in the interview . as twenty - three out of the twenty - five subjects were under 15 years old , the retrospective information collected during the interview was provided by close relatives , usually the mother . both long and short term retrospective recall can provide an accurate report of occurrence of health related events . also , family members playing a caring role , particularly mothers , are in position to provide accurate proxy reports of disease and symptoms . distance from the provincial capital city , santiago del estero , to acute cases ' houses ranged from 12 to 300 km , in villages distributed across 11 departamentos , a subprovincial administrative level ( figures 1 and 2 ) . most of the acute cases were located in rural areas of particularly difficult access : unpaved and dirt roads , narrow and fainting trails , and with no means of public transportation . the average time between the outbreak and the interview was 11 months , ranging from 3 to 22 months . mostly , the study population was from poor rural families with minimal access to water and electricity . most homes were made out of mud , with cracks and crevices that were ideal to accommodate t. infestans ( known in argentina as vinchuca ) . homes that had a bathroom , access to electricity , and easy access to water were considered as having standard living conditions ( table 1 ) . those who had health posts in their area mostly had irregular or minimal attendance of health practitioners or did not have the resources to deal with the health problems presented by the families . the most common symptom of the acute cases was a swollen eye ( 17 out of 25 ) which is a clinical symptom specific to cd . some families were unaware that these symptoms could be a result of cd and these nonspecific symptoms led them to think they acquired other conditions , which influenced their method of care . others were not aware of the existence of cd and how it was caused ; there were few families who were knowledgeable about the transmission of the disease . direct parasitological methods , that is , micromethod in children and strout method in adults , were used to diagnose trypanosoma cruzi infection at the acute phase . there were five different health care seeking behavior pathways the families took in order to finally get the subject diagnosed with acute cd . in 2000 , 51 hospitals and 254 primary health care units ( phcus ) organized with different levels of complexity were part of the provincial health care system . at the bottom , the phcus which were mostly located in rural areas offered outpatient services . at the top of the system , in the capital city it had a specialized center for cd diagnosis and treatment as well as a laboratory equipped for specific studies . in an intermediate position , smaller and lower complexity hospitals , with permanent on - call doctors , delivered basic health services . at the onset of symptoms , subjects searched for care at different levels of the system . one individual directly attended the hospital due to the absence of a primary health care unit ( phcu ) , four dismissed the local phcu as the first line of care , and one was taken to gh by health personnel working in the field where they were diagnosed . there are many reasons for which subjects dismissed the phcu as the lack of consistency in the schedule , e : but it 's nothing but a tale because sometimes the nurse is there and sometimes he is n't ( ) because in this zone we are disadvantaged ( subject 11 ) ; even with a stable schedule , patients had no choice but to seek care at the hospital when doctors attended the post infrequently , e : yes there is a health post here ( does n't the doctor come ? ) in some cases , it had been so long since a doctor had attended the health post they did not expect one to be present , ( do you have a health post ? ) those who had access to a primary health care unit were not always able to be cared for due to policies regarding the need for a doctor 's reference : ( you do n't go to the health post ? e : yes , sometimes but you need a doctor 's referral , otherwise they will not attend to you ) in addition to the limitations mentioned in these four cases , the health care seeking behaviors were driven by the proximity to the hospital . the family of the fifth case was an exception as they chose to be transferred to the capital city ( 135 km ) as a relative lived there . obtaining a diagnosis was not free of complications ; an 11-year - old boy was diagnosed with an eye infection . as the first line of care , twelve individuals attended a hospital , one visited a phcu , and one consulted a healer . from the total of the 12 individuals that made it to different hospitals for their first consult , 5 were diagnosed with cd and were transferred to gh for treatment , six cases were transferred directly to gh without diagnosis , with the presumption of cd , and one attended the gh not by transfer but guided by the informal advice of a nurse . of the 12 cases , 10 travelled between 3 and 25 km and attended hospitals located outside their area of residence , while two attended a hospital in their area of residence . among those who travelled outside of their locality , three did not have a phcu in their area of residence and went to nearby hospitals . in the rest of the seven cases , the families had a phcu in their locality ; however these were dismissed , as their first consult . five of the cases had a negative view on the care provided by the phcu in regard to the lack of supplies and the lack of consistency in care yes , ( ) the post is for decoration because we do n't have medications , nothing and now the nurse is going on leave and we no longer have one ( a post ) ( ) until january . although satisfied with the closest phcu in another village because it was a source of milk and vaccination , and doctors attended regularly , decided to attend a hospital . the only subject that chose the phcu as first line of care lived in a small area with difficult access , 13 km away . faced with the symptoms , the mother attended the post where the acute case was transferred to gh , 127 km away , with the presumption of cd . distrusting the health system , this subject sought care from a healer who had cured one of the adults in the family from cd and had also treated symptoms of parasitic infections in children . the healer suggested transferring the girl to gh : e : he is a healer in the countryside that heals then i took her there , and he tells me ( ) this is a vinchuca bite . of the 14 acute cases , the four that received a misdiagnosis attended diverse hospitals . these wrongfully diagnosed cases returned to their homes and with the persistence of the acute symptoms went back for another consult . the fourth case , unsatisfied with a second diagnosis , attended gh by own decision , without referral . the first step for two of them was to attend the phcu while the remaining was unidentifiable . in the second line of care , the cases attended different hospitals , until they ended their pathway in gh . in one of the cases , the urgency to respond to the symptoms of the acute case prevailed over the negative view of the care offered by the phcu . the other case that attended the phcu belonged to a small rural area without any services for this family , the closest phcu was located at 19 km and offered unsatisfactory care . the three subjects received false diagnoses in their first consult and returned home : e : she became worse , she did n't sleep and at night she would cry and cry , on top of that she had fever from there , we started to get worried and from there i took her to ( hospital ) , but they gave her drops , but for the eye . similarly to other misdiagnosis , the persistence and the worsening of symptoms was a source of worry for the family , where the acute cases felt an impulse to search for other answers : e : and she started to ( ) have inflammation in the eye , and since i did n't know what chagas was ( ) , and i took her to the doctor and they gave me a lot of drops to give her . e : he said that it was conjunctivitis , and from there a nurse told me : no , i do n't think it 's conjunctivitis because it needs to be in both eyes . at first , the mother attributed the inflammation of the eye erroneously to an accident involving a nail . she treated him with edible oil , but once the symptoms worsened she searched for help : e : well , there was the health train that came from buenos aires , ( ) here to the village ( ) i took him ( ) and there they did nothing but transfer me urgently to f ( zonal hospital ) , because they thought that since i had told them it was from a nail from f they transferred me urgently to santiago , to hospital from the zonal hospital located 52 km from their place of residence , the acute case was transferred to the hospital the mother , due to past family history , was knowledgeable about cd . during her interactions with health personnel she communicated her suspicion that grew stronger as the days passed and the symptoms persisted : e : i told her , look doctor , please i ask you to do an analysis of chagas . since the boy did not get better , the mother requested the director of the hospital to transfer him to gh : ( ) finally , i had to get angry ; i had no other choice ( ) . the director authorized the transfer to gh in an ambulance accompanied by a nurse and the doctor who had treated him . the acute symptom initially appeared as general inflammation of the body , discomfort , crying , and sleeping problems . , the boy received a diagnosis for a throat condition , for which he was prescribed an antibiotic treatment : e : he started to swell up ( ) and he cried all night , we took him and the doctors did n't give him anything . faced with persistent symptoms , the parents consulted a healer , who determined that the boy suffered from a scare , which he had treated successfully before , and thereafter , the discomfort and crying disappeared . however , within a few days , the boy presented with a lesion in the arm , which prompted a new consult at the hospital confirmed that it was a burn , although the parents insisted that the boy had not gotten burned . the trajectories of the 25 subjects bring to light active health care seeking behaviors , of which there are five identifiable pathways . these pathways include phcus , different levels of hospitals , the referral hospital ( gh ) , and natural healers . as the number of steps increased , so did the cost of the transfers that came with the search and the disruption that took place in the everyday lives of the families . attending health centers was not delayed either , despite the distances that had to be travelled , as was the case in rutebemberwa et al . various studies have shown that payments for medical services cause a differentiation in health care seeking [ 15 , 23 ] . although there are three families with more stable incomes , the majority have precarious financial means with incomes consisting of governmental cash transfers , remittances from family members , and occasional paid work . the insecurity of income explains the predominance of substandard dwellings and with that a favorable habitat for t. infestans . in contrast to what is noted in other studies , the relative socioeconomic conditions of the acute cases studied did not influence the number of steps the households took to seek care . the difficulty to diagnose the disease during the acute phase is shown in various studies and countries due to the heterogeneity of symptoms that tend to overlap with other diseases . in accordance with the guidelines for patients ' care , the weak surveillance systems hindered the capacity of catching new cases . according to a study conducted in venezuela the acute - phase patient is only detected by chance or by extending sero - parasitologic diagnosis to people living in the vicinity of an identified acute case . coincidently , in the study population , only one case was detected as a result of a blood extraction campaign at the population level . as the first symptoms appeared , in many cases seeking care was oriented by the expectation of an effective answer from the system . in 12 cases , the first decision consisted of not attending the phcu due to a negative view on the care they offer , which is consistent with other studies . despite a negative opinion , 's study on the latino immigrant population in the united states reports a widespread use of home remedies . in the study population , only two individuals used home remedies as the first response to symptoms , which delayed seeking care in the health system . similar to the findings reported in mexico , the study subjects were familiar with triatomine bugs but were not able to connect it with the disease , nor did they have the necessary information for its prevention . the reported acute cases ' pathways show the effort and the perseverance of families to overcome obstacles presented by vulnerable social and economic conditions .

cardiac resynchronization therapy ( crt ) is a well accepted therapy for patients with heart failure ( hf ) , left ventricular ( lv ) systolic dysfunction and qrs prolongation . previously , prospective , randomized trials demonstrated that crt improves quality of life ( qol ) , exercise capacity , lv systolic function , and decreases hospitalizations for hf . with longer follow - up , large randomized studies have now shown a reduction in mortality with crt in both mild and advanced hf . subgroup analyses of these trials have frequently identified qrs duration and morphology as independent predictors of outcomes . specifically , patients with left bundle branch block ( lbbb ) and more prolonged qrs duration ( 150 ms ) tend to have better response rates , whether measured by acute haemodynamics , reverse remodelling , or clinical outcomes . this has reinforced the concept that lv electrical delay or electrical dyssynchrony is an important factor for predicting benefit from crt . to investigate this further , we evaluated the relationship between such electrical delay defined by time interval from the first deflection on a surface ecg to local intrinsic activation at the lv stimulation site ( qlv ) and reverse remodelling in a prospectively designed substudy of the smart - av trial . details of the design and primary results of the smart - av study have been published previously . briefly , this was a multicentre , randomized trial of atrioventricular ( av ) optimization techniques among patients with advanced hf undergoing crt defibrillator implantation . a subset of 426 ( 50.4% ) of the 846 patients in smart - av was included in the qlv substudy . at the final lead positions , surface lead ii , right ventricular ( rv ) and lv egm were recorded simultaneously on paper strips at a sweep speed of 100 mm / s . qlv was measured by a blinded core lab with no knowledge of lead position or clinical outcomes . the qlv interval was measured in sinus rhythm and in the absence of pacing as the interval from the onset of qrs from the surface ecg to the first large positive or negative peak of the lv egm during a cardiac cycle with the resolution of 5 ms ( figure 1 ) . the amplitude of the first large peak needed to be > 50% of the amplitude of the largest peak in the same cardiac cycle . the qlv interval measured for every patient was reduced by 30 ms to account for the average variable latency ( or noise ) between the alignment of surface ecg and the egm channels in device programmers . core lab measurements were performed independently by two reviewers , and a sample of 15 egms were reviewed by both to assess reproducibility of the results . the calipers are aligned with the onset of qrs and peak of the left venticular electrogram . the qlv was calculated as 90 ms for the patient in ( a ) and 165 ms for the patient in ( b ) . the calipers are aligned with the onset of qrs and peak of the left venticular electrogram . the qlv was calculated as 90 ms for the patient in ( a ) and 165 ms for the patient in ( b ) . the primary endpoint of the smart - av trial was left ventricular end - systolic volume ( lvesv ) . secondary endpoints included left ventricular end - diastolic volume ( lvedv ) , lv ejection fraction ( ef ) , qol score , as assessed by the minnesota living with heart failure questionnaire , new york heart association ( nyha ) functional class and 6-min hall walk distance . all deaths , hf - related hospitalizations and emergency room visits were collected as defined previously . the echocardiographic endpoints were analysed blindly by a single echocardiography core laboratory unaware of group assignment . off - line software ( pro - solv version 3.0 or ge echo pac version 6.0 ) was used for measurements . two - dimensional - derived lv volumes were determined in the apical four and two chamber views by the biplane method of discs . in 84% of images , the apical two chamber view image quality was deemed excellent or good with respect to visualization of the anterior wall . intra- and inter - observer core lab reliability for lv volumes was evaluated in a sample of 20 images using lin 's concordance correlation coefficient ( lin 's ccc ) . for both lvedv and lvesv , excellent reliability was observed [ edv intraobserver : ccc = 0.981 95% ci : ( 0.9540.992 ) , edv interobserver : ccc = 0.981 95% ci : ( 0.9560.992 ) , esv intraobserver : ccc = 0.986 95% ci : ( 0.9660.994 ) , esv interobserver : ccc = 0.986 95% ci : ( 0.9640.994 ) ] . in addition to lv volumetric measurements , mechanical dyssynchrony was measured as the delay between the time of peak systolic velocity was achieved at basal septal and basal lateral segments . lifetime % biventricular ( biv ) pacing was determined from the device interrogation disc data collected from the 6-month visit , and was available for 375 ( 88% ) of the 426 patients in the substudy . since no difference was shown in primary or secondary outcomes between randomized treatment groups in the main study , data were pooled for the present analyses . the effect of qlv on crt response was evaluated using univariate and multivariate logistic regression models . stratified models and inclusion of qlv by subgroup interactions in multivariate analysis were utilized to assess for heterogeneity of effect . prespecified response metrics to crt included a > 15% reduction in lvesv , b and a > 10 points reduction in qol response from implant to 6 months . since no difference was shown in primary or secondary outcomes between randomized treatment groups in the main study , data were pooled for the present analyses . the effect of qlv on crt response was evaluated using univariate and multivariate logistic regression models . stratified models and inclusion of qlv by subgroup interactions in multivariate analysis were utilized to assess for heterogeneity of effect . prespecified response metrics to crt included a > 15% reduction in lvesv , b and a > 10 points reduction in qol response from implant to 6 months . the 426 patients included in the qlv substudy were typical of those undergoing crt for advanced hf , including predominately late middle aged males with a reduced ef and advanced hf . a summary of baseline clinical data is presented in table 1 . of note , none of these values differs from the larger full cohort included in the smart - av trial , other than a slightly shorter mean qrs duration in the substudy population ( 151 19 vs. 154 21 ms , p < 0.05 ) . table 1patient characteristics ( n = 426)age , years66 11gender ( male , % ) 66ischaemic heart disease ( % ) 59nyha functional class ( % ) i0 ii3 iii94 iv3lv ejection fraction ( % ) 26 7cardiac medications ( % ) ace / arb84 beta - blocker92 diuretic82 digoxin22ecg characteristics qrs duration ( ms)151 19 lbbb ( % ) 75 rbbb ( % ) 13 ivcd ( % ) 12baseline lvesv ( ml)128 62qol46 26values expressed as mean sd.ace/arb , angiotensin - converting enzyme inhibitor / receptor blocker . patient characteristics ( n = 426 ) values expressed as mean sd.ace/arb , angiotensin - converting enzyme inhibitor / receptor blocker . the qlv measurement was reproducible , as evidenced by a strong concordance among qlv reviewers in the sample of duplicate reviews evaluated . the median value for qlv in this population was 95 ms with inter - quartile range of 70120 ms . the programmed av delay varied depending on whether nominal ( 120 ms ) , smartdelay electrogram optimization or echocardiographic optimization was used . overall , the median sensed av delay was 120 ms with inter - quartile range of 120140 ms . these programmed parameters resulted in a very high rate of lifetime biv pacing during this study with a median of 98.4% ( 95.199.6% ) , which is higher than the 92% threshold often used to insure a maximal crt response . when separated at the median qlv , patients with longer qlv intervals had shorter programmed av delays [ median ( q1q3 ) : 120 ( 110130 ) vs. 120 ( 120140 ) , p = 0.016 ] and slightly higher percentages of biv pacing [ median ( q1q3 ) : 98.8% ( 94.899.7% ) vs. 97.9% ( 95.299.3% ) , p = 0.014 ] . when separated by the qlv median value ( 95 ms ) , lvesv , lvedv , ef , and qol responses all were significantly larger for patients with long vs. short qlv ( figure 2 ) . the responses for lvesv , lvedv , ef , and qol from baseline to 6 months all significantly increased with the increase in the qlv from the shortest quartile ( < 70 ms ) to the longest quartile ( > 120 ms ) ( figure 3 ) . specifically , changes in nyha class , 6-min hall walk distance and hf events are shown for each qlv quartile . although these relationships were only statistically significant for nyha , the largest response figure 2comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the two qlv groups separated by the median value . figure 3comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the qlv quartiles . the data were presented as median inter - quartile range ( box ) . table 2relationship of qlv with clinical outcomesqlv quartilesq1 : 070 ms ( % ) q2 : 7095 ms ( % ) q3 : 95120 ms ( % ) q4 : 120195 ms ( % ) total ( % ) overall p - valueq4 vs. q1 p - valuepatients with hf events15 ( 12.1)7 ( 7.1)7 ( 6.4)6 ( 6.3)35 ( 8.2)0.370.17nyha improved89 ( 73.0)79 ( 80.6)76 ( 71.0)77 ( 83.7)321 ( 76.6)0.040.04 no change33 ( 27.1)16 ( 16.3)30 ( 28.0)14 ( 15.2)93 ( 22.2 ) worsened0 ( 0.0)3 ( 3.1)1 ( 0.9)1 ( 1.1)5 ( 1.2)six minute walk delta52 11868 9150 10470 9359 1030.360.13 comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the two qlv groups separated by the median value . comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the qlv quartiles . relationship of qlv with clinical outcomes the overall response rates were 50% for lvesv and 60% for qol in this population , which is typical for response rate for these parameters to crt . the response rates increased progressively from the shortest quartile to the longest quartile of qlv for both lvesv ( 38.768.4% ) and qol ( 5072% ) criteria . it is noteworthy that the median value ( 95 ms ) was also the optimal cut - point for qlv based on roc analysis using the point on the curve closest to the upper left corner for both lvesv and qol . for lvesv endpoint , auc = 0.634 95% ci : ( 0.6070.660 ) p < 0.001 for test of ho : auc = 0.5 by equivalent wilcoxon test . the sensitivity and specificity at 95 ms cut - off were 0.63 and 0.61 , respectively . for qol endpoint , auc = 0.600 95% ci : ( 0.5730.627 ) p < 0.001 for test of ho : auc = 0.5 by equivalent wilcoxon test . the sensitivity and specificity at 95 ms cut - off were 0.58 and 0.60 , respectively . table 3the left ventricular end - systolic volume and qol response rates for the qlv quartilesqlvnlvesv response rate ( % ) qol response rate ( % ) 070 ms12438.750.07095 ms9839.854.695120 ms10957.865.1120195 ms9568.472.0pearson <0.0010.004 the left ventricular end - systolic volume and qol response rates for the qlv quartiles qlv is expected to be longer with increased qrs duration as well as lbbb . for qrs duration > 150 ms , qlv was 113 33 ms , compared with 78 30 ms for qrs < 150 ms ( p < 0.001 ) . however , qrs duration was not strongly correlated with qlv , as it accounted for only 35% of the variability observed ( r = 0.35 , p < 0.001 ) . similarly , among lbbb patients qlv was 100 35 ms compared with 73 30 ms for non - lbbb patients ( p < 0.001 ) . median mechanical dyssynchrony was 35 ms with inter - quartile range from 13 to 68 ms . in contrast to the relationships between qlv and qrs duration and morphology , mechanical dyssynchrony did not vary by qlv quartile ( p = 0.55 , kruskal wallis test ) . in the multivariate regression models , qlv added significant predictive value for crt responses , assessed by either reductions of lvesv or qol , after accounting for baseline covariates ( table 4 ) . patients in the highest quartile of qlv had a greater than three - fold increase in their odds of lvesv response vs. the shortest quartile . for the endpoint of a > 15% reduction in lvesv , qlv , gender and aetiology of hf were the only independent variables that were significantly associated with the response . for the qol endpoint , only qlv and gender were independently associated with the response . it is noteworthy that , as expected , both qrs and lbbb were univariately associated with both lvesv response ( qrs : p = 0.04 ; lbbb : p < 0.001 ) and qol response ( qrs : p = 0.004 ; lbbb : p = 0.024 ) . table 4multivariate logistic regression model resultscovariateodds ratio ( 95% ci ) , p - valuelvesv responseqol responseqlv 2nd quartile vs. 1st quartile1.10 ( 0.621.95 ) , 0.7431.30 ( 0.752.26 ) , 0.355qlv 3rd quartile vs. 1st quartile1.86 ( 1.043.31 ) , 0.0361.86 ( 1.053.31 ) , 0.033qlv 4th quartile vs. 1st quartile3.21 ( 1.586.50 ) , 0.0012.73 ( 1.355.54 ) , 0.005age ( per 1 year increase)1.00 ( 0.981.02 ) , 0.8010.99 ( 0.971.01 ) , 0.209lvef ( per 1% increase)0.98 ( 0.941.01 ) , 0.1861.00 ( 0.961.03 ) , 0.83ischaemic vs. non - ischaemic0.58 ( 0.370.91 ) , 0.0191.05 ( 0.671.64 ) , 0.846qrs ( > 150 ms vs. 150 ms)0.86 ( 0.531.40 ) , 0.5430.88 ( 0.551.43 ) , 0.611lbbb vs. non - lbbb1.20 ( 0.722.01 ) , 0.481.17 ( 0.711.93 ) , 0.526male vs. female0.53 ( 0.330.85 ) , 0.0080.56 ( 0.340.91 ) , 0.018nyha class iv vs. i iii1.67 ( 0.446.29 ) , 0.453.41 ( 0.6916.92 ) , 0.133lvesv1.00 ( 0.991.01 ) , 0.981.00 ( 0.991.00 ) , 0.682adjusted for baseline ef , lvesv , aetiology of hf , lbbb , gender , nyha , qrs and age . multivariate logistic regression model results adjusted for baseline ef , lvesv , aetiology of hf , lbbb , gender , nyha , qrs and age . the association between longer qlv and crt response was assessed in several pre - specified subgroups . the univariate relationship between qlv and crt response was consistent in all groups . this was the case for both lvesv ( figure 4a ) and qol ( figure 4b ) response criteria . no significant interactions with qlv were observed in the logistic regression models for both endpoints . figure 4univariate logistic regression results for left ventricular end - systolic volume ( a ) and quality of life ( b ) by subgroups . univariate logistic regression results for left ventricular end - systolic volume ( a ) and quality of life ( b ) by subgroups . the location of the lv lead was not controlled in this study . however , as expected , most leads were placed in the anterolateral or posterolateral veins , as reported by the implanting physicians . in fact , only 46 of 426 patients had leads placed apically and only 13 had them placed in an anterior or septal location . these small numbers preclude any meaningful analysis of the impact of lead location on qlv or response rate . however , even in similar locations , there was marked variation in qlv . for instance , the qlv interval ranged from 10 to 195 ms in the mid - anterolateral location ( n = 89 ) , and from 15 to 195 ms in the mid - posterolateral location ( n = 230 ) . the majority of patients ( 76% ) had bipolar leads , 19% patients had unipolar leads , and for 5% patients the lead type was not available . the sensing configuration was true bipolar in 68% of patients , extended bipolar in 28% of patients , while the remaining 4% used unipolar sensing . the 426 patients included in the qlv substudy were typical of those undergoing crt for advanced hf , including predominately late middle aged males with a reduced ef and advanced hf . a summary of baseline clinical data is presented in table 1 . of note , none of these values differs from the larger full cohort included in the smart - av trial , other than a slightly shorter mean qrs duration in the substudy population ( 151 19 vs. 154 21 ms , p < 0.05 ) . table 1patient characteristics ( n = 426)age , years66 11gender ( male , % ) 66ischaemic heart disease ( % ) 59nyha functional class ( % ) i0 ii3 iii94 iv3lv ejection fraction ( % ) 26 7cardiac medications ( % ) ace / arb84 beta - blocker92 diuretic82 digoxin22ecg characteristics qrs duration ( ms)151 19 lbbb ( % ) 75 rbbb ( % ) 13 ivcd ( % ) 12baseline lvesv ( ml)128 62qol46 26values expressed as mean sd.ace/arb , angiotensin - converting enzyme inhibitor / receptor blocker . patient characteristics ( n = 426 ) values expressed as mean sd.ace/arb , angiotensin - converting enzyme inhibitor / receptor blocker . the qlv measurement was reproducible , as evidenced by a strong concordance among qlv reviewers in the sample of duplicate reviews evaluated . ( lin 's ccc , 95% ci : 0.93 , 0.820.98 ) . the median value for qlv in this population was 95 ms with inter - quartile range of 70120 ms . the programmed av delay varied depending on whether nominal ( 120 ms ) , smartdelay electrogram optimization or echocardiographic optimization was used . overall , the median sensed av delay was 120 ms with inter - quartile range of 120140 ms . these programmed parameters resulted in a very high rate of lifetime biv pacing during this study with a median of 98.4% ( 95.199.6% ) , which is higher than the 92% threshold often used to insure a maximal crt response . when separated at the median qlv , patients with longer qlv intervals had shorter programmed av delays [ median ( q1q3 ) : 120 ( 110130 ) vs. 120 ( 120140 ) , p = 0.016 ] and slightly higher percentages of biv pacing [ median ( q1q3 ) : 98.8% ( 94.899.7% ) vs. 97.9% ( 95.299.3% ) , p = 0.014 ] . when separated by the qlv median value ( 95 ms ) , lvesv , lvedv , ef , and qol responses all were significantly larger for patients with long vs. short qlv ( figure 2 ) . the responses for lvesv , lvedv , ef , and qol from baseline to 6 months all significantly increased with the increase in the qlv from the shortest quartile ( < 70 ms ) to the longest quartile ( > 120 ms ) ( figure 3 ) . specifically , changes in nyha class , 6-min hall walk distance and hf events are shown for each qlv quartile . although these relationships were only statistically significant for nyha , the largest response was consistently observed in the fourth quartile . figure 2comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the two qlv groups separated by the median value . figure 3comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the qlv quartiles . the data were presented as median inter - quartile range ( box ) . table 2relationship of qlv with clinical outcomesqlv quartilesq1 : 070 ms ( % ) q2 : 7095 ms ( % ) q3 : 95120 ms ( % ) q4 : 120195 ms ( % ) total ( % ) overall p - valueq4 vs. q1 p - valuepatients with hf events15 ( 12.1)7 ( 7.1)7 ( 6.4)6 ( 6.3)35 ( 8.2)0.370.17nyha improved89 ( 73.0)79 ( 80.6)76 ( 71.0)77 ( 83.7)321 ( 76.6)0.040.04 no change33 ( 27.1)16 ( 16.3)30 ( 28.0)14 ( 15.2)93 ( 22.2 ) worsened0 ( 0.0)3 ( 3.1)1 ( 0.9)1 ( 1.1)5 ( 1.2)six minute walk delta52 11868 9150 10470 9359 1030.360.13 comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the two qlv groups separated by the median value . the data were presented as median inter - quartile range . comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the qlv quartiles . the overall response rates were 50% for lvesv and 60% for qol in this population , which is typical for response rate for these parameters to crt . the response rates increased progressively from the shortest quartile to the longest quartile of qlv for both lvesv ( 38.768.4% ) and qol ( 5072% ) criteria . it is noteworthy that the median value ( 95 ms ) was also the optimal cut - point for qlv based on roc analysis using the point on the curve closest to the upper left corner for both lvesv and qol . for lvesv endpoint , auc = 0.634 95% ci : ( 0.6070.660 ) p < 0.001 for test of ho : auc = 0.5 by equivalent wilcoxon test . the sensitivity and specificity at 95 ms cut - off were 0.63 and 0.61 , respectively . for qol endpoint , auc = 0.600 95% ci : ( 0.5730.627 ) p < 0.001 for test of ho : auc = 0.5 by equivalent wilcoxon test . the sensitivity and specificity at 95 ms cut - off were 0.58 and 0.60 , respectively . table 3the left ventricular end - systolic volume and qol response rates for the qlv quartilesqlvnlvesv response rate ( % ) qol response rate ( % ) 070 ms12438.750.07095 ms9839.854.695120 ms10957.865.1120195 ms9568.472.0pearson <0.0010.004 the left ventricular end - systolic volume and qol response rates for the qlv quartiles qlv is expected to be longer with increased qrs duration as well as lbbb . for qrs duration > 150 ms , qlv was 113 33 ms , compared with 78 30 ms for qrs < 150 ms ( p < 0.001 ) . however , qrs duration was not strongly correlated with qlv , as it accounted for only 35% of the variability observed ( r = 0.35 , p < 0.001 ) . similarly , among lbbb patients qlv was 100 35 ms compared with 73 30 ms for non - lbbb patients ( p < 0.001 ) . median mechanical dyssynchrony was 35 ms with inter - quartile range from 13 to 68 ms . in contrast to the relationships between qlv and qrs duration and morphology , mechanical dyssynchrony did not vary by qlv quartile ( p = 0.55 , kruskal wallis test ) . in the multivariate regression models , qlv added significant predictive value for crt responses , assessed by either reductions of lvesv or qol , after accounting for baseline covariates ( table 4 ) . patients in the highest quartile of qlv had a greater than three - fold increase in their odds of lvesv response vs. the shortest quartile . for the endpoint of a > 15% reduction in lvesv , qlv , gender and aetiology of hf were the only independent variables that were significantly associated with the response . for the qol endpoint , only qlv and gender were independently associated with the response . it is noteworthy that , as expected , both qrs and lbbb were univariately associated with both lvesv response ( qrs : p = 0.04 ; lbbb : p < 0.001 ) and qol response ( qrs : p = 0.004 ; lbbb : p = 0.024 ) . table 4multivariate logistic regression model resultscovariateodds ratio ( 95% ci ) , p - valuelvesv responseqol responseqlv 2nd quartile vs. 1st quartile1.10 ( 0.621.95 ) , 0.7431.30 ( 0.752.26 ) , 0.355qlv 3rd quartile vs. 1st quartile1.86 ( 1.043.31 ) , 0.0361.86 ( 1.053.31 ) , 0.033qlv 4th quartile vs. 1st quartile3.21 ( 1.586.50 ) , 0.0012.73 ( 1.355.54 ) , 0.005age ( per 1 year increase)1.00 ( 0.981.02 ) , 0.8010.99 ( 0.971.01 ) , 0.209lvef ( per 1% increase)0.98 ( 0.941.01 ) , 0.1861.00 ( 0.961.03 ) , 0.83ischaemic vs. non - ischaemic0.58 ( 0.370.91 ) , 0.0191.05 ( 0.671.64 ) , 0.846qrs ( > 150 ms vs. 150 ms)0.86 ( 0.531.40 ) , 0.5430.88 ( 0.551.43 ) , 0.611lbbb vs. non - lbbb1.20 ( 0.722.01 ) , 0.481.17 ( 0.711.93 ) , 0.526male vs. female0.53 ( 0.330.85 ) , 0.0080.56 ( 0.340.91 ) , 0.018nyha class iv vs. i iii1.67 ( 0.446.29 ) , 0.453.41 ( 0.6916.92 ) , 0.133lvesv1.00 ( 0.991.01 ) , 0.981.00 ( 0.991.00 ) , 0.682adjusted for baseline ef , lvesv , aetiology of hf , lbbb , gender , nyha , qrs and age . multivariate logistic regression model results adjusted for baseline ef , lvesv , aetiology of hf , lbbb , gender , nyha , qrs and age . the association between longer qlv and crt response was assessed in several pre - specified subgroups . this was the case for both lvesv ( figure 4a ) and qol ( figure 4b ) response criteria . no significant interactions with qlv were observed in the logistic regression models for both endpoints . figure 4univariate logistic regression results for left ventricular end - systolic volume ( a ) and quality of life ( b ) by subgroups . univariate logistic regression results for left ventricular end - systolic volume ( a ) and quality of life ( b ) by subgroups . the location of the lv lead was not controlled in this study . however , as expected , most leads were placed in the anterolateral or posterolateral veins , as reported by the implanting physicians . in fact , only 46 of 426 patients had leads placed apically and only 13 had them placed in an anterior or septal location . these small numbers preclude any meaningful analysis of the impact of lead location on qlv or response rate . however , even in similar locations , there was marked variation in qlv . for instance , the qlv interval ranged from 10 to 195 ms in the mid - anterolateral location ( n = 89 ) , and from 15 to 195 ms in the mid - posterolateral location ( n = 230 ) . the majority of patients ( 76% ) had bipolar leads , 19% patients had unipolar leads , and for 5% patients the lead type was not available . the sensing configuration was true bipolar in 68% of patients , extended bipolar in 28% of patients , while the remaining 4% used unipolar sensing . the present study is the first comprehensive evaluation of the relationship between electrical delay or dyssynchrony as measured by qlv and crt outcomes in a large clinical trial . the results demonstrate that the qlv was strongly associated with reverse remodelling and qol improvement . longer qlv at the lv stimulation site was associated with better crt responses even after adjusting for baseline covariates , including qrs duration , bundle branch block , and aetiology of hf . previously , in small preliminary studies of qlv , this measure was shown to correlate directly with acute haemodynamic response as assessed by invasive measurement of lv dp / dtmax . subsequently , in a study of 71 patients , singh et al . reported that the percentage of lv delay as a function of qrs duration predicted not only acute haemodynamic response but also chronic clinical outcomes after 12 months of follow - up . the present trial extends these findings by showing the qlv was strongly associated with chronic reverse remodelling and qol in a much larger prospective , multicentre trial . the role of pacing site to influence crt outcomes is an area of active research . however , analyses of multicentre trials have failed to support this strategy . in the companion study , lead position had little effect on outcomes , whereas in madit crt apical positions were associated with worse outcomes , rather than non - lateral positions . however , other studies have showed that 30% of patients had optimal pacing site in the apical regions . these discrepant findings suggest that anatomic lead position alone is unlikely to be a sufficient guide for optimization of crt . in support of this notion , studies of mechanical dyssynchrony to guide lead position showed that optimal pacing site varies from patient to patient . however , current measurement of mechanical dyssynchrony requires echocardiography or other imaging modalities that may be hard to correlate with fluoroscopic imaging at the time of device implantation and have shown to have a high degree of variability . further study will be needed to test the hypothesis that positioning lv leads at the site of maximal qlv rather than simply by anatomical location improves the response rate to crt and clinical outcomes . one mechanism of the benefit of crt is to restore electrical synchrony by pre - exciting the delayed lv area to achieve more synchronous electrical activation and thus contraction within the left ventricle . to identify patients with such conduction delays , thus , patients with right bundle branch block ( rbbb ) can also have a prolonged qrs duration , yet typically will have delayed right but not lv activation . patients with rbbb show little or no response to crt . to reduce the contribution of activation time from the rv , sweeny et al . used left ventricular activation time ( lvat ) , which is the qrs width after substracting the early part that corresponds to the rvat . moreover , lvat also reflects the overall left ventricular activation time , and does not identify regions with latest delay . therefore , it identifies the potential for a good clinical response to crt by measuring electrical dyssyncrhony . to insure good crt responses , it is desirable to have an indicator that reflects the degree of delayed lv activation at the pacing site . thus qlv reflects the time that it takes for the ventricular depolarization wavefront to reach the lv electrode site , and thus the resynchronization that will occur with pacing . it is intriguing to speculate that this can be utilized during the implant procedure to determine an area of late activation by repositioning the lv electrode and examining the qlv value at different locations . in contrast to the strong relationship between qlv and qrs and lbbb , no correlation was observed with mechanical dyssynchrony . this may be due to the fact that qlv reflects electrical dyssynchrony at the stimulation site which is not well correlated with global measures of mechanical dyssynchrony , which are weak predictors of crt response at best . in this regard , qrs duration , morphology ( i.e. lbbb ) , aetiology of hf and gender are consistently associated with outcomes . these variables were also strongly associated with outcomes in the present study . however , lbbb and qrs duration were no longer predictive of crt response after adjusting for qlv . this suggests that qlv or ventricular electrical delay at the stimulation site is a fundamental mechanism for the enhanced crt response in the presence of lbbb or more prolonged qrs duration . non - responders continue to be a challenge for crt therapy despite optimized device programming . recent studies suggest that a purely anatomic approach to lead position will be of limited value . however , the qlv interval is strongly associated with response and helps identify good lv pacing sites . thus , it seems reasonable to consider repositioning the pacing lead either within a vein or in a different vein towards a larger qlv value , particularly if an initial qlv value is 95 ms or less , which could be associated with poor outcome . the intra - patient data may not be the same as cross - patient data for guiding lead placement . in addition , the choice of lead position was not controlled with a marked preponderance on the lateral wall . this limits the ability to evaluate fully the relationship between qlv and lead position , as only 11% of subjects had apical and only 3% had true anterior positions . furthermore , since the qlv is currently measured from electrodes that are placed epicardially through a coronary vein branch , it may not be able to identify intramural or endocardial latest activated regions . advancement in lv lead placement technology may improve this shortcoming when the lead is able to be placed via endocardial approach . finally , an echocardiographic measure of reverse remodelling ( lvesv ) at 6 months was the primary endpoint of this study . whereas reverse remodelling is a good predictor of survival and clinical outcomes with crt , harder endpoints such as hospitalization and survival were not powered endpoints in the smart - av trial and the short follow - up precluded more complete assessment of hf events . in summary , electrical dyssynchrony , as measured by qlv , was strongly and independently associated with chronic outcomes with crt . the best outcomes were observed with a qlv > 95 ms , so this target should be considered when selecting lv lead position at the time of crt implantation . further study is warranted to assess the value of using qlv rather than anatomic location to guide lead positioning to improve response rates with crt . one mechanism of the benefit of crt is to restore electrical synchrony by pre - exciting the delayed lv area to achieve more synchronous electrical activation and thus contraction within the left ventricle . to identify patients with such conduction delays , thus , patients with right bundle branch block ( rbbb ) can also have a prolonged qrs duration , yet typically will have delayed right but not lv activation . patients with rbbb show little or no response to crt . to reduce the contribution of activation time from the rv , sweeny et al . used left ventricular activation time ( lvat ) , which is the qrs width after substracting the early part that corresponds to the rvat . moreover , lvat also reflects the overall left ventricular activation time , and does not identify regions with latest delay . therefore , it identifies the potential for a good clinical response to crt by measuring electrical dyssyncrhony . to insure good crt responses , it is desirable to have an indicator that reflects the degree of delayed lv activation at the pacing site . thus qlv reflects the time that it takes for the ventricular depolarization wavefront to reach the lv electrode site , and thus the resynchronization that will occur with pacing . it is intriguing to speculate that this can be utilized during the implant procedure to determine an area of late activation by repositioning the lv electrode and examining the qlv value at different locations . in contrast to the strong relationship between qlv and qrs and lbbb , no correlation was observed with mechanical dyssynchrony . this may be due to the fact that qlv reflects electrical dyssynchrony at the stimulation site which is not well correlated with global measures of mechanical dyssynchrony , which are weak predictors of crt response at best . regard , qrs duration , morphology ( i.e. lbbb ) , aetiology of hf and gender are consistently associated with outcomes . these variables were also strongly associated with outcomes in the present study . however , lbbb and qrs duration were no longer predictive of crt response after adjusting for qlv . this suggests that qlv or ventricular electrical delay at the stimulation site is a fundamental mechanism for the enhanced crt response in the presence of lbbb or more prolonged qrs duration . non - responders continue to be a challenge for crt therapy despite optimized device programming . recent studies suggest that a purely anatomic approach to lead position will be of limited value . however , the qlv interval is strongly associated with response and helps identify good lv pacing sites . thus , it seems reasonable to consider repositioning the pacing lead either within a vein or in a different vein towards a larger qlv value , particularly if an initial qlv value is 95 ms or less , which could be associated with poor outcome . the intra - patient data may not be the same as cross - patient data for guiding lead placement . in addition , the choice of lead position was not controlled with a marked preponderance on the lateral wall . this limits the ability to evaluate fully the relationship between qlv and lead position , as only 11% of subjects had apical and only 3% had true anterior positions . furthermore , since the qlv is currently measured from electrodes that are placed epicardially through a coronary vein branch , it may not be able to identify intramural or endocardial latest activated regions . advancement in lv lead placement technology may improve this shortcoming when the lead is able to be placed via endocardial approach . finally , an echocardiographic measure of reverse remodelling ( lvesv ) at 6 months was the primary endpoint of this study . whereas reverse remodelling is a good predictor of survival and clinical outcomes with crt , harder endpoints such as hospitalization and survival were not powered endpoints in the smart - av trial and the short follow - up precluded more complete assessment of hf events . in summary , electrical dyssynchrony , as measured by qlv , was strongly and independently associated with chronic outcomes with crt . the best outcomes were observed with a qlv > 95 ms , so this target should be considered when selecting lv lead position at the time of crt implantation . further study is warranted to assess the value of using qlv rather than anatomic location to guide lead positioning to improve response rates with crt . this study was funded by boston scientific corporation . funding to pay the open access publication charges for this article was provided by boston scientific corporation . conflict of interest : m.g . reported receiving research grants from medtronic , boston scientific and st jude , fees for fellowship support from medtronic and biotronik , honoraria and consulting fees from medtronic , boston scientific , st jude and biotronik . u.b . reported receiving research grants from medtronic , boston scientific and st jude , honoraria / speakers bureau from medtronic , boston scientific and st jude . j.s . reported receiving research grants from st jude , medtronic , boston scientific and biotronik , fees for advisory board / steering committee / consultant from boston scientific , biotronik , st jude , medtronic , cardioinsight , thoratec and biosense webster . reported receiving research grants from medtronic , boston scientific , st jude , fees for fellowship support from medtronic , boston scientific , and biotronik , honoraria and consulting fees from medtronic , boston scientific , st jude and biotronik .

aimsthe aim of the present study was to evaluate the relationship between left ventricular ( lv ) electrical delay , as measured by the qlv interval , and outcomes in a prospectively designed substudy of the smart - av trial.methods and resultsthis was a multicentre study of patients with advanced heart failure undergoing cardiac resynchronization therapy ( crt ) defibrillator implantation . in 426 subjects , qlv was measured as the interval from the onset of the qrs from the surface ecg to the first large peak of the lv electrogram . left ventricular volumes were measured by echocardiography at baseline and after 6 months of crt by a blinded core laboratory . quality of life ( qol ) was assessed by a standardized questionnaire . when separated by quartiles based on qlv duration , reverse remodelling response rates ( > 15% reduction in lv end systolic volume ) increased progressively from 38.7 to 68.4% and qol response rate ( > 10 points reduction ) increased from 50 to 72% . patients in the highest quartile of qlv had a 3.21-fold increase ( 1.586.50 , p = 0.001 ) in their odds of a reverse remodelling response after correcting for qrs duration , bundle branch block type , and clinical characteristics by multivariate logistic regression analysis.conclusionelectrical dyssynchrony , as measured by qlv , was strongly and independently associated with reverse remodelling and qol with crt . acute measurements of qlv may be useful to guide lv lead placement .

Introduction Methods Statistical analysis Results Patient population QLV response Biventricular pacing CRT responses CRT response rate Relationship between baseline parameters and QLV Relationship between electrical intervals and anatomical locations Discussion Electrical dyssynchrony measures and CRT responses Clinical implications Limitations Funding

cardiac resynchronization therapy ( crt ) is a well accepted therapy for patients with heart failure ( hf ) , left ventricular ( lv ) systolic dysfunction and qrs prolongation . previously , prospective , randomized trials demonstrated that crt improves quality of life ( qol ) , exercise capacity , lv systolic function , and decreases hospitalizations for hf . specifically , patients with left bundle branch block ( lbbb ) and more prolonged qrs duration ( 150 ms ) tend to have better response rates , whether measured by acute haemodynamics , reverse remodelling , or clinical outcomes . to investigate this further , we evaluated the relationship between such electrical delay defined by time interval from the first deflection on a surface ecg to local intrinsic activation at the lv stimulation site ( qlv ) and reverse remodelling in a prospectively designed substudy of the smart - av trial . a subset of 426 ( 50.4% ) of the 846 patients in smart - av was included in the qlv substudy . qlv was measured by a blinded core lab with no knowledge of lead position or clinical outcomes . the qlv interval was measured in sinus rhythm and in the absence of pacing as the interval from the onset of qrs from the surface ecg to the first large positive or negative peak of the lv egm during a cardiac cycle with the resolution of 5 ms ( figure 1 ) . the primary endpoint of the smart - av trial was left ventricular end - systolic volume ( lvesv ) . secondary endpoints included left ventricular end - diastolic volume ( lvedv ) , lv ejection fraction ( ef ) , qol score , as assessed by the minnesota living with heart failure questionnaire , new york heart association ( nyha ) functional class and 6-min hall walk distance . prespecified response metrics to crt included a > 15% reduction in lvesv , b and a > 10 points reduction in qol response from implant to 6 months . prespecified response metrics to crt included a > 15% reduction in lvesv , b and a > 10 points reduction in qol response from implant to 6 months . of note , none of these values differs from the larger full cohort included in the smart - av trial , other than a slightly shorter mean qrs duration in the substudy population ( 151 19 vs. 154 21 ms , p < 0.05 ) . when separated at the median qlv , patients with longer qlv intervals had shorter programmed av delays [ median ( q1q3 ) : 120 ( 110130 ) vs. 120 ( 120140 ) , p = 0.016 ] and slightly higher percentages of biv pacing [ median ( q1q3 ) : 98.8% ( 94.899.7% ) vs. 97.9% ( 95.299.3% ) , p = 0.014 ] . when separated by the qlv median value ( 95 ms ) , lvesv , lvedv , ef , and qol responses all were significantly larger for patients with long vs. short qlv ( figure 2 ) . the responses for lvesv , lvedv , ef , and qol from baseline to 6 months all significantly increased with the increase in the qlv from the shortest quartile ( < 70 ms ) to the longest quartile ( > 120 ms ) ( figure 3 ) . although these relationships were only statistically significant for nyha , the largest response figure 2comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the two qlv groups separated by the median value . figure 3comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the qlv quartiles . table 2relationship of qlv with clinical outcomesqlv quartilesq1 : 070 ms ( % ) q2 : 7095 ms ( % ) q3 : 95120 ms ( % ) q4 : 120195 ms ( % ) total ( % ) overall p - valueq4 vs. q1 p - valuepatients with hf events15 ( 12.1)7 ( 7.1)7 ( 6.4)6 ( 6.3)35 ( 8.2)0.370.17nyha improved89 ( 73.0)79 ( 80.6)76 ( 71.0)77 ( 83.7)321 ( 76.6)0.040.04 no change33 ( 27.1)16 ( 16.3)30 ( 28.0)14 ( 15.2)93 ( 22.2 ) worsened0 ( 0.0)3 ( 3.1)1 ( 0.9)1 ( 1.1)5 ( 1.2)six minute walk delta52 11868 9150 10470 9359 1030.360.13 comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the two qlv groups separated by the median value . comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the qlv quartiles . the response rates increased progressively from the shortest quartile to the longest quartile of qlv for both lvesv ( 38.768.4% ) and qol ( 5072% ) criteria . table 3the left ventricular end - systolic volume and qol response rates for the qlv quartilesqlvnlvesv response rate ( % ) qol response rate ( % ) 070 ms12438.750.07095 ms9839.854.695120 ms10957.865.1120195 ms9568.472.0pearson <0.0010.004 the left ventricular end - systolic volume and qol response rates for the qlv quartiles qlv is expected to be longer with increased qrs duration as well as lbbb . for qrs duration > 150 ms , qlv was 113 33 ms , compared with 78 30 ms for qrs < 150 ms ( p < 0.001 ) . however , qrs duration was not strongly correlated with qlv , as it accounted for only 35% of the variability observed ( r = 0.35 , p < 0.001 ) . patients in the highest quartile of qlv had a greater than three - fold increase in their odds of lvesv response vs. the shortest quartile . for the endpoint of a > 15% reduction in lvesv , qlv , gender and aetiology of hf were the only independent variables that were significantly associated with the response . it is noteworthy that , as expected , both qrs and lbbb were univariately associated with both lvesv response ( qrs : p = 0.04 ; lbbb : p < 0.001 ) and qol response ( qrs : p = 0.004 ; lbbb : p = 0.024 ) . table 4multivariate logistic regression model resultscovariateodds ratio ( 95% ci ) , p - valuelvesv responseqol responseqlv 2nd quartile vs. 1st quartile1.10 ( 0.621.95 ) , 0.7431.30 ( 0.752.26 ) , 0.355qlv 3rd quartile vs. 1st quartile1.86 ( 1.043.31 ) , 0.0361.86 ( 1.053.31 ) , 0.033qlv 4th quartile vs. 1st quartile3.21 ( 1.586.50 ) , 0.0012.73 ( 1.355.54 ) , 0.005age ( per 1 year increase)1.00 ( 0.981.02 ) , 0.8010.99 ( 0.971.01 ) , 0.209lvef ( per 1% increase)0.98 ( 0.941.01 ) , 0.1861.00 ( 0.961.03 ) , 0.83ischaemic vs. non - ischaemic0.58 ( 0.370.91 ) , 0.0191.05 ( 0.671.64 ) , 0.846qrs ( > 150 ms vs. 150 ms)0.86 ( 0.531.40 ) , 0.5430.88 ( 0.551.43 ) , 0.611lbbb vs. non - lbbb1.20 ( 0.722.01 ) , 0.481.17 ( 0.711.93 ) , 0.526male vs. female0.53 ( 0.330.85 ) , 0.0080.56 ( 0.340.91 ) , 0.018nyha class iv vs. i iii1.67 ( 0.446.29 ) , 0.453.41 ( 0.6916.92 ) , 0.133lvesv1.00 ( 0.991.01 ) , 0.981.00 ( 0.991.00 ) , 0.682adjusted for baseline ef , lvesv , aetiology of hf , lbbb , gender , nyha , qrs and age . figure 4univariate logistic regression results for left ventricular end - systolic volume ( a ) and quality of life ( b ) by subgroups . univariate logistic regression results for left ventricular end - systolic volume ( a ) and quality of life ( b ) by subgroups . of note , none of these values differs from the larger full cohort included in the smart - av trial , other than a slightly shorter mean qrs duration in the substudy population ( 151 19 vs. 154 21 ms , p < 0.05 ) . when separated at the median qlv , patients with longer qlv intervals had shorter programmed av delays [ median ( q1q3 ) : 120 ( 110130 ) vs. 120 ( 120140 ) , p = 0.016 ] and slightly higher percentages of biv pacing [ median ( q1q3 ) : 98.8% ( 94.899.7% ) vs. 97.9% ( 95.299.3% ) , p = 0.014 ] . when separated by the qlv median value ( 95 ms ) , lvesv , lvedv , ef , and qol responses all were significantly larger for patients with long vs. short qlv ( figure 2 ) . the responses for lvesv , lvedv , ef , and qol from baseline to 6 months all significantly increased with the increase in the qlv from the shortest quartile ( < 70 ms ) to the longest quartile ( > 120 ms ) ( figure 3 ) . figure 2comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the two qlv groups separated by the median value . figure 3comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the qlv quartiles . table 2relationship of qlv with clinical outcomesqlv quartilesq1 : 070 ms ( % ) q2 : 7095 ms ( % ) q3 : 95120 ms ( % ) q4 : 120195 ms ( % ) total ( % ) overall p - valueq4 vs. q1 p - valuepatients with hf events15 ( 12.1)7 ( 7.1)7 ( 6.4)6 ( 6.3)35 ( 8.2)0.370.17nyha improved89 ( 73.0)79 ( 80.6)76 ( 71.0)77 ( 83.7)321 ( 76.6)0.040.04 no change33 ( 27.1)16 ( 16.3)30 ( 28.0)14 ( 15.2)93 ( 22.2 ) worsened0 ( 0.0)3 ( 3.1)1 ( 0.9)1 ( 1.1)5 ( 1.2)six minute walk delta52 11868 9150 10470 9359 1030.360.13 comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the two qlv groups separated by the median value . comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the qlv quartiles . the response rates increased progressively from the shortest quartile to the longest quartile of qlv for both lvesv ( 38.768.4% ) and qol ( 5072% ) criteria . table 3the left ventricular end - systolic volume and qol response rates for the qlv quartilesqlvnlvesv response rate ( % ) qol response rate ( % ) 070 ms12438.750.07095 ms9839.854.695120 ms10957.865.1120195 ms9568.472.0pearson <0.0010.004 the left ventricular end - systolic volume and qol response rates for the qlv quartiles qlv is expected to be longer with increased qrs duration as well as lbbb . for qrs duration > 150 ms , qlv was 113 33 ms , compared with 78 30 ms for qrs < 150 ms ( p < 0.001 ) . however , qrs duration was not strongly correlated with qlv , as it accounted for only 35% of the variability observed ( r = 0.35 , p < 0.001 ) . in contrast to the relationships between qlv and qrs duration and morphology , mechanical dyssynchrony did not vary by qlv quartile ( p = 0.55 , kruskal wallis test ) . patients in the highest quartile of qlv had a greater than three - fold increase in their odds of lvesv response vs. the shortest quartile . for the endpoint of a > 15% reduction in lvesv , qlv , gender and aetiology of hf were the only independent variables that were significantly associated with the response . it is noteworthy that , as expected , both qrs and lbbb were univariately associated with both lvesv response ( qrs : p = 0.04 ; lbbb : p < 0.001 ) and qol response ( qrs : p = 0.004 ; lbbb : p = 0.024 ) . table 4multivariate logistic regression model resultscovariateodds ratio ( 95% ci ) , p - valuelvesv responseqol responseqlv 2nd quartile vs. 1st quartile1.10 ( 0.621.95 ) , 0.7431.30 ( 0.752.26 ) , 0.355qlv 3rd quartile vs. 1st quartile1.86 ( 1.043.31 ) , 0.0361.86 ( 1.053.31 ) , 0.033qlv 4th quartile vs. 1st quartile3.21 ( 1.586.50 ) , 0.0012.73 ( 1.355.54 ) , 0.005age ( per 1 year increase)1.00 ( 0.981.02 ) , 0.8010.99 ( 0.971.01 ) , 0.209lvef ( per 1% increase)0.98 ( 0.941.01 ) , 0.1861.00 ( 0.961.03 ) , 0.83ischaemic vs. non - ischaemic0.58 ( 0.370.91 ) , 0.0191.05 ( 0.671.64 ) , 0.846qrs ( > 150 ms vs. 150 ms)0.86 ( 0.531.40 ) , 0.5430.88 ( 0.551.43 ) , 0.611lbbb vs. non - lbbb1.20 ( 0.722.01 ) , 0.481.17 ( 0.711.93 ) , 0.526male vs. female0.53 ( 0.330.85 ) , 0.0080.56 ( 0.340.91 ) , 0.018nyha class iv vs. i iii1.67 ( 0.446.29 ) , 0.453.41 ( 0.6916.92 ) , 0.133lvesv1.00 ( 0.991.01 ) , 0.981.00 ( 0.991.00 ) , 0.682adjusted for baseline ef , lvesv , aetiology of hf , lbbb , gender , nyha , qrs and age . figure 4univariate logistic regression results for left ventricular end - systolic volume ( a ) and quality of life ( b ) by subgroups . univariate logistic regression results for left ventricular end - systolic volume ( a ) and quality of life ( b ) by subgroups . the present study is the first comprehensive evaluation of the relationship between electrical delay or dyssynchrony as measured by qlv and crt outcomes in a large clinical trial . the results demonstrate that the qlv was strongly associated with reverse remodelling and qol improvement . longer qlv at the lv stimulation site was associated with better crt responses even after adjusting for baseline covariates , including qrs duration , bundle branch block , and aetiology of hf . the present trial extends these findings by showing the qlv was strongly associated with chronic reverse remodelling and qol in a much larger prospective , multicentre trial . whereas reverse remodelling is a good predictor of survival and clinical outcomes with crt , harder endpoints such as hospitalization and survival were not powered endpoints in the smart - av trial and the short follow - up precluded more complete assessment of hf events . in summary , electrical dyssynchrony , as measured by qlv , was strongly and independently associated with chronic outcomes with crt . these variables were also strongly associated with outcomes in the present study . whereas reverse remodelling is a good predictor of survival and clinical outcomes with crt , harder endpoints such as hospitalization and survival were not powered endpoints in the smart - av trial and the short follow - up precluded more complete assessment of hf events . in summary , electrical dyssynchrony , as measured by qlv , was strongly and independently associated with chronic outcomes with crt .

to investigate this further , we evaluated the relationship between such electrical delay defined by time interval from the first deflection on a surface ecg to local intrinsic activation at the lv stimulation site ( qlv ) and reverse remodelling in a prospectively designed substudy of the smart - av trial . the qlv interval was measured in sinus rhythm and in the absence of pacing as the interval from the onset of qrs from the surface ecg to the first large positive or negative peak of the lv egm during a cardiac cycle with the resolution of 5 ms ( figure 1 ) . secondary endpoints included left ventricular end - diastolic volume ( lvedv ) , lv ejection fraction ( ef ) , qol score , as assessed by the minnesota living with heart failure questionnaire , new york heart association ( nyha ) functional class and 6-min hall walk distance . for both lvedv and lvesv , excellent reliability was observed [ edv intraobserver : ccc = 0.981 95% ci : ( 0.9540.992 ) , edv interobserver : ccc = 0.981 95% ci : ( 0.9560.992 ) , esv intraobserver : ccc = 0.986 95% ci : ( 0.9660.994 ) , esv interobserver : ccc = 0.986 95% ci : ( 0.9640.994 ) ] . lifetime % biventricular ( biv ) pacing was determined from the device interrogation disc data collected from the 6-month visit , and was available for 375 ( 88% ) of the 426 patients in the substudy . of note , none of these values differs from the larger full cohort included in the smart - av trial , other than a slightly shorter mean qrs duration in the substudy population ( 151 19 vs. 154 21 ms , p < 0.05 ) . table 1patient characteristics ( n = 426)age , years66 11gender ( male , % ) 66ischaemic heart disease ( % ) 59nyha functional class ( % ) i0 ii3 iii94 iv3lv ejection fraction ( % ) 26 7cardiac medications ( % ) ace / arb84 beta - blocker92 diuretic82 digoxin22ecg characteristics qrs duration ( ms)151 19 lbbb ( % ) 75 rbbb ( % ) 13 ivcd ( % ) 12baseline lvesv ( ml)128 62qol46 26values expressed as mean sd.ace/arb , angiotensin - converting enzyme inhibitor / receptor blocker . these programmed parameters resulted in a very high rate of lifetime biv pacing during this study with a median of 98.4% ( 95.199.6% ) , which is higher than the 92% threshold often used to insure a maximal crt response . when separated at the median qlv , patients with longer qlv intervals had shorter programmed av delays [ median ( q1q3 ) : 120 ( 110130 ) vs. 120 ( 120140 ) , p = 0.016 ] and slightly higher percentages of biv pacing [ median ( q1q3 ) : 98.8% ( 94.899.7% ) vs. 97.9% ( 95.299.3% ) , p = 0.014 ] . the responses for lvesv , lvedv , ef , and qol from baseline to 6 months all significantly increased with the increase in the qlv from the shortest quartile ( < 70 ms ) to the longest quartile ( > 120 ms ) ( figure 3 ) . although these relationships were only statistically significant for nyha , the largest response figure 2comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the two qlv groups separated by the median value . figure 3comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the qlv quartiles . table 2relationship of qlv with clinical outcomesqlv quartilesq1 : 070 ms ( % ) q2 : 7095 ms ( % ) q3 : 95120 ms ( % ) q4 : 120195 ms ( % ) total ( % ) overall p - valueq4 vs. q1 p - valuepatients with hf events15 ( 12.1)7 ( 7.1)7 ( 6.4)6 ( 6.3)35 ( 8.2)0.370.17nyha improved89 ( 73.0)79 ( 80.6)76 ( 71.0)77 ( 83.7)321 ( 76.6)0.040.04 no change33 ( 27.1)16 ( 16.3)30 ( 28.0)14 ( 15.2)93 ( 22.2 ) worsened0 ( 0.0)3 ( 3.1)1 ( 0.9)1 ( 1.1)5 ( 1.2)six minute walk delta52 11868 9150 10470 9359 1030.360.13 comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the two qlv groups separated by the median value . comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the qlv quartiles . relationship of qlv with clinical outcomes the overall response rates were 50% for lvesv and 60% for qol in this population , which is typical for response rate for these parameters to crt . for lvesv endpoint , auc = 0.634 95% ci : ( 0.6070.660 ) p < 0.001 for test of ho : auc = 0.5 by equivalent wilcoxon test . table 3the left ventricular end - systolic volume and qol response rates for the qlv quartilesqlvnlvesv response rate ( % ) qol response rate ( % ) 070 ms12438.750.07095 ms9839.854.695120 ms10957.865.1120195 ms9568.472.0pearson <0.0010.004 the left ventricular end - systolic volume and qol response rates for the qlv quartiles qlv is expected to be longer with increased qrs duration as well as lbbb . in contrast to the relationships between qlv and qrs duration and morphology , mechanical dyssynchrony did not vary by qlv quartile ( p = 0.55 , kruskal wallis test ) . in the multivariate regression models , qlv added significant predictive value for crt responses , assessed by either reductions of lvesv or qol , after accounting for baseline covariates ( table 4 ) . patients in the highest quartile of qlv had a greater than three - fold increase in their odds of lvesv response vs. the shortest quartile . for the endpoint of a > 15% reduction in lvesv , qlv , gender and aetiology of hf were the only independent variables that were significantly associated with the response . it is noteworthy that , as expected , both qrs and lbbb were univariately associated with both lvesv response ( qrs : p = 0.04 ; lbbb : p < 0.001 ) and qol response ( qrs : p = 0.004 ; lbbb : p = 0.024 ) . table 4multivariate logistic regression model resultscovariateodds ratio ( 95% ci ) , p - valuelvesv responseqol responseqlv 2nd quartile vs. 1st quartile1.10 ( 0.621.95 ) , 0.7431.30 ( 0.752.26 ) , 0.355qlv 3rd quartile vs. 1st quartile1.86 ( 1.043.31 ) , 0.0361.86 ( 1.053.31 ) , 0.033qlv 4th quartile vs. 1st quartile3.21 ( 1.586.50 ) , 0.0012.73 ( 1.355.54 ) , 0.005age ( per 1 year increase)1.00 ( 0.981.02 ) , 0.8010.99 ( 0.971.01 ) , 0.209lvef ( per 1% increase)0.98 ( 0.941.01 ) , 0.1861.00 ( 0.961.03 ) , 0.83ischaemic vs. non - ischaemic0.58 ( 0.370.91 ) , 0.0191.05 ( 0.671.64 ) , 0.846qrs ( > 150 ms vs. 150 ms)0.86 ( 0.531.40 ) , 0.5430.88 ( 0.551.43 ) , 0.611lbbb vs. non - lbbb1.20 ( 0.722.01 ) , 0.481.17 ( 0.711.93 ) , 0.526male vs. female0.53 ( 0.330.85 ) , 0.0080.56 ( 0.340.91 ) , 0.018nyha class iv vs. i iii1.67 ( 0.446.29 ) , 0.453.41 ( 0.6916.92 ) , 0.133lvesv1.00 ( 0.991.01 ) , 0.981.00 ( 0.991.00 ) , 0.682adjusted for baseline ef , lvesv , aetiology of hf , lbbb , gender , nyha , qrs and age . multivariate logistic regression model results adjusted for baseline ef , lvesv , aetiology of hf , lbbb , gender , nyha , qrs and age . of note , none of these values differs from the larger full cohort included in the smart - av trial , other than a slightly shorter mean qrs duration in the substudy population ( 151 19 vs. 154 21 ms , p < 0.05 ) . table 1patient characteristics ( n = 426)age , years66 11gender ( male , % ) 66ischaemic heart disease ( % ) 59nyha functional class ( % ) i0 ii3 iii94 iv3lv ejection fraction ( % ) 26 7cardiac medications ( % ) ace / arb84 beta - blocker92 diuretic82 digoxin22ecg characteristics qrs duration ( ms)151 19 lbbb ( % ) 75 rbbb ( % ) 13 ivcd ( % ) 12baseline lvesv ( ml)128 62qol46 26values expressed as mean sd.ace/arb , angiotensin - converting enzyme inhibitor / receptor blocker . these programmed parameters resulted in a very high rate of lifetime biv pacing during this study with a median of 98.4% ( 95.199.6% ) , which is higher than the 92% threshold often used to insure a maximal crt response . when separated at the median qlv , patients with longer qlv intervals had shorter programmed av delays [ median ( q1q3 ) : 120 ( 110130 ) vs. 120 ( 120140 ) , p = 0.016 ] and slightly higher percentages of biv pacing [ median ( q1q3 ) : 98.8% ( 94.899.7% ) vs. 97.9% ( 95.299.3% ) , p = 0.014 ] . when separated by the qlv median value ( 95 ms ) , lvesv , lvedv , ef , and qol responses all were significantly larger for patients with long vs. short qlv ( figure 2 ) . the responses for lvesv , lvedv , ef , and qol from baseline to 6 months all significantly increased with the increase in the qlv from the shortest quartile ( < 70 ms ) to the longest quartile ( > 120 ms ) ( figure 3 ) . figure 2comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the two qlv groups separated by the median value . figure 3comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the qlv quartiles . table 2relationship of qlv with clinical outcomesqlv quartilesq1 : 070 ms ( % ) q2 : 7095 ms ( % ) q3 : 95120 ms ( % ) q4 : 120195 ms ( % ) total ( % ) overall p - valueq4 vs. q1 p - valuepatients with hf events15 ( 12.1)7 ( 7.1)7 ( 6.4)6 ( 6.3)35 ( 8.2)0.370.17nyha improved89 ( 73.0)79 ( 80.6)76 ( 71.0)77 ( 83.7)321 ( 76.6)0.040.04 no change33 ( 27.1)16 ( 16.3)30 ( 28.0)14 ( 15.2)93 ( 22.2 ) worsened0 ( 0.0)3 ( 3.1)1 ( 0.9)1 ( 1.1)5 ( 1.2)six minute walk delta52 11868 9150 10470 9359 1030.360.13 comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the two qlv groups separated by the median value . comparisons of the changes in left ventricular end - systolic volume , left ventricular end - diastolic volume , ejection fraction , and quality of life from implant baseline to 6 months for the qlv quartiles . it is noteworthy that the median value ( 95 ms ) was also the optimal cut - point for qlv based on roc analysis using the point on the curve closest to the upper left corner for both lvesv and qol . table 3the left ventricular end - systolic volume and qol response rates for the qlv quartilesqlvnlvesv response rate ( % ) qol response rate ( % ) 070 ms12438.750.07095 ms9839.854.695120 ms10957.865.1120195 ms9568.472.0pearson <0.0010.004 the left ventricular end - systolic volume and qol response rates for the qlv quartiles qlv is expected to be longer with increased qrs duration as well as lbbb . in the multivariate regression models , qlv added significant predictive value for crt responses , assessed by either reductions of lvesv or qol , after accounting for baseline covariates ( table 4 ) . patients in the highest quartile of qlv had a greater than three - fold increase in their odds of lvesv response vs. the shortest quartile . it is noteworthy that , as expected , both qrs and lbbb were univariately associated with both lvesv response ( qrs : p = 0.04 ; lbbb : p < 0.001 ) and qol response ( qrs : p = 0.004 ; lbbb : p = 0.024 ) . table 4multivariate logistic regression model resultscovariateodds ratio ( 95% ci ) , p - valuelvesv responseqol responseqlv 2nd quartile vs. 1st quartile1.10 ( 0.621.95 ) , 0.7431.30 ( 0.752.26 ) , 0.355qlv 3rd quartile vs. 1st quartile1.86 ( 1.043.31 ) , 0.0361.86 ( 1.053.31 ) , 0.033qlv 4th quartile vs. 1st quartile3.21 ( 1.586.50 ) , 0.0012.73 ( 1.355.54 ) , 0.005age ( per 1 year increase)1.00 ( 0.981.02 ) , 0.8010.99 ( 0.971.01 ) , 0.209lvef ( per 1% increase)0.98 ( 0.941.01 ) , 0.1861.00 ( 0.961.03 ) , 0.83ischaemic vs. non - ischaemic0.58 ( 0.370.91 ) , 0.0191.05 ( 0.671.64 ) , 0.846qrs ( > 150 ms vs. 150 ms)0.86 ( 0.531.40 ) , 0.5430.88 ( 0.551.43 ) , 0.611lbbb vs. non - lbbb1.20 ( 0.722.01 ) , 0.481.17 ( 0.711.93 ) , 0.526male vs. female0.53 ( 0.330.85 ) , 0.0080.56 ( 0.340.91 ) , 0.018nyha class iv vs. i iii1.67 ( 0.446.29 ) , 0.453.41 ( 0.6916.92 ) , 0.133lvesv1.00 ( 0.991.01 ) , 0.981.00 ( 0.991.00 ) , 0.682adjusted for baseline ef , lvesv , aetiology of hf , lbbb , gender , nyha , qrs and age . multivariate logistic regression model results adjusted for baseline ef , lvesv , aetiology of hf , lbbb , gender , nyha , qrs and age . longer qlv at the lv stimulation site was associated with better crt responses even after adjusting for baseline covariates , including qrs duration , bundle branch block , and aetiology of hf . whereas reverse remodelling is a good predictor of survival and clinical outcomes with crt , harder endpoints such as hospitalization and survival were not powered endpoints in the smart - av trial and the short follow - up precluded more complete assessment of hf events . whereas reverse remodelling is a good predictor of survival and clinical outcomes with crt , harder endpoints such as hospitalization and survival were not powered endpoints in the smart - av trial and the short follow - up precluded more complete assessment of hf events .

privatization of public services has been mentioned as a way to solve public sector s traditional problems such as inefficiency and lack of motives among its staff ( 13 ) . hence , preference of private over public units has been raised by many theories such as agency , property - right as well as public choice ( 47 ) , although some have argued that public sector has been attacked by false assumptions ( 8) . some governments have used private sector s mechanisms in their public sector entities under the new public management ( 9 ) . health sector , as one of whom traditionally managed by governments , has also been subject to some extent of privatization as well . even nations such as the uk with longest running public health systems has shown some interests in diluting the role of the central government at their health services especially across hospitals ( 10 ) . privatization or liberalization has resulted in improved efficiency in some nations such as the usa ( 11 ) and germany ( 12 ) . although privatization may resolve inefficiency problem in the public sector , evidence suggest that sudden and mass liberalization of public sector could lead to dysfunctional consequences , as caused a massive fiscal shock across the former soviet nations after the dissolution ( 13 ) . moreover , some governments believe that privatization of health sector would mean as shirking their duty of saving and improving public health stated and supported through 1978 alma - ata and 2004 mumbai declarations ( 14 ) . based on such evidence and believes some models especially one developed by harding and preker ( 15 ) , suggest that liberalization of hospitals could be considered as a spectrum , with some intermediate steps ( fig . 1 ) . based on this model , at the first stage of privatization public facilities would be acknowledged as autonomous units , distinguished from the budgetary units by having greater freedom at financial management , recruitment and promotion of staff . harding and preker s ( 15 ) model of organizational reform towards corporatization in iran , the economy has been mainly owned and administered by the public sector . the privatization efforts were taken more seriously after 2003 , when the government was allowed to privatize or decentralize of 80 percent of the state assets according to the article 44 of the iranian constitution . based on such a general policy , the ministry of health and medical education ( mohme ) gradually started moving toward liberalization of public university owned hospitals in 2006 ( encompassing about two - third of all 900 hospitals in iran ) , with granting autonomy to 18 hospitals . then mohme asked all medical universities - which act on behalf of the mohme in each province and are responsible for delivering health care , academic education at medical sciences and monitoring public and private healthcare organizations - to announce at least one public hospital from their catchment area as autonomous , delegating them the appropriate management and autonomy in 2009 . mohme stated the goals of the reform as ( i ) continuous quality improvement ; ( ii ) productivity improvement ; ( iii ) acceleration of health services delivery and ( iv ) increase of patient satisfaction with services across hospitals ( 16 ) . in parallel , the mohme added another incentive for hospitals switched into autonomous with increasing their annual budget compared to the regular public hospitals . this extra budget was committed by the mohme and two main basic insurance organizations in iran , social security insurance organization ( ssio ) and medical services insurance organization ( msio ) . the insurance organizations committed to pay a double bill to autonomous hospitals . in addition , the mohme committed to increase the budget from 1.2 times of the bill to 1.6 . hence , altogether the budget would increase by about 64 percent for converted autonomous hospitals while patients would pay only as equal as regular public hospital charges . in response to mohme s order , the iranian medical universities all over the country announced 36 teaching hospitals to undergo the reform and convert into the board of trustees - operated or autonomous entities , which increased the total number of the autonomous hospitals to 54 . granting autonomy to hospitals would give a wider range of freedom to the hospitals . table 1 compares a regular public hospital with an autonomous one based on the autonomy and responsibilities delegated by the government to the latter . comparison of autonomous and regular public hospitals in terms of autonomy the iranian government granted them the board of trustees includes the chancellor of the affiliating medical university ( as the head of the board ) , hospital head , an expert in management recommended by the hospital head , a representative of charitable people recommended by the province s charity society , two consultants from the hospital recommended by the hospital head , a representative recommended by the provincial governor or mayor , and two members from the basic insurance organizations . these members are assigned by the chancellor for a two - year period and can be selected again . the board meetings should be held at least every 3 months with two - thirds of the members . all approved regulations by the board should be confirmed by the chancellor to be executed . although granting autonomy to the hospitals was started by the formal announcement of 54 hospitals as autonomous , the implementation of the reform encountered some obstacles . as a result , the reform did not spread to the remaining 500 university hospitals . some news warned the failure of the reform ( 17 ) and even suggested cessation of the reform due to its poor implementation and misuse of regulations ( 18 ) . very small numbers of studies have conducted on autonomous hospitals reform in iran , among which one concluded that the necessary autonomy was not transferred to the hospitals , especially at the areas of strategic management , human and physical resources management , and governance arrangement and accountability ( 19 ) . hence , this study aimed to explore the obstacles of establishing autonomous hospitals in the iranian public health sector and to figure out how the obstacles hindered the reform . we used a qualitative approach through 2013 for our study with two phases . at the first phase , we posted a questionnaire with open - ended questions to all medical universities and all 54-university hospitals that had been granted autonomy in iran . the questionnaire was developed by the authors and its validity checked through content analysis by a group of experts . the questionnaire included three questions : ( i ) if the reform had been implemented well ; ( ii ) what the obstacles were ( if any ) ; and ( iii ) how the obstacles affected the reform . the respondents were hospital heads and hospital managers ( members of the board of trustees ) , and quality improvement officer at each hospital , plus one staff from each medical university in charge of administrative issues of the reform and some other key informants at the universities . consequently , 276 paper based questionnaires were sent with a stamped empty envelope of which 202 were returned after two rounds of telephone follow up ( response rate = 73% ) . the completed questionnaires were assessed primarily for the development of themes through thematic analysis ( 20 ) . at the second phase of data collection , we contacted selected respondents from the first phase for telephone interview . the selection of interviewees was based on the nine themes developed through the first phase . for each general theme , we contacted at least three respondents who had given answers that are more comprehensive in order to obtain in - depth information . therefore 23 telephone interviews were undertaken ( some people were interviewed for more than one theme ) by one of the authors ( nm ) . most interviewees discussed other themes beside their own theme as well because the themes were generally connected . the foci of the interview questions were : ( i ) what the exact obstacle(s ) were for implementation of the reform at the levels of mohme , university or hospital ; ( ii ) when , how , and why they occurred ; ( iii ) what the impact was . all interviews were recorded after the interviewees agreement and a verbal consent was obtained and recorded at the start of each interview . the general frame of themes was developed at the first phase , so we used framework method ( 20 ) for analysis of the transcripts . nevertheless , some themes were amended . at the final stage , we contacted some interviewees , as member checking ( 21 ) , in order to make sure that the developed meanings and findings were consistent with the interviewees original meanings . all organizations and interviewees name were kept private at all stages of the study and only the researchers could access to them . the names of medical universities and interviewees were switched to codes to avoid any accidental release of identities through the study . the themes or barriers were not at a same level and some might be at a higher degree of importance and trigger other barriers , but for greater clarity , we did not merge them and reported all at a same level . we used walt and gilson s ( 22 ) framework for analysis of health sector policies which incorporates our developed themes for structuring our findings . therefore the themes , which counts as barriers , are reported under three headings of policy content , process ( generally policy implementation ) , and context . actors or stakeholders were not reported under a separate heading but discussed through the three - abovementioned headings . nevertheless , we could not avoid overlaps and some themes may seem appropriate to be listed under other headings as well . the board composition many barriers that challenged the reform were due to the regulations set at the policy content . the main problem mentioned by some respondents was related to the members of the board of trustees . this meant that the affiliating medical university would have the ultimate power at the board , so the hospital would lack real autonomy . indeed the autonomous hospitals would probably be run through the university instead of making their policies inside the hospital . this decreases motivation among other board members , as the university is still the boss . what is our autonomy then ? another concern was mohme s negligence in appointing some key members at the board . the structure lacked an expert in budgeting . as hospitals would have greater degree of financial autonomy , the board would need an expert in budgeting to handle the financial issue of the hospital . respondents believed that the board had certain members to support and lead physicians and administrative staff at hospital , but no one for nurses . mohme s delay in announcing formal charges to autonomous hospitals unclearness about the charges and physicians share from them ( fee for service ) appeared as a serious problem at the first stages of implementation of the reform . in iran , a list of charges for services is announced annually by the mohme at the start of each year , but the mohme did not announce any special list for the autonomous hospitals , but only for the regular public ones . this caused some problems in the autonomous hospitals in paying physicians share and in contracting the insurance organizations , which were supposed to reimburse the autonomous hospitals . we contacted the mohme for the list of charges but they have not developed that . we have to charge like a regular hospital and now are in problem with our physicians fee for service . lack of financing by the committed organizations as explained in the introduction , the autonomous hospitals were supposed to be benefited from higher budgets , as an incentive to work autonomously . the main insurance organizations did not pay their appointed share ( 2 times as they paid to the regular public hospitals ) . their payment to the autonomous hospitals was delayed and then just as same they paid the regular public hospitals . moreover , the mohme also could not pay its own share - 1.6 times of the bill - to the hospitals . these caused hospitals could not pay fee for to their physicians services and ask their affiliating medical universities to assist on the delayed payments , which challenged the medical universities . what a disastrous situation for the medical universities ! poor follow up for implementation of the reform the reform lacked a leading body at the mohme to follow the execution . they just asked for announcing a hospital as autonomous and then no follow up was made by the mohme . hence , if the medical universities did not wish to establish the reform at the announced hospital , no obligation forced them to proceed . we did not feel any force from their side , so everything we did was almost on the paper . indeed some key informant even mentioned that the university chancellors typically did not wish the reform proceeds because granting autonomy to hospitals could decrease the chancellors autonomy . university chancellors say if the reform proceeds and all their hospitals get autonomy then what they would have to do ! irregular board meetings and absence of members this barrier may have been resulted partly from the abovementioned factor of lack of mohme s follow up . hospitals board of trustees should meet every three months with at least two - third of their members . however , this was not the case at most hospitals and the meetings were held less frequently and with fewer members . in some provinces , due to the members absence and irregular meetings , the affiliating medical university had to hold joint meetings for all its autonomous hospitals , which questions hospitals autonomy . obviously , certain members , especially those beyond the hospitals , did not have enough motives to attend the meetings and get involved at hospitals policymaking . lack of an external overseer ; uncertainty to proceed on the reform some interviewees believed that letting hospitals have more autonomy would dilute supervision on autonomous hospitals . public hospitals are under strict supervision of the legal auditors , especially in terms of their financial issues , but for autonomous hospitals , no external overseer was appointed . this caused some hospitals make illegal decisions , so medical university chancellors get cautious about the reform implementation that slowed the reform down even more . lack of any governmental supervision on [ autonomous hospital ] boards decisions is obvious . some for example have decided to change their wards to private wards , with no insurance coverage , which ultimately limits patients access . therefore , the chancellor that would take all responsibility should be very cautious on carrying out the reform issues . they may get reluctant shortage of full - time physicians at public hospitals the autonomous hospitals were supposed to change all their physicians to the full - time state , by changing their contracts or employing new physicians as full - time . however , this proved to be impossible for hospitals in practice . first , most physicians who worked at public hospitals , especially in big cities , were simultaneously working at private sector as well , so did not like to change to full - time at public hospitals . this was caused by lack of any serious rule to prohibit physicians from dual practice and the huge difference between private and public hospitals charges that would make private hospitals payment to their physicians much more than that of the public hospitals . second , hospitals could not cease their contract with the employed physicians , because ( i ) they were not allowed legally , and ( ii ) they could not replace physicians with new ones , due to the lack of physicians who would be happy to work full - time . moreover , employing physicians at certain specialties was not possible due to shortage of physicians in some specialties at the country . therefore , the mohme s goal of changing 100 percent of hospitals physicians to full - time ones at autonomous hospitals by 2013 would be practically impossible . who is willing to work full - time at public hospitals with such a low and delayed payments . most physicians like to have their private sector activities and work only as part - time physicians at autonomous hospitals . lack of management stability at public hospitals public hospitals usually are very prone to changes at their top levels of managerial positions and this causes difficulties in the implementation of the reform . the changes happen usually after elections or after any major change at the mohme level . in the last 4 years our hospital has had 4 different heads and 3 managers . the managers are not confident enough to undergo the reform , because they do not know whether they would stay at their position . health insurance organizations delayed payments to the public hospitals health insurance organizations are traditionally very late in paying hospital bills . two main health insurance organizations usually have a delay of at least six months , which makes autonomous hospitals more vulnerable ( 23 ) . most interviewees believed that with such a delay the autonomous hospitals could not afford their heavy human resources payments and so would not survive . although all public hospitals are paid late by the insurance organizations , autonomous hospitals are more vulnerable . the board composition many barriers that challenged the reform were due to the regulations set at the policy content . the main problem mentioned by some respondents was related to the members of the board of trustees . this meant that the affiliating medical university would have the ultimate power at the board , so the hospital would lack real autonomy . indeed the autonomous hospitals would probably be run through the university instead of making their policies inside the hospital . this decreases motivation among other board members , as the university is still the boss . what is our autonomy then ? another concern was mohme s negligence in appointing some key members at the board . the structure lacked an expert in budgeting . as hospitals would have greater degree of financial autonomy , the board would need an expert in budgeting to handle the financial issue of the hospital . respondents believed that the board had certain members to support and lead physicians and administrative staff at hospital , but no one for nurses . mohme s delay in announcing formal charges to autonomous hospitals unclearness about the charges and physicians share from them ( fee for service ) appeared as a serious problem at the first stages of implementation of the reform . in iran , a list of charges for services is announced annually by the mohme at the start of each year , but the mohme did not announce any special list for the autonomous hospitals , but only for the regular public ones . this caused some problems in the autonomous hospitals in paying physicians share and in contracting the insurance organizations , which were supposed to reimburse the autonomous hospitals . we contacted the mohme for the list of charges but they have not developed that . we have to charge like a regular hospital and now are in problem with our physicians fee for service . lack of financing by the committed organizations as explained in the introduction , the autonomous hospitals were supposed to be benefited from higher budgets , as an incentive to work autonomously . the main insurance organizations did not pay their appointed share ( 2 times as they paid to the regular public hospitals ) . their payment to the autonomous hospitals was delayed and then just as same they paid the regular public hospitals . moreover , the mohme also could not pay its own share - 1.6 times of the bill - to the hospitals . these caused hospitals could not pay fee for to their physicians services and ask their affiliating medical universities to assist on the delayed payments , which challenged the medical universities . what a disastrous situation for the medical universities ! poor follow up for implementation of the reform the reform lacked a leading body at the mohme to follow the execution . they just asked for announcing a hospital as autonomous and then no follow up was made by the mohme . hence , if the medical universities did not wish to establish the reform at the announced hospital , no obligation forced them to proceed . we did not feel any force from their side , so everything we did was almost on the paper . indeed some key informant even mentioned that the university chancellors typically did not wish the reform proceeds because granting autonomy to hospitals could decrease the chancellors autonomy . university chancellors say if the reform proceeds and all their hospitals get autonomy then what they would have to do ! irregular board meetings and absence of members this barrier may have been resulted partly from the abovementioned factor of lack of mohme s follow up . hospitals board of trustees should meet every three months with at least two - third of their members . however , this was not the case at most hospitals and the meetings were held less frequently and with fewer members . in some provinces , due to the members absence and irregular meetings , the affiliating medical university had to hold joint meetings for all its autonomous hospitals , which questions hospitals autonomy . obviously , certain members , especially those beyond the hospitals , did not have enough motives to attend the meetings and get involved at hospitals policymaking . usually no one attends on behalf of the mayor or commander . they have no motives . lack of an external overseer ; uncertainty to proceed on the reform some interviewees believed that letting hospitals have more autonomy would dilute supervision on autonomous hospitals . public hospitals are under strict supervision of the legal auditors , especially in terms of their financial issues , but for autonomous hospitals , no external overseer was appointed . this caused some hospitals make illegal decisions , so medical university chancellors get cautious about the reform implementation that slowed the reform down even more . lack of any governmental supervision on [ autonomous hospital ] boards decisions is obvious . sometimes they convince the chancellor for very dramatic changes . some for example have decided to change their wards to private wards , with no insurance coverage , which ultimately limits patients access . therefore , the chancellor that would take all responsibility should be very cautious on carrying out the reform issues . shortage of full - time physicians at public hospitals the autonomous hospitals were supposed to change all their physicians to the full - time state , by changing their contracts or employing new physicians as full - time . however , this proved to be impossible for hospitals in practice . first , most physicians who worked at public hospitals , especially in big cities , were simultaneously working at private sector as well , so did not like to change to full - time at public hospitals . this was caused by lack of any serious rule to prohibit physicians from dual practice and the huge difference between private and public hospitals charges that would make private hospitals payment to their physicians much more than that of the public hospitals . second , hospitals could not cease their contract with the employed physicians , because ( i ) they were not allowed legally , and ( ii ) they could not replace physicians with new ones , due to the lack of physicians who would be happy to work full - time . moreover , employing physicians at certain specialties was not possible due to shortage of physicians in some specialties at the country . therefore , the mohme s goal of changing 100 percent of hospitals physicians to full - time ones at autonomous hospitals by 2013 would be practically impossible . who is willing to work full - time at public hospitals with such a low and delayed payments . most physicians like to have their private sector activities and work only as part - time physicians at autonomous hospitals lack of management stability at public hospitals public hospitals usually are very prone to changes at their top levels of managerial positions and this causes difficulties in the implementation of the reform . the changes happen usually after elections or after any major change at the mohme level . in the last 4 years our hospital has had 4 different heads and 3 managers . the managers are not confident enough to undergo the reform , because they do not know whether they would stay at their position . health insurance organizations delayed payments to the public hospitals health insurance organizations are traditionally very late in paying hospital bills . two main health insurance organizations usually have a delay of at least six months , which makes autonomous hospitals more vulnerable ( 23 ) . most interviewees believed that with such a delay the autonomous hospitals could not afford their heavy human resources payments and so would not survive . although all public hospitals are paid late by the insurance organizations , autonomous hospitals are more vulnerable . in this qualitative study , we explored the obstacles that the iranian health system encountered through liberalization of the public university owned hospitals . the findings showed different obstacles , categorized in three general headings of policy content , policymaking process ( policy implementation ) and the context . as a serious obstacle categorized under policy content , composition of the autonomous hospitals board role of the university chancellor as the head of the board would mean that the autonomous hospitals could have no real freedom and their policies would be imposed by the affiliating university and the mohme ultimately . this shows the central state s concerns over the first stages of liberalization and its cautious policy in delegating responsibilities to the local health sectors . similar pattern has been seen even among developed nations such as the uk where foundation trusts , as autonomous entities , were indeed controlled by primary care trusts that could dictate the central government s priorities ( 24 ) . nevertheless , the whole reform was not exhausted in the uk ( 25 ) and most developed nations such as germany ( 12 ) . although one may argue that the chancellor is only one of the members and he / she would not necessarily impose the university s policies on the hospital board , sensitivity to the fact that the chancellor is the head and has the ultimate power would decrease inspiration among hospital - based members . moreover , chancellors concern about decrease of their authority and power probably has slowed down the process of accomplishment of the reform . furthermore , the university would not charge a 5% overhead cost from autonomous hospitals , which again counts as a competing incentive against the reform . therefore , there might be some degree of veiled resistance against the reform at the iranian medical universities and even the ministry level . although resistance against liberalization of public hospitals is not unprecedented even among less - centralized health systems such as germany s due to the nature of health that should not be treated such as a commodity ( 26 ) , the resistance in iran was due to a different reason ; losing power among some authorities . at the implementation stage , the reform encountered various barriers that made the establishment of real autonomous hospitals almost impossible in iran . this was probably because the ministry knew that it could not afford autonomous hospitals bills ( 27 ) , and/or the insurance organizations had warned the mohme they could not afford the costs , although they had agreed the reform . hence , the charges were announced late and then neither the mohme nor the insurance organizations paid hospitals the amounts they had guaranteed to pay . the mohme could pay just partially and the insurance organizations paid the autonomous hospitals just as same as regular public hospitals . we had no finding about why the insurance organizations did not pay their committed payments to autonomous hospitals while they had agreed this in advance , but a possible explanation could be that the basic insurance organizations had experienced many changes after signing the agreement and their heads did not and could not pay what their predecessors had committed . moreover , it is possible that the negotiations between the mohme and insurance organizations had conducted only at their supreme levels , not tailored first among their experts and middle level managers . the delayed or reduced funding when governments decentralize their facilities and delegate some authority has been reported in similar reforms such as the indonesian health sector reform ( 28 ) . poor follow up from the mohme part was obvious and so the medical universities did not take the implementation serious . we did not examine directly why this was not taken serious by asking direct questions , because we could not access the mohme s top managers . however , our other findings might answer this ; the mohme could not afford higher costs of the autonomous hospitals at least at short term . therefore hospital boards would not have enough motives to hold their meetings regularly due to lack of receiving committed budgets form the mohme and insurance organizations . moreover the board s head , the university chancellor , was reluctant to lead the reform because the reform would limit his / her power , both in terms of managerial authority and financial , due to missing a 5% university share of hospitals revenue . moreover , lack of any external overseer and certain ambiguities in rules and regulations would make the chancellor very cautious in implementation of the reform because he / she would have to take all the responsibilities for the consequences . lack of any regulation against physicians mobility between public and private hospitals and physicians natural and traditional interest in working at both sectors hindered the reform . the public sector s safety and private income would shape a model of dual practice that is obvious in the iranian health sector although it could be seen across many other nations ( 29 ) . therefore , hospitals could not change all their clinicians into full - time ones as the reform required . moreover , at some rare specialties changing physicians to full - time would not be appropriate as this can limit patients access . lack of managerial stability at most public organizations in iran , seen at hospitals as well , was the other contextual barrier , which slowed down the reform . most managers were not sure if they could establish the reform at their hospitals during their mission at hospitals and so did not proceed on reforms . even where the autonomous hospitals reform was starting to proceed , it restarted after new managers came . lack of managerial stability through insurance organizations , discussed earlier , also would probably have obstructed working of the reform , by delayed and decreased payments to the hospitals . our first phase of study included all iranian medical universities and all autonomous hospitals , to catch their views on obstacles and barriers of the reform . by this comprehensive sample , we potentially have all barriers and experiences of the reform across all possible locations with their specific context . nevertheless , we could not interview the mohme authorities who knew policy issues at the highest level of the health sector . the findings had some similarities and differences with those of at different countries where the world bank s autonomy hospitals reform was established . in pakistan , the reform was proved very political and not straightforward ( 30 ) . it was run generally based on the donors will rather than the government s own , while in iran no external will obstructed the reform . in pakistan , street bureaucrats as was in iran where insurance organizations , university chancellors and physicians did this . in south - east asia , the reform has proceeded in philippines , indonesia and vietnam but no significant transformation at their public stewardship has occurred ( 31 ) , and the reform could not improve efficiency and quality of care ( 32 ) . in iran , no study has examined the impact of the reform on hospitals clinical indicators . the story was a bit different where the autonomous hospitals were implemented at developed countries . foundation trusts are good example of successful liberalization of public hospitals in order to increase business - like practices ( 25 ) . liberalization of public hospitals , especially in the settings with a dominant public sector , would be a political reform rather than just a managerial delegation . we saw that the iranian reform was not supported very well by the mohme as the main policy - maker and its introducer . the mohme should have taken into account all its capacities and thought about all necessary tools , such as lobbying the key stakeholders or establishing necessary regulations ( say for dual practice ) , and then decide to implement the reform , or at the other case , cease it totally . the iranian health insurance organizations as the key stakeholders and the most influential bodies in financing the autonomous hospitals did not stand on their commitment , probably because the mohme s initial agreement with them was poor and could not satisfy their benefits . more detail policy formulation issues , such as revising the board structure , should have been approved by all direct and indirect stakeholders . in iran , the board should not be dominated by the affiliating university , but by the local authorities and the hospital if the reform is really going to be implemented . instead the reform was agreed by the mohme and insurance organizations , however when it was the insurance organizations turn to pay the hospitals based on the agreed amount , they denied their commitment . the reasons behind such behavior are not clear for us and so could be investigated through further research . our first phase of study included all iranian medical universities and all autonomous hospitals , to catch their views on obstacles and barriers of the reform . by this comprehensive sample , we potentially have all barriers and experiences of the reform across all possible locations with their specific context . nevertheless , we could not interview the mohme authorities who knew policy issues at the highest level of the health sector . the findings had some similarities and differences with those of at different countries where the world bank s autonomy hospitals reform was established . in pakistan , it was run generally based on the donors will rather than the government s own , while in iran no external will obstructed the reform . in pakistan , also the reform was altered or obstructed by street bureaucrats as was in iran where insurance organizations , university chancellors and physicians did this . in south - east asia , the reform has proceeded in philippines , indonesia and vietnam but no significant transformation at their public stewardship has occurred ( 31 ) , and the reform could not improve efficiency and quality of care ( 32 ) . in iran , no study has examined the impact of the reform on hospitals clinical indicators . the story was a bit different where the autonomous hospitals were implemented at developed countries . uk s foundation trusts are good example of successful liberalization of public hospitals in order to increase business - like practices ( 25 ) . liberalization of public hospitals , especially in the settings with a dominant public sector , would be a political reform rather than just a managerial delegation . we saw that the iranian reform was not supported very well by the mohme as the main policy - maker and its introducer . the mohme should have taken into account all its capacities and thought about all necessary tools , such as lobbying the key stakeholders or establishing necessary regulations ( say for dual practice ) , and then decide to implement the reform , or at the other case , cease it totally . the iranian health insurance organizations as the key stakeholders and the most influential bodies in financing the autonomous hospitals did not stand on their commitment , probably because the mohme s initial agreement with them was poor and could not satisfy their benefits . more detail policy formulation issues , such as revising the board structure , should have been approved by all direct and indirect stakeholders . in iran , the board should not be dominated by the affiliating university , but by the local authorities and the hospital if the reform is really going to be implemented . instead the reform was agreed by the mohme and insurance organizations , however when it was the insurance organizations turn to pay the hospitals based on the agreed amount , they denied their commitment . the reasons behind such behavior are not clear for us and so could be investigated through further research . the findings had some similarities and differences with those of at different countries where the world bank s autonomy hospitals reform was established . in pakistan , the reform was proved very political and not straightforward ( 30 ) . it was run generally based on the donors will rather than the government s own , while in iran no external will obstructed the reform . in pakistan , also the reform was altered or obstructed by street bureaucrats as was in iran where insurance organizations , university chancellors and physicians did this . in south - east asia , the reform has proceeded in philippines , indonesia and vietnam but no significant transformation at their public stewardship has occurred ( 31 ) , and the reform could not improve efficiency and quality of care ( 32 ) . in iran , no study has examined the impact of the reform on hospitals clinical indicators . the story was a bit different where the autonomous hospitals were implemented at developed countries . uk s foundation trusts are good example of successful liberalization of public hospitals in order to increase business - like practices ( 25 ) . liberalization of public hospitals , especially in the settings with a dominant public sector , would be a political reform rather than just a managerial delegation . we saw that the iranian reform was not supported very well by the mohme as the main policy - maker and its introducer . the mohme should have taken into account all its capacities and thought about all necessary tools , such as lobbying the key stakeholders or establishing necessary regulations ( say for dual practice ) , and then decide to implement the reform , or at the other case , cease it totally . the iranian health insurance organizations as the key stakeholders and the most influential bodies in financing the autonomous hospitals did not stand on their commitment , probably because the mohme s initial agreement with them was poor and could not satisfy their benefits . more detail policy formulation issues , such as revising the board structure , should have been approved by all direct and indirect stakeholders . in iran , the board should not be dominated by the affiliating university , but by the local authorities and the hospital if the reform is really going to be implemented . instead the reform was agreed by the mohme and insurance organizations , however when it was the insurance organizations turn to pay the hospitals based on the agreed amount , they denied their commitment . the reasons behind such behavior are not clear for us and so could be investigated through further research . the iranian autonomous hospitals reform , as a major public sector liberalization attempts , was challenged by different factors such as poor policy formulation where the potential stakeholders interest were not met . even the policymakers at the top levels of the mohme and the key stake - holders seem to have reached no consensus on the implementation and have moved back from their commitments . therefore , the medical universities and hospitals were struggled at execution of the reform . all our findings bring us to this conclusion that the stages of policy formulation and policy implementation were performed separately in iran and were not thought simultaneously and in common with those who formulate a policy and those who have to implement and execute it . in addition , the contextual factors , such as prevalent dual practice among physicians , were not considered by the policy makers in order to alter the policy accordingly . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .

background : liberalization and decentralization of public sector has been triggered in some developing countries and in iran by the ministry of health and medical education ( mohme ) that granted autonomy to 54 public hospitals . however , establishment of such a complex organizational reform was rather unsuccessful . we aimed to explore the obstacles and barriers caused such a failure and their mechanisms.methods:using a qualitative approach in 2013 , we consulted key informants at the autonomous hospitals and their affiliating universities . data collection was done within two phases : ( i ) 276 unstructured questionnaires asking respondents of barriers , and ( ii ) 23 semi - structured interviews from the first phase s key respondents . the first phase data were analyzed using thematic analysis and the second s by framework approach based on the frame shaped at the first phase.results:nine obstacles were recognized including autonomous hospitals board composition , delay in announcing autonomous hospitals charges by the mohme , lack of financing by the committed organizations , poor follow up for implementation of the reform , irregular board meetings , lack of an external overseer , shortage of full - time physicians , lack of management stability , and health insurance organizations delayed payments.conclusion:the mohme and insurance organizations did not pay the reform expenses . there were some competing motives as well to slow the reform or to shut it down . the stages of policy formulation and implementation were done separately in iran , so this big organizational reform encountered serious obstacles .

Introduction Materials & Methods Results Policy Content Process (Policy implementation) Context (Iranian health Sector inherent problems) Discussion Strengths/Limitations Comparison with other reforms Policy and research implications Conclusion Ethical considerations

privatization of public services has been mentioned as a way to solve public sector s traditional problems such as inefficiency and lack of motives among its staff ( 13 ) . hence , preference of private over public units has been raised by many theories such as agency , property - right as well as public choice ( 47 ) , although some have argued that public sector has been attacked by false assumptions ( 8) . based on this model , at the first stage of privatization public facilities would be acknowledged as autonomous units , distinguished from the budgetary units by having greater freedom at financial management , recruitment and promotion of staff . harding and preker s ( 15 ) model of organizational reform towards corporatization in iran , the economy has been mainly owned and administered by the public sector . based on such a general policy , the ministry of health and medical education ( mohme ) gradually started moving toward liberalization of public university owned hospitals in 2006 ( encompassing about two - third of all 900 hospitals in iran ) , with granting autonomy to 18 hospitals . mohme stated the goals of the reform as ( i ) continuous quality improvement ; ( ii ) productivity improvement ; ( iii ) acceleration of health services delivery and ( iv ) increase of patient satisfaction with services across hospitals ( 16 ) . this extra budget was committed by the mohme and two main basic insurance organizations in iran , social security insurance organization ( ssio ) and medical services insurance organization ( msio ) . in response to mohme s order , the iranian medical universities all over the country announced 36 teaching hospitals to undergo the reform and convert into the board of trustees - operated or autonomous entities , which increased the total number of the autonomous hospitals to 54 . table 1 compares a regular public hospital with an autonomous one based on the autonomy and responsibilities delegated by the government to the latter . comparison of autonomous and regular public hospitals in terms of autonomy the iranian government granted them the board of trustees includes the chancellor of the affiliating medical university ( as the head of the board ) , hospital head , an expert in management recommended by the hospital head , a representative of charitable people recommended by the province s charity society , two consultants from the hospital recommended by the hospital head , a representative recommended by the provincial governor or mayor , and two members from the basic insurance organizations . although granting autonomy to the hospitals was started by the formal announcement of 54 hospitals as autonomous , the implementation of the reform encountered some obstacles . very small numbers of studies have conducted on autonomous hospitals reform in iran , among which one concluded that the necessary autonomy was not transferred to the hospitals , especially at the areas of strategic management , human and physical resources management , and governance arrangement and accountability ( 19 ) . hence , this study aimed to explore the obstacles of establishing autonomous hospitals in the iranian public health sector and to figure out how the obstacles hindered the reform . at the first phase , we posted a questionnaire with open - ended questions to all medical universities and all 54-university hospitals that had been granted autonomy in iran . the questionnaire included three questions : ( i ) if the reform had been implemented well ; ( ii ) what the obstacles were ( if any ) ; and ( iii ) how the obstacles affected the reform . the respondents were hospital heads and hospital managers ( members of the board of trustees ) , and quality improvement officer at each hospital , plus one staff from each medical university in charge of administrative issues of the reform and some other key informants at the universities . at the second phase of data collection , we contacted selected respondents from the first phase for telephone interview . the selection of interviewees was based on the nine themes developed through the first phase . the foci of the interview questions were : ( i ) what the exact obstacle(s ) were for implementation of the reform at the levels of mohme , university or hospital ; ( ii ) when , how , and why they occurred ; ( iii ) what the impact was . the general frame of themes was developed at the first phase , so we used framework method ( 20 ) for analysis of the transcripts . the themes or barriers were not at a same level and some might be at a higher degree of importance and trigger other barriers , but for greater clarity , we did not merge them and reported all at a same level . therefore the themes , which counts as barriers , are reported under three headings of policy content , process ( generally policy implementation ) , and context . mohme s delay in announcing formal charges to autonomous hospitals unclearness about the charges and physicians share from them ( fee for service ) appeared as a serious problem at the first stages of implementation of the reform . in iran , a list of charges for services is announced annually by the mohme at the start of each year , but the mohme did not announce any special list for the autonomous hospitals , but only for the regular public ones . this caused some problems in the autonomous hospitals in paying physicians share and in contracting the insurance organizations , which were supposed to reimburse the autonomous hospitals . lack of financing by the committed organizations as explained in the introduction , the autonomous hospitals were supposed to be benefited from higher budgets , as an incentive to work autonomously . the main insurance organizations did not pay their appointed share ( 2 times as they paid to the regular public hospitals ) . these caused hospitals could not pay fee for to their physicians services and ask their affiliating medical universities to assist on the delayed payments , which challenged the medical universities . poor follow up for implementation of the reform the reform lacked a leading body at the mohme to follow the execution . they just asked for announcing a hospital as autonomous and then no follow up was made by the mohme . irregular board meetings and absence of members this barrier may have been resulted partly from the abovementioned factor of lack of mohme s follow up . however , this was not the case at most hospitals and the meetings were held less frequently and with fewer members . lack of an external overseer ; uncertainty to proceed on the reform some interviewees believed that letting hospitals have more autonomy would dilute supervision on autonomous hospitals . public hospitals are under strict supervision of the legal auditors , especially in terms of their financial issues , but for autonomous hospitals , no external overseer was appointed . they may get reluctant shortage of full - time physicians at public hospitals the autonomous hospitals were supposed to change all their physicians to the full - time state , by changing their contracts or employing new physicians as full - time . first , most physicians who worked at public hospitals , especially in big cities , were simultaneously working at private sector as well , so did not like to change to full - time at public hospitals . this was caused by lack of any serious rule to prohibit physicians from dual practice and the huge difference between private and public hospitals charges that would make private hospitals payment to their physicians much more than that of the public hospitals . second , hospitals could not cease their contract with the employed physicians , because ( i ) they were not allowed legally , and ( ii ) they could not replace physicians with new ones , due to the lack of physicians who would be happy to work full - time . therefore , the mohme s goal of changing 100 percent of hospitals physicians to full - time ones at autonomous hospitals by 2013 would be practically impossible . who is willing to work full - time at public hospitals with such a low and delayed payments . most physicians like to have their private sector activities and work only as part - time physicians at autonomous hospitals . lack of management stability at public hospitals public hospitals usually are very prone to changes at their top levels of managerial positions and this causes difficulties in the implementation of the reform . health insurance organizations delayed payments to the public hospitals health insurance organizations are traditionally very late in paying hospital bills . although all public hospitals are paid late by the insurance organizations , autonomous hospitals are more vulnerable . the board composition many barriers that challenged the reform were due to the regulations set at the policy content . mohme s delay in announcing formal charges to autonomous hospitals unclearness about the charges and physicians share from them ( fee for service ) appeared as a serious problem at the first stages of implementation of the reform . in iran , a list of charges for services is announced annually by the mohme at the start of each year , but the mohme did not announce any special list for the autonomous hospitals , but only for the regular public ones . this caused some problems in the autonomous hospitals in paying physicians share and in contracting the insurance organizations , which were supposed to reimburse the autonomous hospitals . lack of financing by the committed organizations as explained in the introduction , the autonomous hospitals were supposed to be benefited from higher budgets , as an incentive to work autonomously . the main insurance organizations did not pay their appointed share ( 2 times as they paid to the regular public hospitals ) . their payment to the autonomous hospitals was delayed and then just as same they paid the regular public hospitals . moreover , the mohme also could not pay its own share - 1.6 times of the bill - to the hospitals . these caused hospitals could not pay fee for to their physicians services and ask their affiliating medical universities to assist on the delayed payments , which challenged the medical universities . poor follow up for implementation of the reform the reform lacked a leading body at the mohme to follow the execution . indeed some key informant even mentioned that the university chancellors typically did not wish the reform proceeds because granting autonomy to hospitals could decrease the chancellors autonomy . irregular board meetings and absence of members this barrier may have been resulted partly from the abovementioned factor of lack of mohme s follow up . in some provinces , due to the members absence and irregular meetings , the affiliating medical university had to hold joint meetings for all its autonomous hospitals , which questions hospitals autonomy . lack of an external overseer ; uncertainty to proceed on the reform some interviewees believed that letting hospitals have more autonomy would dilute supervision on autonomous hospitals . public hospitals are under strict supervision of the legal auditors , especially in terms of their financial issues , but for autonomous hospitals , no external overseer was appointed . shortage of full - time physicians at public hospitals the autonomous hospitals were supposed to change all their physicians to the full - time state , by changing their contracts or employing new physicians as full - time . first , most physicians who worked at public hospitals , especially in big cities , were simultaneously working at private sector as well , so did not like to change to full - time at public hospitals . this was caused by lack of any serious rule to prohibit physicians from dual practice and the huge difference between private and public hospitals charges that would make private hospitals payment to their physicians much more than that of the public hospitals . second , hospitals could not cease their contract with the employed physicians , because ( i ) they were not allowed legally , and ( ii ) they could not replace physicians with new ones , due to the lack of physicians who would be happy to work full - time . therefore , the mohme s goal of changing 100 percent of hospitals physicians to full - time ones at autonomous hospitals by 2013 would be practically impossible . who is willing to work full - time at public hospitals with such a low and delayed payments . most physicians like to have their private sector activities and work only as part - time physicians at autonomous hospitals lack of management stability at public hospitals public hospitals usually are very prone to changes at their top levels of managerial positions and this causes difficulties in the implementation of the reform . health insurance organizations delayed payments to the public hospitals health insurance organizations are traditionally very late in paying hospital bills . although all public hospitals are paid late by the insurance organizations , autonomous hospitals are more vulnerable . as a serious obstacle categorized under policy content , composition of the autonomous hospitals board role of the university chancellor as the head of the board would mean that the autonomous hospitals could have no real freedom and their policies would be imposed by the affiliating university and the mohme ultimately . although resistance against liberalization of public hospitals is not unprecedented even among less - centralized health systems such as germany s due to the nature of health that should not be treated such as a commodity ( 26 ) , the resistance in iran was due to a different reason ; losing power among some authorities . at the implementation stage , the reform encountered various barriers that made the establishment of real autonomous hospitals almost impossible in iran . this was probably because the ministry knew that it could not afford autonomous hospitals bills ( 27 ) , and/or the insurance organizations had warned the mohme they could not afford the costs , although they had agreed the reform . the mohme could pay just partially and the insurance organizations paid the autonomous hospitals just as same as regular public hospitals . we had no finding about why the insurance organizations did not pay their committed payments to autonomous hospitals while they had agreed this in advance , but a possible explanation could be that the basic insurance organizations had experienced many changes after signing the agreement and their heads did not and could not pay what their predecessors had committed . moreover , it is possible that the negotiations between the mohme and insurance organizations had conducted only at their supreme levels , not tailored first among their experts and middle level managers . poor follow up from the mohme part was obvious and so the medical universities did not take the implementation serious . however , our other findings might answer this ; the mohme could not afford higher costs of the autonomous hospitals at least at short term . therefore hospital boards would not have enough motives to hold their meetings regularly due to lack of receiving committed budgets form the mohme and insurance organizations . moreover , lack of any external overseer and certain ambiguities in rules and regulations would make the chancellor very cautious in implementation of the reform because he / she would have to take all the responsibilities for the consequences . lack of any regulation against physicians mobility between public and private hospitals and physicians natural and traditional interest in working at both sectors hindered the reform . therefore , hospitals could not change all their clinicians into full - time ones as the reform required . lack of managerial stability at most public organizations in iran , seen at hospitals as well , was the other contextual barrier , which slowed down the reform . lack of managerial stability through insurance organizations , discussed earlier , also would probably have obstructed working of the reform , by delayed and decreased payments to the hospitals . our first phase of study included all iranian medical universities and all autonomous hospitals , to catch their views on obstacles and barriers of the reform . by this comprehensive sample , we potentially have all barriers and experiences of the reform across all possible locations with their specific context . nevertheless , we could not interview the mohme authorities who knew policy issues at the highest level of the health sector . it was run generally based on the donors will rather than the government s own , while in iran no external will obstructed the reform . in pakistan , street bureaucrats as was in iran where insurance organizations , university chancellors and physicians did this . in iran , no study has examined the impact of the reform on hospitals clinical indicators . we saw that the iranian reform was not supported very well by the mohme as the main policy - maker and its introducer . the mohme should have taken into account all its capacities and thought about all necessary tools , such as lobbying the key stakeholders or establishing necessary regulations ( say for dual practice ) , and then decide to implement the reform , or at the other case , cease it totally . the iranian health insurance organizations as the key stakeholders and the most influential bodies in financing the autonomous hospitals did not stand on their commitment , probably because the mohme s initial agreement with them was poor and could not satisfy their benefits . in iran , the board should not be dominated by the affiliating university , but by the local authorities and the hospital if the reform is really going to be implemented . instead the reform was agreed by the mohme and insurance organizations , however when it was the insurance organizations turn to pay the hospitals based on the agreed amount , they denied their commitment . our first phase of study included all iranian medical universities and all autonomous hospitals , to catch their views on obstacles and barriers of the reform . by this comprehensive sample , we potentially have all barriers and experiences of the reform across all possible locations with their specific context . nevertheless , we could not interview the mohme authorities who knew policy issues at the highest level of the health sector . in pakistan , it was run generally based on the donors will rather than the government s own , while in iran no external will obstructed the reform . in pakistan , also the reform was altered or obstructed by street bureaucrats as was in iran where insurance organizations , university chancellors and physicians did this . in south - east asia , the reform has proceeded in philippines , indonesia and vietnam but no significant transformation at their public stewardship has occurred ( 31 ) , and the reform could not improve efficiency and quality of care ( 32 ) . in iran , no study has examined the impact of the reform on hospitals clinical indicators . we saw that the iranian reform was not supported very well by the mohme as the main policy - maker and its introducer . the mohme should have taken into account all its capacities and thought about all necessary tools , such as lobbying the key stakeholders or establishing necessary regulations ( say for dual practice ) , and then decide to implement the reform , or at the other case , cease it totally . the iranian health insurance organizations as the key stakeholders and the most influential bodies in financing the autonomous hospitals did not stand on their commitment , probably because the mohme s initial agreement with them was poor and could not satisfy their benefits . in iran , the board should not be dominated by the affiliating university , but by the local authorities and the hospital if the reform is really going to be implemented . instead the reform was agreed by the mohme and insurance organizations , however when it was the insurance organizations turn to pay the hospitals based on the agreed amount , they denied their commitment . it was run generally based on the donors will rather than the government s own , while in iran no external will obstructed the reform . in pakistan , also the reform was altered or obstructed by street bureaucrats as was in iran where insurance organizations , university chancellors and physicians did this . in iran , no study has examined the impact of the reform on hospitals clinical indicators . liberalization of public hospitals , especially in the settings with a dominant public sector , would be a political reform rather than just a managerial delegation . we saw that the iranian reform was not supported very well by the mohme as the main policy - maker and its introducer . the mohme should have taken into account all its capacities and thought about all necessary tools , such as lobbying the key stakeholders or establishing necessary regulations ( say for dual practice ) , and then decide to implement the reform , or at the other case , cease it totally . the iranian health insurance organizations as the key stakeholders and the most influential bodies in financing the autonomous hospitals did not stand on their commitment , probably because the mohme s initial agreement with them was poor and could not satisfy their benefits . in iran , the board should not be dominated by the affiliating university , but by the local authorities and the hospital if the reform is really going to be implemented . instead the reform was agreed by the mohme and insurance organizations , however when it was the insurance organizations turn to pay the hospitals based on the agreed amount , they denied their commitment . the iranian autonomous hospitals reform , as a major public sector liberalization attempts , was challenged by different factors such as poor policy formulation where the potential stakeholders interest were not met . even the policymakers at the top levels of the mohme and the key stake - holders seem to have reached no consensus on the implementation and have moved back from their commitments . all our findings bring us to this conclusion that the stages of policy formulation and policy implementation were performed separately in iran and were not thought simultaneously and in common with those who formulate a policy and those who have to implement and execute it .

hence , preference of private over public units has been raised by many theories such as agency , property - right as well as public choice ( 47 ) , although some have argued that public sector has been attacked by false assumptions ( 8) . some governments have used private sector s mechanisms in their public sector entities under the new public management ( 9 ) . health sector , as one of whom traditionally managed by governments , has also been subject to some extent of privatization as well . even nations such as the uk with longest running public health systems has shown some interests in diluting the role of the central government at their health services especially across hospitals ( 10 ) . although privatization may resolve inefficiency problem in the public sector , evidence suggest that sudden and mass liberalization of public sector could lead to dysfunctional consequences , as caused a massive fiscal shock across the former soviet nations after the dissolution ( 13 ) . moreover , some governments believe that privatization of health sector would mean as shirking their duty of saving and improving public health stated and supported through 1978 alma - ata and 2004 mumbai declarations ( 14 ) . based on such evidence and believes some models especially one developed by harding and preker ( 15 ) , suggest that liberalization of hospitals could be considered as a spectrum , with some intermediate steps ( fig . based on this model , at the first stage of privatization public facilities would be acknowledged as autonomous units , distinguished from the budgetary units by having greater freedom at financial management , recruitment and promotion of staff . harding and preker s ( 15 ) model of organizational reform towards corporatization in iran , the economy has been mainly owned and administered by the public sector . the privatization efforts were taken more seriously after 2003 , when the government was allowed to privatize or decentralize of 80 percent of the state assets according to the article 44 of the iranian constitution . based on such a general policy , the ministry of health and medical education ( mohme ) gradually started moving toward liberalization of public university owned hospitals in 2006 ( encompassing about two - third of all 900 hospitals in iran ) , with granting autonomy to 18 hospitals . then mohme asked all medical universities - which act on behalf of the mohme in each province and are responsible for delivering health care , academic education at medical sciences and monitoring public and private healthcare organizations - to announce at least one public hospital from their catchment area as autonomous , delegating them the appropriate management and autonomy in 2009 . mohme stated the goals of the reform as ( i ) continuous quality improvement ; ( ii ) productivity improvement ; ( iii ) acceleration of health services delivery and ( iv ) increase of patient satisfaction with services across hospitals ( 16 ) . in parallel , the mohme added another incentive for hospitals switched into autonomous with increasing their annual budget compared to the regular public hospitals . this extra budget was committed by the mohme and two main basic insurance organizations in iran , social security insurance organization ( ssio ) and medical services insurance organization ( msio ) . in addition , the mohme committed to increase the budget from 1.2 times of the bill to 1.6 . in response to mohme s order , the iranian medical universities all over the country announced 36 teaching hospitals to undergo the reform and convert into the board of trustees - operated or autonomous entities , which increased the total number of the autonomous hospitals to 54 . table 1 compares a regular public hospital with an autonomous one based on the autonomy and responsibilities delegated by the government to the latter . comparison of autonomous and regular public hospitals in terms of autonomy the iranian government granted them the board of trustees includes the chancellor of the affiliating medical university ( as the head of the board ) , hospital head , an expert in management recommended by the hospital head , a representative of charitable people recommended by the province s charity society , two consultants from the hospital recommended by the hospital head , a representative recommended by the provincial governor or mayor , and two members from the basic insurance organizations . these members are assigned by the chancellor for a two - year period and can be selected again . although granting autonomy to the hospitals was started by the formal announcement of 54 hospitals as autonomous , the implementation of the reform encountered some obstacles . some news warned the failure of the reform ( 17 ) and even suggested cessation of the reform due to its poor implementation and misuse of regulations ( 18 ) . very small numbers of studies have conducted on autonomous hospitals reform in iran , among which one concluded that the necessary autonomy was not transferred to the hospitals , especially at the areas of strategic management , human and physical resources management , and governance arrangement and accountability ( 19 ) . hence , this study aimed to explore the obstacles of establishing autonomous hospitals in the iranian public health sector and to figure out how the obstacles hindered the reform . at the first phase , we posted a questionnaire with open - ended questions to all medical universities and all 54-university hospitals that had been granted autonomy in iran . the questionnaire included three questions : ( i ) if the reform had been implemented well ; ( ii ) what the obstacles were ( if any ) ; and ( iii ) how the obstacles affected the reform . the respondents were hospital heads and hospital managers ( members of the board of trustees ) , and quality improvement officer at each hospital , plus one staff from each medical university in charge of administrative issues of the reform and some other key informants at the universities . consequently , 276 paper based questionnaires were sent with a stamped empty envelope of which 202 were returned after two rounds of telephone follow up ( response rate = 73% ) . at the second phase of data collection , we contacted selected respondents from the first phase for telephone interview . the selection of interviewees was based on the nine themes developed through the first phase . for each general theme , we contacted at least three respondents who had given answers that are more comprehensive in order to obtain in - depth information . therefore 23 telephone interviews were undertaken ( some people were interviewed for more than one theme ) by one of the authors ( nm ) . the foci of the interview questions were : ( i ) what the exact obstacle(s ) were for implementation of the reform at the levels of mohme , university or hospital ; ( ii ) when , how , and why they occurred ; ( iii ) what the impact was . all interviews were recorded after the interviewees agreement and a verbal consent was obtained and recorded at the start of each interview . the general frame of themes was developed at the first phase , so we used framework method ( 20 ) for analysis of the transcripts . at the final stage , we contacted some interviewees , as member checking ( 21 ) , in order to make sure that the developed meanings and findings were consistent with the interviewees original meanings . all organizations and interviewees name were kept private at all stages of the study and only the researchers could access to them . the themes or barriers were not at a same level and some might be at a higher degree of importance and trigger other barriers , but for greater clarity , we did not merge them and reported all at a same level . we used walt and gilson s ( 22 ) framework for analysis of health sector policies which incorporates our developed themes for structuring our findings . therefore the themes , which counts as barriers , are reported under three headings of policy content , process ( generally policy implementation ) , and context . the board composition many barriers that challenged the reform were due to the regulations set at the policy content . mohme s delay in announcing formal charges to autonomous hospitals unclearness about the charges and physicians share from them ( fee for service ) appeared as a serious problem at the first stages of implementation of the reform . in iran , a list of charges for services is announced annually by the mohme at the start of each year , but the mohme did not announce any special list for the autonomous hospitals , but only for the regular public ones . this caused some problems in the autonomous hospitals in paying physicians share and in contracting the insurance organizations , which were supposed to reimburse the autonomous hospitals . we contacted the mohme for the list of charges but they have not developed that . lack of financing by the committed organizations as explained in the introduction , the autonomous hospitals were supposed to be benefited from higher budgets , as an incentive to work autonomously . moreover , the mohme also could not pay its own share - 1.6 times of the bill - to the hospitals . poor follow up for implementation of the reform the reform lacked a leading body at the mohme to follow the execution . hence , if the medical universities did not wish to establish the reform at the announced hospital , no obligation forced them to proceed . irregular board meetings and absence of members this barrier may have been resulted partly from the abovementioned factor of lack of mohme s follow up . however , this was not the case at most hospitals and the meetings were held less frequently and with fewer members . in some provinces , due to the members absence and irregular meetings , the affiliating medical university had to hold joint meetings for all its autonomous hospitals , which questions hospitals autonomy . obviously , certain members , especially those beyond the hospitals , did not have enough motives to attend the meetings and get involved at hospitals policymaking . public hospitals are under strict supervision of the legal auditors , especially in terms of their financial issues , but for autonomous hospitals , no external overseer was appointed . they may get reluctant shortage of full - time physicians at public hospitals the autonomous hospitals were supposed to change all their physicians to the full - time state , by changing their contracts or employing new physicians as full - time . first , most physicians who worked at public hospitals , especially in big cities , were simultaneously working at private sector as well , so did not like to change to full - time at public hospitals . this was caused by lack of any serious rule to prohibit physicians from dual practice and the huge difference between private and public hospitals charges that would make private hospitals payment to their physicians much more than that of the public hospitals . second , hospitals could not cease their contract with the employed physicians , because ( i ) they were not allowed legally , and ( ii ) they could not replace physicians with new ones , due to the lack of physicians who would be happy to work full - time . moreover , employing physicians at certain specialties was not possible due to shortage of physicians in some specialties at the country . therefore , the mohme s goal of changing 100 percent of hospitals physicians to full - time ones at autonomous hospitals by 2013 would be practically impossible . lack of management stability at public hospitals public hospitals usually are very prone to changes at their top levels of managerial positions and this causes difficulties in the implementation of the reform . the board composition many barriers that challenged the reform were due to the regulations set at the policy content . mohme s delay in announcing formal charges to autonomous hospitals unclearness about the charges and physicians share from them ( fee for service ) appeared as a serious problem at the first stages of implementation of the reform . in iran , a list of charges for services is announced annually by the mohme at the start of each year , but the mohme did not announce any special list for the autonomous hospitals , but only for the regular public ones . this caused some problems in the autonomous hospitals in paying physicians share and in contracting the insurance organizations , which were supposed to reimburse the autonomous hospitals . we contacted the mohme for the list of charges but they have not developed that . lack of financing by the committed organizations as explained in the introduction , the autonomous hospitals were supposed to be benefited from higher budgets , as an incentive to work autonomously . moreover , the mohme also could not pay its own share - 1.6 times of the bill - to the hospitals . poor follow up for implementation of the reform the reform lacked a leading body at the mohme to follow the execution . hence , if the medical universities did not wish to establish the reform at the announced hospital , no obligation forced them to proceed . irregular board meetings and absence of members this barrier may have been resulted partly from the abovementioned factor of lack of mohme s follow up . however , this was not the case at most hospitals and the meetings were held less frequently and with fewer members . in some provinces , due to the members absence and irregular meetings , the affiliating medical university had to hold joint meetings for all its autonomous hospitals , which questions hospitals autonomy . obviously , certain members , especially those beyond the hospitals , did not have enough motives to attend the meetings and get involved at hospitals policymaking . public hospitals are under strict supervision of the legal auditors , especially in terms of their financial issues , but for autonomous hospitals , no external overseer was appointed . shortage of full - time physicians at public hospitals the autonomous hospitals were supposed to change all their physicians to the full - time state , by changing their contracts or employing new physicians as full - time . first , most physicians who worked at public hospitals , especially in big cities , were simultaneously working at private sector as well , so did not like to change to full - time at public hospitals . this was caused by lack of any serious rule to prohibit physicians from dual practice and the huge difference between private and public hospitals charges that would make private hospitals payment to their physicians much more than that of the public hospitals . second , hospitals could not cease their contract with the employed physicians , because ( i ) they were not allowed legally , and ( ii ) they could not replace physicians with new ones , due to the lack of physicians who would be happy to work full - time . therefore , the mohme s goal of changing 100 percent of hospitals physicians to full - time ones at autonomous hospitals by 2013 would be practically impossible . most physicians like to have their private sector activities and work only as part - time physicians at autonomous hospitals lack of management stability at public hospitals public hospitals usually are very prone to changes at their top levels of managerial positions and this causes difficulties in the implementation of the reform . in this qualitative study , we explored the obstacles that the iranian health system encountered through liberalization of the public university owned hospitals . the findings showed different obstacles , categorized in three general headings of policy content , policymaking process ( policy implementation ) and the context . as a serious obstacle categorized under policy content , composition of the autonomous hospitals board role of the university chancellor as the head of the board would mean that the autonomous hospitals could have no real freedom and their policies would be imposed by the affiliating university and the mohme ultimately . this shows the central state s concerns over the first stages of liberalization and its cautious policy in delegating responsibilities to the local health sectors . similar pattern has been seen even among developed nations such as the uk where foundation trusts , as autonomous entities , were indeed controlled by primary care trusts that could dictate the central government s priorities ( 24 ) . although one may argue that the chancellor is only one of the members and he / she would not necessarily impose the university s policies on the hospital board , sensitivity to the fact that the chancellor is the head and has the ultimate power would decrease inspiration among hospital - based members . therefore , there might be some degree of veiled resistance against the reform at the iranian medical universities and even the ministry level . although resistance against liberalization of public hospitals is not unprecedented even among less - centralized health systems such as germany s due to the nature of health that should not be treated such as a commodity ( 26 ) , the resistance in iran was due to a different reason ; losing power among some authorities . at the implementation stage , the reform encountered various barriers that made the establishment of real autonomous hospitals almost impossible in iran . we had no finding about why the insurance organizations did not pay their committed payments to autonomous hospitals while they had agreed this in advance , but a possible explanation could be that the basic insurance organizations had experienced many changes after signing the agreement and their heads did not and could not pay what their predecessors had committed . moreover , it is possible that the negotiations between the mohme and insurance organizations had conducted only at their supreme levels , not tailored first among their experts and middle level managers . moreover , lack of any external overseer and certain ambiguities in rules and regulations would make the chancellor very cautious in implementation of the reform because he / she would have to take all the responsibilities for the consequences . lack of any regulation against physicians mobility between public and private hospitals and physicians natural and traditional interest in working at both sectors hindered the reform . the public sector s safety and private income would shape a model of dual practice that is obvious in the iranian health sector although it could be seen across many other nations ( 29 ) . lack of managerial stability at most public organizations in iran , seen at hospitals as well , was the other contextual barrier , which slowed down the reform . our first phase of study included all iranian medical universities and all autonomous hospitals , to catch their views on obstacles and barriers of the reform . by this comprehensive sample , we potentially have all barriers and experiences of the reform across all possible locations with their specific context . nevertheless , we could not interview the mohme authorities who knew policy issues at the highest level of the health sector . in south - east asia , the reform has proceeded in philippines , indonesia and vietnam but no significant transformation at their public stewardship has occurred ( 31 ) , and the reform could not improve efficiency and quality of care ( 32 ) . in iran , no study has examined the impact of the reform on hospitals clinical indicators . we saw that the iranian reform was not supported very well by the mohme as the main policy - maker and its introducer . the iranian health insurance organizations as the key stakeholders and the most influential bodies in financing the autonomous hospitals did not stand on their commitment , probably because the mohme s initial agreement with them was poor and could not satisfy their benefits . instead the reform was agreed by the mohme and insurance organizations , however when it was the insurance organizations turn to pay the hospitals based on the agreed amount , they denied their commitment . our first phase of study included all iranian medical universities and all autonomous hospitals , to catch their views on obstacles and barriers of the reform . by this comprehensive sample , we potentially have all barriers and experiences of the reform across all possible locations with their specific context . nevertheless , we could not interview the mohme authorities who knew policy issues at the highest level of the health sector . in south - east asia , the reform has proceeded in philippines , indonesia and vietnam but no significant transformation at their public stewardship has occurred ( 31 ) , and the reform could not improve efficiency and quality of care ( 32 ) . in iran , no study has examined the impact of the reform on hospitals clinical indicators . the iranian health insurance organizations as the key stakeholders and the most influential bodies in financing the autonomous hospitals did not stand on their commitment , probably because the mohme s initial agreement with them was poor and could not satisfy their benefits . in south - east asia , the reform has proceeded in philippines , indonesia and vietnam but no significant transformation at their public stewardship has occurred ( 31 ) , and the reform could not improve efficiency and quality of care ( 32 ) . in iran , no study has examined the impact of the reform on hospitals clinical indicators . the iranian health insurance organizations as the key stakeholders and the most influential bodies in financing the autonomous hospitals did not stand on their commitment , probably because the mohme s initial agreement with them was poor and could not satisfy their benefits . even the policymakers at the top levels of the mohme and the key stake - holders seem to have reached no consensus on the implementation and have moved back from their commitments . all our findings bring us to this conclusion that the stages of policy formulation and policy implementation were performed separately in iran and were not thought simultaneously and in common with those who formulate a policy and those who have to implement and execute it .

adult t - cell leukemia - lymphoma ( atl ) was first described in 1977 by uchiyama et al . as a distinct clinico - pathological entity with a suspected viral etiology because of the clustering of the disease in the southwest region of japan . subsequently , a novel rna retrovirus , human t - cell leukemia / lymphotropic virus type i ( htlv-1 ) , was isolated from a cell line established from leukemic cells of an atl patient , and the finding of a clear association with atl led to its inclusion among human carcinogenic pathogens [ 25 ] . in the mid-1980s and 1990s , several inflammatory diseases were reported to be associated with htlv-1 [ 610 ] . at the same time , endemic areas for the virus and diseases have been found ( reviewed in [ 1113 ] ) . diversity in atl has been recognized and a classification of clinical subtypes of the disease was proposed . this chapter will review the current recognition of atl focusing on treatment of the disease . atl patients show a variety of clinical manifestations because of various complications of organ involvement by atl cells , opportunistic infections and/or hypercalcemia [ 1114 ] . lymph node , liver , spleen , and skin lesions are frequently observed . though less frequently , digestive tract , lungs , central nervous system , bone , and/or other organs may be involved . large nodules , plaques , ulcers , and erythroderma are common skin lesions [ 1517 ] . approximately 26% of 854 patients with atl had active infections at diagnosis in a prior nationwide study in japan . the incidence was highest in the chronic and smoldering types ( 36% ) and lower in the acute ( 27% ) and lymphoma types ( 11% ) . the infections were bacterial in 43% , fungal in 31% , protozoal in 18% , and viral in 8% of patients . individuals with indolent atl might have no manifestation of the disease and are identified only by health checkups and laboratory examinations . atl cells are usually detected quite easily in the blood of affected individuals except for the smoldering type with mainly skin manifestations and lymphoma type . these so - called flower cells have highly indented or lobulated nuclei with condensed chromatin , small or absent nucleoli , and a agranular and basophilic cytoplasm . the histological analysis of aberrant cutaneous lesions or lymph nodes is essential for the diagnosis of the smoldering type with mainly skin manifestations and lymphoma type of atl , respectively . because atl cells in the skin and lymph node can vary in size from small to large and in form from pleomorphic to anaplastic and hodgkin - like cell with no specific histological pattern of involvement , differentiating between sezary syndrome , other peripheral t - cell lymphomas and hodgkin lymphoma versus atl can at times be difficult without examinations for htlv-1 serotype / genotype [ 13 , 19 ] . hypercalcemia is the most distinctive laboratory abnormality in atl as compared to other lymphoid malignancies and is observed in 31% of patients ( 50% in acute type , 17% in lymphoma type , and 0% in the other two types ) at onset . parathyroid hormone - related protein or receptor activator of nuclear factor kappa b ligand ( rankl ) produced by atl cells is considered the main factor causing hypercalcemia [ 20 , 21 ] . similar to serum ldh , 2-microglobulin , and serum thymidine kinase levels reflecting disease bulk / activity , the level of the soluble form of interleukin ( il)-2 receptor alpha - chain is elevated in the order of acute / lymphoma - type atl , smoldering / chronic - type atl , and htlv-1 carriers as compared with normal individuals , perhaps with better accuracy than the other markers [ 2224 ] . these serum markers are useful for detecting the acute transformation of indolent atl as well as the early relapse of atl after achieving responses by therapy . prototypical atl cells have a mature alpha - beta t - cell phenotype , that is , they are terminal deoxynucleotidyl transferase- ( tdt-)negative , cluster of differentiation ( cd ) 1a - negative , t - cell receptor alpha - beta positive , cd2-positive and cd5 , cd45ro , and cd29-positive , and frequently do not express cd7 and cd26 . a decline in the cd3 level with the appearance of cd25 indicates that the atl cells are in an activated state . most atl cells are cd52-positive but some are negative , and this may correlate with the coexpression of cd30 . about 90% of cases are cd4-positive and cd8-negative , and in rare cases either coexpress cd4 and cd8 , are negative for both markers , or are only cd8-positive . cc chemokine receptor 4 ( ccr4 ) is expressed in more than 90% of cases and associated with a poor prognosis . recent studies have suggested that the cells of some atl may be the equivalent of regulatory t - cells because of the high frequency of expression of cd25/ccr4 and about half of foxp3 [ 2628 ] . the diagnosis of typical atl is not difficult and is based on clinical features , atl cell morphology , mature helper - t - cell phenotype , and anti - htlv-1 antibody in most cases . those rare cases , which might be difficult to diagnose , can be shown to have the monoclonal integration of htlv-1 proviral dna in the malignant cells as determined by southern blotting . however , the monoclonal integration of htlv-1 is also detected in some ham / tsp patients and htlv-1 carriers [ 29 , 30 ] . after the diagnosis of atl , subtype classification of the disease is necessary for the selection of appropriate treatment [ 14 , 31 ] . atl is a distinct peripheral t - lymphocytic malignancy associated with a retrovirus designated human t - cell leukemia virus type i or human t - cell lymphotropic virus type i ( htlv-1 ) [ 1 , 1114 , 31 ] . major prognostic indicators for atl , which have been elucidated in 854 patients with atl in japan , the lymphoma study group ( lsg ) of the japan clinical oncology group ( jcog ) by multivariate analysis , were advanced performance status ( ps ) , high lactic dehydrogenase ( ldh ) level , age of 40 years or more , more than 3 involved lesions , and hypercalcemia . also a classification of clinical subtypes into acute , lymphoma , chronic , and smoldering types was proposed based on prognostic factors and clinical features of the disease . the leukemic subtypes include all of the chronic type and most of the acute and smoldering types . the acute type has a rapid course with leukemic manifestation ( 2% atl cells ) mostly , with or without lymphocytosis ( > 4 109/l ) including atl cells and most of the characteristic features of atl - generalized lymphadenopathy , hepatosplenomegaly , skin involvement , other organ involvement , a high ldh value , and hypercalcemia . the symptoms and signs include abdominal pain , diarrhea , ascites , jaundice , unconsciousness , dyspnea , pleural effusion , cough , sputum , and chest x - ray abnormalities because of organ involvement , hypercalcemia , and/or opportunistic infections . the smoldering type shows an indolent course and 5% or more of leukemic cells in the peripheral blood without lymphocytosis but may include skin / lung involvement . the calcium level is less than the upper limit , and ldh level is less than 1.5 times the upper limit in smoldering atl . the chronic type , with absolute lymphocytosis ( 4 109/l ) less frequently showing flower cell morphology than the acute type , is frequently and occasionally associated with skin involvement and lymphadenopathy , respectively , and also usually shows a relatively indolent course . the calcium level is less than the upper limit , and the ldh level is less than double the upper limit of the chronic type . the lymphoma type presents with the manifestations of a nodal - lymphoma without leukemic cells , frequently with high ldh / ca levels , a rapid course , and symptoms and signs similar to the acute type . in case of atl , clinical subtype is more important than ann arbor stage for predicting prognosis and deciding treatment because of frequent leukemic manifestation defined as stage iv . additional factors associated with a poor prognosis include thrombocytopenia , eosinophilia , bone marrow involvement , a high interleukin ( il)-5 serum - level , c - c chemokine receptor 4 ( ccr4 ) expression , lung resistance - related protein ( lrp ) , p53 mutation , and p16 deletion by multivariate analysis [ 26 , 27 , 3337 ] . specific for the chronic type of atl , high ldh , high blood urea nitrogen ( bun ) , and low albumin levels were identified as factors for a poor prognosis by multivariate analysis . primary cutaneous tumoral type although generally included among smoldering atl had a poor prognosis in univariate analysis . treatment decisions should be based on the atl subtype - classification and the prognostic factors at onset including those related with atl and comorbidity . as mentioned above , subtype - classification of this disease has been proposed based on the prognosis and clinical manifestations . without treatment , most patients with acute-/lymphoma / type atl die of the disease or infections within weeks or months . more than half of patients with smoldering atl survive for more than 5 years without chemotherapy and transformation to aggressive atl . chronic atl has the most diverse prognosis among the subtypes and could be divided into favorable and unfavorable by clinical parameters ( serum albumin , bun , and ldh levels ) after a multivariate analysis . current treatment options for atl include watchful waiting until the disease progresses , interferon alpha ( ifn ) and zidovudine ( azt ) therapy , multiagent chemotherapy , allogeneic hematopoietic stem cell transplantation ( allo - hsct ) , and a new agent . at present , no standard treatment for atl exists . therefore , patients with the smoldering or favorable chronic type , who may survive one or more years without chemotherapy , excluding topical therapy for cutaneous lesions , should be observed and therapy should be delayed until progression of the disease . however , it was recently found that the long - term prognosis of such patients was poorer than expected . in a long - term followup study for 78 patients with indolent atl ( favorable chronic- or smoldering - type ) with a policy of watchful waiting until disease progression at a single institution , the median survival time was 5.3 years with no plateau in the survival curve . twelve patients remained alive for > 10 years , 32 progressed to acute atl , and 51 died . recently , the striking benefit of early intervention to indolent atl by ifn and an antiretroviral agent was reported by a meta - analysis . this modality should be extensively evaluated by larger clinical trials to establish appropriate management practices for indolent atl . since 1978 , chemotherapy trials have been consecutively conducted for patients newly diagnosed with atl by jcog 's lymphoma study group ( lsg ) ( table 1 ) [ 4045 ] . between 1981 and 1983 , jcog conducted a phase iii trial ( jcog8101 ) to evaluate lsg1-vepa ( vincristine , cyclophosphamide , prednisone , and doxorubicin ) versus lsg2-vepa - m ( vepa plus methotrexate ( mtx ) ) for advanced non - hodgkin lymphoma ( nhl ) , including atl [ 40 , 41 ] . the complete response ( cr ) rate of lsg2-vepa - m for atl ( 37% ) was higher than that of lsg1-vepa ( 17% ; p = .09 ) . however , the cr rate was significantly lower for atl than for b - cell nhl and peripheral t - cell lymphoma ( ptcl ) other than atl ( p < .001 ) . the median survival time of the 54 patients with atl was 6 months , and the estimated 4-year survival rate was 8% . in 1987 , jcog initiated a multicenter phase ii study ( jcog8701 ) of a multiagent combination chemotherapy ( lsg4 ) for advanced aggressive nhl ( including atl ) . lsg4 consisted of three regimens : ( 1 ) vepa - b ( vepa plus bleomycin ) , ( 2 ) m - fepa ( methotrexate , vindesine , cyclophosphamide , prednisone , and doxorubicin ) , and ( 3 ) vepp - b , ( vincristine , etoposide , procarbazine , prednisone , and bleomycin ) . the cr rate for atl patients was improved from 28% ( jcog8101 ) to 43% ( jcog8701 ) ; however , the cr rate was significantly lower in atl than in b - cell nhl and ptcl ( p < .01 ) . patients with atl still showed a poor prognosis , with a median survival time of 8 months and a 4-year survival rate of 12% . the disappointing results with conventional chemotherapies have led to a search for new active agents . multicenter phase i and ii studies of pentostatin ( 2-deoxycoformycin , an inhibitor of adenosine deaminase ) were conducted against atl in japan . the phase ii study revealed a response rate of 32% ( 10 of 31 ) in cases of relapsed or refractory atl ( 2crs and 8prs ) . these encouraging results prompted the investigators to conduct a phase ii trial ( jcog9109 ) with a pentostatin - containing combination ( lsg11 ) as the initial chemotherapy . patients with aggressive atl that is , of the acute , lymphoma , or unfavorable chronic type were eligible for this study . unfavorable chronic - type atl , defined as having at least 1 of 3 unfavorable prognostic factors ( low serum albumin level , high ldh level , or high bun ) , has an unfavorable prognosis similar to that for acute- and lymphoma - type atl . a total of 62 untreated patients with aggressive atl ( 34 acute , 21 lymphoma , and 7 unfavorable chronic type ) were enrolled . a regimen of 1 mg / m vincristine on days 1 and 8 , 40 mg / m doxorubicin on day 1 , 100 mg / m etoposide on days 1 through 3 , 40 mg / m prednisolone ( psl ) on days 1 and 2 , and 5 mg / m pentostatin on days 8 , 15 , and 22 was administered every 28 days for 10 cycles . among the 61 patients evaluable for toxicity , four patients ( 7% ) died of infections , two from septicemia , and two from cytomegalovirus pneumonia . among the 60 eligible patients , there were 17crs ( 28% ) and 14 partial responses ( prs ) ( overall response rate [ orr ] = 52% ) . the median survival time was 7.4 months , and the estimated 2-year survival rate was 17% . the prognosis in patients with atl remained poor , even though they were treated with a pentostatin - containing combination chemotherapy . in 1994 , jcog initiated a phase ii trial ( jcog9303 ) of an eight - drug regimen ( lsg15 ) consisting of vincristine , cyclophosphamide , doxorubicin , prednisone , ranimustine , vindesine , etoposide , and carboplatin for untreated atl . dose intensification was attempted with the prophylactic use of granulocyte colony - stimulating factor ( g - csf ) . in addition , non - cross - resistant agents , such as ranimustine and carboplatin , and intrathecal prophylaxis with mtx and psl were incorporated . ninety - six previously untreated patients with aggressive atl were enrolled : 58 acute , 28 lymphoma , and 10 unfavorable chronic types . approximately 81% of the 93 eligible patients responded ( 75/93 ) , with 33 patients obtaining a cr ( 35% ) . the overall survival rate of the 93 patients at 2 years was estimated to be 31% , with a median survival time of 13 months . grade 4 neutropenia and thrombocytopenia were observed in 65% and 53% of the patients , respectively , whereas grade 4 nonhematologic toxicity was observed in only one patient . dose intensification of chop with prophylactic use of g - csf was expected to improve survival among patients with aggressive nhl , and our randomized phase ii study ( jcog9505 ) comparing chop-14 ( lsg19 ) and dose - escalated chop ( lsg20 ) to treat aggressive nhl excluding atl revealed biweekly chop to be more promising . therefore , we regarded biweekly chop as a standard treatment for nhl including aggressive atl at the time of designing this phase iii study . to confirm whether the lsg15 regimen is a new standard for the treatment of aggressive atl , jcog conducted a phase iii trial comparing modified ( m)-lsg15 with biweekly chop ( cyclophosphamide , hydroxy - doxorubicin , vincristine [ oncovin ] , and prednisone ) , both supported with g - csf and intrathecal prophylaxis . mlsg19 , a modified version of lsg19 , consisted of eight cycles of chop [ cpa 750 mg / m , adm 50 mg / m , vcr 1.4 mg / m(maximum 2 mg ) on day 1 and psl 100 mg on days 1 to 5 ] every 2 weeks . mlsg15 in jcog9801 was a modified version of lsg15 in jcog9303 , consisting of three regimens : vcap [ vcr 1 mg / m ( maximum 2 mg ) , cpa 350 mg / m , adm 40 mg / m , psl 40 mg / m ] on day 1 , amp [ adm 30 mg / m , mcnu 60 mg / m , psl 40 mg / m ] on day 8 , and vecp [ vds 2.4 mg / m on day 15 , etp 100 mg / m on days 15 to 17 , cbdca 250 mg / m on day15 , psl 40 mg / m on days 15 to 17 ] on days 1517 , and the next course was to be started on day 29 ( figure 1 ) . the modifications in mlsg15 as compared to lsg15 were as follows : ( 1 ) the total number of cycles was reduced from 7 to 6 because of progressive cytopenia , especially thrombocytopenia , after repeating the lsg15 therapy . ( 2 ) cytarabine 40 mg was used with mtx 15 mg and psl 10 mg for prophylactic intrathecal administration , at the recovery phases of courses 1 , 3 , and 5 because of the high frequency of central nervous system relapse in the jcog9303 study . untreated patients with aggressive atl were assigned to receive either six courses of mlsg15 every 4 weeks or eight courses of biweekly chop . the cr rate was higher in the mlsg15 arm than in the biweekly chop arm ( 40% versus 25% , resp . ; p = .020 ) . as shown in table 1 , the median survival time and os rate at 3 years were 12.7 months and 24% in the mlsg15 arm and 10.9 months and 13% in the biweekly chop arm [ two - sided p = .169 , and the hazard ratio was 0.75 ; 95% confidence interval ( ci ) , 0.50 to 1.13 ] . a cox regression analysis with performance status ( ps 0 versus 1 versus 24 ) as the stratum for baseline hazard functions was performed to evaluate the effect on overall survival of age , b - symptoms , subtypes of atl , ldh , bun , bulky mass , and treatment arms . according to this analysis , the hazard ratio and two - sided p value for the treatment arms were 0.62 ( 95% ci , 0.38 to 1.01 ) and .056 , respectively . the difference between the crude analysis and this result was because of unbalanced prognostic factors , such as ps 0 versus 1 , and the presence or absence of bulky lesions between the treatment arms . the progression - free survival rate at 1 year was 28% in the mlsg15 arm compared with 16% in the biweekly chop arm ( two - sided p = .20 ) . in mlsg15 versus mlsg19 , rate of grade 4 neutropenia , grade 4 thrombocytopenia , and grade 3/4 infection were 98% versus 83% , 74% versus 17% , and 32% versus 15% , respectively . three treatment - related deaths ( trds ) , two from sepsis and one from interstitial pneumonitis related to neutropenia , were reported in the mlsg15 arm . the longer survival at 3 years and higher cr rate with mlsg15 compared with mlsg19 suggest that mlsg15 is a more effective regimen at the expense of higher toxicity , providing the basis for future investigations in the treatment of atl . the superiority of vcap - amp - vecp in mlsg15 to biweekly chop in mlsg19 may be explained by the more prolonged , dose dense schedule of therapy in addition to 4 more drugs . in addition , agents such as carboplatin and ranimustine not affected by multidrug - resistance ( mdr ) related genes , which were frequently expressed in atl cells at onset , were incorporated . intrathecal prophylaxis , which was incorporated in both arms of the phase iii study , should be considered for patients with aggressive atl even in the absence of clinical symptoms because a previous analysis revealed that more than half of relapses at new sites after chemotherapy occurred in the cns . however , the median survival time of 13 months in vcap - amp - vecp ( lsg15/mlsg15 ) still compares unfavorably to other hematological malignancies , requiring further effort to improve the outcome . a small phase ii trial in japan of ifn alpha against relapsed / refractory atl showed a response rate ( all pr ) of 33% ( 8/24 ) , including 5 out of 9 ( 56% ) chronic - type atl . in 1995 , gill and associates reported that 11 of 19 patients with acute- or lymphoma - type atl showed major responses ( 5 cr and 6 pr ) to a combination of interferon - alpha ( ifn ) and zidovudine ( azt ) . the efficacy of this combination was also observed by hermine and associates ; major objective responses were obtained in all five patients with atl ( four with acute type and one with smoldering type ) . although these results are encouraging , the os of previously untreated patients with atl was relatively short ( 4.8 months ) compared with the survival of those in the chemotherapy trials conducted by the jcog - lsg ( 7 to 8 months ) . after that , numerous small phase ii studies using azt and ifn have shown responses in atl patients [ 5456 ] . high doses of both agents are recommended : 69 million units of ifn in combination with daily divided azt doses of 8001000 mg / day . therapeutic effect of azt and ifn is not through a direct cytotoxic effect of these drugs on the leukemic cells . enduring azt treatment of atl cell lines results in inhibition of telomerase which reprograms the cells to p53-dependent senescence . recently , the results of a meta - analysis on the use of ifn and azt for atl were reported . the orr was 66% , with a 43% cr rate . in this worldwide retrospective analysis , the median survival time was 24 months and the 5-year survival rate was 50% for first - line ifn and azt , versus 7 months and 20% for 84 patients who received first - line chemotherapy . the median survival time of patients with acute - type atl treated with first - line ifn / azt and chemotherapy was 12 and 9 months , respectively . in addition , first - line ifn / azt therapy in chronic- and smoldering - type atl resulted in a 100% survival rate at a median followup of 5 years . however , because of the retrospective nature of this meta - analysis based on medical records at each hospital , the decision process to select the therapeutic modality for each patient and the possibility of interference with os by second - line treatment remains unknown . while the results for ifn / azt in indolent atl appear to be promising compared to those with watchful - waiting policy until disease progression , recently reported from japan , the possibility of selection bias can not be ruled out . a prospective multicenter phase iii study evaluating the efficacy of ifn / azt as compared to watchful - waiting for indolent atl is to be initiated in japan . recently , a phase ii study of the combination of arsenic trioxide , ifn , and azt for chronic atl revealed an impressive response rate and moderate toxicity . although the results appeared promising , the addition of arsenic trioxide to ifn / azt , which might be sufficient for the treatment of chronic atl as described above , caused more toxicity and should be evaluated with caution . allo - hsct is now recommended for the treatment of young patients with aggressive atl [ 31 , 59 ] . despite higher treatment - related mortality including graft versus host disease in a retrospective multicenter analysis of myeloablative allo - hsct , the estimated 3-year os of 33% is promising , possibly reflecting a graft versus atl effect . to evaluate the efficacy of allo - hsct more accurately , especially in view of a comparison with intensive chemotherapy , a prospective multicenter phase ii study of lsg15 chemotherapy followed by allo - hsct is ongoing ( jcog0907 ) . feasibility studies of allo - hsct with reduced intensity conditioning for relatively aged patients with atl also revealed promising results , and subsequent multicenter trials are being conducted in japan [ 61 , 62 ] . the minimal residual disease after allo - hsct detected as htlv-1 proviral load was much less than that after chemotherapy or azt / ifn therapy , suggesting the presence of a graft - versus - atl effect as well as graft - versus - htlv-1 activity . it remains unclear which type of allo - hsct ( myeloablative or reduced intensity conditioning ) is more suitable for the treatment of atl . furthermore , selection criteria with respect to responses to previous treatments , sources of stem cells , and htlv-1 viral status of the donor remain to be determined . recently , a patient in whom atl derived from donor cells developed four months after transplantation of stem cells from a sibling with htlv - i was reported . however , several other retrospective studies as well as those mentioned above on allo - hsct showed a promising long - term survival rate of 20 to 40% with an apparent plateau phase despite significant treatment - related mortality . the prevention of opportunistic infections is essential in the management of atl patients , nearly half of whom develop severe infections during chemotherapy . sulfamethoxazole / trimethoprim and antifungal agents have been recommended as prophylaxes for pneumocystis jiroveci pneumonia and fungal infections , respectively , in the jcog trials [ 4345 ] . while cytomegalovirus infections are not infrequent among atl patients , ganciclovir is not usually recommended as a prophylaxis . in addition , in patients not receiving chemotherapy or allo - hsct , antifungal prophylaxis may not be critical . an antistrongyloides agent , such as ivermectin or albendazole , should be considered to avoid systemic infections in patients with a history of exposure to the parasite in the tropics . treatment with steroids and proton pump inhibitors may precipitate a fulminant strongyloides infestation and warrants testing before these agents are used in endemic areas . hypercalcemia associated with aggressive atl can be corrected using chemotherapy in combination with hydration and bisphosphonate even when the performance status of the patient is poor . the complex nature of atl , often with both leukemic and lymphomatous components , makes response assessment difficult . a modification of the jcog response criteria was suggested by atl consensus - meeting reflecting those for cll and nhl which had been published later [ 31 , 64 , 65 ] . new guidelines were presented incorporating positron emission tomography ( pet ) , especially for the assessment of cr . it is well known and described in the criteria that several kinds of lymphoma including peripheral t - cell lymphomas were variably [ 18f ] fluorodeoxyglucose ( fdg ) avid . meanwhile , pet or pet / ct is recommended for evaluations of response when the tumorous lesions are fdg - avid at diagnosis . several purine analogs have been evaluated for atl . among them , pentostatin ( deoxycoformycin ) has been most extensively evaluated as a single agent and in combination as described above [ 43 , 46 ] . fludarabine is among standard treatments for b - chronic lymphocytic leukemia and other lymphoid malignancies . in a phase i study of fludarabine in japan , 5 atl patients and 10 b - cll patients with refractory or relapsed - disease were enrolled . pr was achieved only in one of the 5 atl patients and the duration was short . a phase ii study of cladribine for relapsed / refractory aggressive - atl in 15 patients revealed only one pr . forodesine , a purine nucleotide phosphorylase ( pnp ) inhibitor , is among purine nucleotide analogs . pnp is an enzyme in the purine salvage pathway that phosphorolysis 2deoxyguanosine ( dguo ) . purine nucleoside phosphorylase ( pnp ) deficiency in humans results in a severe combined immunodeficiency phenotype and the selective depletion of t cells associated with high plasma deoxyguanosine ( dguo ) and high intracellular deoxyguanosine triphosphate levels in those cells with high deoxynucleoside kinase activity such as t cells , leading to cell death . inhibitors of pnp , such as forodesine , mimic scid in vitro and in vivo , suggesting a new targeting agent specific for t cell malignancies . a dose escalating phase i study of forodesine is being conducted in japan for t cell malignancies including atl . histone deacetylases ( hdacs ) are enzymes involved in the remodeling of chromatin and play a key role in the epigenetic regulation of gene expression . deacetylase inhibitors ( dacis ) induce the hyperacetylation of nonhistone proteins as well as nucleosomal histones resulting in the expression of repressed genes involved in growth arrest , terminal differentiation , and/or apoptosis among cancer cells . several classes of hdaci have been found to have potent anticancer effects in preclinical studies . hdacis such as vorinostat ( suberoylanilide hydroxamic acid : saha ) , romidepsin ( depsipeptide ) , and panobinostat ( lbh589 ) have also shown promise in preclinical and/or clinical studies against t - cell malignancies including atl [ 70 , 71 ] . vorinostat and romidepsin have been approved for cutaneous t - cell lymphoma ( ctcl ) by the food and drug administration in the usa . however , a phase ii study for ctcl and indolent atl in japan was terminated because of severe infections associated with the shrinkage of skin tumors and formation of ulcers in patients with atl . further study is required to evaluate the efficacy of hdacis for ptcl / ctcl including atl . monoclonal antibodies ( moab ) and toxin fusion proteins targeting several molecules expressed on the surface of atl cells and other lymphoid malignant cells , such as cd25 , cd2 , cd52 , and chemokine receptor 4 ( ccr4 ) , have shown promise in recent clinical trials . because most atl cells express the alpha - chain of il-2r ( cd25 ) , waldmann et al . treated patients with atl using monoclonal antibodies to cd25 . six ( 32% ) of 19 patients treated with anti - tac showed objective responses lasting from 9 weeks to longer than 3 years . one impediment to this approach is the quantity of soluble il-2r shed by the tumor cells into the circulation . another strategy for targeting il-2r is conjugation with an immunotoxin ( pseudomonas exotoxin ) or radioisotope ( yttrium-90 ) . developed a stable conjugate of anti - tac with yttrium-90 . among the 16 patients with atl who received 5- to 15-mci doses , 9 ( 56% ) showed objective responses . the response lasted longer than that obtained with unconjugated anti - tac antibody [ 73 , 74 ] . lmb-2 , composed of the anti - cd25 murine moab fused to the truncated form of pseudomonas toxin , was cytotoxic to cd25-expressing cells including atl cells in vitro and in mice . phase i / ii trials of this agent showed some effect against hairy cell leukemia , ctcl , and atl . six of 35 patients in the phase i study had significant levels of neutralizing antibodies after the first cycle . denileukin diftitox ( dd ; dab(389)-interleukin-2 [ il-2 ] ) , an interleukin-2-diphtheria toxin fusion protein targeting il-2 receptor - expressing malignant t lymphocytes , shows efficacy as a single agent against ctcl and peripheral t - cell lymphoma ( ptcl ) . also the combination of this agent with multiagent chemotherapy , chop , was promising for ptcl . atl cells frequently and highly express cd25 as described above , and several atl cases successfully treated with this agent have been reported . , however , cd52 expression varies among patients , with an overall expression rate lower than 50% in one study but not in another [ 78 , 79 ] . the humanized anti - cd52 monoclonal antibody alemtuzumab is active against cll and ptcl as a single agent . the combination of alemtuzumab with a standard - dose cyclophosphamide / doxorubicin / vincristine / prednisone ( chop ) regimen as a first - line treatment for 24 patients with ptcl showed promising results with cr in 17 ( 71% ) patients ; 1 had a partial remission , with an overall median duration of response of 11 months and was associated with mostly manageable infections but including cmv reactivation . major infections were jacob - creutzfeldt virus reactivation , pulmonary invasive aspergillosis , and staphylococcus sepsis . atl cells express cd52 , the target of alemtuzumab , which was active in a preclinical model of atl and toxic to p53-deficient cells , and several atl cases successfully treated with this agent have been reported [ 8183 ] . siplizumab is a humanized moab targeting cd2 and showed efficacy in a murine atl model . p1 dose - escalating study of this agent in 22 patients with several kinds of t / nk - cell malignancy revealed 6 responses ( 2 cr in lgl leukemia , 3 pr in atl , and 1 pr in ctcl ) . the broad specificity of this agent may eliminate both cd4- and cd8-positive t cells as well as nk cells without effecting b cells and predispose individuals to the development of ebv lymphoproliferative syndrome . cc chemokine receptor 4 ( ccr4 ) is expressed on normal t helper type 27 and regulatory t ( treg ) cells and on certain types of t - cell neoplasms [ 20 , 21 , 35 ] . kw-0761 , a next generation humanized anti - ccr4 mab , with a defucosylated fc region , exerts strong antibody - dependent cellular cytotoxicity ( adcc ) due to increased binding to the fc receptor on effecter cells . a phase i study of dose escalation with 4 weekly intravenous infusions of kw-0761 in 16 patients with relapsed ccr4-positive t cell malignancy ( 13 atl and 3 ptcl ) revealed that one patient , at the maximum dose ( 1.0 mg / kg ) , developed grade ( g ) 3 dose - limiting toxic effects , namely , skin rashes and febrile neutropenia and g4 neutropenia . other treatment - related g3 - 4 toxic effects were lymphopenia ( n = 10 ) , neutropenia ( n = 3 ) , leukopenia ( n = 2 ) , herpes zoster ( n = 1 ) , and acute infusion reaction / cytokine release syndrome ( n = 1 ) . neither the frequency nor severity of these effects increased with dose escalation or the plasma concentration of the agent . five patients ( 31% ; 95% ci , 11% to 59% ) achieved objective responses : 2 complete ( 0.1 ; 1.0 mg / kg ) and 3 partial ( 0.01 ; 2 at 1.0 mg / kg ) responses . three out of 13 patients with atl ( 31% ) achieved a response ( 2 cr and 1 pr ) . responses in each lesion were diverse , that is , good in pb ( 6 cr and 1 pr/7 evaluable cases ) , intermediate in skin ( 3 cr and 1 pr/8 evaluable cases ) , and poor in ln ( 1 cr and 2 pr/11 evaluable cases ) . kw-0761 was well tolerated at all the doses tested , demonstrating potential efficacy against relapsed ccr4-positive atl or ptcl . recently , results of subsequent phase ii studies at the 1.0 mg / kg in relapsed atl , showing 50% of response rate with acceptable toxicity profiles , reported . a phase ii trial of single agent kw-0761 at the 1.0 mg / kg in relapsed ptcl / ctcl and a phase ii trial of vcap - amp - vecp combined with kw-0761 for untreated aggressive atl are ongoing . a proteasome inhibitor , bortezomib ( velcade ) , and an immunomodulatory agent , lenalidomide ( revlimid ) , both have potent preclinical and clinical activity in t - cell malignancies including atl , are now under clinical trials for relapsed atl in japan [ 8890 ] . other potential drugs for atl include pralatrexate ( folotyn ) , a new agent with clinical activity in t - cell malignancies including atl [ 9193 ] . the agent is a novel antifolate with improved membrane transport and polyglutamylation in tumor cells and high affinity for the reduced folate carrier ( rfc ) highly expressed in malignant cells and has been approved by fda recently for t - cell lymphoma including atl . this has been achieved in some endemic areas in japan by screening for htlv-1 among blood donors and asking mothers who are carriers to refrain from breast feeding . for several decades , before initiation of the interventions , the prevalence of htlv-1 has declined drastically in endemic areas in japan , probably because of birth cohort effects . the elimination of htlv-1 in endemic areas is now considered possible due to the natural decrease in the prevalence as well as the intervention of transmission through blood transfusion and breast feeding . this has not been achieved partly because only about 5% of htlv-1 carriers develop the disease in their life time although several risk factors have been identified by a cohort study of htlv-1 carriers ( joint study of predisposing factors for atl development ) . also , no agent has been found to be effective in preventing the development of atl among htlv-1 carriers . clinical trials have been paramount to the recent advances in atl treatment , including assessments of chemotherapy , azt / ifn , and allo - hsct . recently , a strategy for atl treatment , stratified by subtype - classification , prognostic factors , and the response to initial treatment as well as response criteria , was proposed . the recommended treatment algorithm for atl however , atl still has a worse prognosis than the other t - cell malignancies .there is no plateau with an initial steep slope and subsequent gentle slope without a plateau in the survival curve for aggressive or indolent atl treated by watchful waiting and with chemotherapy , respectively , although the prognosis is much better in the latter . a prognostic model for each subgroup should be elucidated to properly identify the candidate for allo - hsct which can achieve a cure of atl despite considerable treatment - related mortality . although several small phase ii trials and a recent metaanalysis suggested ifn / azt therapy to be promising , no confirmative phase iii study has been conducted . furthermore , as described in the other chapters in detail , more than ten promising new agents for ptcl / ctcl including atl are now in clinical trials or preparation . future clinical trials on atl as described above should be incorporated to ensure that the consensus is continually updated to establish evidence - based practical guidelines .

atl is a distinct peripheral t - lymphocytic malignancy associated with human t - cell lymphotropic virus type i ( htlv-1 ) . the diversity in clinical features and prognosis of patients with this disease has led to its subtype - classification into four categories , acute , lymphoma , chronic , and smoldering types , defined by organ involvement , and ldh and calcium values . in case of acute , lymphoma , or unfavorable chronic subtypes ( aggressive atl ) , intensive chemotherapy like the lsg15 regimen ( vcap - amp - vecp ) is usually recommended if outside of clinical trials , based on the results of a phase 3 trial . in case of favorable chronic or smoldering atl ( indolent atl ) , watchful waiting until disease progression has been recommended , although the long - term prognosis was inferior to those of , for instance , chronic lymphoid leukemia . retrospective analysis suggested that the combination of interferon alpha and zidovudine was apparently promising for the treatment of atl , especially for types with leukemic manifestation . allogeneic hematopoietic stem cell transplantation ( allo - hsct ) is also promising for the treatment of aggressive atl possibly reflecting graft versus atl effect . several new agent trials for atl are ongoing and in preparation , including a defucosylated humanized anti - cc chemokine receptor 4 monoclonal antibody , il2-fused with diphtheria toxin , histone deacetylase inhibitors , a purine nucleoside phosphorylase inhibitor , a proteasome inhibitor , and lenalidomide .

1. Introduction 2. Clinical Features and Laboratory Findings of ATL 3. Diagnosis of ATL 4. Definition, Prognostic Factors, and Subtype Classification of ATL 5. Clinical Course, Treatment, and Response Criteria of ATL 6. Conclusions

adult t - cell leukemia - lymphoma ( atl ) was first described in 1977 by uchiyama et al . subsequently , a novel rna retrovirus , human t - cell leukemia / lymphotropic virus type i ( htlv-1 ) , was isolated from a cell line established from leukemic cells of an atl patient , and the finding of a clear association with atl led to its inclusion among human carcinogenic pathogens [ 25 ] . diversity in atl has been recognized and a classification of clinical subtypes of the disease was proposed . the histological analysis of aberrant cutaneous lesions or lymph nodes is essential for the diagnosis of the smoldering type with mainly skin manifestations and lymphoma type of atl , respectively . because atl cells in the skin and lymph node can vary in size from small to large and in form from pleomorphic to anaplastic and hodgkin - like cell with no specific histological pattern of involvement , differentiating between sezary syndrome , other peripheral t - cell lymphomas and hodgkin lymphoma versus atl can at times be difficult without examinations for htlv-1 serotype / genotype [ 13 , 19 ] . similar to serum ldh , 2-microglobulin , and serum thymidine kinase levels reflecting disease bulk / activity , the level of the soluble form of interleukin ( il)-2 receptor alpha - chain is elevated in the order of acute / lymphoma - type atl , smoldering / chronic - type atl , and htlv-1 carriers as compared with normal individuals , perhaps with better accuracy than the other markers [ 2224 ] . prototypical atl cells have a mature alpha - beta t - cell phenotype , that is , they are terminal deoxynucleotidyl transferase- ( tdt-)negative , cluster of differentiation ( cd ) 1a - negative , t - cell receptor alpha - beta positive , cd2-positive and cd5 , cd45ro , and cd29-positive , and frequently do not express cd7 and cd26 . about 90% of cases are cd4-positive and cd8-negative , and in rare cases either coexpress cd4 and cd8 , are negative for both markers , or are only cd8-positive . cc chemokine receptor 4 ( ccr4 ) is expressed in more than 90% of cases and associated with a poor prognosis . recent studies have suggested that the cells of some atl may be the equivalent of regulatory t - cells because of the high frequency of expression of cd25/ccr4 and about half of foxp3 [ 2628 ] . the diagnosis of typical atl is not difficult and is based on clinical features , atl cell morphology , mature helper - t - cell phenotype , and anti - htlv-1 antibody in most cases . after the diagnosis of atl , subtype classification of the disease is necessary for the selection of appropriate treatment [ 14 , 31 ] . atl is a distinct peripheral t - lymphocytic malignancy associated with a retrovirus designated human t - cell leukemia virus type i or human t - cell lymphotropic virus type i ( htlv-1 ) [ 1 , 1114 , 31 ] . major prognostic indicators for atl , which have been elucidated in 854 patients with atl in japan , the lymphoma study group ( lsg ) of the japan clinical oncology group ( jcog ) by multivariate analysis , were advanced performance status ( ps ) , high lactic dehydrogenase ( ldh ) level , age of 40 years or more , more than 3 involved lesions , and hypercalcemia . also a classification of clinical subtypes into acute , lymphoma , chronic , and smoldering types was proposed based on prognostic factors and clinical features of the disease . the acute type has a rapid course with leukemic manifestation ( 2% atl cells ) mostly , with or without lymphocytosis ( > 4 109/l ) including atl cells and most of the characteristic features of atl - generalized lymphadenopathy , hepatosplenomegaly , skin involvement , other organ involvement , a high ldh value , and hypercalcemia . the symptoms and signs include abdominal pain , diarrhea , ascites , jaundice , unconsciousness , dyspnea , pleural effusion , cough , sputum , and chest x - ray abnormalities because of organ involvement , hypercalcemia , and/or opportunistic infections . the calcium level is less than the upper limit , and ldh level is less than 1.5 times the upper limit in smoldering atl . the lymphoma type presents with the manifestations of a nodal - lymphoma without leukemic cells , frequently with high ldh / ca levels , a rapid course , and symptoms and signs similar to the acute type . in case of atl , clinical subtype is more important than ann arbor stage for predicting prognosis and deciding treatment because of frequent leukemic manifestation defined as stage iv . additional factors associated with a poor prognosis include thrombocytopenia , eosinophilia , bone marrow involvement , a high interleukin ( il)-5 serum - level , c - c chemokine receptor 4 ( ccr4 ) expression , lung resistance - related protein ( lrp ) , p53 mutation , and p16 deletion by multivariate analysis [ 26 , 27 , 3337 ] . specific for the chronic type of atl , high ldh , high blood urea nitrogen ( bun ) , and low albumin levels were identified as factors for a poor prognosis by multivariate analysis . treatment decisions should be based on the atl subtype - classification and the prognostic factors at onset including those related with atl and comorbidity . as mentioned above , subtype - classification of this disease has been proposed based on the prognosis and clinical manifestations . more than half of patients with smoldering atl survive for more than 5 years without chemotherapy and transformation to aggressive atl . current treatment options for atl include watchful waiting until the disease progresses , interferon alpha ( ifn ) and zidovudine ( azt ) therapy , multiagent chemotherapy , allogeneic hematopoietic stem cell transplantation ( allo - hsct ) , and a new agent . however , it was recently found that the long - term prognosis of such patients was poorer than expected . in a long - term followup study for 78 patients with indolent atl ( favorable chronic- or smoldering - type ) with a policy of watchful waiting until disease progression at a single institution , the median survival time was 5.3 years with no plateau in the survival curve . between 1981 and 1983 , jcog conducted a phase iii trial ( jcog8101 ) to evaluate lsg1-vepa ( vincristine , cyclophosphamide , prednisone , and doxorubicin ) versus lsg2-vepa - m ( vepa plus methotrexate ( mtx ) ) for advanced non - hodgkin lymphoma ( nhl ) , including atl [ 40 , 41 ] . however , the cr rate was significantly lower for atl than for b - cell nhl and peripheral t - cell lymphoma ( ptcl ) other than atl ( p < .001 ) . patients with aggressive atl that is , of the acute , lymphoma , or unfavorable chronic type were eligible for this study . unfavorable chronic - type atl , defined as having at least 1 of 3 unfavorable prognostic factors ( low serum albumin level , high ldh level , or high bun ) , has an unfavorable prognosis similar to that for acute- and lymphoma - type atl . a total of 62 untreated patients with aggressive atl ( 34 acute , 21 lymphoma , and 7 unfavorable chronic type ) were enrolled . in 1994 , jcog initiated a phase ii trial ( jcog9303 ) of an eight - drug regimen ( lsg15 ) consisting of vincristine , cyclophosphamide , doxorubicin , prednisone , ranimustine , vindesine , etoposide , and carboplatin for untreated atl . ninety - six previously untreated patients with aggressive atl were enrolled : 58 acute , 28 lymphoma , and 10 unfavorable chronic types . to confirm whether the lsg15 regimen is a new standard for the treatment of aggressive atl , jcog conducted a phase iii trial comparing modified ( m)-lsg15 with biweekly chop ( cyclophosphamide , hydroxy - doxorubicin , vincristine [ oncovin ] , and prednisone ) , both supported with g - csf and intrathecal prophylaxis . the modifications in mlsg15 as compared to lsg15 were as follows : ( 1 ) the total number of cycles was reduced from 7 to 6 because of progressive cytopenia , especially thrombocytopenia , after repeating the lsg15 therapy . a cox regression analysis with performance status ( ps 0 versus 1 versus 24 ) as the stratum for baseline hazard functions was performed to evaluate the effect on overall survival of age , b - symptoms , subtypes of atl , ldh , bun , bulky mass , and treatment arms . the longer survival at 3 years and higher cr rate with mlsg15 compared with mlsg19 suggest that mlsg15 is a more effective regimen at the expense of higher toxicity , providing the basis for future investigations in the treatment of atl . the superiority of vcap - amp - vecp in mlsg15 to biweekly chop in mlsg19 may be explained by the more prolonged , dose dense schedule of therapy in addition to 4 more drugs . intrathecal prophylaxis , which was incorporated in both arms of the phase iii study , should be considered for patients with aggressive atl even in the absence of clinical symptoms because a previous analysis revealed that more than half of relapses at new sites after chemotherapy occurred in the cns . however , the median survival time of 13 months in vcap - amp - vecp ( lsg15/mlsg15 ) still compares unfavorably to other hematological malignancies , requiring further effort to improve the outcome . a small phase ii trial in japan of ifn alpha against relapsed / refractory atl showed a response rate ( all pr ) of 33% ( 8/24 ) , including 5 out of 9 ( 56% ) chronic - type atl . in 1995 , gill and associates reported that 11 of 19 patients with acute- or lymphoma - type atl showed major responses ( 5 cr and 6 pr ) to a combination of interferon - alpha ( ifn ) and zidovudine ( azt ) . recently , the results of a meta - analysis on the use of ifn and azt for atl were reported . while the results for ifn / azt in indolent atl appear to be promising compared to those with watchful - waiting policy until disease progression , recently reported from japan , the possibility of selection bias can not be ruled out . recently , a phase ii study of the combination of arsenic trioxide , ifn , and azt for chronic atl revealed an impressive response rate and moderate toxicity . although the results appeared promising , the addition of arsenic trioxide to ifn / azt , which might be sufficient for the treatment of chronic atl as described above , caused more toxicity and should be evaluated with caution . allo - hsct is now recommended for the treatment of young patients with aggressive atl [ 31 , 59 ] . despite higher treatment - related mortality including graft versus host disease in a retrospective multicenter analysis of myeloablative allo - hsct , the estimated 3-year os of 33% is promising , possibly reflecting a graft versus atl effect . to evaluate the efficacy of allo - hsct more accurately , especially in view of a comparison with intensive chemotherapy , a prospective multicenter phase ii study of lsg15 chemotherapy followed by allo - hsct is ongoing ( jcog0907 ) . feasibility studies of allo - hsct with reduced intensity conditioning for relatively aged patients with atl also revealed promising results , and subsequent multicenter trials are being conducted in japan [ 61 , 62 ] . the minimal residual disease after allo - hsct detected as htlv-1 proviral load was much less than that after chemotherapy or azt / ifn therapy , suggesting the presence of a graft - versus - atl effect as well as graft - versus - htlv-1 activity . it remains unclear which type of allo - hsct ( myeloablative or reduced intensity conditioning ) is more suitable for the treatment of atl . however , several other retrospective studies as well as those mentioned above on allo - hsct showed a promising long - term survival rate of 20 to 40% with an apparent plateau phase despite significant treatment - related mortality . new guidelines were presented incorporating positron emission tomography ( pet ) , especially for the assessment of cr . it is well known and described in the criteria that several kinds of lymphoma including peripheral t - cell lymphomas were variably [ 18f ] fluorodeoxyglucose ( fdg ) avid . forodesine , a purine nucleotide phosphorylase ( pnp ) inhibitor , is among purine nucleotide analogs . purine nucleoside phosphorylase ( pnp ) deficiency in humans results in a severe combined immunodeficiency phenotype and the selective depletion of t cells associated with high plasma deoxyguanosine ( dguo ) and high intracellular deoxyguanosine triphosphate levels in those cells with high deoxynucleoside kinase activity such as t cells , leading to cell death . hdacis such as vorinostat ( suberoylanilide hydroxamic acid : saha ) , romidepsin ( depsipeptide ) , and panobinostat ( lbh589 ) have also shown promise in preclinical and/or clinical studies against t - cell malignancies including atl [ 70 , 71 ] . however , a phase ii study for ctcl and indolent atl in japan was terminated because of severe infections associated with the shrinkage of skin tumors and formation of ulcers in patients with atl . monoclonal antibodies ( moab ) and toxin fusion proteins targeting several molecules expressed on the surface of atl cells and other lymphoid malignant cells , such as cd25 , cd2 , cd52 , and chemokine receptor 4 ( ccr4 ) , have shown promise in recent clinical trials . lmb-2 , composed of the anti - cd25 murine moab fused to the truncated form of pseudomonas toxin , was cytotoxic to cd25-expressing cells including atl cells in vitro and in mice . denileukin diftitox ( dd ; dab(389)-interleukin-2 [ il-2 ] ) , an interleukin-2-diphtheria toxin fusion protein targeting il-2 receptor - expressing malignant t lymphocytes , shows efficacy as a single agent against ctcl and peripheral t - cell lymphoma ( ptcl ) . also the combination of this agent with multiagent chemotherapy , chop , was promising for ptcl . the humanized anti - cd52 monoclonal antibody alemtuzumab is active against cll and ptcl as a single agent . the combination of alemtuzumab with a standard - dose cyclophosphamide / doxorubicin / vincristine / prednisone ( chop ) regimen as a first - line treatment for 24 patients with ptcl showed promising results with cr in 17 ( 71% ) patients ; 1 had a partial remission , with an overall median duration of response of 11 months and was associated with mostly manageable infections but including cmv reactivation . atl cells express cd52 , the target of alemtuzumab , which was active in a preclinical model of atl and toxic to p53-deficient cells , and several atl cases successfully treated with this agent have been reported [ 8183 ] . p1 dose - escalating study of this agent in 22 patients with several kinds of t / nk - cell malignancy revealed 6 responses ( 2 cr in lgl leukemia , 3 pr in atl , and 1 pr in ctcl ) . cc chemokine receptor 4 ( ccr4 ) is expressed on normal t helper type 27 and regulatory t ( treg ) cells and on certain types of t - cell neoplasms [ 20 , 21 , 35 ] . kw-0761 , a next generation humanized anti - ccr4 mab , with a defucosylated fc region , exerts strong antibody - dependent cellular cytotoxicity ( adcc ) due to increased binding to the fc receptor on effecter cells . a phase i study of dose escalation with 4 weekly intravenous infusions of kw-0761 in 16 patients with relapsed ccr4-positive t cell malignancy ( 13 atl and 3 ptcl ) revealed that one patient , at the maximum dose ( 1.0 mg / kg ) , developed grade ( g ) 3 dose - limiting toxic effects , namely , skin rashes and febrile neutropenia and g4 neutropenia . a phase ii trial of single agent kw-0761 at the 1.0 mg / kg in relapsed ptcl / ctcl and a phase ii trial of vcap - amp - vecp combined with kw-0761 for untreated aggressive atl are ongoing . a proteasome inhibitor , bortezomib ( velcade ) , and an immunomodulatory agent , lenalidomide ( revlimid ) , both have potent preclinical and clinical activity in t - cell malignancies including atl , are now under clinical trials for relapsed atl in japan [ 8890 ] . other potential drugs for atl include pralatrexate ( folotyn ) , a new agent with clinical activity in t - cell malignancies including atl [ 9193 ] . the agent is a novel antifolate with improved membrane transport and polyglutamylation in tumor cells and high affinity for the reduced folate carrier ( rfc ) highly expressed in malignant cells and has been approved by fda recently for t - cell lymphoma including atl . clinical trials have been paramount to the recent advances in atl treatment , including assessments of chemotherapy , azt / ifn , and allo - hsct . recently , a strategy for atl treatment , stratified by subtype - classification , prognostic factors , and the response to initial treatment as well as response criteria , was proposed . the recommended treatment algorithm for atl however , atl still has a worse prognosis than the other t - cell malignancies .there is no plateau with an initial steep slope and subsequent gentle slope without a plateau in the survival curve for aggressive or indolent atl treated by watchful waiting and with chemotherapy , respectively , although the prognosis is much better in the latter . a prognostic model for each subgroup should be elucidated to properly identify the candidate for allo - hsct which can achieve a cure of atl despite considerable treatment - related mortality . furthermore , as described in the other chapters in detail , more than ten promising new agents for ptcl / ctcl including atl are now in clinical trials or preparation .

subsequently , a novel rna retrovirus , human t - cell leukemia / lymphotropic virus type i ( htlv-1 ) , was isolated from a cell line established from leukemic cells of an atl patient , and the finding of a clear association with atl led to its inclusion among human carcinogenic pathogens [ 25 ] . the incidence was highest in the chronic and smoldering types ( 36% ) and lower in the acute ( 27% ) and lymphoma types ( 11% ) . the histological analysis of aberrant cutaneous lesions or lymph nodes is essential for the diagnosis of the smoldering type with mainly skin manifestations and lymphoma type of atl , respectively . because atl cells in the skin and lymph node can vary in size from small to large and in form from pleomorphic to anaplastic and hodgkin - like cell with no specific histological pattern of involvement , differentiating between sezary syndrome , other peripheral t - cell lymphomas and hodgkin lymphoma versus atl can at times be difficult without examinations for htlv-1 serotype / genotype [ 13 , 19 ] . hypercalcemia is the most distinctive laboratory abnormality in atl as compared to other lymphoid malignancies and is observed in 31% of patients ( 50% in acute type , 17% in lymphoma type , and 0% in the other two types ) at onset . similar to serum ldh , 2-microglobulin , and serum thymidine kinase levels reflecting disease bulk / activity , the level of the soluble form of interleukin ( il)-2 receptor alpha - chain is elevated in the order of acute / lymphoma - type atl , smoldering / chronic - type atl , and htlv-1 carriers as compared with normal individuals , perhaps with better accuracy than the other markers [ 2224 ] . prototypical atl cells have a mature alpha - beta t - cell phenotype , that is , they are terminal deoxynucleotidyl transferase- ( tdt-)negative , cluster of differentiation ( cd ) 1a - negative , t - cell receptor alpha - beta positive , cd2-positive and cd5 , cd45ro , and cd29-positive , and frequently do not express cd7 and cd26 . recent studies have suggested that the cells of some atl may be the equivalent of regulatory t - cells because of the high frequency of expression of cd25/ccr4 and about half of foxp3 [ 2628 ] . the diagnosis of typical atl is not difficult and is based on clinical features , atl cell morphology , mature helper - t - cell phenotype , and anti - htlv-1 antibody in most cases . atl is a distinct peripheral t - lymphocytic malignancy associated with a retrovirus designated human t - cell leukemia virus type i or human t - cell lymphotropic virus type i ( htlv-1 ) [ 1 , 1114 , 31 ] . major prognostic indicators for atl , which have been elucidated in 854 patients with atl in japan , the lymphoma study group ( lsg ) of the japan clinical oncology group ( jcog ) by multivariate analysis , were advanced performance status ( ps ) , high lactic dehydrogenase ( ldh ) level , age of 40 years or more , more than 3 involved lesions , and hypercalcemia . the acute type has a rapid course with leukemic manifestation ( 2% atl cells ) mostly , with or without lymphocytosis ( > 4 109/l ) including atl cells and most of the characteristic features of atl - generalized lymphadenopathy , hepatosplenomegaly , skin involvement , other organ involvement , a high ldh value , and hypercalcemia . the chronic type , with absolute lymphocytosis ( 4 109/l ) less frequently showing flower cell morphology than the acute type , is frequently and occasionally associated with skin involvement and lymphadenopathy , respectively , and also usually shows a relatively indolent course . the lymphoma type presents with the manifestations of a nodal - lymphoma without leukemic cells , frequently with high ldh / ca levels , a rapid course , and symptoms and signs similar to the acute type . additional factors associated with a poor prognosis include thrombocytopenia , eosinophilia , bone marrow involvement , a high interleukin ( il)-5 serum - level , c - c chemokine receptor 4 ( ccr4 ) expression , lung resistance - related protein ( lrp ) , p53 mutation , and p16 deletion by multivariate analysis [ 26 , 27 , 3337 ] . specific for the chronic type of atl , high ldh , high blood urea nitrogen ( bun ) , and low albumin levels were identified as factors for a poor prognosis by multivariate analysis . therefore , patients with the smoldering or favorable chronic type , who may survive one or more years without chemotherapy , excluding topical therapy for cutaneous lesions , should be observed and therapy should be delayed until progression of the disease . in a long - term followup study for 78 patients with indolent atl ( favorable chronic- or smoldering - type ) with a policy of watchful waiting until disease progression at a single institution , the median survival time was 5.3 years with no plateau in the survival curve . between 1981 and 1983 , jcog conducted a phase iii trial ( jcog8101 ) to evaluate lsg1-vepa ( vincristine , cyclophosphamide , prednisone , and doxorubicin ) versus lsg2-vepa - m ( vepa plus methotrexate ( mtx ) ) for advanced non - hodgkin lymphoma ( nhl ) , including atl [ 40 , 41 ] . the complete response ( cr ) rate of lsg2-vepa - m for atl ( 37% ) was higher than that of lsg1-vepa ( 17% ; p = .09 ) . however , the cr rate was significantly lower for atl than for b - cell nhl and peripheral t - cell lymphoma ( ptcl ) other than atl ( p < .001 ) . lsg4 consisted of three regimens : ( 1 ) vepa - b ( vepa plus bleomycin ) , ( 2 ) m - fepa ( methotrexate , vindesine , cyclophosphamide , prednisone , and doxorubicin ) , and ( 3 ) vepp - b , ( vincristine , etoposide , procarbazine , prednisone , and bleomycin ) . the cr rate for atl patients was improved from 28% ( jcog8101 ) to 43% ( jcog8701 ) ; however , the cr rate was significantly lower in atl than in b - cell nhl and ptcl ( p < .01 ) . unfavorable chronic - type atl , defined as having at least 1 of 3 unfavorable prognostic factors ( low serum albumin level , high ldh level , or high bun ) , has an unfavorable prognosis similar to that for acute- and lymphoma - type atl . a regimen of 1 mg / m vincristine on days 1 and 8 , 40 mg / m doxorubicin on day 1 , 100 mg / m etoposide on days 1 through 3 , 40 mg / m prednisolone ( psl ) on days 1 and 2 , and 5 mg / m pentostatin on days 8 , 15 , and 22 was administered every 28 days for 10 cycles . dose intensification of chop with prophylactic use of g - csf was expected to improve survival among patients with aggressive nhl , and our randomized phase ii study ( jcog9505 ) comparing chop-14 ( lsg19 ) and dose - escalated chop ( lsg20 ) to treat aggressive nhl excluding atl revealed biweekly chop to be more promising . to confirm whether the lsg15 regimen is a new standard for the treatment of aggressive atl , jcog conducted a phase iii trial comparing modified ( m)-lsg15 with biweekly chop ( cyclophosphamide , hydroxy - doxorubicin , vincristine [ oncovin ] , and prednisone ) , both supported with g - csf and intrathecal prophylaxis . mlsg19 , a modified version of lsg19 , consisted of eight cycles of chop [ cpa 750 mg / m , adm 50 mg / m , vcr 1.4 mg / m(maximum 2 mg ) on day 1 and psl 100 mg on days 1 to 5 ] every 2 weeks . mlsg15 in jcog9801 was a modified version of lsg15 in jcog9303 , consisting of three regimens : vcap [ vcr 1 mg / m ( maximum 2 mg ) , cpa 350 mg / m , adm 40 mg / m , psl 40 mg / m ] on day 1 , amp [ adm 30 mg / m , mcnu 60 mg / m , psl 40 mg / m ] on day 8 , and vecp [ vds 2.4 mg / m on day 15 , etp 100 mg / m on days 15 to 17 , cbdca 250 mg / m on day15 , psl 40 mg / m on days 15 to 17 ] on days 1517 , and the next course was to be started on day 29 ( figure 1 ) . ( 2 ) cytarabine 40 mg was used with mtx 15 mg and psl 10 mg for prophylactic intrathecal administration , at the recovery phases of courses 1 , 3 , and 5 because of the high frequency of central nervous system relapse in the jcog9303 study . as shown in table 1 , the median survival time and os rate at 3 years were 12.7 months and 24% in the mlsg15 arm and 10.9 months and 13% in the biweekly chop arm [ two - sided p = .169 , and the hazard ratio was 0.75 ; 95% confidence interval ( ci ) , 0.50 to 1.13 ] . a cox regression analysis with performance status ( ps 0 versus 1 versus 24 ) as the stratum for baseline hazard functions was performed to evaluate the effect on overall survival of age , b - symptoms , subtypes of atl , ldh , bun , bulky mass , and treatment arms . according to this analysis , the hazard ratio and two - sided p value for the treatment arms were 0.62 ( 95% ci , 0.38 to 1.01 ) and .056 , respectively . in mlsg15 versus mlsg19 , rate of grade 4 neutropenia , grade 4 thrombocytopenia , and grade 3/4 infection were 98% versus 83% , 74% versus 17% , and 32% versus 15% , respectively . the longer survival at 3 years and higher cr rate with mlsg15 compared with mlsg19 suggest that mlsg15 is a more effective regimen at the expense of higher toxicity , providing the basis for future investigations in the treatment of atl . intrathecal prophylaxis , which was incorporated in both arms of the phase iii study , should be considered for patients with aggressive atl even in the absence of clinical symptoms because a previous analysis revealed that more than half of relapses at new sites after chemotherapy occurred in the cns . however , the median survival time of 13 months in vcap - amp - vecp ( lsg15/mlsg15 ) still compares unfavorably to other hematological malignancies , requiring further effort to improve the outcome . in 1995 , gill and associates reported that 11 of 19 patients with acute- or lymphoma - type atl showed major responses ( 5 cr and 6 pr ) to a combination of interferon - alpha ( ifn ) and zidovudine ( azt ) . although these results are encouraging , the os of previously untreated patients with atl was relatively short ( 4.8 months ) compared with the survival of those in the chemotherapy trials conducted by the jcog - lsg ( 7 to 8 months ) . in this worldwide retrospective analysis , the median survival time was 24 months and the 5-year survival rate was 50% for first - line ifn and azt , versus 7 months and 20% for 84 patients who received first - line chemotherapy . however , because of the retrospective nature of this meta - analysis based on medical records at each hospital , the decision process to select the therapeutic modality for each patient and the possibility of interference with os by second - line treatment remains unknown . while the results for ifn / azt in indolent atl appear to be promising compared to those with watchful - waiting policy until disease progression , recently reported from japan , the possibility of selection bias can not be ruled out . a prospective multicenter phase iii study evaluating the efficacy of ifn / azt as compared to watchful - waiting for indolent atl is to be initiated in japan . although the results appeared promising , the addition of arsenic trioxide to ifn / azt , which might be sufficient for the treatment of chronic atl as described above , caused more toxicity and should be evaluated with caution . despite higher treatment - related mortality including graft versus host disease in a retrospective multicenter analysis of myeloablative allo - hsct , the estimated 3-year os of 33% is promising , possibly reflecting a graft versus atl effect . to evaluate the efficacy of allo - hsct more accurately , especially in view of a comparison with intensive chemotherapy , a prospective multicenter phase ii study of lsg15 chemotherapy followed by allo - hsct is ongoing ( jcog0907 ) . the minimal residual disease after allo - hsct detected as htlv-1 proviral load was much less than that after chemotherapy or azt / ifn therapy , suggesting the presence of a graft - versus - atl effect as well as graft - versus - htlv-1 activity . purine nucleoside phosphorylase ( pnp ) deficiency in humans results in a severe combined immunodeficiency phenotype and the selective depletion of t cells associated with high plasma deoxyguanosine ( dguo ) and high intracellular deoxyguanosine triphosphate levels in those cells with high deoxynucleoside kinase activity such as t cells , leading to cell death . hdacis such as vorinostat ( suberoylanilide hydroxamic acid : saha ) , romidepsin ( depsipeptide ) , and panobinostat ( lbh589 ) have also shown promise in preclinical and/or clinical studies against t - cell malignancies including atl [ 70 , 71 ] . monoclonal antibodies ( moab ) and toxin fusion proteins targeting several molecules expressed on the surface of atl cells and other lymphoid malignant cells , such as cd25 , cd2 , cd52 , and chemokine receptor 4 ( ccr4 ) , have shown promise in recent clinical trials . denileukin diftitox ( dd ; dab(389)-interleukin-2 [ il-2 ] ) , an interleukin-2-diphtheria toxin fusion protein targeting il-2 receptor - expressing malignant t lymphocytes , shows efficacy as a single agent against ctcl and peripheral t - cell lymphoma ( ptcl ) . the combination of alemtuzumab with a standard - dose cyclophosphamide / doxorubicin / vincristine / prednisone ( chop ) regimen as a first - line treatment for 24 patients with ptcl showed promising results with cr in 17 ( 71% ) patients ; 1 had a partial remission , with an overall median duration of response of 11 months and was associated with mostly manageable infections but including cmv reactivation . atl cells express cd52 , the target of alemtuzumab , which was active in a preclinical model of atl and toxic to p53-deficient cells , and several atl cases successfully treated with this agent have been reported [ 8183 ] . p1 dose - escalating study of this agent in 22 patients with several kinds of t / nk - cell malignancy revealed 6 responses ( 2 cr in lgl leukemia , 3 pr in atl , and 1 pr in ctcl ) . a phase i study of dose escalation with 4 weekly intravenous infusions of kw-0761 in 16 patients with relapsed ccr4-positive t cell malignancy ( 13 atl and 3 ptcl ) revealed that one patient , at the maximum dose ( 1.0 mg / kg ) , developed grade ( g ) 3 dose - limiting toxic effects , namely , skin rashes and febrile neutropenia and g4 neutropenia . other treatment - related g3 - 4 toxic effects were lymphopenia ( n = 10 ) , neutropenia ( n = 3 ) , leukopenia ( n = 2 ) , herpes zoster ( n = 1 ) , and acute infusion reaction / cytokine release syndrome ( n = 1 ) . five patients ( 31% ; 95% ci , 11% to 59% ) achieved objective responses : 2 complete ( 0.1 ; 1.0 mg / kg ) and 3 partial ( 0.01 ; 2 at 1.0 mg / kg ) responses . responses in each lesion were diverse , that is , good in pb ( 6 cr and 1 pr/7 evaluable cases ) , intermediate in skin ( 3 cr and 1 pr/8 evaluable cases ) , and poor in ln ( 1 cr and 2 pr/11 evaluable cases ) . a phase ii trial of single agent kw-0761 at the 1.0 mg / kg in relapsed ptcl / ctcl and a phase ii trial of vcap - amp - vecp combined with kw-0761 for untreated aggressive atl are ongoing . a proteasome inhibitor , bortezomib ( velcade ) , and an immunomodulatory agent , lenalidomide ( revlimid ) , both have potent preclinical and clinical activity in t - cell malignancies including atl , are now under clinical trials for relapsed atl in japan [ 8890 ] . the agent is a novel antifolate with improved membrane transport and polyglutamylation in tumor cells and high affinity for the reduced folate carrier ( rfc ) highly expressed in malignant cells and has been approved by fda recently for t - cell lymphoma including atl . this has not been achieved partly because only about 5% of htlv-1 carriers develop the disease in their life time although several risk factors have been identified by a cohort study of htlv-1 carriers ( joint study of predisposing factors for atl development ) . the recommended treatment algorithm for atl however , atl still has a worse prognosis than the other t - cell malignancies .there is no plateau with an initial steep slope and subsequent gentle slope without a plateau in the survival curve for aggressive or indolent atl treated by watchful waiting and with chemotherapy , respectively , although the prognosis is much better in the latter .

leishmaniasis is a debilitating disease that is prevalent across the globe with 350 million people in 88 countries at risk of acquiring leishmaniasis . a recent effort by the world health organization to provide a current estimate of the incidence of leishmaniasis concluded that between 0.2 and 0.4 million cases of visceral leishmaniasis occur each year , while from 0.7 to 1.2 million cases of cutaneous leishmaniasis occur each year . this study also estimated that from 20 000 to 40 000 deaths occur each year due to leishmaniasis . leishmaniasis manifests itself in numerous forms depending on which parasite species infects the host . the parasites are transferred from host to host by about 30 species of female sandfly vectors of the genera phlebotomus and lutzomyia that infect the host when taking a blood meal . symptoms of leishmaniasis include unsightly spontaneously healing ulcers on the skin when cutaneous leishmaniasis is present , nonhealing lesions in the mucosa when mucocutaneous leishmaniasis is the affliction , and chronic , debilitating infection of the reticuloendothelial system that is fatal if left untreated due to visceral leishmaniasis . the majority of cases of visceral leishmaniasis are caused by l. donovani in east africa and asia , l. infantum in the mediterranean region , and l. chagasi in latin america . infantum and l. chagasi mainly affect children and immunocompromised individuals and are zoonotic parasites with canines being a major reservoir.l . donovani , on the other hand , is an anthroponotic parasite that affects a broad range of ages . for over 100 years , antimonials have been the drug of choice to combat leishmaniasis . in 1912 gaspar vianna first reported the use of the trivalent antimonial tartar emetic for the treatment of cutaneous leishmaniasis caused by l. braziliensis in brazil . shortly thereafter mccombie young and upendranath brahmachari used trivalent and pentavalent antimonials to treat visceral leishmaniasis in india with great success , decreasing the mortality rate of 95% to just 10% in 10 years ( figure 1a ) . pentavalent antimonials such as meglumine antimoniate and sodium stibogluconate are currently the first line antileishmanial drugs in many areas . problems with this treatment include a high rate of resistance that has been encountered in india , especially the state of bihar , where up to 60% of infected individuals do not improve with treatment . the high rate of resistance to pentavalent antimonials in india has led to the increasing use of amphotericin b and miltefosine against visceral leishmaniasis . since the 1960s it has a cure rate of over 90% but is often accompanied by severe side effects such as nephrotoxicity that require administration in a hospital setting . lipid formulations of amphotericin b have fewer side effects and are safer to use with the same cure rate . depending on the dose and formulation , the treatment regimen varies from 3 to 5 days to 8 weeks of administration on alternate days . miltefosine is the first oral drug to be released for leishmaniasis and is currently available in india , germany , and colombia . miltefosine is not recommended for women who are pregnant or may become pregnant because it is teratogenic . miltefosine resistance has been demonstrated in vitro , and its long half - life in the body , the 28-day treatment regimen , and it previously being available over the counter in india have led to concerns of clinical resistance . a recent study of 567 individuals in the bihar state of india has been performed to determine the efficacy of miltefosine since its introduction in 2002 . the 6-month cure rate was found to be roughly 90% and gastrointestinal intolerance was encountered in 64.5% of the cases with two deaths related to drug toxicity . patients who did not improve with treatment were cured using amphotericin b. the authors of this study concluded that the failure rate of miltefosine has increased in the 10 years since its introduction for the treatment of visceral leishmaniasis in india . a recent study also showed that 20% of the visceral leishmaniasis patients in nepal who were treated with miltefosine relapsed 12 months after treatment . because of increased parasite resistance , toxicity issues , increasing failure rates of current treatments , and the lack of effective clinical agents against cutaneous leishmaniasis , new drugs are needed to have an effective strategy for treating leishmaniasis . berman et al . reported a class of 2,4-diaminoquinazolines with ec50 as low as 0.04 nm against l. major amastigotes in human monocyte - derived macrophages ( a , figure 1b ) ; however the development of this compound series was abandoned because of toxicity issues . this class of compounds has been reported as being dihydrofolate reductase ( dhfr ) inhibitors , although another mechanism of action may be involved with leishmania . recently , we tested a small library of structurally diverse compounds , originally designed as potential anticancer probes , for antileishmanial activity in a l. mexicana axenic amastigote assay . among this library were n , n - disubstituted quinazolines 1 and 2 ( figure 1b ) , which are structurally different from quinazoline series a e . antileishmanial testing of quinazolines 1 and 2 against l. mexicana axenic amastigotes revealed ec50 values in the single digit micromolar range , motivating us to investigate whether quinazolines structurally related to the hits 1 and 2 have potential to display potent antileishmanial activity . herein , we report a detailed structure activity relationship ( sar ) study focusing on the 2-position , the 4-position , and the quinazoline s benzenoid ring . all compounds were initially examined in l. donovani and l. amazonensis intracellular amastigote assays to preselect quinazoline candidates active against parasites responsible for causing visceral leishmaniasis and cutaneous leishmaniasis , respectively . promising compounds have subsequently been tested for efficacy in a murine model of visceral leishmaniasis . antileishmanial compounds : ( a ) structures of currently used antileishmanial drugs ; ( b ) reported structures of quinazolines diplaying antileishmanial activity and including the hit compounds 1 and 2 and sar studies targeting the major quinazoline sites . commercially available anthranilic acids ( a ) were cyclized with urea , and the resulting quinazoline-2,4-dione ( b ) was reacted with phosphorus oxychloride to give the 2,4-dichloroquinazoline ( c ) . substitution with amines occurred selectively at position 4 , yielding 4-amino-2-chloroquinazoline ( d ) followed by substitution at position 2 to give the 2,4-diamino - substituted quinazoline . in this synthetic sequence , only 4-amino-2-chloroquinazoline ( d ) and the final n , n - disubstituted quinazoline-2,4-diamine have been purified and characterized . all target compounds were tested against l. donovani and l. amazonensis intracellular amastigotes to identify molecules that are broadly active against these medically important leishmania species . the testing involved murine peritoneal macrophages as host cells , since compounds with potential for clinical use must be able to penetrate the infected macrophage in a human host . antileishmanial activity was determined in an assay using transgenic parasites expressing a -lactamase gene as outlined previously . concentration response data for each compound was fitted by a nonlinear regression model , and the concentration that induces 50% inhibition was calculated as the effective concentration ec50 ( l. donovani or l. amazonensis . ) . additionally , cytotoxicity against the macrophage cell line j774a.1 was determined as the effective concentration ec50 ( j774a.1 ) and the selectivity index si was calculated as the ratio of ec50 value for j774a.1 and the value for l. donovani ( si = ec50(j774a.1)/ec50(l . donovani ) ) . to validate and optimize the antileishmanial activity of n , n - disubstituted quinazoline-2,4-diamines , two compound subseries were prepared and tested . the first subseries focused mainly on the optimization of the n- and n - moieties ( table 1 ) , whereas the second subseries was designed to investigate whether analogues being substituted at the quinazoline s benzenoid ring display improved antileishmanial activity ( table 2 ) . starting from hit compound 2 , n - furfuryl analogues 3 and 4 were prepared in which the n - isopropyl group was replaced by a short trifluoroalkyl or hydroxyalkyl group . while the 2,2,2-trifluoroethyl - substituted quinazoline 3 was slightly less potent than compound 2 , alcohol 4 lost potency against l. donovani and l. amazonensis by a factor of 10 and > 13 , respectively . testing of a small set of quinazoline-2,4-diamines 59 with n - monosubstituted or n - disubstituted with alkyl groups differing in size and polarity did not identify a particular structural motif improving the potency over 2 . replacement of the furfuryl and isopropyl groups in 2 by two benzyl or two n - butyl groups yielded quinazolines 10 and 11 , of which both analogues were more potent than reference 2 . bis - benzyl - substituted quinazoline 10 displayed ec50 values of 670 nm against l. donovani and 1.4 m against l. amazonensis , whereas analogue 11 was approximately 2-fold less potent in comparison to compound 10 . consequently , a following set of six quinazolines 1217 was designed in which one of the 2- and 4-positions was substituted by one benzylamine , while the remaining position was derivatized with an aniline , n - butylamine , or methylamine . interestingly , with the exception of the n - benzyl - n - methylquinazoline-2,4-diamine 12 , all of these compounds demonstrated submicromolar ec50 values against l. donovani . among these six quinazolines tested , the n - benzylquinazoline-2,4-diamines 1517 appeared to be at least 2-fold more potent against l. donovani than the n - benzyl counterparts 1214 , suggesting that an n - benzyl is more favorable than an n - benzyl for antileishmanial activity . compound 15 was the most potent compound with an ec50 of 150 nm against l. donovani . for l. amazonensis , the set of 1217 displayed a similar activity trend , with n - benzylquinazolines 15 and 16 being the most potent compounds with submicromolar or single digit micromolar ec50 values . amphotericin b is the internal control for the in vitro antileishmanial activity assay with ec50 = 40 9 nm against l. donovani and ec50 = 89 16 nm against l. amazonensis . podophyllotoxin is the internal control for the in vitro cytotoxicity assay with ec50 = 250 10 nm against j774a.1 . nfurfuryl - n - isopropyl - substituted quinazoline 2 , with ec50 values of 2.5 m against l. donovani and 3.7 m against l. amazonensis , was considered to be well suited as the key scaffold for a sar study focusing on the benzenoid moiety of the quinazoline core . in a systematic approach following the topliss operational scheme , compound 2 was monosubstituted with a chlorine atom , a methyl group , or a methoxy group in the 5- , 6- , 7- , and 8-positions to probe the benzenoid ring for steric and electronic effects . overall , substitution on the benzenoid ring provided compounds with ec50 values in the single digit micromolar or submicromolar range against l. donovani . among the chloro - substituted subseries , 5- and 6-substituted analogues 18 and 19 were less potent by a factor of 2 or more compared to reference 2 , while the 7- and 8-substituted quinazolines 20 and 21 displayed an activity on par with compound 2 . in contrast , the majority of the methyl- and methoxy - substituted quinazolines demonstrated a modest potency improvement over quinazoline 2 . for the methoxy - substituted quinazolines , potency increased according to substitutions in 8-position < 7-position < 5-position 6-position , whereas a potency dependence in the order of 8-position 6-position < 7-position 5-position was observed for the methyl - substituted compounds . 7-methoxyquinazoline 28 with an ec50 of 740 nm against l. donovani was the most potent analogue of the compound subseries substituted at the benzenoid ring . in contrast , the testing of the benzenoid ring substituted quinazolines against l. amazonensis gave insight into a sar , which differed from the one observed for l. donovani . 8-methoxy - substituted quinazoline 29 was the only analogue that was marginally more potent than reference compound 2 , whereas all of the other analogues were equally or less potent than quinazoline 2 . amphotericin b is the internal control for the in vitro antileishmanial activity assay with ec50 = 40 9 nm against l. donovani and ec50 = 89 16 nm against l. amazonensis . podophyllotoxin is the internal control for the in vitro cytotoxicity assay with ec50 = 250 10 nm against j774a.1 . an important quality of the quinazolines is their selectivity to inhibit parasite growth over mammalian cells . generally , the majority of the quinazolines 229 exhibited ec50 of 20 m or higher against j774a.1 ( tables 1 and 2 ) . quinazolines 1820 and 2224 , whose benzenoid ring is substituted with one chloride or one methyl in 5- , 6- , or 7-position , were significantly less toxic than their reference 2 . the quinazolines displaying potent antileishmanial activity , especially the n - benzylquinazoline-2,4-diamines 1517 , were shown to have respectable si values of 10 and larger , indicating that they are relatively selective , nontoxic chemotypes . the si of 100 for compound 15 was particularly enticing and led to this compound being tested in vivo . the potency of selected quinazolines against antimony - resistant leishmania was assessed using murine peritoneal macrophages infected with two l. donovani clinical strains , bpk206/0 and bpk164/1 ( table 3 ) . strain bpk164/1 was isolated from a bone marrow aspirate taken from a nepalese visceral leishmaniasis patient not responding to antimonial therapy ; the bpk164/1 strain was originally found to be 6-fold resistant to sodium stibogluconate compared to the antimony - susceptible reference strain bpk206/0 . compounds 15 , 16 , and 23 displayed similar activity against the antimony - susceptible and antimony - resistant parasites . the ec50 values against the clinical strains were 5- to 19-fold higher than their ec50 values against -lactamase expressing l. donovani ( table 1 ) . there are several possible reasons for the differences in susceptibility observed between the clinical l. donovani isolates and the -lactamase expressing genetically modified lab strain : ( 1 ) the former strains are from the indian subcontinent , while the lab strain is of african origin . antileishmanial drugs display differences in efficacy for treating indian visceral leishmaniasis compared to african visceral leishmaniasis ; strain differences could account for such differential susceptibility ; ( 2 ) the clinical strains could have a greater fitness in an in vitro intracellular amastigote model than the lab adapted strain which has been in axenic culture for many passages , leading to lower compound susceptibility in the former ; ( 3 ) the clinical strains were assayed using a different method under different conditions compared to the -lactamase expressing lab strain , potentially contributing to distinctions in compound susceptibility . nevertheless , these data confirm the in vitro activity of compounds 15 , 16 , and 23 against clinical strains currently circulating in endemic regions of the indian subcontinent . values for sodium stibogluconate are given in micrograms of pentavalent antimony per milliliter . we investigated the possibility that quinazolines inhibit folate metabolism in the parasite , since previous studies have shown dhfr inhibition with similar structures.l . donovani axenic amastigotes and j774a.1 ( mouse macrophage cell line ) were adapted to grow in media deficient in p - aminobenzoic acid ( paba ) and folic acid prior to susceptibility testing . susceptibility to quinazolines and miltefosine , as well as the known antifolate drugs methotrexate and pyrimethamine as positive controls , was assessed in the presence or absence of folinic acid ( figure 3 ) . the presence of folinic acid dramatically increased the ec50 values of each of the quinazolines as well as the methotrexate and pyrimethamine . for example , quinazoline 23 was 6.4-fold less potent in the presence of 488 nm folinic acid than in completely deficient media . conversely , efficacy of miltefosine was not affected by addition of folinic acid to the media . interestingly , we saw no antagonism of activity of quinazolines 10 , 12 , 13 , 15 , 16 , or 23 for the mammalian cell line ( j774a.1 ) in the presence of folinic acid . for example , the ec50 of quinazoline 10 was 84.8 2.2 and 84.2 2.8 m in the presence or absence of 488 nm folinic acid , respectively . in contrast , the efficacy of methotrexate and pyrimethamine was antagonized significantly by folinic acid in j774a.1 . these results are consistent with the hypothesis that the quinazolines interfere with folate metabolism in l. donovani . in vitro efficacy of quinazolines , methotrexate ( mtx ) , pyrimethamine ( pyr ) , and miltefosine ( milt ) for axenic amastigotes of leishmania donovani ( a c ) and j774.a1 macrophages ( d f ) in the absence or presence of d , l - folinic acid ( fna ) . results are presented as the ec50 ( m ) in the absence or presence of increasing concentrations of fna . in parallel to the testing of the compounds for antileishmanial activity in vitro , a structure property relationship ( spr ) study focusing on log d , aqueous solubility , and permeability has been conducted with all compounds to assess potential physicochemical liabilities ( table 4 ) . the log d3.0 and log d7.4 , the distribution coefficient between octanol and water at ph 3.0 and ph 7.4 , were experimentally determined via a previously described hplc - based method . solubility at ph 7.4 was determined using biomek fx lab automation workstation with pion sol evolution software as reported previously and at ph 2.0 using an in - house hplc assay based on uv absorption . passive transcellular permeability was assessed in a standard parallel artificial membrane permeability assay ( pampa ) at ph 7.4 and ph 4.0 . generally , the aqueous solubility , the distribution coefficient log d , and the permeability of all quinazolines display a ph dependence ( exemplified on log d or permeability in table 4 ) . the permeability is enhanced at neutral ph , while the aqueous solubility and log d are better at lower ph ranges . however , since the aqueous solubility , the distribution coefficient , and the permeability are within the acceptable ranges ( solubility of > 20 m , pe > 10 10 cms , 1 < log d < 4 ) , the quinazoline compound series is considered to be suitable for the development of bioavailable antileishmanial compounds . ( ) for solubility 30 m . for the determination of the pe values , the following internal controls were utilized : carbazepine ph 4.0 permeability pe = 108 10 cm / s and ph 7.4 permeability pe = 130 10 cm / s ; ranitidinehcl ph 4.0 permeability pe = 5.2 10 cm / s and ph 7.4 permeability pe = 2.2 10 cm / s ; verapamilhcl ph 4.0 permeability pe = 20.6 10 cm / s and ph 7.4 permeability pe = 1360 10 cm / s . n / d : not determined . for the selection of the candidates for in vivo efficacy against l. donovani , only compounds that displayed submicromolar in vitro activity against l. donovani with a si value greater than 10 and a balanced combination of good physicochemical properties were chosen . among the benzyl - substituted quinazolines 1217 , 15 and 16 appeared to be the best candidates for in vivo evaluation because of their combination of potency , selectivity , and favorable physicochemical properties . from the compound subseries substituted at the benzenoid ring , the physicochemical properties were not discriminatory , and hence , compound 23 was chosen as a viable candidate because of its submicromolar ec50 against l. donovani as well as the outstanding si value of this compound ( > 40 ) . the three compounds mentioned above were dissolved in an appropriate vehicle ( 15 and 23 dissolved in 0.5% methylcellulose and 0.1% tween 80 in water ; 16 dissolved in 45% ( w / v ) ( 2-hydroxypropyl)--cyclodextrin solution ( hpcd ) ) and administered to uninfected balb / c mice intraperitoneally to determine a tolerated dose for in vivo efficacy studies . while 15 was well tolerated when given at 30 mg / kg ip for 5 consecutive days , 16 ( slowed breathing ) and 23 ( hypoactivity ) were toxic to animals when given at the same dosing regimen . considering the toxicity of 16 and 23 when given at 30 mg / kg ip , lower doses of these compounds were administered in subsequent in vivo efficacy studies in a murine visceral leishmaniasis model ( figure 4 ) . when tested at 5 15 mg / kg ip , 23 inhibited liver parasitemia by 37% compared to the vehicle control . however , 15 did not show significant antileishmanial efficacy when tested at 5 30 mg / kg ip . there was also no significant difference in the parasite burden between mice in the group treated with compound 16 ( 5 10 mg / kg ip ) and the vehicle control group ( p > 0.05 ) . as expected , the 45% hpcd vehicle used to solubilize 16 itself resulted in 18% inhibition of liver parasitemia , consistent with our previous report . in vivo efficacy of quinazolines against lv82 in l. donovani infected balb / c mice . results are presented as the liver parasitemia ( ldu ) for each mouse ( ) , and the average ldu in each group ( ) was determined by microscopy ( n = 4 ) : ( a ) ldu for mice treated with 16 and 23 ; ( b ) ldu for mice treated with 15 . compounds 15 and 23 were dissolved in 0.5% methylcellulose and 0.1% tween 80 ( mc ) , while compound 16 was dissolved in 45% ( w / v ) ( 2-hydroxypropyl)--cyclodextrin solution ( hpcd ) . pharmacokinetic studies were conducted to determine the systemic and target tissue exposures of 16 and 23 . the mean plasma and tissue concentration time profiles of 16 and 23 after po and ip administration are shown in figures 5 and 6 , respectively . after po administration at 100 mol / kg ( or approximately 30 mg / kg ) , both compounds were absorbed from the gastrointestinal tract of mice and plasma concentration reached a cmax of 0.44 and 0.25 m for 16 and 23 , respectively . the systemic and tissue exposure of 16 ( auc and cmax ) were considerably greater than those of 23 ( table 4 ) . after ip administration , plasma concentration reached a cmax of 5.2 and 2.7 m for 16 and 23 , respectively , before decreasing rapidly in the first 4 h , followed by a slower elimination process until 24 h. the rapid decline in plasma concentration was likely due to extensive tissue redistribution after absorption , as indicated by the high target tissue concentrations . the plasma and tissue exposures after ip administration were markedly greater than those after po administration ( table 4 ) , suggesting significant first - pass metabolism and/or partial gastrointestinal absorption after oral administration of 16 and 23 . the terminal elimination half - life ranged from 5 to 20 h. minor reversible overt toxicity ( hypoactivity ) was observed after ip administration of 16 ; however , more severe toxicity was observed , unexpectedly , after a single ip administration of 23 , which warranted euthanization of some mice within 15 min postdose . as efficacy was evaluated at lower doses than 30 mg / kg to avoid toxicity and dose linearity for pharmacokinetic outcomes were unknown , it is not possible to directly correlate drug exposure with antileishmanial activity in mice . in addition , it is worth pointing out that the terminal half - life of 23 after ip administration was considerably shorter than that after po administration ( 5 h vs 20 h ; table 5 ) , and the liver concentration of 23 was detectable at 12 and 24 h after po administration , whereas it was below the detection limit after ip administration ( figure 6 ) . these observations suggest flip - flop pharmacokinetics , where the rate of gastrointestinal absorption is slower than the rate of elimination due to dosage formulations ( e.g. , sustained release ) , excipients , physiological factors ( e.g. , intestinal mobility and ph ) , and/or drug characteristics . the dramatic decrease ( 12-fold ) in the permeability of 23 from neutral to acidic ph ( table 4 ) could contribute to the slowed absorption of this compound in the upper gastrointestinal tract . in contrast , the permeability of 16 decreased only 3-fold from neutral to acidic ph ( table 4 ) . furthermore , 23 was metabolized quickly in the mouse liver microsomes ( t1/2 = 9.4 min ; table 5 ) , suggesting that it is possible that the rate of elimination was greater than the rate of gastrointestinal absoption for 23 . plasma ( open circles ) and tissue ( squares for liver and triangles for spleen ) concentration time profiles after po ( a ) and ip ( b ) administration of 16 in mice at a dose level of 100 mol / kg ( 30 mg / kg ) . symbols and error bars represent the mean and standard error of triplicate determinations except for those labeled with asterisks where only one or two determinations were obtained because of sample loss . plasma ( open circles ) and tissue ( squares for liver and triangles for spleen ) concentration time profiles after po ( a ) and ip ( b ) administration of 23 in mice at a dose level of 100 mol / kg ( 30 mg / kg ) . symbols and error bars represent the mean and standard error of triplicate determinations except for those labeled with asterisks where only one or two determinations were obtained because of sample loss . 23 was below the detection limit ( 0.1 m ) in the liver and spleen 12 and 24 h after ip administration . auc ( 024 h ) was calculated for plasma using noncompartmental analysis , whereas auc ( 124 h ) was calculated for tissues using trapezoid rule . nc , not calculable because of lack of a data point . in vitro mouse liver microsomeal half - life . n , n - disubstituted quinazoline-2,4-diamines 1 and 2 were found to display antileishmanial activity in the single digit micromolar range . subsequently a total of 28 molecules have been synthesized systematically by varying the substitutions in the 2- , 4- , 5- , 6- , 7- , and/or 8-positions . all quinazolines have been tested with the aim to further optimize hit compounds 1 and 2 and to conduct a detailed sar study against l. donovani and l. amazonensis . the most potent activities with ec50 values in the submicromolar range against l. donovani were obtained with quinazoline-2,4-diamine scaffolds bearing either a n - benzyl - n - alkyl / phenyl or a n - ispropyl - n - furfuryl substituent combination . furthermore , although the benzenoid ring of the quinazoline-2,4-diamine scaffold has been identified to play a secondary role for efficacy , quinazolines substituted at the 5- or 6-position with one methyl or one methoxy group have also been identified to possess submicromolar ec50 values against l. donovani . although the quinazoline-2,4-diamines appear to display some cytotoxicity against the macrophage cell line j774a.1 yielding modest si values , the si values of the best antileishmanial quinazolines were larger than 10 . in addition , assessment of key physicochemical properties confirmed that the quinazoline s aqueous solubility , distribution coefficient , and passive transcellular permeability were in acceptable ranges . these promising results led to efficacy testing of the lead compounds 15 , 16 , and 23 in an in vivo murine visceral leishmaniasis model . while compounds 15 and 16 did not have activity translate from in vitro to in vivo , quinazoline 23 reduced parasitemia by 37% when 15 mg kg day was given via the intraperitoneal route for 5 consecutive days . pharmocokinetic studies of compound 23 revealed a maximum plasma concentration that was 3-fold higher than the ec50 and a terminal half - life of 5 h after ip administration . although a clear correlation between in vitro activity , in vitro physicochemical properties , and in vivo activity is not clearly observed , the potencies of front - runner compounds 15 , 16 , and 23 in conjunction with favorable physicochemical properties make n , n - disubstituted quinazoline-2,4-diamines a suitable platform for the future development of antileishmanial agents . for future compound design to be successful , optimization will focus not only on improving antileishmanial activity and key physicochemical properties but also on improving the pharmacokinetics and si values for the entire quinazoline compound series . all reagents and solvents were obtained from aldrich chemical co. and used without further purification . anthranilic acids were purchased from sigma - aldrich , oakwood products , inc . , or tci america . nmr spectra were recorded at ambient temperature on a 250 mhz bruker , 400 mhz varian , or 500 mhz varian nmr spectrometer in the solvent indicated . all h nmr experiments are reported in units , parts per million ( ppm ) downfield of tms , and were measured relative to the signals for chloroform ( 7.26 ppm ) , methanol ( 3.31 ppm ) , and dimethylsulfoxide ( 2.50 ppm ) . all c nmr spectra were reported in ppm relative to the signals for chloroform ( 77 ppm ) , methanol ( 49 ppm ) , and dimethylsulfoxide ( 39.5 ppm ) with h decoupled observation . data for h nmr are reported as follows : chemical shift ( ppm ) , multiplicity ( s = singlet , d = doublet , t = triplet , q = quartet , p = pentet , sext = sextet , sept = septet , oct = octet , m = multiplet ) , integration , and coupling constant ( hz ) , whereas c nmr analyses were reported in terms of chemical shift . the purity of the final compounds was determined to be 95% by high pressure liquid chromatography ( hplc ) using an agilent 1100 lc / msd - vl with electrospray ionization . melting points were determined using a mel - temp 3.0 instrument and are uncorrected . low resolution mass spectrometry was performed on an agilent 1100 lc / msd - vl with electrospray ionization . high resolution mass spectrometry ( hrms ) was performed on an agilent lc / msd tof system g3250aa . analytical thin layer chromatography ( tlc ) was performed on silica gel 60 f254 precoated plates ( 0.25 mm ) from emd chemical inc . , and components were visualized by ultraviolet light ( 254 nm ) . silicycle silica gel 230400 ( particle size 4063 m ) mesh was used for all flash column chromatography experiments . 1 equiv of anthranilic acid and 3.5 equiv of urea were mortar and pestled to a powder and heated to 200 c in a round - bottom flask open to the atmosphere . after 2 h , the mixture was cooled , triturated with water , and filtered to give the product as crude . no further purification was performed . 1 equiv of quinazoline-2,4-dione and 1 equiv of n , n - dimethylaniline were mixed in 12 equiv of phosphorus oxychloride , and the mixture was refluxed under an argon atmosphere until starting material was no longer present by tlc ( 316 h ) . the mixture was then cooled and added to ice in the amount of 10 times the reaction volume . 1.1 equiv of amine and sodium acetate were mixed with 1 equiv of 2,4-dichloroquinazoline at 0.1 m concentration in a 3 to 1 mix of tetrahydrofuran and water and heated to 65 c . when the reaction was observed to be finished by tlc , the solution was diluted with ethyl acetate , the layers were separated , and the organic phase was washed three times with an equal amount of water and dried over na2so4 . the crude was then purified by either method ca or cb : the compound was recrystallized with ethanol and water , filtered , and rinsed with cold ethanol to yield pure product . 1.5 equiv of amine was mixed with 1 equiv of 4-amino - substituted 2-chloroquinazoline at 0.2 m concentration in ethanol in a sealed tube and heated to 150 c . when the reaction was finished as observed by tlc , the compound was purified by either method da or db : the compound crystallized out of the cool solution and was filtered and rinsed with cold ethanol to yield pure product . the solvent was evaporated , and the crude mixture was purified by flash chromatography using dichloromethane and methanol . commercially available benzoyleneurea ( 0.12 mol ) and n , n - dimethylaniline ( 0.12 mol ) were mixed in 60 ml of phosphorus oxychloride and heated to reflux under an atmosphere of argon . after 5 h the solution was cooled and slowly added to 300 ml of ice . once quenching was finished , the compound was extracted with chloroform ( 4 125 ml ) and purified by flash chromatography using hexanes and ethyl acetate to yield the title compound in 61% yield ( 14.5 g , 73 mmol ) . h nmr ( 500 mhz , cdcl3 ) 8.298.24 ( m , 1h ) , 8.027.99 ( m , 2h ) , 7.74 ( ddd , j = 6.3 , 5.0 , 3.2 , 1h ) . c nmr ( 126 mhz , cdcl3 ) 164.25 , 155.36 , 152.60 , 136.41 , 129.61 , 128.25 , 126.42 , 122.59 . mass ( esi ) : [ m + h ] 199 , 201 ; found 198.9 ( 100% ) , 200.9 ( 64% ) . 1 g ( 5.0 mmol ) of 1c was reacted with furfurylamine and purified according to method ca to furnish 1.21 g ( 4.7 mmol ) of the title compound in 93% yield . h nmr ( 250 mhz , cdcl3 ) 7.757.55 ( m , 3h ) , 7.34 ( ddd , j = 8.1 , 5.4 , 2.9 hz , 1h ) , 6.48 ( s , 1h ) , 6.306.22 ( m , 2h ) , 4.77 ( d , j = 5.1 hz , 2h ) . c nmr ( 63 mhz , cdcl3 ) 160.57 , 157.54 , 150.82 , 150.24 , 142.56 , 133.62 , 127.66 , 126.32 , 121.09 , 113.24 , 110.65 , 108.66 , 38.43 . mass ( esi ) : [ m + h ] 260 ; found 260.1 . 1.05 g ( 4.04 mmol ) of 2d was reacted with isopropylamine and purified according to method da to furnish 0.47 g ( 1.66 mmol ) of the title compound as a light yellow solid in 41% yield . h nmr ( 500 mhz , cd3od ) 7.94 ( d , j = 8.1 hz , 1h ) , 7.537.46 ( m , 1h ) , 7.447.40 ( m , 1h ) , 7.31 ( s , 1h ) , 7.177.09 ( m , 1h ) , 6.386.31 ( m , 2h ) , 4.78 ( s , 2h ) , 4.26 ( sept , j = 6.5 hz , 1h ) , 1.27 ( d , j = 6.5 hz , 6h ) . c nmr ( 126 mhz , cd3od ) 160.14 , 154.22 , 151.24 , 142.01 , 141.97 , 134.05 , 123.14 , 123.04 , 118.38 , 110.17 , 109.87 , 107.44 , 43.29 , 37.65 , 21.62 . hrms : m / z calcd for c16h19n4o [ m + h ] 283.1553 ; found 283.1558 . 0.045 g ( 0.17 mmol ) of 2d was reacted with 2,2,2-trifluoroethylamine and purified according to method da to furnish 0.041 g ( 0.13 mmol ) of the title compound as a white crystalline solid in 76% yield . h nmr ( 500 mhz , cd3od ) 8.17 ( d , j = 8.2 , 1h ) , 7.79 ( dd , j = 8.4 , 7.3 , 1h ) , 7.51 ( d , j = 7.1 , 1h ) , 7.487.45 ( m , 1h ) , 7.44 ( dd , j = 7.3 , 1.0 , 1h ) , 6.39 ( m , 2h ) , 4.89 ( s , 2h ) , 4.38 ( q , j = 9.0 , 2h ) . c nmr ( 126 mhz , cd3od ) 160.64 , 153.51 , 150.23 , 142.31 , 138.72 , 135.45 , 125.29 , 124.36 ( q , j = 278.71 ) , 123.64 , 116.87 , 110.16 , 110.05 , 107.89 , 41.73 ( q , j = 35.03 ) , 38.11 . hrms : m / z calcd for c15h14f3n4o g ( 0.46 mmol ) of 2d was reacted with 2-aminoethanol and purified according to method da to furnish 0.048 g ( 0.17 mmol ) of the title compound in 37% yield . h nmr ( 500 mhz , cd3od ) 8.11 ( d , j = 7.6 hz , 1h ) , 7.76 ( ddd , j = 8.4 , 7.3 , 1.3 hz , 1h ) , 7.46 ( d , j = 0.8 hz , 1h ) , 7.40 ( t , j = 7.5 hz , 2h ) , 6.41 ( d , j = 2.9 hz , 1h ) , 6.406.36 ( m , 1h ) , 3.77 ( s , 2h ) , 3.70 ( s , 2h ) . c nmr ( 101 mhz , cdcl3 ) 159.87 , 159.63 , 151.51 , 150.07 , 142.05 , 132.90 , 124.21 , 121.55 , 121.39 , 110.88 , 110.45 , 107.64 , 63.77 , 44.88 , 37.89 . hrms : m / z calcd for c15h17n4o2 [ m + h ] 285.1346 ; found 285.1350 . 0.050 g ( 0.25 mmol ) of 1c was reacted with 2,2,2-trifluoroethylamine and purified according to method cb to furnish 0.017 g ( 0.065 mmol ) of the title compound in 26% yield . h nmr ( 400 mhz , cd3od ) 8.16 ( d , j = 7.2 hz , 1h ) , 7.82 ( t , j = 7.4 hz , 1h ) , 7.68 ( d , j = 7.5 hz , 1h ) , 7.58 ( t , j = 7.7 hz , 1h ) , 4.39 ( q , j = 8.9 hz , 2h ) . mass ( esi ) : [ m + h ] 235 , 237 ; found 235.0 ( 100% ) , 237.1 ( 33% ) . 0.017 g ( 0.065 mmol ) of 5d was reacted with isopropylamine and purified according to method db to furnish 0.005 g ( 0.018 mmol ) of the title compound as a white crystalline solid in 28% yield . h nmr ( 500 mhz , cd3od ) 7.91 ( dd , j = 8.2 , 1.1 , 1h ) , 7.60 ( ddd , j = 8.4 , 7.0 , 1.4 , 1h ) , 7.38 ( d , j = 8.4 , 1h ) , 7.17 ( ddd , j = 8.1 , 7.0 , 1.0 , 1h ) , 4.38 ( q , j = 9.3 , 2h ) , 4.24 ( sept , j = 6.5 , 1h ) , 1.26 ( d , j = 6.5 , 6h ) . c nmr ( 126 mhz , cd3od ) 160.87 , 157.71 , 149.57 , 133.28 , 124.83 ( q , j = 278.55 ) , 122.38 , 122.15 , 121.53 , 110.32 , 42.57 , 40.98 ( q , j = 34.34 ) , 21.71 . hrms : m / z calcd for c13h16f3n4 [ m + h ] 285.1322 ; found 285.1325 . melting point 103108 c . 0.20 g ( 1.0 mmol ) of 1c was reacted with 2-(methylthio)ethylamine and purified according to method cb to furnish 0.15 g ( 0.59 mmol ) of the title compound in 59% yield . h nmr ( 250 mhz , cd3od ) 7.93 ( dd , j = 8.3 , 0.8 hz , 1h ) , 7.65 ( ddd , j = 8.4 , 7.0 , 1.4 hz , 1h ) , 7.507.44 ( m , 1h ) , 7.38 ( ddd , j = 8.3 , 7.0 , 1.3 hz , 1h ) , 3.70 ( dd , j = 7.5 , 6.6 hz , 2h ) , 2.752.65 ( m , 2h ) , 2.08 ( s , 3h ) . mass ( esi ) : [ m + h ] 254 , 256 ; found 254.0 ( 100% ) , 256.0 ( 33% ) . 0.12 g ( 0.47 mmol ) of 6d was reacted with isopropylamine and purified according to method db to furnish 0.056 g ( 0.20 mmol ) of the title compound as a white solid in 43% yield . h nmr ( 500 mhz , cd3od ) 7.81 ( d , j = 8.1 hz , 1h ) , 7.47 ( t , j = 7.5 hz , 1h ) , 7.28 ( d , j = 8.2 hz , 1h ) , 7.06 ( t , j = 7.6 hz , 1h ) , 4.20 ( sept , j = 6.5 hz , 1h ) , 3.763.71 ( m , 2h ) , 2.792.74 ( m , 2h ) , 2.11 ( s , 3h ) , 1.22 ( d , j = 6.5 hz , 6h ) . c nmr ( 126 mhz , cd3od ) 160.39 , 157.49 , 148.19 , 132.93 , 122.28 , 121.64 , 121.48 , 110.56 , 42.62 , 40.15 , 32.36 , 22.03 , 14.11 . hrms : m / z calcd for c14h21n4s [ m + h ] 277.1481 ; found 277.1488 . rf = 0.52 ( dichloromethane to methanol 9:1 ) . melting point 8790 c . 0.198 g ( 0.99 mmol ) of 1c was reacted with glycine methyl ester and purified according to method ca to furnish 0.18 g ( 0.72 mmol ) of the title compound in 73% yield . h nmr ( 400 mhz , cd3od ) 7.85 ( d , j = 8.1 hz , 1h ) , 7.62 ( dd , j = 11.3 , 4.1 hz , 1h ) , 7.45 ( d , j = 8.3 hz , 1h ) , 7.33 ( t , j = 7.6 hz , 1h ) , 4.26 ( s , 2h ) , 3.71 ( s , 3h ) . c nmr ( 101 mhz , cd3od ) 170.77 , 161.67 , 157.12 , 150.04 , 133.76 , 126.38 , 125.93 , 122.25 , 113.24 , 51.72 , 42.35 . 0.12 g ( 0.47 mmol ) of 7d was reacted with isopropylamine and purified according to method db to furnish 0.060 g ( 0.21 mmol ) of the title compound as a white crystalline solid in 44% yield . h nmr ( 400 mhz , cd3od ) 7.84 ( d , j = 8.2 hz , 1h ) , 7.577.51 ( t , j = 7.8 hz , 1h ) , 7.32 ( d , j = 8.2 hz , 1h ) , 7.10 ( t , j = 7.8 hz , 1h ) , 4.264.14 ( m , 5h ) , 1.25 ( t , j = 7.1 hz , 3h ) , 1.21 ( d , j = 6.5 hz , 6h ) . c nmr ( 101 mhz , cd3od ) 172.23 , 162.13 , 159.29 , 150.86 , 134.31 , 123.71 , 123.57 , 122.61 , 111.84 , 62.22 , 43.87 , 43.66 , 23.25 , 14.52 . hrms : m / z calcd for c15h21n4o2 [ m + h ] 289.1659 ; found 289.1668 . 0.16 g ( 0.80 mmol ) of 1c was reacted with cyclohexylamine and purified according to method cb to furnish 0.15 g ( 0.55 mmol ) of the title compound in 69% yield . h nmr ( 250 mhz , cd3od ) 8.057.99 ( m , 1h ) , 7.60 ( ddd , j = 8.4 , 7.0 , 1.4 hz , 1h ) , 7.467.41 ( m , 1h ) , 7.33 ( ddd , j = 8.3 , 7.0 , 1.3 hz , 1h ) , 4.174.01 ( m , 1h ) , 1.971.84 ( m , 2h ) , 1.70 ( d , j = 2.6 hz , 2h ) , 1.651.52 ( m , 1h ) , 1.381.25 ( m , 4h ) , 1.201.00 ( m , 1h ) . c nmr ( 63 mhz , cd3od ) 161.94 , 159.05 , 151.35 , 134.67 , 127.25 , 126.90 , 123.83 , 114.81 , 51.68 , 33.32 , 26.71 , 26.48 . mass ( esi ) : [ m + h ] 262 , 264 ; found 262.1 ( 100% ) , 264.1 ( 33% ) . 0.10 g ( 0.40 mmol ) of 8d was reacted with isopropylamine and purified according to method db to furnish 0.060 g ( 0.21 mmol ) of the title compound as a white crystalline solid in 53% yield . h nmr ( 400 mhz , cd3od ) 7.95 ( d , j = 8.0 hz , 1h ) , 7.55 ( t , j = 7.5 hz , 1h ) , 7.31 ( d , j = 8.3 hz , 1h ) , 7.14 ( t , j = 7.6 hz , 1h ) , 4.20 ( sept , j = 6.5 hz , 2h ) , 2.04 ( d , j = 2.2 hz , 2h ) , 1.83 ( d , j = 4.5 hz , 2h ) , 1.70 ( d , j = 12.4 hz , 1h ) , 1.41 ( m , 5h ) , 1.25 ( d , j = 6.5 hz , 6h ) . c nmr ( 101 mhz , cd3od ) 159.82 , 157.34 , 147.57 , 133.16 , 122.65 , 121.83 , 121.25 , 110.84 , 50.46 , 42.93 , 32.26 , 25.37 , 21.93 . hrms : m / z calcd for c17h25n4 [ m + h ] 285.2074 ; found 285.2077 . rf = 0.38 ( dichloromethane to methanol 9:1 ) . decomposed at 345 c . 0.054 g ( 0.27 mmol ) of 1c was reacted with dimethylamine and purified according to method ca to furnish 0.037 g ( 0.18 mmol ) of the title compound in 69% yield . h nmr ( 400 mhz , cd3od ) 8.06 ( d , j = 8.5 hz , 1h ) , 7.687.62 ( m , 1h ) , 7.52 ( d , j = 8.4 hz , 1h ) , 7.397.33 ( m , 1h ) , 3.35 ( s , 6h ) . c nmr ( 101 mhz , cd3od ) 163.76 , 156.03 , 152.57 , 133.10 , 126.41 , 125.89 , 124.83 , 114.07 , 41.20 . 0.030 g ( 0.14 mmol ) of 9d was reacted with isopropylamine and purified according to method db to furnish 0.015 g ( 0.065 mmol ) of the title compound as a white solid in 46% yield . h nmr ( 400 mhz , cd3od ) 8.11 ( d , j = 8.3 hz , 1h ) , 7.69 ( t , j = 8.2 hz , 1h ) , 7.44 ( d , j = 8.2 hz , 1h ) , 7.31 ( t , j = 8.2 hz , 1h ) , 4.314.19 ( m , 1h ) , 3.48 ( s , 6h ) , 1.29 ( d , j = 6.5 hz , 6h ) . c nmr ( 101 mhz , cd3od ) 162.97 , 152.20 , 143.16 , 134.25 , 127.59 , 122.97 , 117.82 , 110.60 , 43.69 , 41.45 , 21.46 . hrms : m / z calcd for c13h19n4 [ m + h ] 231.1604 ; found 231.1608 . 0.30 g ( 1.5 mmol ) of 1c was reacted with benzylamine and purified according to method ca to furnish 0.345 g ( 1.28 mmol ) of the title compound in 85% yield . h nmr ( 400 mhz , cdcl3 ) 7.70 ( ddd , j = 11.3 , 8.4 , 4.9 hz , 3h ) , 7.487.26 ( m , 5h ) , 6.27 ( s , 1h ) , 4.83 ( d , j = 5.3 hz , 2h ) . c nmr ( 101 mhz , cdcl3 ) 160.93 , 157.93 , 151.09 , 137.48 , 133.76 , 129.11 , 128.51 , 128.21 , 128.00 , 126.46 , 121.07 , 113.38 , 45.94 . 0.10 g ( 0.37 mmol ) of 10d was reacted with benzylamine and purified according to method da to furnish 0.101 g ( 0.297 mmol ) of the title compound as a white crystalline solid in 80% yield . h nmr ( 500 mhz , dmso - d6 ) 10.46 ( s , nh ) , 8.64 ( s , nh ) , 8.45 ( d , j = 8.0 hz , 1h ) , 7.74 ( dt , j = 7.8 , 1.5 hz , 1h ) , 7.44 ( d , j = 8.5 hz , 1h ) , 7.36 ( dt , j = 7.8 , 1.3 hz , 1h ) , 7.367.12 ( m , 10h ) , 4.75 ( d , j = 5.5 hz , 2h ) , 4.63 ( d , j = 4.8 hz , 2h ) . c nmr ( 126 mhz , dmso - d6 ) 160.26 , 153.49 , 139.44 , 138.85 , 138.29 , 135.67 , 129.93 , 128.85 , 128.80 , 128.24 , 128.12 , 127.72 , 127.57 , 124.93 , 124.64 , 117.19 , 110.22 , 110.12 , 44.86 , 44.35 . hrms : m / z calcd for c22h21n4 [ m + h ] 341.1761 ; found 341.1751 . 0.48 g ( 2.4 mmol ) of 1c was reacted with n - butylamine and purified according to method ca to furnish 0.49 g ( 2.1 mmol ) of the title compound in 86% yield . h nmr ( 250 mhz , cdcl3 ) 7.777.53 ( m , 3h ) , 7.447.31 ( m , 1h ) , 5.82 ( s , nh ) , 3.61 ( td , j = 7.1 , 5.6 , 2h ) , 1.64 ( dt , j = 14.7 , 7.2 , 2h ) , 1.40 ( td , j = 14.5 , 7.2 , 2h ) , 0.92 ( t , j = 7.3 , 3h ) . c nmr ( 101 mhz , cdcl3 ) 161.14 , 150.99 , 133.58 , 128.10 , 126.29 , 121.19 , 120.79 , 113.42 , 41.57 , 31.47 , 20.35 , 14.02 . mass ( esi ) : [ m + h ] 236 , 238 ; found 236.0 ( 100% ) , 238.0 ( 34% ) . 0.080 g ( 0.34 mmol ) of 11d was reacted with n - butylamine and purified according to method da to furnish 0.031 g ( 0.114 mmol ) of the title compound as a white solid in 34% yield . h nmr ( 500 mhz , dmso - d6 ) 9.78 ( s , nh ) , 8.36 ( d , j = 8.5 hz , 1h ) , 8.14 ( s , nh ) , 7.76 ( t , j = 8.0 hz , 1h ) , 7.42 ( d , j = 6.6 hz , 1h ) , 7.37 ( t , j = 6.2 hz , 1h ) , 3.58 ( m , 2h ) , 3.45 ( m , 2h ) , 1.65 ( p , j = 7.5 hz , 2h ) , 1.57 ( p , j = 7.5 , 2h ) , 1.36 ( sept , j = 7.3 hz , 4h ) , 0.92 ( td , j = 7.3 , 4.7 hz , 6h ) . c nmr ( 101 mhz , dmso - d6 ) 160.10 , 153.50 , 139.26 , 135.43 , 124.83 , 124.36 , 116.97 , 110.02 , 41.37 , 40.72 , 31.49 , 30.69 , 20.16 , 19.93 , 14.12 , 14.07 . hrms : m / z calcd for c16h25n4 [ m + h ] 273.2074 ; found 273.2072 . 0.088 g ( 0.326 mmol ) of 10d was reacted with 2.0 m methylamine in methanol and purified according to method da to furnish 0.085 g ( 0.321 mmol ) of the title compound as a cream colored solid in 98% yield . h nmr ( 400 mhz , cdcl3 ) 8.29 ( s , 1h ) , 7.42 ( d , j = 7.7 hz , 1h ) , 7.37 ( d , j = 8.0 hz , 2h ) , 7.27 ( d , j = 9.0 hz , 1h ) , 7.207.11 ( m , 3h ) , 7.06 ( t , j = 7.6 hz , 1h ) , 4.80 ( s , 2h ) , 2.93 ( s , 3h ) . c nmr ( 101 mhz , cdcl3 ) 160.11 , 156.03 , 142.69 , 138.07 , 133.96 , 128.35 , 127.94 , 127.26 , 123.74 , 123.10 , 119.20 , 110.28 , 45.06 , 28.01 . hrms : m / z calcd for c16h17n4 [ m + h ] 265.1448 ; found 265.1454 . 0.10 g ( 0.37 mmol ) of 10d was reacted with n - butylamine and purified according to method da to furnish 0.047 g ( 0.15 mmol ) of the title compound as a white solid in 41% yield . h nmr ( 400 mhz , cdcl3 ) 9.54 ( s , 1h ) , 8.43 ( d , j = 8.1 hz , 1h ) , 7.82 ( s , 1h ) , 7.447.37 ( m , 3h ) , 7.257.11 ( m , 5h ) , 4.83 ( d , j = 5.7 hz , 2h ) , 3.39 ( q , j = 6.8 hz , 2h ) , 1.49 ( p , j = 7.3 hz , 2h ) , 1.31 ( sext , j = 7.3 hz , 2h ) , 0.85 ( t , j = 7.3 hz , 3h ) . c nmr ( 101 mhz , cdcl3 ) 160.08 , 153.46 , 137.43 , 134.65 , 128.41 , 127.83 , 127.40 , 124.41 , 124.12 , 116.62 , 109.99 , 109.68 , 45.31 , 41.02 , 31.30 , 19.95 , 13.66 . hrms : m / z calcd for c19h23n4 [ m + h ] 307.1917 ; found 307.1926 . g ( 0.37 mmol ) of 10d was reacted with aniline and purified according to method da to furnish 0.117 g ( 0.358 mmol ) of the title compound as a white crystalline solid in 97% yield . h nmr ( 500 mhz , dmso - d6 ) 10.46 ( s , 2h ) , 8.46 ( d , j = 8.3 hz , 1h ) , 7.84 ( ddd , j = 8.4 , 7.3 , 1.1 hz , 1h ) , 7.56 ( d , j = 8.3 hz , 1h ) , 7.48 ( td , j = 7.8 , 1.0 hz , 1h ) , 7.457.41 ( m , 2h ) , 7.377.29 ( m , 6h ) , 7.297.22 ( m , 1h ) , 7.18 ( t , j = 7.4 hz , 1h ) , 4.76 ( d , j = 5.9 hz , 2h ) . c nmr ( 126 mhz , dmso - d6 ) 160.71 , 151.94 , 139.36 , 138.02 , 137.13 , 135.93 , 129.41 , 128.89 , 127.78 , 127.67 , 125.45 , 125.31 , 124.81 , 122.71 , 117.94 , 110.70 , 45.14 . hrms : m / z calcd for c21h19n4 [ m + h ] 327.1604 ; found 327.1613 . rf = 0.56 ( dichloromethane to methanol 9:1 ) . decomposed at 310 c . 1.0 g ( 5.0 mmol ) of 1c was reacted with methylamine and purified according to method ca to furnish 0.89 g ( 4.6 mmol ) of the title compound in 92% yield . h nmr ( 400 mhz , cdcl3 ) 7.747.66 ( m , 3h ) , 7.41 ( t , j = 6.9 hz , 1h ) , 6.28 ( s , nh ) , 3.20 ( d , j = 4.7 hz , 3h ) . c nmr ( 101 mhz , cdcl3 ) 161.82 , 158.02 , 150.80 , 133.64 , 127.90 , 126.41 , 121.02 , 113.60 , 28.74 . 0.287 g ( 1.48 mmol ) of 15d was reacted with benzylamine and purified according to method da to furnish 0.193 g ( 0.730 mmol ) of the title compound as a white crystalline solid in 49% yield . h nmr ( 250 mhz , cd3od ) 8.06 ( d , j = 8.1 hz , 1h ) , 7.73 ( t , j = 8.4 hz , 1h ) , 7.507.20 ( m , 7h ) , 4.75 ( s , 2h ) , 3.14 ( s , 3h ) . c nmr ( 63 mhz , cd3od ) 162.17 , 154.54 , 140.08 , 139.51 , 136.27 , 129.70 , 128.69 , 128.59 , 125.92 , 124.85 , 117.86 , 111.30 , 45.91 , 29.04 . hrms : m / z calcd for c16h17n4 [ m + h ] 265.1453 ; found 265.1457 . 0.48 g ( 2.0 mmol ) of 11d was reacted with benzylamine and purified according to method da to furnish 0.40 g ( 1.3 mmol ) of the title compound as a white crystalline solid in 65% yield . h nmr ( 500 mhz , dmso - d6 ) 9.84 ( s , 1h ) , 8.64 ( s , 1h ) , 8.37 ( d , j = 7.7 hz , 1h ) , 7.76 ( ddd , j = 8.4 , 7.3 , 1.1 hz , 1h ) , 7.46 ( d , j = 7.9 hz , 1h ) , 7.47.3 ( m , 5h ) , 7.24 ( t , j = 7.1 hz , 1h ) , 4.66 ( d , j = 4.4 hz , 2h ) , 3.49 ( d , j = 6.1 hz , 2h ) , 1.611.43 ( m , 2h ) , 1.331.17 ( m , 2h ) , 0.80 ( t , j = 6.9 hz , 3h ) . c nmr ( 126 mhz , dmso - d6 ) 160.11 , 153.48 , 139.24 , 139.11 , 135.49 , 128.83 , 127.61 , 127.51 , 124.83 , 124.54 , 117.14 , 110.15 , 44.46 , 41.46 , 30.62 , 20.11 , 14.09 . hrms : m / z calcd for c19h23n4 [ m + h ] 307.1923 ; found 307.1921 . 0.30 g ( 1.5 mmol ) of 1c was reacted with aniline and purified according to method ca to furnish 0.36 g ( 1.4 mmol ) of the title compound as a white solid in 93% yield . h nmr ( 400 mhz , cd3od ) 8.33 ( d , j = 8.3 hz , 1h ) , 7.80 ( ddd , j = 8.3 , 7.0 , 1.2 hz , 1h ) , 7.787.73 ( m , 2h ) , 7.64 ( dd , j = 8.4 , 0.9 hz , 1h ) , 7.56 ( ddd , j = 8.2 , 7.0 , 1.2 hz , 1h ) , 7.407.34 ( m , 2h ) , 7.17 ( t , j = 7.4 hz , 1h ) . c nmr ( 101 mhz , cd3od ) 159.61 , 156.96 , 150.58 , 137.99 , 133.70 , 128.28 , 126.50 , 126.00 , 124.67 , 122.55 , 122.40 , 113.65 . 0.10 g ( 0.39 mmol ) of 17d was reacted with benzylamine and purified according to method da to furnish 0.010 g ( 0.031 mmol ) of the title compound as a white crystalline solid in 10% yield . h nmr ( 500 mhz , dmso - d6 ) 9.56 ( s , 1h ) , 9.13 ( s , 1h ) , 8.37 ( d , j = 7.5 hz , 1h ) , 7.92 ( d , j = 7.5 hz , 2h ) , 7.86 ( d , j = 8.1 hz , 2h ) , 7.66 ( ddd , j = 8.3 , 6.9 , 1.3 hz , 1h ) , 7.48 ( d , j = 8.4 hz , 1h ) , 7.39 ( dd , j = 8.4 , 7.4 hz , 2h ) , 7.27 ( ddd , j = 8.1 , 7.0 , 1.2 hz , 1h ) , 7.22 ( dd , j = 8.4 , 7.4 hz , 2h ) , 7.13 ( t , j = 7.4 hz , 1h ) , 6.89 ( t , j = 7.3 hz , 1h ) , 1.89 ( s , 2h ) . c nmr ( 101 mhz , dmso - d6 ) 159.01 , 157.09 , 152.33 , 141.80 , 140.10 , 133.64 , 129.12 , 128.92 , 126.26 , 124.14 , 123.77 , 123.10 , 122.52 , 121.45 , 119.58 , 112.43 , 21.76 . hrms : m / z calcd for c21h19n4 [ m + h ] 327.1604 ; found 327.1614 . 0.5 g ( 2.9 mmol ) of commercially available 2-amino-3-chlorobenzoic acid was reacted according to general procedure a to give crude 18b . without further purification , 18b was reacted according to general procedure b to give 0.15 g ( 0.6 mmol ) of the crude title compound ( 20% over two steps ) . mass ( esi ) : [ m + h ] 233 , 235 ; found 232.9 ( 100% ) , 235.0 ( 86% ) . 0.10 g ( 0.43 mmol ) of 18c was reacted with furfurylamine according to general procedure cb to give 0.11 g ( 0.37 mmol ) of 18d in 87% yield . h nmr ( 250 mhz , cd3od ) 7.89 ( dd , j = 8.3 , 1.2 hz , 1h ) , 7.69 ( dd , j = 7.7 , 1.2 hz , 1h ) , 7.33 ( dd , j = 1.7 , 1.0 hz , 1h ) , 7.307.21 ( m , 1h ) , 6.286.21 ( m , 2h ) , 4.67 ( s , 2h ) . c nmr ( 63 mhz , cd3od ) 162.61 , 159.88 , 152.48 , 148.51 , 143.44 , 134.77 , 132.04 , 127.17 , 122.63 , 116.31 , 111.47 , 109.13 , 39.00 . mass ( esi ) : [ m + h ] 294 , 296 ; found 294.0 ( 100% ) , 296.0 ( 64% ) . 0.047 g ( 0.16 mmol ) of 18d was reacted with isopropylamine according to general procedure db to give 0.030 g ( 0.095 mmol ) of the title compound as a yellow solid in 59% yield . h nmr ( 400 mhz , dmso - d6 ) 8.38 ( s , nh ) , 7.97 ( d , j = 8.0 hz , 1h ) , 7.63 ( d , j = 7.5 hz , 1h ) , 7.56 ( s , 1h ) , 6.95 ( t , j = 7.8 hz , 1h ) , 6.65 ( s , nh ) , 6.426.36 ( m , 1h ) , 6.32 ( s , 1h ) , 4.70 ( d , j = 5.5 hz , 2h ) , 4.284.14 ( m , 1h ) , 1.17 ( d , j = 5.5 hz , 6h ) . c nmr ( 101 mhz , cdcl3 ) 159.87 , 158.98 , 151.43 , 148.83 , 142.16 , 132.55 , 128.98 , 119.93 , 119.78 , 111.79 , 110.46 , 107.66 , 42.93 , 38.15 , 22.99.hrms : m / z calcd for c16h17cln4o [ m + h ] 317.1164 ; found 317.1173 0.75 g ( 4.4 mmol ) of commercially available 2-amino-4-chlorobenzoic acid was reacted according to general procedure a to give crude 19b . without further purification , 19b was reacted according to general procedure b to give 0.64 g ( 2.7 mmol ) of the crude title compound ( 61% over two steps ) . mass ( esi ) : [ m + h ] 233 , 235 ; found 232.9 ( 100% ) , 235.0 ( 94% ) . 0.30 g ( 1.3 mmol ) of 19c was reacted with furfurylamine according to general procedure cb to give 0.35 g ( 1.2 mmol ) of 19d in 92% yield . h nmr ( 250 mhz , cd3od ) 8.138.06 ( m , 1h ) , 7.627.58 ( m , 1h ) , 7.49 ( d , j = 2.1 hz , 1h ) , 7.477.42 ( m , 1h ) , 6.376.35 ( m , 2h ) , 4.80 ( s , 2h ) . c nmr ( 63 mhz , cd3od ) 163.24 , 160.01 , 156.93 , 152.44 , 143.49 , 140.90 , 128.01 , 126.34 , 125.69 , 113.46 , 111.47 , 109.11 , 38.87 . mass ( esi ) : [ m + h ] 294 , 296 ; found 294.0 ( 100% ) , 296.0 ( 63% ) . 0.113 g ( 0.38 mmol ) of 19d was reacted with isopropylamine according to general procedure db to give 0.085 g ( 0.27 mmol ) of the title compound in 71% yield . h nmr ( 500 mhz , dmso - d6 ) 8.37 ( s , nh ) , 8.03 ( d , j = 8.7 hz , 1h ) , 7.567.55 ( m , 1h ) , 7.23 ( s , 1h ) , 7.02 ( dd , j = 8.7 , 1.8 hz , 1h ) , 6.57 ( s , nh ) , 6.466.37 ( m , 1h ) , 6.34 ( s , 1h ) , 4.70 ( d , j = 5.6 hz , 2h ) , 4.16 ( oct , j = 6.6 hz , 1h ) , 1.15 ( d , j = 6.5 hz , 6h ) . c nmr ( 126 mhz , dmso - d6 ) 159.87 , 153.82 , 152.94 , 142.36 , 142.32 , 137.40 , 125.34 , 123.78 , 120.26 , 110.95 , 110.90 , 107.56 , 42.22 , 37.32 , 23.15 . hrms : m / z calcd for c16h18cln4o [ m + h ] 317.1164 ; found 317.1161 . 1 g ( 5.8 mmol ) of commercially available 2-amino-5-chlorobenzoic acid was reacted according to general procedure a to give crude 20b . without further purification , 20b was reacted according to general procedure b to give 0.55 g ( 2.7 mmol ) of the crude title compound ( 47% over two steps ) . h nmr ( 250 mhz , cdcl3 ) 8.17 ( d , j = 8.9 hz , 1h ) , 7.94 ( d , j = 2.0 hz , 1h ) , 7.66 ( dd , j = 8.9 , 2.0 hz , 1h ) . c nmr ( 63 mhz , cdcl3 ) 163.85 , 156.31 , 152.68 , 142.97 , 130.45 , 127.37 , 127.07 , 120.77 . mass ( esi ) : [ m + h ] 233 , 235 ; found 232.9 ( 100% ) , 235.0 ( 98% ) . 0.30 g ( 1.3 mmol ) of 20c was reacted with furfurylamine according to general procedure cb to give 0.072 g ( 0.24 mmol ) of 20d in 18% yield . h nmr ( 400 mhz , dmso - d6 ) 9.23 ( t , j = 5.1 hz , nh ) , 8.44 ( d , j = 2.1 hz , 1h ) , 7.78 ( dd , j = 8.9 , 2.1 hz , 1h ) , 7.657.54 ( m , 2h ) , 6.426.34 ( m , 2h ) , 4.69 ( d , j = 5.4 hz , 2h ) . c nmr ( 63 mhz , cdcl3 ) 160.42 , 158.82 , 151.67 , 139.79 , 136.95 , 128.95 , 128.39 , 128.14 , 127.04 , 126.91 , 122.42 , 111.58 , 45.86 . mass ( esi ) : [ m + h ] 294 , 296 ; found 294.0 ( 100% ) , 296.0 ( 67% ) . 0.041 g ( 0.14 mmol ) of 20d was reacted with isopropylamine according to general procedure db to give 0.026 g ( 0.082 mmol ) of the title compound in 59% yield . h nmr ( 400 mhz , dmso - d6 ) 8.36 ( s , nh ) , 8.15 ( d , j = 2.3 hz , 1h ) , 7.57 ( d , j = 0.7 hz , 1h ) , 7.46 ( dd , j = 8.9 , 2.3 hz , 1h ) , 7.23 ( d , j = 8.4 hz , 1h ) , 6.56 ( s , nh ) , 6.39 ( dd , j = 3.0 , 1.9 hz , 1h ) , 6.34 ( s , 1h ) , 4.68 ( d , j = 5.4 hz , 2h ) , 4.14 ( oct , j = 6.6 hz , 1h ) , 1.14 ( d , j = 6.5 hz , 6h ) . c nmr ( 101 mhz , cdcl3 ) 159.22 , 158.96 , 151.64 , 150.51 , 142.01 , 133.02 , 126.36 , 125.48 , 120.96 , 111.58 , 110.43 , 107.59 , 42.72 , 37.84 , 23.16 . hrms : m / z calcd for c16h17cln4o [ m + h ] 317.1164 ; found 317.1165 . 0.36 g ( 2.1 mmol ) of commercially available 2-amino-6-chlorobenzoic acid was reacted according to general procedure a to give crude 21b . without further purification , 21b was reacted according to general procedure b to give 0.12 g ( 0.51 mmol ) of the crude title compound ( 24% over two steps ) . mass ( esi ) : [ m + h ] 233 , 235 ; found 235.0 ( 100% ) , 232.9 ( 84% ) . 0.045 g ( 0.15 mmol ) of 21c was reacted with furfurylamine according to general procedure cb . 21d was reacted with isopropylamine according to general procedure db to give 0.006 g ( 0.019 mmol ) of the title compound in 13% yield over two steps . h nmr ( 400 mhz , cdcl3 ) 7.78 ( s , 1h ) , 7.35 ( dd , j = 1.8 , 0.8 hz , 1h ) , 7.287.24 ( m , 2h ) , 6.986.88 ( m , 1h ) , 6.31 ( dd , j = 3.2 , 1.9 hz , 1h ) , 6.26 ( dd , j = 3.2 , 0.8 hz , 1h ) , 4.93 ( s , 1h ) , 4.73 ( d , j = 5.0 hz , 2h ) , 4.22 ( oct , j = 6.5 hz , 1h ) , 1.21 ( d , j = 6.5 hz , 6h ) . c nmr ( 101 mhz , cdcl3 ) 159.17 , 158.25 , 155.04 , 151.58 , 142.07 , 131.75 , 128.92 , 124.92 , 122.93 , 110.40 , 108.71 , 107.17 , 42.66 , 38.54 , 23.17 . hrms : m / z calcd for c16h18cln4o [ m + h ] 317.1164 ; found 317.1169 . 1.0 g ( 6.6 mmol ) of commercially available 2-amino-3-methylbenzoic acid was reacted according to general procedure a to give crude 22b . without further purification , 22b was reacted according to general procedure b to give 1.1 g ( 5.2 mmol ) of the crude title compound ( 78% over two steps ) . mass ( esi ) : [ m + h ] 213 , 215 ; found 213.0 ( 100% ) , 215.0 ( 73% ) . 0.50 g ( 2.3 mmol ) of 22c was reacted with furfurylamine according to general procedure cb to give 0.43 g ( 1.6 mmol ) of 22d in 70% yield . h nmr ( 400 mhz , dmso - d6 ) 9.08 ( t , j = 5.5 hz , nh ) , 8.07 ( d , j = 8.2 hz , 1h ) , 7.587.53 ( m , 2h ) , 7.33 ( t , j = 7.7 hz , 1h ) , 6.396.35 ( m , 1h ) , 6.32 ( d , j = 3.0 hz , 1h ) , 4.70 ( d , j = 5.5 hz , 2h ) , 2.45 ( s , 3h ) . c nmr ( 101 mhz , dmso - d6 ) 161.92 , 156.75 , 152.08 , 149.93 , 142.85 , 135.39 , 134.26 , 126.11 , 121.26 , 113.88 , 111.21 , 108.30 , 38.10 , 17.88 . mass ( esi ) : [ m + h ] 274 , 276 ; found 274.1 ( 100% ) , 276.1 ( 35% ) . 0.13 g ( 0.47 mmol ) of 22d was reacted with isopropylamine according to general procedure db to give 0.10 g ( 0.34 mmol ) of the title compound as a yellow solid in 74% yield . h nmr ( 400 mhz , cdcl3 ) 8.87 ( s , 1h ) , 8.53 ( s , 1h ) , 7.94 ( s , 1h ) , 7.35 ( d , j = 7.3 hz , 1h ) , 7.227.21 ( m , 1h ) , 7.04 ( t , j = 7.7 hz , 1h ) , 6.276.19 ( m , 2h ) , 4.80 ( d , j = 5.2 hz , 2h ) , 4.22 ( oct , j = 6.7 hz , 1h ) , 1.25 ( d , j = 6.6 hz , 6h ) . c nmr ( 101 mhz , cdcl3 ) 160.37 , 153.04 , 150.27 , 142.20 , 138.12 , 135.91 , 126.12 , 123.57 , 121.16 , 110.46 , 109.19 , 108.09 , 43.89 , 38.50 , 22.34 , 18.07 . hrms : m / z calcd for c17h21n4o [ m + h ] 297.1710 ; found 297.1712 . 0.5 g ( 3.3 mmol ) of commercially available 2-amino-4-methylbenzoic acid was reacted according to general procedure a to give crude 23b . without further purification , 23b was reacted according to general procedure b to give 0.34 g ( 1.6 mmol ) of the crude title compound ( 48% over two steps ) . mass ( esi ) : [ m + h ] 213 , 215 ; found 213.0 ( 100% ) , 215.0 ( 63% ) . 0.20 g ( 0.94 mmol ) of 23c was reacted with furfurylamine according to general procedure cb to give 0.091 g ( 0.33 mmol ) of 23d in 35% yield . h nmr ( 400 mhz , cd3od ) 7.91 ( d , j = 8.4 hz , 1h ) , 7.40 ( s , 1h ) , 7.34 ( s , 1h ) , 7.27 ( d , j = 8.5 hz , 1h ) , 6.32 ( d , j = 1.2 hz , 2h ) , 4.74 ( s , 2h ) , 2.43 ( s , 3h ) . c nmr ( 101 mhz , cd3od ) 161.28 , 157.55 , 151.56 , 150.46 , 144.94 , 142.11 , 128.03 , 125.11 , 122.27 , 111.37 , 110.21 , 107.66 , 37.53 , 20.59 . mass ( esi ) : [ m + h ] 274 , 276 ; found 274.1 ( 100% ) , 276.1 ( 36% ) . 0.068 g ( 0.25 mmol ) of 23d was reacted with isopropylamine according to general procedure db to give 0.030 g ( 0.10 mmol ) of the title compound in 40% yield . h nmr ( 250 mhz , dmso - d6 ) 10.00 ( s , nh ) , 8.21 ( d , j = 7.6 hz , 1h ) , 8.04 ( s , 1h ) , 7.60 ( d , j = 1.3 hz , 1h ) , 7.16 ( d , j = 8.0 hz , 1h ) , 6.436.32 ( m , 2h ) , 4.77 ( d , j = 4.9 hz , 2h ) , 4.324.16 ( m , 1h ) , 2.41 ( s , 3h ) , 1.22 ( d , j = 6.5 hz , 6h ) . c nmr ( 63 mhz , dmso - d6 ) 159.59 , 151.02 , 145.75 , 142.30 , 138.22 , 135.88 , 125.02 , 124.02 , 116.51 , 112.23 , 110.54 , 107.67 , 42.88 , 37.55 , 22.21 , 21.34 . hrms : m / z calcd for c17h21n4o [ m + h ] 297.1710 ; found 297.1716 . 1 g ( 6.6 mmol ) of commercially available 2-amino-5-methylbenzoic acid was reacted according to general procedure a to give crude 24b . without further purification , 24b was reacted according to general procedure b to give 0.34 g ( 1.6 mmol ) of the crude title compound ( 24% over two steps ) . 0.32 g ( 1.5 mmol ) of 24c was reacted with furfurylamine according to general procedure cb to give 0.19 g ( 0.69 mmol ) of 24d in 46% yield . h nmr ( 400 mhz , cd3od ) 7.79 ( s , 1h ) , 7.50 ( dd , j = 8.5 , 1.3 hz , 1h ) , 7.41 ( d , j = 8.3 hz , 2h ) , 6.33 ( d , j = 1.0 hz , 2h ) , 4.74 ( s , 2h ) , 2.41 ( s , 3h ) . c nmr ( 101 mhz , cd3od ) 161.06 , 156.69 , 151.55 , 148.37 , 142.11 , 136.74 , 135.25 , 125.55 , 121.59 , 113.35 , 110.21 , 107.68 , 37.56 , 20.30 . mass ( esi ) : [ m + h ] 274 , 276 ; found 274.1 ( 100% ) , 276.1 ( 33% ) . 0.114 g ( 0.42 mmol ) of 24d was reacted with isopropylamine according to general procedure db to give 0.105 g ( 0.35 mmol ) of the title compound as a yellow solid in 83% yield . h nmr ( 400 mhz , cdcl3 ) 7.51 ( s , 1h ) , 7.407.29 ( m , 3h ) , 6.33 ( dd , j = 3.2 , 1.9 hz , 1h ) , 6.31 ( d , j = 3.1 hz , 1h ) , 4.82 ( s , 2h ) , 4.27 ( oct , j = 6.6 hz , 1h ) , 2.36 ( s , 3h ) , 1.27 ( d , j = 6.5 hz , 6h ) . c nmr ( 101 mhz , cdcl3 ) 159.79 , 154.12 , 150.67 , 142.21 , 135.81 , 133.04 , 122.06 , 119.30 , 110.46 , 109.44 , 107.96 , 43.45 , 38.27 , 22.63 , 21.05.hrms : m / z calcd for c17h21n4o [ m + h ] 297.1710 ; found 297.1712 . rf = 0.43 ( dichloromethane to methanol 9:1 ) . melting point 200204 c . 1 g ( 6.6 mmol ) of commercially available 2-amino-6-methylbenzoic acid was reacted according to general procedure a to give crude 25b . without further purification , 25b was reacted according to general procedure b to give 0.60 g ( 2.8 mmol ) of the crude title compound ( 42% over two steps ) . mass ( esi ) : [ m + h ] 213 , 215 ; found 213.0 ( 100% ) , 215.0 ( 71% ) . 0.30 g ( 1.4 mmol ) of 25c was reacted with furfurylamine according to general procedure cb to give 0.055 g ( 0.20 mmol ) of 25d in 14% yield . h nmr ( 400 mhz , cd3od ) 7.597.54 ( m , 1h ) , 7.457.34 ( m , 2h ) , 7.25 ( d , j = 7.1 hz , 1h ) , 6.36 ( s , 2h ) , 4.79 ( s , 2h ) , 2.82 ( s , 3h ) . c nmr ( 101 mhz , cd3od ) 162.25 , 152.30 , 142.08 , 134.99 , 134.03 , 132.97 , 129.84 , 129.37 , 126.90 , 124.28 , 110.25 , 107.61 , 38.32 , 22.20 . mass ( esi ) : [ m + h ] 274 , 276 ; found 274.1 ( 100% ) , 276.1 ( 33% ) . 0.10 g ( 0.37 mmol ) of 25d was reacted with isopropylamine according to general procedure db to give 0.066 g ( 0.22 mmol ) of the title compound as a yellow solid in 60% yield . h nmr ( 400 mhz , cd3od ) 7.41 ( d , j = 1.0 hz , 1h ) , 7.28 ( t , j = 7.8 hz , 1h ) , 7.14 ( d , j = 8.3 hz , 1h ) , 6.78 ( d , j = 7.1 hz , 1h ) , 6.34 ( dd , j = 3.0 , 1.9 hz , 1h ) , 6.27 ( d , j = 2.8 hz , 1h ) , 4.74 ( s , 2h ) , 4.21(sept , j = 6.5 hz , 1h ) , 2.69 ( s , 3h ) , 1.21 ( d , j = 6.5 hz , 6h ) . c nmr ( 101 mhz , cd3od ) 161.42 , 158.32 , 153.28 , 152.67 , 141.75 , 134.10 , 131.88 , 124.40 , 122.13 , 111.02 , 110.20 , 106.52 , 42.50 , 38.16 , 22.56 , 22.30 . hrms : m / z calcd for c17h21n4o [ m + h ] 297.1710 ; found 297.1710 . 1 g ( 6.0 mmol ) of commercially available 2-amino-3-methoxybenzoic acid was reacted according to general procedure a to give crude 26b . without further purification , 26b was reacted according to general procedure b to give 0.15 g ( 0.65 mmol ) of the crude title compound ( 11% over two steps ) . mass ( esi ) : [ m + h ] 229 , 231 ; found 229.0 ( 100% ) , 230.9 ( 68% ) . 0.10 g ( 0.46 mmol ) of 26c was reacted with furfurylamine according to general procedure cb to give 0.083 g ( 0.29 mmol ) of 20d in 63% yield . mass ( esi ) : [ m + h ] 290 , 292 ; found 290.1 ( 100% ) , 292.0 ( 68% ) . 0.069 g ( 0.24 mmol ) of 26d was reacted with isopropylamine according to general procedure db to give 0.023 g ( 0.073 mmol ) of the title compound as a yellow crystalline solid in 30% yield . h nmr ( 500 mhz , cd3od ) 7.54 ( dd , j = 7.2 , 1.8 hz , 1h ) , 7.447.42 ( m , 1h ) , 7.177.09 ( m , 2h ) , 6.376.33 ( m , 2h ) , 4.81 ( s , 2h ) , 4.30 ( hept , j = 6.6 hz , 1h ) , 3.92 ( s , 3h ) , 1.29 ( d , j = 6.6 hz , 6h ) . c nmr ( 126 mhz , cd3od ) 160.21 , 153.10 , 150.93 , 147.67 , 142.12 , 131.49 , 123.46 , 114.22 , 113.55 , 110.15 , 109.86 , 107.53 , 55.58 , 43.46 , 37.85 , 21.51 . hrms : m / z calcd for c17h21n4o2 [ m + h ] 313.1659 ; found 313.1654 . 0.98 g ( 5.9 mmol ) of commercially available 2-amino-4-methoxybenzoic acid was reacted according to general procedure a to give crude 27b . without further purification , 27b was reacted according to general procedure b to give 0.09 g ( 0.39 mmol ) of the crude title compound ( 7% over two steps ) . mass ( esi ) : [ m + h ] 229 , 231 ; found 228.9 ( 100% ) , 231.0 ( 68% ) . 0.075 g ( 0.33 mmol ) of 27c was reacted with furfurylamine according to general procedure cb to give 0.083 g ( 0.29 mmol ) of 27d in 88% yield . h nmr ( 500 mhz , cd3od ) 7.92 ( d , j = 9.1 hz , 1h ) , 7.43 ( d , j = 1.0 hz , 1h ) , 7.01 ( dd , j = 9.1 , 2.5 hz , 1h ) , 6.91 ( d , j = 2.3 hz , 1h ) , 6.35 ( s , 2h ) , 4.76 ( s , 2h ) , 3.88 ( s , 3h ) . c nmr ( 126 mhz , cd3od ) 163.95 , 160.75 , 157.72 , 152.39 , 151.46 , 141.90 , 123.70 , 117.16 , 109.99 , 107.40 , 107.26 , 105.06 , 54.76 , 37.32 . mass ( esi ) : [ m + h ] 290 , 292 ; found 290.1 ( 100% ) , 292.0 ( 33% ) . 0.065 g ( 0.29 mmol ) of 27d was reacted with isopropylamine according to general procedure db to give 0.007 g ( 0.022 mmol ) of the title compound in 8% yield . h nmr ( 500 mhz , cdcl3 ) 7.70 ( d , j = 21.0 hz , 1h ) , 7.377.31 ( m , 1h ) , 6.77 ( s , 1h ) , 6.70 ( dd , j = 9.0 , 2.4 hz , 1h ) , 6.32 ( dd , j = 3.1 , 1.9 hz , 1h ) , 6.29 ( d , j = 3.2 hz , 1h ) , 4.80 ( s , 2h ) , 4.27 ( oct , j = 6.6 hz , 1h ) , 3.81 ( s , 3h ) , 1.27 ( d , j = 6.6 hz , 6h ) . c nmr ( 101 mhz , cdcl3 ) 163.99 , 159.57 , 156.47 , 151.19 , 142.15 , 127.72 , 123.50 , 113.25 , 110.44 , 107.73 , 103.92 , 101.78 , 55.57 , 43.17 , 38.07 , 22.83 . hrms : m / z calcd for c17h21n4o2 [ m + h ] 313.1659 ; found 313.1667 . 0.20 g ( 1.2 mmol ) of commercially available 2-amino-4-methoxybenzoic acid was reacted according to general procedure a to give crude 28b . without further purification , 28b was reacted according to general procedure b to give 0.14 g ( 0.61 mmol ) of the crude title compound ( 50% over two steps ) . mass ( esi ) : [ m + h ] 229 , 231 ; found 229.0 ( 100% ) , 231.0 ( 67% ) . 0.061 g ( 0.27 mmol ) of 28c was reacted with furfurylamine according to general procedure cb to give 0.031 g ( 0.11 mmol ) of 28d in 41% yield . h nmr ( 500 mhz , cd3od ) 7.45 ( m , 2h ) , 7.40 ( s , 1h ) , 7.28 ( d , j = 9.0 hz , 1h ) , 6.37 ( d , j = 3.8 hz , 2h ) , 4.77 ( s , 2h ) , 3.86 ( s , 3h ) . c nmr ( 126 mhz , cd3od ) 160.52 , 158.00 , 155.06 , 151.38 , 144.99 , 141.90 , 127.01 , 124.51 , 113.88 , 109.99 , 107.59 , 101.40 , 54.99 , 37.43 . mass ( esi ) : [ m + h ] 290 , 292 ; found 290.0 ( 100% ) , 292.0 ( 33% ) . 0.14 g ( 0.48 mmol ) of 28d was reacted with isopropylamine according to general procedure db to give 0.045 g ( 0.14 mmol ) of the title compound as a yellow solid in 29% yield . h nmr ( 400 mhz , dmso - d6 ) 8.42 ( s , 1h ) , 7.58 ( dd , j = 1.8 , 0.9 hz , 1h ) , 7.54 ( d , j = 2.4 hz , 1h ) , 7.24 ( d , j = 9.0 hz , 1h ) , 7.18 ( dd , j = 9.0 , 2.5 hz , 1h ) , 6.40 ( dd , j = 3.1 , 1.8 hz , 1h ) , 6.35 ( d , j = 2.8 hz , 1h ) , 4.72 ( d , j = 5.3 hz , 2h ) , 4.13 ( oct , j = 6.5 hz , 1h ) , 3.79 ( s , 3h ) , 1.15 ( d , j = 6.5 hz , 6h ) . c nmr ( 101 mhz , dmso - d6 ) 159.63 , 157.27 , 153.87 , 152.97 , 145.81 , 142.36 , 125.50 , 123.66 , 110.91 , 107.59 , 103.66 , 56.05 , 42.25 , 37.36 , 23.19 . hrms : m / z calcd for c17h21n4o2 [ m + h ] 313.1659 ; found 313.1659 . 1.0 g ( 6.2 mmol ) of commercially available 2-amino-4-methoxybenzoic acid was reacted according to general procedure a to give crude 29b . without further purification , 29b was reacted according to general procedure b to give 0.036 g ( 0.16 mmol ) of the crude title compound ( 3% over two steps ) . mass ( esi ) : [ m + h ] 229 , 231 ; found 229.0 ( 100% ) , 231.0 ( 70% ) . 0.0094 g ( 41 mol ) of 29c was reacted with furfurylamine according to general procedure cb to give 0.0045 g ( 0.11 mmol ) of 29d in 38% yield . h nmr ( 250 mhz , cd3od ) 7.58 ( t , j = 8.3 hz , 1h ) , 7.36 ( d , j = 1.0 hz , 1h ) , 7.08 ( d , j = 8.2 hz , 1h ) , 6.93 ( d , j = 7.9 hz , 1h ) , 6.336.20 ( m , 2h ) , 4.70 ( s , 2h ) , 3.95 ( s , 3h ) . mass ( esi ) : [ m + h ] 290 , 292 ; found 290.0 ( 100% ) , 292.0 ( 37% ) . 0.0019 g ( 6.6 mol ) of 29d was reacted with isopropylamine according to general procedure db to give 0.0012 g ( 3.8 mol ) of the title compound in 58% yield . h nmr ( 400 mhz , cd3od ) 7.53 ( t , j = 8.3 hz , 1h ) , 7.41 ( t , j = 6.7 hz , 1h ) , 6.93 ( d , j = 8.1 hz , 1h ) , 6.78 ( d , j = 8.2 hz , 1h ) , 6.386.32 ( m , 1h ) , 6.29 ( s , 1h ) , 4.79 ( s , 2h ) , 4.304.16 ( m , 1h ) , 4.00 ( s , 3h ) , 1.23 ( d , j = 6.5 hz , 6h ) . c nmr ( 101 mhz , cdcl3 ) 160.20 , 157.19 , 150.24 , 142.45 , 142.31 , 134.94 , 127.73 , 113.87 , 110.56 , 107.75 , 104.07 , 100.31 , 56.50 , 43.55 , 38.44 , 22.48 . hrms : m / z calcd for c17h21n4o2 [ m + h ] 313.1659 ; found 313.1654 . permeability pe has been determined by a standard parallel artificial membrane permeability assay ( pampa by pion ) as reported previously . solubility at ph 7.4 has been determined using biomek fx lab automation workstation with pion sol evolution software as reported previously and at ph 2.0 using an in - house hplc assay . for ph 2.0 , a calibration curve was made by plotting the area under the curve at 254 nm ( uv by hplc ) against the concentration of each compound injected after performing a serial dilution ( 250.781 m ) using a solvent in which the compound is soluble ( dmso ) . a 100 m solution was then made for each compound in a buffer at 2.0 by performing a 1:100 serial dilution using a 10 mm dmso stock solution of each compound . this solution was incubated at 21 c for 18 h , filtered using a filter plate , and injected into the hplc to compare the area found at wavelength 254 nm with the previously made calibration curve . the log d was also determined in - house via an hplc method adapted from the strategy reported by donovan and pescatore . two buffers were made at a concentration of 50 m each : ammonium acetate at ph 7.4 and ammonium formate at ph 3.0 . a set of compounds with known log d values between 0.36 and 5.68 were used to make a calibration curve at each ph by using a linear gradient between 0% and 100% acetonitrile , with buffer at the specific ph used as the second solvent . the curve was made by plotting the log d value against the retention time . quinazolines were then injected into the hplc instrument , and the retention time was compared to the calibration curve previously determined . in vitro antileishmanial potency of compounds against intracellular l. donovani mhom / et/67/l82 and l. amazonensis lv78 parasites was measured using the colorimetric assay as described previously . the potency of compounds 15 , 16 , and 23 against intracellular antimony - resistant clinical isolate l. donovani mhom / np/02/bpk164/1 and antimony - susceptible isolate l. donovani mhom / np/03/bpk206/0 was also assessed . briefly , second to third day stationary phase promastigotes grown in homem ( invitrogen ) supplemented with 20% fetal calf serum were used to infect murine primary peritoneal macrophages at a ratio of 10 parasites to 1 host cell and subsequently exposed to four concentrations ranging between 0.31 and 25 m for compound 15 or 0.3110 m for compounds 16 and 23 . after 4 days of exposure to compounds , slides were fixed with methanol and stained with giemsa to manually assess parasite survival ( the percentage of infected macrophages ) using conventional light microscopy . a standardized antimony - susceptibility test was performed in parallel to confirm the validity of the test and the clinical isolates antimony - susceptibility profiles . toxicity of compounds against the murine macrophage cell line j774a.1 was conducted using the 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assay as previously described except that cells were incubated with compounds for 72 h. the in vivo antileishmanial efficacy of compounds was evaluated in a murine model of visceral leishmaniasis by the general method described earlier with minor modifications . briefly , balb / c mice were inoculated with lv82 promastigotes on day 0 and then weighed and marked individually on day 6 in order to calculate the volume of solution to be administered to each mouse . in these studies , 45% ( w / v ) ( 2-hydroxypropyl)--cyclodextrin ( hpcd ) vehicle was used to formulate 16 , and 0.5% methylcellulose/0.1% tween 80 in water ( mc ) was used to dissolve 15 and 23 ; all compounds and vehicles were administered intraperitoneally . after 5 consecutive days of treatment from day 7 to day 11 , animals were sacrificed on day 14 . liver smear slides from each animal were made and stained , and the liver parasitemia expressed in leishman donovan unit ( ldu ) was determined by microscopy . protocols for pharmacokinetics studies in mice were approved by the institutional animal care and use committee of the university of kansas . male swiss webster mice ( weighing 2025 g ) were purchased from the charles river laboratories ( raleigh , nc ) . mice were housed in a clean - room under filtered , pathogen - free air , in a 12 h light / dark cycle , and with food pellets and water available ad libitum . 16 ( dissolved in 50% dma ) and 23 [ dissolved in peg400/dma / water = 5:3:2 ( v / v ) ] were administered at a dose of 100 mol / kg body weight ( or approximately 30 mg / kg ; dose volume = 5 ml / kg ) via oral gavage ( po ) or intraperitoneal ( ip ) administration to swiss webster mice . blood sampling time schedules were 1 , 2 , 4 , 8 , 12 , and 24 h ( n = 3 at each blood sampling time point ) after dosing . blood and tissue samples were collected and processed for drug concentration determination as previously described . metabolic stability of 16 and 23 was evaluated using pooled mouse liver microsomes ( xenotech llc , lenexa , ks ) supplemented with nadph . substrate and microsomal protein concentrations during incubations were 3 m and 0.5 mg / ml , respectively . substrate depletion over a 60 min incubation time was quantified using high - performance liquid chromatography tandem mass spectrometry ( hplc ms / ms ) as described below . the processed samples ( plasma , tissue homogenates , and microsomal incubation mixtures ) were analyzed for drug concentration using an api3000 triple quadrupole mass spectrometer equipped with a turbo ionspray interface ( ab sciex , foster city , ca ) . samples ( 5 l ) were introduced to the mass spectrometer using a thermostatic ( 4 c ) autosampler ( ctc pal , leap technologies , carrboro , nc ) and a shimadzu hplc system ( shimadzu , kyoto , japan ) . compounds were separated on a zorbax bonus - rp c18 hplc column ( 2.1 mm 50 mm , 3.5 m particle size ; agilent , milford , ma ) . hplc mobile phases consisted of water containing 0.1% formic acid ( a ) and methanol containing 0.1% formic acid ( b ) with a flow rate of 0.35 ml / min . after a 0.4 min initial hold at 15% b , mobile phase composition started with 15% b and was increased to 95% b over 1.1 min . then the column was washed with 95% b for 1.5 min and was re - equilibrated with 15% b for 1.0 min before injecting the next sample . the characteristic single reaction monitoring ( srm ) transitions for 16 and 23 were m / z 297.1 81.0 and 307.1 91.0 under positive ion mode . the calibration curves for 16 and 23 ranged from 0.01 to 10 m for plasma and from 0.10 to 50 m for tissue homogenates . for microsomal stability samples , substrate depletion was calculated based on the relative ms signal of the analyte normalized by the internal standard . microsomal half - life ( mic t1/2 ) values were obtained by fitting the one - phase exponential decay equation to the percentage of substrate remaining versus time curves ( graphpad prism , version 5.04 , san diego , ca ) . noncompartmental pharmacokinetic analysis of plasma concentration versus time curves was performed to obtain the area under the concentration time curve ( auc ) , cmax , tmax , and terminal half - life ( t1/2 ) ( winnonlin 6.3 , pharsight , mountain view , ca ) . tissue auc was calculated using the trapezoid rule from 1 h ( first sampling time ) to the last time point with quantifiable drug levels without extrapolating back to t = 0 h or beyond the last quantifiable time point ( graphpad prism , version 5.04 ) .

a series of n2,n4-disubstituted quinazoline-2,4-diamines has been synthesized and tested against leishmania donovani and l. amazonensis intracellular amastigotes . a structure activity and structure property relationship study was conducted in part using the topliss operational scheme to identify new lead compounds . this study led to the identification of quinazolines with ec50 values in the single digit micromolar or high nanomolar range in addition to favorable physicochemical properties . quinazoline 23 also displayed efficacy in a murine model of visceral leishmaniasis , reducing liver parasitemia by 37% when given by the intraperitoneal route at 15 mg kg1 day1 for 5 consecutive days . their antileishmanial efficacy , ease of synthesis , and favorable physicochemical properties make the n2,n4-disubstituted quinazoline-2,4-diamine compound series a suitable platform for future development of antileishmanial agents .

Introduction Results and Discussion Conclusions Experimental Section

a recent effort by the world health organization to provide a current estimate of the incidence of leishmaniasis concluded that between 0.2 and 0.4 million cases of visceral leishmaniasis occur each year , while from 0.7 to 1.2 million cases of cutaneous leishmaniasis occur each year . symptoms of leishmaniasis include unsightly spontaneously healing ulcers on the skin when cutaneous leishmaniasis is present , nonhealing lesions in the mucosa when mucocutaneous leishmaniasis is the affliction , and chronic , debilitating infection of the reticuloendothelial system that is fatal if left untreated due to visceral leishmaniasis . the majority of cases of visceral leishmaniasis are caused by l. donovani in east africa and asia , l. infantum in the mediterranean region , and l. chagasi in latin america . infantum and l. chagasi mainly affect children and immunocompromised individuals and are zoonotic parasites with canines being a major reservoir.l . the high rate of resistance to pentavalent antimonials in india has led to the increasing use of amphotericin b and miltefosine against visceral leishmaniasis . miltefosine resistance has been demonstrated in vitro , and its long half - life in the body , the 28-day treatment regimen , and it previously being available over the counter in india have led to concerns of clinical resistance . a recent study of 567 individuals in the bihar state of india has been performed to determine the efficacy of miltefosine since its introduction in 2002 . patients who did not improve with treatment were cured using amphotericin b. the authors of this study concluded that the failure rate of miltefosine has increased in the 10 years since its introduction for the treatment of visceral leishmaniasis in india . because of increased parasite resistance , toxicity issues , increasing failure rates of current treatments , and the lack of effective clinical agents against cutaneous leishmaniasis , new drugs are needed to have an effective strategy for treating leishmaniasis . reported a class of 2,4-diaminoquinazolines with ec50 as low as 0.04 nm against l. major amastigotes in human monocyte - derived macrophages ( a , figure 1b ) ; however the development of this compound series was abandoned because of toxicity issues . antileishmanial testing of quinazolines 1 and 2 against l. mexicana axenic amastigotes revealed ec50 values in the single digit micromolar range , motivating us to investigate whether quinazolines structurally related to the hits 1 and 2 have potential to display potent antileishmanial activity . herein , we report a detailed structure activity relationship ( sar ) study focusing on the 2-position , the 4-position , and the quinazoline s benzenoid ring . all compounds were initially examined in l. donovani and l. amazonensis intracellular amastigote assays to preselect quinazoline candidates active against parasites responsible for causing visceral leishmaniasis and cutaneous leishmaniasis , respectively . promising compounds have subsequently been tested for efficacy in a murine model of visceral leishmaniasis . antileishmanial compounds : ( a ) structures of currently used antileishmanial drugs ; ( b ) reported structures of quinazolines diplaying antileishmanial activity and including the hit compounds 1 and 2 and sar studies targeting the major quinazoline sites . commercially available anthranilic acids ( a ) were cyclized with urea , and the resulting quinazoline-2,4-dione ( b ) was reacted with phosphorus oxychloride to give the 2,4-dichloroquinazoline ( c ) . all target compounds were tested against l. donovani and l. amazonensis intracellular amastigotes to identify molecules that are broadly active against these medically important leishmania species . the testing involved murine peritoneal macrophages as host cells , since compounds with potential for clinical use must be able to penetrate the infected macrophage in a human host . concentration response data for each compound was fitted by a nonlinear regression model , and the concentration that induces 50% inhibition was calculated as the effective concentration ec50 ( l. donovani or l. amazonensis . ) while the 2,2,2-trifluoroethyl - substituted quinazoline 3 was slightly less potent than compound 2 , alcohol 4 lost potency against l. donovani and l. amazonensis by a factor of 10 and > 13 , respectively . bis - benzyl - substituted quinazoline 10 displayed ec50 values of 670 nm against l. donovani and 1.4 m against l. amazonensis , whereas analogue 11 was approximately 2-fold less potent in comparison to compound 10 . for l. amazonensis , the set of 1217 displayed a similar activity trend , with n - benzylquinazolines 15 and 16 being the most potent compounds with submicromolar or single digit micromolar ec50 values . amphotericin b is the internal control for the in vitro antileishmanial activity assay with ec50 = 40 9 nm against l. donovani and ec50 = 89 16 nm against l. amazonensis . nfurfuryl - n - isopropyl - substituted quinazoline 2 , with ec50 values of 2.5 m against l. donovani and 3.7 m against l. amazonensis , was considered to be well suited as the key scaffold for a sar study focusing on the benzenoid moiety of the quinazoline core . in a systematic approach following the topliss operational scheme , compound 2 was monosubstituted with a chlorine atom , a methyl group , or a methoxy group in the 5- , 6- , 7- , and 8-positions to probe the benzenoid ring for steric and electronic effects . overall , substitution on the benzenoid ring provided compounds with ec50 values in the single digit micromolar or submicromolar range against l. donovani . amphotericin b is the internal control for the in vitro antileishmanial activity assay with ec50 = 40 9 nm against l. donovani and ec50 = 89 16 nm against l. amazonensis . strain bpk164/1 was isolated from a bone marrow aspirate taken from a nepalese visceral leishmaniasis patient not responding to antimonial therapy ; the bpk164/1 strain was originally found to be 6-fold resistant to sodium stibogluconate compared to the antimony - susceptible reference strain bpk206/0 . antileishmanial drugs display differences in efficacy for treating indian visceral leishmaniasis compared to african visceral leishmaniasis ; strain differences could account for such differential susceptibility ; ( 2 ) the clinical strains could have a greater fitness in an in vitro intracellular amastigote model than the lab adapted strain which has been in axenic culture for many passages , leading to lower compound susceptibility in the former ; ( 3 ) the clinical strains were assayed using a different method under different conditions compared to the -lactamase expressing lab strain , potentially contributing to distinctions in compound susceptibility . for example , quinazoline 23 was 6.4-fold less potent in the presence of 488 nm folinic acid than in completely deficient media . interestingly , we saw no antagonism of activity of quinazolines 10 , 12 , 13 , 15 , 16 , or 23 for the mammalian cell line ( j774a.1 ) in the presence of folinic acid . for example , the ec50 of quinazoline 10 was 84.8 2.2 and 84.2 2.8 m in the presence or absence of 488 nm folinic acid , respectively . in vitro efficacy of quinazolines , methotrexate ( mtx ) , pyrimethamine ( pyr ) , and miltefosine ( milt ) for axenic amastigotes of leishmania donovani ( a c ) and j774.a1 macrophages ( d f ) in the absence or presence of d , l - folinic acid ( fna ) . in parallel to the testing of the compounds for antileishmanial activity in vitro , a structure property relationship ( spr ) study focusing on log d , aqueous solubility , and permeability has been conducted with all compounds to assess potential physicochemical liabilities ( table 4 ) . generally , the aqueous solubility , the distribution coefficient log d , and the permeability of all quinazolines display a ph dependence ( exemplified on log d or permeability in table 4 ) . however , since the aqueous solubility , the distribution coefficient , and the permeability are within the acceptable ranges ( solubility of > 20 m , pe > 10 10 cms , 1 < log d < 4 ) , the quinazoline compound series is considered to be suitable for the development of bioavailable antileishmanial compounds . among the benzyl - substituted quinazolines 1217 , 15 and 16 appeared to be the best candidates for in vivo evaluation because of their combination of potency , selectivity , and favorable physicochemical properties . from the compound subseries substituted at the benzenoid ring , the physicochemical properties were not discriminatory , and hence , compound 23 was chosen as a viable candidate because of its submicromolar ec50 against l. donovani as well as the outstanding si value of this compound ( > 40 ) . while 15 was well tolerated when given at 30 mg / kg ip for 5 consecutive days , 16 ( slowed breathing ) and 23 ( hypoactivity ) were toxic to animals when given at the same dosing regimen . considering the toxicity of 16 and 23 when given at 30 mg / kg ip , lower doses of these compounds were administered in subsequent in vivo efficacy studies in a murine visceral leishmaniasis model ( figure 4 ) . when tested at 5 15 mg / kg ip , 23 inhibited liver parasitemia by 37% compared to the vehicle control . there was also no significant difference in the parasite burden between mice in the group treated with compound 16 ( 5 10 mg / kg ip ) and the vehicle control group ( p > 0.05 ) . results are presented as the liver parasitemia ( ldu ) for each mouse ( ) , and the average ldu in each group ( ) was determined by microscopy ( n = 4 ) : ( a ) ldu for mice treated with 16 and 23 ; ( b ) ldu for mice treated with 15 . after ip administration , plasma concentration reached a cmax of 5.2 and 2.7 m for 16 and 23 , respectively , before decreasing rapidly in the first 4 h , followed by a slower elimination process until 24 h. the rapid decline in plasma concentration was likely due to extensive tissue redistribution after absorption , as indicated by the high target tissue concentrations . in addition , it is worth pointing out that the terminal half - life of 23 after ip administration was considerably shorter than that after po administration ( 5 h vs 20 h ; table 5 ) , and the liver concentration of 23 was detectable at 12 and 24 h after po administration , whereas it was below the detection limit after ip administration ( figure 6 ) . the dramatic decrease ( 12-fold ) in the permeability of 23 from neutral to acidic ph ( table 4 ) could contribute to the slowed absorption of this compound in the upper gastrointestinal tract . n , n - disubstituted quinazoline-2,4-diamines 1 and 2 were found to display antileishmanial activity in the single digit micromolar range . subsequently a total of 28 molecules have been synthesized systematically by varying the substitutions in the 2- , 4- , 5- , 6- , 7- , and/or 8-positions . all quinazolines have been tested with the aim to further optimize hit compounds 1 and 2 and to conduct a detailed sar study against l. donovani and l. amazonensis . the most potent activities with ec50 values in the submicromolar range against l. donovani were obtained with quinazoline-2,4-diamine scaffolds bearing either a n - benzyl - n - alkyl / phenyl or a n - ispropyl - n - furfuryl substituent combination . furthermore , although the benzenoid ring of the quinazoline-2,4-diamine scaffold has been identified to play a secondary role for efficacy , quinazolines substituted at the 5- or 6-position with one methyl or one methoxy group have also been identified to possess submicromolar ec50 values against l. donovani . in addition , assessment of key physicochemical properties confirmed that the quinazoline s aqueous solubility , distribution coefficient , and passive transcellular permeability were in acceptable ranges . these promising results led to efficacy testing of the lead compounds 15 , 16 , and 23 in an in vivo murine visceral leishmaniasis model . while compounds 15 and 16 did not have activity translate from in vitro to in vivo , quinazoline 23 reduced parasitemia by 37% when 15 mg kg day was given via the intraperitoneal route for 5 consecutive days . although a clear correlation between in vitro activity , in vitro physicochemical properties , and in vivo activity is not clearly observed , the potencies of front - runner compounds 15 , 16 , and 23 in conjunction with favorable physicochemical properties make n , n - disubstituted quinazoline-2,4-diamines a suitable platform for the future development of antileishmanial agents . for future compound design to be successful , optimization will focus not only on improving antileishmanial activity and key physicochemical properties but also on improving the pharmacokinetics and si values for the entire quinazoline compound series . all h nmr experiments are reported in units , parts per million ( ppm ) downfield of tms , and were measured relative to the signals for chloroform ( 7.26 ppm ) , methanol ( 3.31 ppm ) , and dimethylsulfoxide ( 2.50 ppm ) . all c nmr spectra were reported in ppm relative to the signals for chloroform ( 77 ppm ) , methanol ( 49 ppm ) , and dimethylsulfoxide ( 39.5 ppm ) with h decoupled observation . 1 equiv of anthranilic acid and 3.5 equiv of urea were mortar and pestled to a powder and heated to 200 c in a round - bottom flask open to the atmosphere . 1.1 equiv of amine and sodium acetate were mixed with 1 equiv of 2,4-dichloroquinazoline at 0.1 m concentration in a 3 to 1 mix of tetrahydrofuran and water and heated to 65 c . the crude was then purified by either method ca or cb : the compound was recrystallized with ethanol and water , filtered , and rinsed with cold ethanol to yield pure product . this solution was incubated at 21 c for 18 h , filtered using a filter plate , and injected into the hplc to compare the area found at wavelength 254 nm with the previously made calibration curve . quinazolines were then injected into the hplc instrument , and the retention time was compared to the calibration curve previously determined . in vitro antileishmanial potency of compounds against intracellular l. donovani mhom / et/67/l82 and l. amazonensis lv78 parasites was measured using the colorimetric assay as described previously . toxicity of compounds against the murine macrophage cell line j774a.1 was conducted using the 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assay as previously described except that cells were incubated with compounds for 72 h. the in vivo antileishmanial efficacy of compounds was evaluated in a murine model of visceral leishmaniasis by the general method described earlier with minor modifications . after 5 consecutive days of treatment from day 7 to day 11 , animals were sacrificed on day 14 . liver smear slides from each animal were made and stained , and the liver parasitemia expressed in leishman donovan unit ( ldu ) was determined by microscopy . mice were housed in a clean - room under filtered , pathogen - free air , in a 12 h light / dark cycle , and with food pellets and water available ad libitum . blood sampling time schedules were 1 , 2 , 4 , 8 , 12 , and 24 h ( n = 3 at each blood sampling time point ) after dosing . for microsomal stability samples , substrate depletion was calculated based on the relative ms signal of the analyte normalized by the internal standard . tissue auc was calculated using the trapezoid rule from 1 h ( first sampling time ) to the last time point with quantifiable drug levels without extrapolating back to t = 0 h or beyond the last quantifiable time point ( graphpad prism , version 5.04 ) .

a recent effort by the world health organization to provide a current estimate of the incidence of leishmaniasis concluded that between 0.2 and 0.4 million cases of visceral leishmaniasis occur each year , while from 0.7 to 1.2 million cases of cutaneous leishmaniasis occur each year . symptoms of leishmaniasis include unsightly spontaneously healing ulcers on the skin when cutaneous leishmaniasis is present , nonhealing lesions in the mucosa when mucocutaneous leishmaniasis is the affliction , and chronic , debilitating infection of the reticuloendothelial system that is fatal if left untreated due to visceral leishmaniasis . shortly thereafter mccombie young and upendranath brahmachari used trivalent and pentavalent antimonials to treat visceral leishmaniasis in india with great success , decreasing the mortality rate of 95% to just 10% in 10 years ( figure 1a ) . miltefosine resistance has been demonstrated in vitro , and its long half - life in the body , the 28-day treatment regimen , and it previously being available over the counter in india have led to concerns of clinical resistance . patients who did not improve with treatment were cured using amphotericin b. the authors of this study concluded that the failure rate of miltefosine has increased in the 10 years since its introduction for the treatment of visceral leishmaniasis in india . because of increased parasite resistance , toxicity issues , increasing failure rates of current treatments , and the lack of effective clinical agents against cutaneous leishmaniasis , new drugs are needed to have an effective strategy for treating leishmaniasis . reported a class of 2,4-diaminoquinazolines with ec50 as low as 0.04 nm against l. major amastigotes in human monocyte - derived macrophages ( a , figure 1b ) ; however the development of this compound series was abandoned because of toxicity issues . recently , we tested a small library of structurally diverse compounds , originally designed as potential anticancer probes , for antileishmanial activity in a l. mexicana axenic amastigote assay . antileishmanial testing of quinazolines 1 and 2 against l. mexicana axenic amastigotes revealed ec50 values in the single digit micromolar range , motivating us to investigate whether quinazolines structurally related to the hits 1 and 2 have potential to display potent antileishmanial activity . herein , we report a detailed structure activity relationship ( sar ) study focusing on the 2-position , the 4-position , and the quinazoline s benzenoid ring . all compounds were initially examined in l. donovani and l. amazonensis intracellular amastigote assays to preselect quinazoline candidates active against parasites responsible for causing visceral leishmaniasis and cutaneous leishmaniasis , respectively . antileishmanial compounds : ( a ) structures of currently used antileishmanial drugs ; ( b ) reported structures of quinazolines diplaying antileishmanial activity and including the hit compounds 1 and 2 and sar studies targeting the major quinazoline sites . additionally , cytotoxicity against the macrophage cell line j774a.1 was determined as the effective concentration ec50 ( j774a.1 ) and the selectivity index si was calculated as the ratio of ec50 value for j774a.1 and the value for l. donovani ( si = ec50(j774a.1)/ec50(l . the first subseries focused mainly on the optimization of the n- and n - moieties ( table 1 ) , whereas the second subseries was designed to investigate whether analogues being substituted at the quinazoline s benzenoid ring display improved antileishmanial activity ( table 2 ) . replacement of the furfuryl and isopropyl groups in 2 by two benzyl or two n - butyl groups yielded quinazolines 10 and 11 , of which both analogues were more potent than reference 2 . consequently , a following set of six quinazolines 1217 was designed in which one of the 2- and 4-positions was substituted by one benzylamine , while the remaining position was derivatized with an aniline , n - butylamine , or methylamine . interestingly , with the exception of the n - benzyl - n - methylquinazoline-2,4-diamine 12 , all of these compounds demonstrated submicromolar ec50 values against l. donovani . among these six quinazolines tested , the n - benzylquinazoline-2,4-diamines 1517 appeared to be at least 2-fold more potent against l. donovani than the n - benzyl counterparts 1214 , suggesting that an n - benzyl is more favorable than an n - benzyl for antileishmanial activity . for l. amazonensis , the set of 1217 displayed a similar activity trend , with n - benzylquinazolines 15 and 16 being the most potent compounds with submicromolar or single digit micromolar ec50 values . amphotericin b is the internal control for the in vitro antileishmanial activity assay with ec50 = 40 9 nm against l. donovani and ec50 = 89 16 nm against l. amazonensis . nfurfuryl - n - isopropyl - substituted quinazoline 2 , with ec50 values of 2.5 m against l. donovani and 3.7 m against l. amazonensis , was considered to be well suited as the key scaffold for a sar study focusing on the benzenoid moiety of the quinazoline core . in a systematic approach following the topliss operational scheme , compound 2 was monosubstituted with a chlorine atom , a methyl group , or a methoxy group in the 5- , 6- , 7- , and 8-positions to probe the benzenoid ring for steric and electronic effects . among the chloro - substituted subseries , 5- and 6-substituted analogues 18 and 19 were less potent by a factor of 2 or more compared to reference 2 , while the 7- and 8-substituted quinazolines 20 and 21 displayed an activity on par with compound 2 . for the methoxy - substituted quinazolines , potency increased according to substitutions in 8-position < 7-position < 5-position 6-position , whereas a potency dependence in the order of 8-position 6-position < 7-position 5-position was observed for the methyl - substituted compounds . the quinazolines displaying potent antileishmanial activity , especially the n - benzylquinazoline-2,4-diamines 1517 , were shown to have respectable si values of 10 and larger , indicating that they are relatively selective , nontoxic chemotypes . the potency of selected quinazolines against antimony - resistant leishmania was assessed using murine peritoneal macrophages infected with two l. donovani clinical strains , bpk206/0 and bpk164/1 ( table 3 ) . strain bpk164/1 was isolated from a bone marrow aspirate taken from a nepalese visceral leishmaniasis patient not responding to antimonial therapy ; the bpk164/1 strain was originally found to be 6-fold resistant to sodium stibogluconate compared to the antimony - susceptible reference strain bpk206/0 . there are several possible reasons for the differences in susceptibility observed between the clinical l. donovani isolates and the -lactamase expressing genetically modified lab strain : ( 1 ) the former strains are from the indian subcontinent , while the lab strain is of african origin . antileishmanial drugs display differences in efficacy for treating indian visceral leishmaniasis compared to african visceral leishmaniasis ; strain differences could account for such differential susceptibility ; ( 2 ) the clinical strains could have a greater fitness in an in vitro intracellular amastigote model than the lab adapted strain which has been in axenic culture for many passages , leading to lower compound susceptibility in the former ; ( 3 ) the clinical strains were assayed using a different method under different conditions compared to the -lactamase expressing lab strain , potentially contributing to distinctions in compound susceptibility . interestingly , we saw no antagonism of activity of quinazolines 10 , 12 , 13 , 15 , 16 , or 23 for the mammalian cell line ( j774a.1 ) in the presence of folinic acid . in vitro efficacy of quinazolines , methotrexate ( mtx ) , pyrimethamine ( pyr ) , and miltefosine ( milt ) for axenic amastigotes of leishmania donovani ( a c ) and j774.a1 macrophages ( d f ) in the absence or presence of d , l - folinic acid ( fna ) . in parallel to the testing of the compounds for antileishmanial activity in vitro , a structure property relationship ( spr ) study focusing on log d , aqueous solubility , and permeability has been conducted with all compounds to assess potential physicochemical liabilities ( table 4 ) . generally , the aqueous solubility , the distribution coefficient log d , and the permeability of all quinazolines display a ph dependence ( exemplified on log d or permeability in table 4 ) . however , since the aqueous solubility , the distribution coefficient , and the permeability are within the acceptable ranges ( solubility of > 20 m , pe > 10 10 cms , 1 < log d < 4 ) , the quinazoline compound series is considered to be suitable for the development of bioavailable antileishmanial compounds . for the determination of the pe values , the following internal controls were utilized : carbazepine ph 4.0 permeability pe = 108 10 cm / s and ph 7.4 permeability pe = 130 10 cm / s ; ranitidinehcl ph 4.0 permeability pe = 5.2 10 cm / s and ph 7.4 permeability pe = 2.2 10 cm / s ; verapamilhcl ph 4.0 permeability pe = 20.6 10 cm / s and ph 7.4 permeability pe = 1360 10 cm / s . for the selection of the candidates for in vivo efficacy against l. donovani , only compounds that displayed submicromolar in vitro activity against l. donovani with a si value greater than 10 and a balanced combination of good physicochemical properties were chosen . among the benzyl - substituted quinazolines 1217 , 15 and 16 appeared to be the best candidates for in vivo evaluation because of their combination of potency , selectivity , and favorable physicochemical properties . from the compound subseries substituted at the benzenoid ring , the physicochemical properties were not discriminatory , and hence , compound 23 was chosen as a viable candidate because of its submicromolar ec50 against l. donovani as well as the outstanding si value of this compound ( > 40 ) . the three compounds mentioned above were dissolved in an appropriate vehicle ( 15 and 23 dissolved in 0.5% methylcellulose and 0.1% tween 80 in water ; 16 dissolved in 45% ( w / v ) ( 2-hydroxypropyl)--cyclodextrin solution ( hpcd ) ) and administered to uninfected balb / c mice intraperitoneally to determine a tolerated dose for in vivo efficacy studies . considering the toxicity of 16 and 23 when given at 30 mg / kg ip , lower doses of these compounds were administered in subsequent in vivo efficacy studies in a murine visceral leishmaniasis model ( figure 4 ) . there was also no significant difference in the parasite burden between mice in the group treated with compound 16 ( 5 10 mg / kg ip ) and the vehicle control group ( p > 0.05 ) . results are presented as the liver parasitemia ( ldu ) for each mouse ( ) , and the average ldu in each group ( ) was determined by microscopy ( n = 4 ) : ( a ) ldu for mice treated with 16 and 23 ; ( b ) ldu for mice treated with 15 . after ip administration , plasma concentration reached a cmax of 5.2 and 2.7 m for 16 and 23 , respectively , before decreasing rapidly in the first 4 h , followed by a slower elimination process until 24 h. the rapid decline in plasma concentration was likely due to extensive tissue redistribution after absorption , as indicated by the high target tissue concentrations . the terminal elimination half - life ranged from 5 to 20 h. minor reversible overt toxicity ( hypoactivity ) was observed after ip administration of 16 ; however , more severe toxicity was observed , unexpectedly , after a single ip administration of 23 , which warranted euthanization of some mice within 15 min postdose . as efficacy was evaluated at lower doses than 30 mg / kg to avoid toxicity and dose linearity for pharmacokinetic outcomes were unknown , it is not possible to directly correlate drug exposure with antileishmanial activity in mice . in addition , it is worth pointing out that the terminal half - life of 23 after ip administration was considerably shorter than that after po administration ( 5 h vs 20 h ; table 5 ) , and the liver concentration of 23 was detectable at 12 and 24 h after po administration , whereas it was below the detection limit after ip administration ( figure 6 ) . plasma ( open circles ) and tissue ( squares for liver and triangles for spleen ) concentration time profiles after po ( a ) and ip ( b ) administration of 16 in mice at a dose level of 100 mol / kg ( 30 mg / kg ) . the most potent activities with ec50 values in the submicromolar range against l. donovani were obtained with quinazoline-2,4-diamine scaffolds bearing either a n - benzyl - n - alkyl / phenyl or a n - ispropyl - n - furfuryl substituent combination . furthermore , although the benzenoid ring of the quinazoline-2,4-diamine scaffold has been identified to play a secondary role for efficacy , quinazolines substituted at the 5- or 6-position with one methyl or one methoxy group have also been identified to possess submicromolar ec50 values against l. donovani . although a clear correlation between in vitro activity , in vitro physicochemical properties , and in vivo activity is not clearly observed , the potencies of front - runner compounds 15 , 16 , and 23 in conjunction with favorable physicochemical properties make n , n - disubstituted quinazoline-2,4-diamines a suitable platform for the future development of antileishmanial agents . for future compound design to be successful , optimization will focus not only on improving antileishmanial activity and key physicochemical properties but also on improving the pharmacokinetics and si values for the entire quinazoline compound series . data for h nmr are reported as follows : chemical shift ( ppm ) , multiplicity ( s = singlet , d = doublet , t = triplet , q = quartet , p = pentet , sext = sextet , sept = septet , oct = octet , m = multiplet ) , integration , and coupling constant ( hz ) , whereas c nmr analyses were reported in terms of chemical shift . 1 equiv of quinazoline-2,4-dione and 1 equiv of n , n - dimethylaniline were mixed in 12 equiv of phosphorus oxychloride , and the mixture was refluxed under an argon atmosphere until starting material was no longer present by tlc ( 316 h ) . the potency of compounds 15 , 16 , and 23 against intracellular antimony - resistant clinical isolate l. donovani mhom / np/02/bpk164/1 and antimony - susceptible isolate l. donovani mhom / np/03/bpk206/0 was also assessed . toxicity of compounds against the murine macrophage cell line j774a.1 was conducted using the 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assay as previously described except that cells were incubated with compounds for 72 h. the in vivo antileishmanial efficacy of compounds was evaluated in a murine model of visceral leishmaniasis by the general method described earlier with minor modifications . 16 ( dissolved in 50% dma ) and 23 [ dissolved in peg400/dma / water = 5:3:2 ( v / v ) ] were administered at a dose of 100 mol / kg body weight ( or approximately 30 mg / kg ; dose volume = 5 ml / kg ) via oral gavage ( po ) or intraperitoneal ( ip ) administration to swiss webster mice . noncompartmental pharmacokinetic analysis of plasma concentration versus time curves was performed to obtain the area under the concentration time curve ( auc ) , cmax , tmax , and terminal half - life ( t1/2 ) ( winnonlin 6.3 , pharsight , mountain view , ca ) .

the integrity and feasibility of all clinical trials should be assured through scientific review of proposals or protocols by a panel of subject experts . such a review includes assessment of particular features relevant to the research proposal , the team , and environment associated with the proposed research , project design , preliminary data , likely relevance of the anticipated outcomes of the research , and budget appropriateness . this process establishes accountability of the study process in terms of commitment of the time , resources , and effort of all parties engaged in clinical trials . although the majority of clinical trials undergo formal review , this is not always the case . commercially sponsored trials , donorsponsored trials , and hospitalsubsidized trials may not receive a scientific peer review separate from an evaluation by the institutional animal care and use committee ( iacuc ) , the mandate of which does not always include such scientific merit review . although iacuc approval currently is mandatory for all research involving animals that are conducted by institutions that receive federal funding , it is primarily designed to protect the welfare of laboratory animals , not privately owned pets.11 the iacuc review process may face challenges in addressing some scientific and ethical concerns that are unique to clientowned animals involved in clinical research . for this type of animal research , particular attention to and critical evaluation of clinical merit this evaluation has similarities to that performed by institutional review boards ( irbs ) charged with evaluating clinical trials involving humans . in addition , postapproval monitoring and research oversight should be an integral component of all veterinary clinical trials , including standardized clinical endpoint assessments , the need for interim evaluations , accrual targets , and adherence to patient enrollment criteria . a rigorous scientific and ethical review of all veterinary clinical trials is warranted to ensure the validity of the research and protection of animal patient welfare , and to maximize translational potential . the adoption of innovative trial designs that decrease the number of patients needed for meaningful results should be vigorously encouraged . conduct of trials so designed is also compatible with the tenets for appropriate use of animals in research : replace , reduce , and refine.12 several recent reports describe standard and modified novel trial designs currently used for veterinary clinical studies in companion animals with cancer.13 , 14 , 15 two examples of these include ( 1 ) adaptive or bayesian designs for phase i iii trials in which a dynamic assessment of data is used to continuously update the probability of outcomes such as safety , efficacy , or both , at the same time in order to decrease early phase patient enrollment16 , 17 ; and ( 2 ) enrichment of the study population to insure the new product will be studied in the correct subset of patients capable of responding appropriately to a specific targeted therapeutic agent and the use of validated biological endpoints that regulate downstream response.18 the continuous evaluation of innovative trial designs should improve risk : benefit evaluation and result in fewer patients at risk and fewer patients needed to make a go / no go decision to the next level of investigation . the assessment of goals , risks and cost are critical determinants of an owner 's decision to enroll in a study . table 2 compares the riskbenefit relationship between the human patient and the study team for phase 0 and phase i trials which represent those with the most controversial ethical concerns in the clinical trials continuum . phase 0 trials are those in which minor procedures are conducted on a patient , and involve minimal clinical impact of either toxicity or efficacy.19 , 20 such trials may include fineneedle aspirates of a patient 's tumor to assess a biological process or target modulation or microdosing of a compound to define preliminary pharmacokinetics without risk of systemic effects . phase 0 and phase i trials often are conducted in both human and animal patients with poor to grave prognoses that have already progressed through multiple conventional or investigational treatment regimens or both . in both phase 0 and phase i studies , there may be minimal to no benefit to the patient . as explained further in the section on informed consent recommendations it is important for the clinical trials team to thoroughly understand and clearly articulate the purpose and potential risks and benefits of the study . phase i trials in human patients.20 similar issues and concerns seemingly apply to animal patients phase 0 : microdosing , target modulation detection with biopsy , biomarkers . phase i : dose escalation trials to determine maximally tolerated dose . some toxicity and perhaps some benefit may result from study . focus : physicianscientist assists the patient with their medical needs . in animal patients , other ethical scenarios arise because standards of care do not exist for many cancers in companion animals . as a result , phase 0 , phase i , and some phase ii trials may be offered early in the treatment course . in addition , even when conventional treatment options are available , these treatments may be delayed while shortterm study interventions ( such as drug microdosing or tumor sampling ) are conducted in exchange for a therapeutic incentive provided by the study sponsor to support subsequent conventional treatment . a potential ethical conflict occurs when an animal is placed at risk , real or perceived , in return for a therapeutic incentive . the owner is placed in a financially vulnerable position when study completion is required before receiving reimbursement for veterinary care . the conduct of randomized clinical trials with a control group ( either a true placebo or a notreatment group ) may be necessary in some situations when there is no known effective conventional treatment for comparison with the investigational product . such trials may be necessary for evaluation of a new animal health product , but placebo or notreatment control groups are only needed rarely for animal patient studies focused on , product development for humans , ( ie , comparative oncology trials ) . trials involving a placebo group should be minimized in patients with cancer and careful evaluation of the risk of tumor progression during placebo treatment period must be considered.21 , 22 , 23 all patients enrolled in clinical trials , whether studied with or without a control group , must receive best supportive care . ( bsc ) is defined as treatment focusing on relieving symptoms ( eg , pain , inappetence ) in order to maintain or improve quality of life , but does not include treatment directed specifically at the cancer . best supportive care may include analgesics , appetite stimulants , antibiotics , and antiemetics as needed to maintain or improve quality of life . as an example , a randomized placebocontrolled trial could divide patients into an investigational new treatment plus bsc group versus a placebo treatment plus bsc group . consequently , because of financial support for enrollment in a clinical study that provides frequent monitoring and bsc management , study participants conceivably could receive improved care regardless of study group assignment . nonetheless , full disclosure of the trial design , study purpose , and all contingencies must be clearly communicated to the owner . in addition to providing bsc for all study patients , strict criteria for release from the study or offer of rescue treatment must be in place if progressive tumor growth occurs or if the patient experiences substantial adverse clinical signs . recommended criteria exist in veterinary oncology including response evaluation criteria in solid tumors ( recist ) measurements , response evaluation criteria for peripheral nodal lymphoma ( recpnl ) , and the veterinary cooperative oncology group common terminology criteria for adverse event ( vcogctcae ) reporting.24 , 25 , 26 it also is recommended that trials incorporate a quality of life scoring system and performance scores throughout the enrollment period and the conduct of the trial to characterize the impact of treatment on these indicators of response.27 , 28 biomarkers and imaging assessments also provide evidence of cancer progression and such outcomes routinely are included in trials . crossover trial designs to the investigational treatment , exit to a distinct therapeutic cohort or management with a noninvestigational finally , weighted randomization ( eg , 2:1 investigational compound : placebo accrual ratio ) frequently is used to compare a smaller group of patients in the placebo group to an investigational product group if determined to be acceptable by a formal statistical assessment of outcome expectations . the selection and evaluation of meaningful and measureable endpoints for each study must be clearly described in the protocol . for example , pharmacologic measurements , targetdrug interactions , and genetic determinants of treatment outcomes frequently are assessed in personalized therapeutic trials . toxicity endpoints are quantified by the use of vcogctcae criteria and are standardized for acute adverse event reporting.24 chronic or delayed toxicity may be subject to reporting bias if followup is not consistent or timed appropriately . reporting bias of efficacy endpoints can occur from imprecise or incomplete collection of clinical data or from interindividual interpretation variability in endpoints such as pathologic assessments or imaging interpretation . efficacy endpoint evaluations after a clinical study should be conducted on a defined schedule ( eg , thoracic radiographs every 2 months ) and any variance from this schedule should be reported . otherwise , the progressionfree interval may be subject to significant error as tumor progression or metastasis may be only intermittently evaluated creating a large margin of error . the interpretation of imaging data or pathologic samples also is subject to variability , and adjudication procedures should be described and followed in the protocol . multiple , independent reviews of clinical endpoint assessments by qualified specialists should be considered best practice for clinical trials.29 , 30 overall survival is a relatively poor efficacy endpoint because of multiple , potentially competing , and confounding issues . owner acceptance of palliative or hospice care of their pets is variable , multiple interventions that are challenging to account for may be attempted to achieve responses after tumor progression and censoring rules for death may not be accurately interpreted . in addition , euthanasia is a wellknown confounder for determination of overall survival in animal patients enrolled in clinical trials.31 elective euthanasia , when a patient in a selfsupportive , clinically stable status is euthanized at the owner 's request , must be considered carefully when examining outcome data from clinical trials . complete financial support for necropsy procedures , cremation , and return of animal remains may minimize concerns about endpoint misinterpretation and optimize owner participation . when clinical trials are conducted for purposes of registration of results for approval of a new therapeutic entity with the center for veterinary medicine of the federal drug administrtion ( fda ) , a separate standard of oversight and documentation are required . multiple data recorders and monitors , data audit requirements , sample custody issues , and secondary biorepository sites to insure sample security are some of the additional responsibilities . the specific requirements and execution for these trials a recent review assessed the coordination of the approval of clinical trials in companion animals between the institutional iacuc committee and an advisory committee that is comprised of veterinary clinical specialists.32 several institutions have adopted the model of a clinical review board ( crb ) to evaluate the merit , feasibility , and compliance with ethical standards for clinical trials in clientowned animals . these boards or committees may be granted the ability to recommend a waiver of full review from the iacuc when appropriate . the iacuc may support the review from the clinical review board or choose to implement a full review . the avma also has recently addressed clinical trial management and approved a policy entitled establishment and use of veterinary clinical studies committees.33 according to this policy , the vcsc would evaluate clinical research that conforms to general standards of care but requires additional biomaterials that would be in excess of residual portions of samples taken only for routine health screening under a clientveterinarian relationship . similar to the function of the crb , the vcsc serves to ensure informed consent and to protect animals from conflict of interest issues . the avma recommends that when the vcsc determines that the protocol of a clinical research study will influence the management of the animal patient , the veterinary clinical studies committee should refer the proposed work for iacuc review . broad representation of clinical expertise is needed for a clinical review board and should include at least 1 member of the iacuc committee as a liaison for communication and clarification . someone with expertise in animal welfare should be included on the iacuc , crb , or both . currently , expertise in veterinary clinical research ethics is frequently not available , but expanding this resource is desirable . specific training for crb members has not been established but should be developed in the future . resources developed to aid clinical irb members for trials involving humans to evaluate the scientific merit of a study , informed consent forms ( icfs ) , and the consenting process can be readily adapted for veterinary clinical trials.34 the usual responsibilities of the vcsc or crb include consideration of study merit , study feasibility , and the informed consent process . a review of the clinical study design by a peer group is particularly useful for those trials that have not undergone a formal scientific review . complex trials may require an expert ad hoc assessment by those best qualified to evaluate the project . the crb may request review by such ad hoc experts . the crb also may be tasked with other responsibilities such as evaluation of conflict of interest , coordination of postapproval monitoring and insuring adverse event reporting for all study participants . . administrative support , educational support for the members of the crb and clinical investigators , and data capture management systems are recommended as best practices . several specific considerations for veterinary cancer clinical trials involving new drugs and devices have been emphasized.32 for example , careful consideration of preliminary data on toxicity in the target animal species is critical before to initiating a clinical study . insufficient information about product safety may result in failure of the clinical trial to be approved under the full review and oversight of the iacuc . oversight of the clinical trial approval process in nonacademic or private veterinary practices also must be addressed and should evolve as such facilities are asked to participate in multisite clinical studies . several issues that may be of concern include oversight of animal welfare assurance and sufficient training for veterinarians and staff . if a study is being conducted under the assurance of an institution with a formal iacuc , that institution is responsible for completion of all requirements for compliance such as the informed consent process and its documentation , communication of adverse events with all other sites involved , and postapproval monitoring procedures . another option is to contract with a professional iacuc associated with a contract research organization ( cro ) that is charged with ensuring that similar responsibilities are met at the nonacademic site . informed consent provides the ethical assurance of patient welfare , owner understanding , and study compliance throughout clinical research . the optimal process of informed consent for humans enrolling in clinical trials has been challenging to achieve effectively . guidelines have been continually revised.35 , 36 , 37 , 38 the guiding principle stated in table 1 [ the consent process must be honest , thorough , wellcommunicated , and the owner must have adequate time to consider participation without real or perceived coercion ] fulfills the intention of the informed consent process from the owner 's perspective and should drive communications relevant to enrollment of an animal patient in clinical oncology trials . recently , the national cancer institute ( nci ) reviewed and simplified its standard informed consent document for human patients in order to ensure better comprehension , and we recommend that veterinary cancer clinical trials adopt a similar template.39 full disclosure of the study purpose , associated risks and benefits , study design and interventions required , funding source for the study , incentives to the owner , and conflict of interest ( coi ) statements from the study staff and investigators are required elements to address during the consent process . the recommended best practice for the consenting process is that it be managed by a neutral individual ( nurse , nonsponsor supported veterinarian ) with a witness present . examples of the intended purpose of a study include whether the study seeks to improve clinical signs associated with cancer or its treatment , if a study represents a safety or efficacy study and whether the study is intended to ultimately benefit animal health , human health , or both . animal patients participating in clinical trials without an efficacy intent must receive extraordinary protection all potential scenarios of risk should be explained , particularly for phase i trials that are designed to identify a safe or maximally tolerated dose when direct patient benefit may not be achieved . the source of funding for each study should be disclosed because it often indicates whether or not any potential coi exists.40 , 41 , 42 conflict of interest concerns should be disclosed and , if necessary , managed in a manner consistent with the office of research integrity guidelines from the department of health and human services.43 all financial subsidies or incentives should be carefully and fully described . , a standard of care does not formally exist in veterinary oncology for many types of cancer . therefore , the enrollment of animal patients , when conventional treatment offers little likelihood of response or success , requires very careful discussion regarding study goals and expectations . even more critical is the communication with pet owners who may be unable to afford an effective , conventional treatment option , and are inclined to enroll their pets into a preliminary clinical trial to receive a subsidy for the conventional treatment . the ethical issues of considering the enrollment of animal patients into clinical trials under these circumstances should be clearly understood by the study team and discussed honestly with the owner . a primary advocate for the owner of an animal patient being considered for enrollment into a clinical trial currently , the principal investigator or member of the clinical team such as the study coordinator or study nurse often is considered the primary advocate if no conflict of interest exists in the study ( or if it is disclosed and managed ) . careful oversight of whether study personnel are in a position of conflict is vital to the trust required of owners to enroll their pets in clinical trials . in addition , there should be a defacto advocate assigned whom the owner can contact if he or she does do not feel comfortable communicating concerns with the pi . for example , a designated ombudsman , counselor , or a clinical trial hotline for anonymous communication could be made available . a copy of the informed consent document should be provided for review and consultation with the owner and the identified patient advocate . if needed , independent psychological , social , and grief counseling support should be identified and utilized for discussions related to clinical study advocacy during the consenting process . the informed consent document should describe the aspects of the study in simple , concise , and easytounderstand language . an evaluation of the quality of patient comprehension after a standard informed consent process identified several features that were statistically associated with high patient recall of study goals and requirements.44 these include the use of the nci template , the extent of the patient 's education , high english language proficiency , and involvement of a nurse familiar with the study and thorough reading of the consent form with adequate time to consider the study consent issues outside of the examination room . oversight and surveillance of clinical trials after approval and during conduct of the study often are negligible in companion animals but should be rigorously applied to ensure accurate interpretation of results . postapproval monitoring commonly applies to more traditional studies employing laboratory animals as a formal policy to observe and ensure good welfare practices and adverse event ( eg , pain ) management . such postapproval monitoring procedures are designed to provide close observation when there is a required action ( eg , addendum to the protocol ) or when a protocol breach is suspected . the postapproval monitoring of clinical trials also should manage animal welfare issues , trial protocol adherence , and owner concerns . in addition , postapproval monitoring should include oversight of data relevant to endpoint assessment , adjudication , and adverse event communication . indications for a postapproval audit may include any concerns regarding study participant eligibility , use of proper informed consent procedures , protocol compliance , and adverse event or compliance reporting . a confidential mechanism for communication of concerns identified by anyone engaged in clinical studies including owners , staff or students should be available . routine or random postapproval monitoring efforts including audits of clinical trial procedures may be recommended to provide ongoing quality assurance and accountability . postapproval monitoring procedures and audits for conventional laboratory animal studies often are conducted by the university veterinarian or delegate , and include a mechanism for study coordinators to respond to or appeal the audit . the process for clinical study postapproval monitoring also could be managed , as with more routine situations , by the institutional veterinarian or could be the responsibility of the clinical review board , iacuc , or the research integrity and compliance review committee at the institutional level or through a cro . the nci has developed audit guidelines that are readily adaptable to veterinary clinical trials.45 the role of the academic hospital administration and facilities in the conduct of clinical research also is a critical component of a clinical trials program in veterinary medicine . the hospital may be the repository for study documents ( eg , consent forms ) , provide trained personnel and facilities to execute sampling and data gathering , offer financial support for animal care , and provide counseling and emotional support for owners . the use of consolidated standards of reporting trials ( consort ) guidelines offers a wellaccepted and standardized method to assess design for clinical trials in humans.46 , 47 many journals now require such reporting guidelines compliance for publication of clinical trials . until an equivalent set of guidelines is developed for companion animal trial reporting , the itemized listing of trial reporting requirements is sufficient , and should ideally be broadly implemented . it provides a catalog of initiated trials that is accessible by clinical investigators and owners considering enrollment in a clinical trial . a clinical trials registry for human participants was initiated in 2000 and is managed through the national library of medicine.48 registration of a clinical trial is required for future publication of trial results . this registry ensures public access to trial information and permits assessment of unnecessary duplication of trials . the veterinary cancer society has developed a voluntary listing of active clinical trials for pets with cancer.49 however , resource limitations preclude archival data collection , data retrieval , and a mechanism for reporting trial results . a comprehensive clinical trial registry system should be considered as an essential component of clinical trials best practices . effective data sharing is considered vital to the success of multiinstitutional clinical trial conduct and was the subject of an iom workshop.50 the scientific and ethical considerations of data sharing are summarized in the following excerpts from that report:the moral and ethical arguments for data sharing center on fulfilling obligations to research participants , minimizing safety risks , and honoring the nature of medical research as a public good . the practical and scientific arguments for data sharing include improving the accuracy of research , informing risk / benefit analysis of treatment options , strengthening collaborations , accelerating biomedical research , and restoring trust in the clinical research enterprise . the moral and ethical arguments for data sharing center on fulfilling obligations to research participants , minimizing safety risks , and honoring the nature of medical research as a public good . the practical and scientific arguments for data sharing include improving the accuracy of research , informing risk / benefit analysis of treatment options , strengthening collaborations , accelerating biomedical research , and restoring trust in the clinical research enterprise . implicit in the concept of data sharing is open access to data , which provides greater transparency of study protocols , study reports , and adverse event documentation . several data management systems have been developed for data sharing , and an effort should be made to develop a usable , costeffective platform that would integrate multiinstitutional trial data , and ideally integrate with data management platforms used in clinical studies in humans . as mentioned previously , publication of results should follow accepted guidelines that describe the design , conduct and analysis of clinical trials . the publication of all trial results , whether associated with a positive or a negative outcome , in a searchable database would address the a recent editorial from the editors of the british journal of pharmacology details a policy of reporting transparency of research involving animals including increased accountability for animal use and open access to all primary data including negative studies.51 such reporting guidelines are likely to be adopted by numerous journals . to fully realize such guidelines , a publication strategy would need to be established for negative study reporting and the release of embargoed data from contractsponsored research after a suitable period . an overarching purpose of the workshop and this document is to encourage continued research and education on clinical trial improvement by outreach to scientists , veterinarians , and the petowning public . biomedical researchers should be educated about the value of translational research in naturally occurring diseases in companion animals and the various resources and opportunities available to do so . veterinary schools need to be informed of the resources available so as to encourage and facilitate the responsible conduct of clinical research and bioethics by their students , faculty , and employees.43 , 52 veterinary specialists need to be aware of research opportunities and how to incorporate them into their practices . general veterinary practitioners need to be educated in the value of clinical trials for their patients and owners . the ability to improve owner comprehension regarding clinical trials is an essential component of this concept . owners need to clearly understand how knowledge gained by enrollment in clinical trials might help companion animals and humans in the future even though trials may not be designed to help their individual pet . equally important is facilitating the continued debate regarding unique ethical considerations of clinical research in companion animals . finally , the value of translational clinical research into cancer care linking animals and humans should be better defined and promoted to all stakeholders . it is implicit in best practice recommendations that regular review and revision will occur as required by the development of resources , regulations , and ethical priorities . such revisions will be driven by both quantitative and qualitative research concerning communication with , and comprehension of , companion animal owners , improved trial design , and compliance . as with the evolution of clinical trial conduct in human oncology , we suspect that owner participation in data gathering and outcome assessment will become better integrated in trial management . the role of social media methods to support trial completion and to educate the public regarding the critical role of studies to improve animal health inevitably will grow . it will likewise be critical to review and adopt best practices as they emerge from other disciplines , institutions , and countries engaged in discussion on the ethical and proper conduct of clinical studies . we anticipate that additional workshops will be forthcoming to determine more best practice recommendations or deepen discussions on current issues and will include multidisciplinary perspectives within oncology and affiliated specialties . when properly addressed by adherence to sound scientific principles , ethical conduct of research , and respect for patients and their owners , better health may result in the future for all members of the family animals and people .

development of effective and safe treatments for companion animals with cancer requires the collaboration of numerous animal health professionals and the full engagement of animal owners . establishing best practice recommendations for clinical trials in veterinary oncology represents an important step toward meeting the goal of rigorous clinical trial design and conduct that is required to establish valid evidence . likewise , optimizing patient welfare and owner education and advocacy is crucial to meet the unique ethical obligations to both owners and animals enrolled in these clinical trials and to ensure trust in the team conducting the research . to date , best practice recommendations for clinical trial conduct have not been reported for veterinary oncology . this document summarizes the consensus of a workshop held in november , 2014 to identify relevant ethical principles and to ensure responsible conduct of clinical research in companion animals with cancer . it is intended as a working document that will be updated as advances in science and ethical considerations require . to the extent possible , existing guidelines for the conduct and oversight of clinical trials in humans have been adapted for veterinary trials to avoid duplicative effort and to facilitate integration of clinical trials such that translational research with benefits for both companion animals and humans are encouraged .

Study Design Best Practices and Ethics Recommendations Clinical Trial Approval Policies Best Practice Recommendations Consenting Process Best Practices And Ethics Recommendations Postapproval Monitoring Best Practices Recommendations Reporting And Publication Best Practice Recommendations Commitment to Improvement of the Clinical Trial Process

the integrity and feasibility of all clinical trials should be assured through scientific review of proposals or protocols by a panel of subject experts . such a review includes assessment of particular features relevant to the research proposal , the team , and environment associated with the proposed research , project design , preliminary data , likely relevance of the anticipated outcomes of the research , and budget appropriateness . this process establishes accountability of the study process in terms of commitment of the time , resources , and effort of all parties engaged in clinical trials . although the majority of clinical trials undergo formal review , this is not always the case . although iacuc approval currently is mandatory for all research involving animals that are conducted by institutions that receive federal funding , it is primarily designed to protect the welfare of laboratory animals , not privately owned pets.11 the iacuc review process may face challenges in addressing some scientific and ethical concerns that are unique to clientowned animals involved in clinical research . for this type of animal research , particular attention to and critical evaluation of clinical merit this evaluation has similarities to that performed by institutional review boards ( irbs ) charged with evaluating clinical trials involving humans . in addition , postapproval monitoring and research oversight should be an integral component of all veterinary clinical trials , including standardized clinical endpoint assessments , the need for interim evaluations , accrual targets , and adherence to patient enrollment criteria . a rigorous scientific and ethical review of all veterinary clinical trials is warranted to ensure the validity of the research and protection of animal patient welfare , and to maximize translational potential . conduct of trials so designed is also compatible with the tenets for appropriate use of animals in research : replace , reduce , and refine.12 several recent reports describe standard and modified novel trial designs currently used for veterinary clinical studies in companion animals with cancer.13 , 14 , 15 two examples of these include ( 1 ) adaptive or bayesian designs for phase i iii trials in which a dynamic assessment of data is used to continuously update the probability of outcomes such as safety , efficacy , or both , at the same time in order to decrease early phase patient enrollment16 , 17 ; and ( 2 ) enrichment of the study population to insure the new product will be studied in the correct subset of patients capable of responding appropriately to a specific targeted therapeutic agent and the use of validated biological endpoints that regulate downstream response.18 the continuous evaluation of innovative trial designs should improve risk : benefit evaluation and result in fewer patients at risk and fewer patients needed to make a go / no go decision to the next level of investigation . table 2 compares the riskbenefit relationship between the human patient and the study team for phase 0 and phase i trials which represent those with the most controversial ethical concerns in the clinical trials continuum . phase 0 trials are those in which minor procedures are conducted on a patient , and involve minimal clinical impact of either toxicity or efficacy.19 , 20 such trials may include fineneedle aspirates of a patient 's tumor to assess a biological process or target modulation or microdosing of a compound to define preliminary pharmacokinetics without risk of systemic effects . in both phase 0 and phase i studies , there may be minimal to no benefit to the patient . as explained further in the section on informed consent recommendations it is important for the clinical trials team to thoroughly understand and clearly articulate the purpose and potential risks and benefits of the study . phase i trials in human patients.20 similar issues and concerns seemingly apply to animal patients phase 0 : microdosing , target modulation detection with biopsy , biomarkers . phase i : dose escalation trials to determine maximally tolerated dose . in animal patients , other ethical scenarios arise because standards of care do not exist for many cancers in companion animals . as a result , phase 0 , phase i , and some phase ii trials may be offered early in the treatment course . the owner is placed in a financially vulnerable position when study completion is required before receiving reimbursement for veterinary care . the conduct of randomized clinical trials with a control group ( either a true placebo or a notreatment group ) may be necessary in some situations when there is no known effective conventional treatment for comparison with the investigational product . such trials may be necessary for evaluation of a new animal health product , but placebo or notreatment control groups are only needed rarely for animal patient studies focused on , product development for humans , ( ie , comparative oncology trials ) . trials involving a placebo group should be minimized in patients with cancer and careful evaluation of the risk of tumor progression during placebo treatment period must be considered.21 , 22 , 23 all patients enrolled in clinical trials , whether studied with or without a control group , must receive best supportive care . nonetheless , full disclosure of the trial design , study purpose , and all contingencies must be clearly communicated to the owner . in addition to providing bsc for all study patients , strict criteria for release from the study or offer of rescue treatment must be in place if progressive tumor growth occurs or if the patient experiences substantial adverse clinical signs . recommended criteria exist in veterinary oncology including response evaluation criteria in solid tumors ( recist ) measurements , response evaluation criteria for peripheral nodal lymphoma ( recpnl ) , and the veterinary cooperative oncology group common terminology criteria for adverse event ( vcogctcae ) reporting.24 , 25 , 26 it also is recommended that trials incorporate a quality of life scoring system and performance scores throughout the enrollment period and the conduct of the trial to characterize the impact of treatment on these indicators of response.27 , 28 biomarkers and imaging assessments also provide evidence of cancer progression and such outcomes routinely are included in trials . crossover trial designs to the investigational treatment , exit to a distinct therapeutic cohort or management with a noninvestigational finally , weighted randomization ( eg , 2:1 investigational compound : placebo accrual ratio ) frequently is used to compare a smaller group of patients in the placebo group to an investigational product group if determined to be acceptable by a formal statistical assessment of outcome expectations . the selection and evaluation of meaningful and measureable endpoints for each study must be clearly described in the protocol . reporting bias of efficacy endpoints can occur from imprecise or incomplete collection of clinical data or from interindividual interpretation variability in endpoints such as pathologic assessments or imaging interpretation . the interpretation of imaging data or pathologic samples also is subject to variability , and adjudication procedures should be described and followed in the protocol . multiple , independent reviews of clinical endpoint assessments by qualified specialists should be considered best practice for clinical trials.29 , 30 overall survival is a relatively poor efficacy endpoint because of multiple , potentially competing , and confounding issues . in addition , euthanasia is a wellknown confounder for determination of overall survival in animal patients enrolled in clinical trials.31 elective euthanasia , when a patient in a selfsupportive , clinically stable status is euthanized at the owner 's request , must be considered carefully when examining outcome data from clinical trials . complete financial support for necropsy procedures , cremation , and return of animal remains may minimize concerns about endpoint misinterpretation and optimize owner participation . when clinical trials are conducted for purposes of registration of results for approval of a new therapeutic entity with the center for veterinary medicine of the federal drug administrtion ( fda ) , a separate standard of oversight and documentation are required . the specific requirements and execution for these trials a recent review assessed the coordination of the approval of clinical trials in companion animals between the institutional iacuc committee and an advisory committee that is comprised of veterinary clinical specialists.32 several institutions have adopted the model of a clinical review board ( crb ) to evaluate the merit , feasibility , and compliance with ethical standards for clinical trials in clientowned animals . the avma also has recently addressed clinical trial management and approved a policy entitled establishment and use of veterinary clinical studies committees.33 according to this policy , the vcsc would evaluate clinical research that conforms to general standards of care but requires additional biomaterials that would be in excess of residual portions of samples taken only for routine health screening under a clientveterinarian relationship . similar to the function of the crb , the vcsc serves to ensure informed consent and to protect animals from conflict of interest issues . the avma recommends that when the vcsc determines that the protocol of a clinical research study will influence the management of the animal patient , the veterinary clinical studies committee should refer the proposed work for iacuc review . broad representation of clinical expertise is needed for a clinical review board and should include at least 1 member of the iacuc committee as a liaison for communication and clarification . currently , expertise in veterinary clinical research ethics is frequently not available , but expanding this resource is desirable . specific training for crb members has not been established but should be developed in the future . resources developed to aid clinical irb members for trials involving humans to evaluate the scientific merit of a study , informed consent forms ( icfs ) , and the consenting process can be readily adapted for veterinary clinical trials.34 the usual responsibilities of the vcsc or crb include consideration of study merit , study feasibility , and the informed consent process . a review of the clinical study design by a peer group is particularly useful for those trials that have not undergone a formal scientific review . . administrative support , educational support for the members of the crb and clinical investigators , and data capture management systems are recommended as best practices . several specific considerations for veterinary cancer clinical trials involving new drugs and devices have been emphasized.32 for example , careful consideration of preliminary data on toxicity in the target animal species is critical before to initiating a clinical study . insufficient information about product safety may result in failure of the clinical trial to be approved under the full review and oversight of the iacuc . oversight of the clinical trial approval process in nonacademic or private veterinary practices also must be addressed and should evolve as such facilities are asked to participate in multisite clinical studies . several issues that may be of concern include oversight of animal welfare assurance and sufficient training for veterinarians and staff . another option is to contract with a professional iacuc associated with a contract research organization ( cro ) that is charged with ensuring that similar responsibilities are met at the nonacademic site . informed consent provides the ethical assurance of patient welfare , owner understanding , and study compliance throughout clinical research . the optimal process of informed consent for humans enrolling in clinical trials has been challenging to achieve effectively . guidelines have been continually revised.35 , 36 , 37 , 38 the guiding principle stated in table 1 [ the consent process must be honest , thorough , wellcommunicated , and the owner must have adequate time to consider participation without real or perceived coercion ] fulfills the intention of the informed consent process from the owner 's perspective and should drive communications relevant to enrollment of an animal patient in clinical oncology trials . recently , the national cancer institute ( nci ) reviewed and simplified its standard informed consent document for human patients in order to ensure better comprehension , and we recommend that veterinary cancer clinical trials adopt a similar template.39 full disclosure of the study purpose , associated risks and benefits , study design and interventions required , funding source for the study , incentives to the owner , and conflict of interest ( coi ) statements from the study staff and investigators are required elements to address during the consent process . the recommended best practice for the consenting process is that it be managed by a neutral individual ( nurse , nonsponsor supported veterinarian ) with a witness present . examples of the intended purpose of a study include whether the study seeks to improve clinical signs associated with cancer or its treatment , if a study represents a safety or efficacy study and whether the study is intended to ultimately benefit animal health , human health , or both . animal patients participating in clinical trials without an efficacy intent must receive extraordinary protection all potential scenarios of risk should be explained , particularly for phase i trials that are designed to identify a safe or maximally tolerated dose when direct patient benefit may not be achieved . , a standard of care does not formally exist in veterinary oncology for many types of cancer . therefore , the enrollment of animal patients , when conventional treatment offers little likelihood of response or success , requires very careful discussion regarding study goals and expectations . even more critical is the communication with pet owners who may be unable to afford an effective , conventional treatment option , and are inclined to enroll their pets into a preliminary clinical trial to receive a subsidy for the conventional treatment . the ethical issues of considering the enrollment of animal patients into clinical trials under these circumstances should be clearly understood by the study team and discussed honestly with the owner . a primary advocate for the owner of an animal patient being considered for enrollment into a clinical trial currently , the principal investigator or member of the clinical team such as the study coordinator or study nurse often is considered the primary advocate if no conflict of interest exists in the study ( or if it is disclosed and managed ) . careful oversight of whether study personnel are in a position of conflict is vital to the trust required of owners to enroll their pets in clinical trials . for example , a designated ombudsman , counselor , or a clinical trial hotline for anonymous communication could be made available . a copy of the informed consent document should be provided for review and consultation with the owner and the identified patient advocate . the informed consent document should describe the aspects of the study in simple , concise , and easytounderstand language . an evaluation of the quality of patient comprehension after a standard informed consent process identified several features that were statistically associated with high patient recall of study goals and requirements.44 these include the use of the nci template , the extent of the patient 's education , high english language proficiency , and involvement of a nurse familiar with the study and thorough reading of the consent form with adequate time to consider the study consent issues outside of the examination room . oversight and surveillance of clinical trials after approval and during conduct of the study often are negligible in companion animals but should be rigorously applied to ensure accurate interpretation of results . postapproval monitoring commonly applies to more traditional studies employing laboratory animals as a formal policy to observe and ensure good welfare practices and adverse event ( eg , pain ) management . such postapproval monitoring procedures are designed to provide close observation when there is a required action ( eg , addendum to the protocol ) or when a protocol breach is suspected . the postapproval monitoring of clinical trials also should manage animal welfare issues , trial protocol adherence , and owner concerns . in addition , postapproval monitoring should include oversight of data relevant to endpoint assessment , adjudication , and adverse event communication . routine or random postapproval monitoring efforts including audits of clinical trial procedures may be recommended to provide ongoing quality assurance and accountability . the process for clinical study postapproval monitoring also could be managed , as with more routine situations , by the institutional veterinarian or could be the responsibility of the clinical review board , iacuc , or the research integrity and compliance review committee at the institutional level or through a cro . the nci has developed audit guidelines that are readily adaptable to veterinary clinical trials.45 the role of the academic hospital administration and facilities in the conduct of clinical research also is a critical component of a clinical trials program in veterinary medicine . the use of consolidated standards of reporting trials ( consort ) guidelines offers a wellaccepted and standardized method to assess design for clinical trials in humans.46 , 47 many journals now require such reporting guidelines compliance for publication of clinical trials . until an equivalent set of guidelines is developed for companion animal trial reporting , the itemized listing of trial reporting requirements is sufficient , and should ideally be broadly implemented . it provides a catalog of initiated trials that is accessible by clinical investigators and owners considering enrollment in a clinical trial . a clinical trials registry for human participants was initiated in 2000 and is managed through the national library of medicine.48 registration of a clinical trial is required for future publication of trial results . the veterinary cancer society has developed a voluntary listing of active clinical trials for pets with cancer.49 however , resource limitations preclude archival data collection , data retrieval , and a mechanism for reporting trial results . a comprehensive clinical trial registry system should be considered as an essential component of clinical trials best practices . effective data sharing is considered vital to the success of multiinstitutional clinical trial conduct and was the subject of an iom workshop.50 the scientific and ethical considerations of data sharing are summarized in the following excerpts from that report:the moral and ethical arguments for data sharing center on fulfilling obligations to research participants , minimizing safety risks , and honoring the nature of medical research as a public good . the practical and scientific arguments for data sharing include improving the accuracy of research , informing risk / benefit analysis of treatment options , strengthening collaborations , accelerating biomedical research , and restoring trust in the clinical research enterprise . the moral and ethical arguments for data sharing center on fulfilling obligations to research participants , minimizing safety risks , and honoring the nature of medical research as a public good . the practical and scientific arguments for data sharing include improving the accuracy of research , informing risk / benefit analysis of treatment options , strengthening collaborations , accelerating biomedical research , and restoring trust in the clinical research enterprise . implicit in the concept of data sharing is open access to data , which provides greater transparency of study protocols , study reports , and adverse event documentation . several data management systems have been developed for data sharing , and an effort should be made to develop a usable , costeffective platform that would integrate multiinstitutional trial data , and ideally integrate with data management platforms used in clinical studies in humans . as mentioned previously , publication of results should follow accepted guidelines that describe the design , conduct and analysis of clinical trials . to fully realize such guidelines , a publication strategy would need to be established for negative study reporting and the release of embargoed data from contractsponsored research after a suitable period . an overarching purpose of the workshop and this document is to encourage continued research and education on clinical trial improvement by outreach to scientists , veterinarians , and the petowning public . biomedical researchers should be educated about the value of translational research in naturally occurring diseases in companion animals and the various resources and opportunities available to do so . veterinary schools need to be informed of the resources available so as to encourage and facilitate the responsible conduct of clinical research and bioethics by their students , faculty , and employees.43 , 52 veterinary specialists need to be aware of research opportunities and how to incorporate them into their practices . general veterinary practitioners need to be educated in the value of clinical trials for their patients and owners . the ability to improve owner comprehension regarding clinical trials is an essential component of this concept . owners need to clearly understand how knowledge gained by enrollment in clinical trials might help companion animals and humans in the future even though trials may not be designed to help their individual pet . equally important is facilitating the continued debate regarding unique ethical considerations of clinical research in companion animals . finally , the value of translational clinical research into cancer care linking animals and humans should be better defined and promoted to all stakeholders . it is implicit in best practice recommendations that regular review and revision will occur as required by the development of resources , regulations , and ethical priorities . such revisions will be driven by both quantitative and qualitative research concerning communication with , and comprehension of , companion animal owners , improved trial design , and compliance . as with the evolution of clinical trial conduct in human oncology , we suspect that owner participation in data gathering and outcome assessment will become better integrated in trial management . the role of social media methods to support trial completion and to educate the public regarding the critical role of studies to improve animal health inevitably will grow . it will likewise be critical to review and adopt best practices as they emerge from other disciplines , institutions , and countries engaged in discussion on the ethical and proper conduct of clinical studies . we anticipate that additional workshops will be forthcoming to determine more best practice recommendations or deepen discussions on current issues and will include multidisciplinary perspectives within oncology and affiliated specialties . when properly addressed by adherence to sound scientific principles , ethical conduct of research , and respect for patients and their owners , better health may result in the future for all members of the family animals and people .

such a review includes assessment of particular features relevant to the research proposal , the team , and environment associated with the proposed research , project design , preliminary data , likely relevance of the anticipated outcomes of the research , and budget appropriateness . this process establishes accountability of the study process in terms of commitment of the time , resources , and effort of all parties engaged in clinical trials . commercially sponsored trials , donorsponsored trials , and hospitalsubsidized trials may not receive a scientific peer review separate from an evaluation by the institutional animal care and use committee ( iacuc ) , the mandate of which does not always include such scientific merit review . although iacuc approval currently is mandatory for all research involving animals that are conducted by institutions that receive federal funding , it is primarily designed to protect the welfare of laboratory animals , not privately owned pets.11 the iacuc review process may face challenges in addressing some scientific and ethical concerns that are unique to clientowned animals involved in clinical research . for this type of animal research , particular attention to and critical evaluation of clinical merit this evaluation has similarities to that performed by institutional review boards ( irbs ) charged with evaluating clinical trials involving humans . in addition , postapproval monitoring and research oversight should be an integral component of all veterinary clinical trials , including standardized clinical endpoint assessments , the need for interim evaluations , accrual targets , and adherence to patient enrollment criteria . a rigorous scientific and ethical review of all veterinary clinical trials is warranted to ensure the validity of the research and protection of animal patient welfare , and to maximize translational potential . the adoption of innovative trial designs that decrease the number of patients needed for meaningful results should be vigorously encouraged . conduct of trials so designed is also compatible with the tenets for appropriate use of animals in research : replace , reduce , and refine.12 several recent reports describe standard and modified novel trial designs currently used for veterinary clinical studies in companion animals with cancer.13 , 14 , 15 two examples of these include ( 1 ) adaptive or bayesian designs for phase i iii trials in which a dynamic assessment of data is used to continuously update the probability of outcomes such as safety , efficacy , or both , at the same time in order to decrease early phase patient enrollment16 , 17 ; and ( 2 ) enrichment of the study population to insure the new product will be studied in the correct subset of patients capable of responding appropriately to a specific targeted therapeutic agent and the use of validated biological endpoints that regulate downstream response.18 the continuous evaluation of innovative trial designs should improve risk : benefit evaluation and result in fewer patients at risk and fewer patients needed to make a go / no go decision to the next level of investigation . the assessment of goals , risks and cost are critical determinants of an owner 's decision to enroll in a study . table 2 compares the riskbenefit relationship between the human patient and the study team for phase 0 and phase i trials which represent those with the most controversial ethical concerns in the clinical trials continuum . phase 0 trials are those in which minor procedures are conducted on a patient , and involve minimal clinical impact of either toxicity or efficacy.19 , 20 such trials may include fineneedle aspirates of a patient 's tumor to assess a biological process or target modulation or microdosing of a compound to define preliminary pharmacokinetics without risk of systemic effects . phase 0 and phase i trials often are conducted in both human and animal patients with poor to grave prognoses that have already progressed through multiple conventional or investigational treatment regimens or both . in both phase 0 and phase i studies , there may be minimal to no benefit to the patient . as explained further in the section on informed consent recommendations it is important for the clinical trials team to thoroughly understand and clearly articulate the purpose and potential risks and benefits of the study . phase i trials in human patients.20 similar issues and concerns seemingly apply to animal patients phase 0 : microdosing , target modulation detection with biopsy , biomarkers . as a result , phase 0 , phase i , and some phase ii trials may be offered early in the treatment course . in addition , even when conventional treatment options are available , these treatments may be delayed while shortterm study interventions ( such as drug microdosing or tumor sampling ) are conducted in exchange for a therapeutic incentive provided by the study sponsor to support subsequent conventional treatment . the conduct of randomized clinical trials with a control group ( either a true placebo or a notreatment group ) may be necessary in some situations when there is no known effective conventional treatment for comparison with the investigational product . such trials may be necessary for evaluation of a new animal health product , but placebo or notreatment control groups are only needed rarely for animal patient studies focused on , product development for humans , ( ie , comparative oncology trials ) . trials involving a placebo group should be minimized in patients with cancer and careful evaluation of the risk of tumor progression during placebo treatment period must be considered.21 , 22 , 23 all patients enrolled in clinical trials , whether studied with or without a control group , must receive best supportive care . ( bsc ) is defined as treatment focusing on relieving symptoms ( eg , pain , inappetence ) in order to maintain or improve quality of life , but does not include treatment directed specifically at the cancer . as an example , a randomized placebocontrolled trial could divide patients into an investigational new treatment plus bsc group versus a placebo treatment plus bsc group . consequently , because of financial support for enrollment in a clinical study that provides frequent monitoring and bsc management , study participants conceivably could receive improved care regardless of study group assignment . nonetheless , full disclosure of the trial design , study purpose , and all contingencies must be clearly communicated to the owner . in addition to providing bsc for all study patients , strict criteria for release from the study or offer of rescue treatment must be in place if progressive tumor growth occurs or if the patient experiences substantial adverse clinical signs . recommended criteria exist in veterinary oncology including response evaluation criteria in solid tumors ( recist ) measurements , response evaluation criteria for peripheral nodal lymphoma ( recpnl ) , and the veterinary cooperative oncology group common terminology criteria for adverse event ( vcogctcae ) reporting.24 , 25 , 26 it also is recommended that trials incorporate a quality of life scoring system and performance scores throughout the enrollment period and the conduct of the trial to characterize the impact of treatment on these indicators of response.27 , 28 biomarkers and imaging assessments also provide evidence of cancer progression and such outcomes routinely are included in trials . crossover trial designs to the investigational treatment , exit to a distinct therapeutic cohort or management with a noninvestigational finally , weighted randomization ( eg , 2:1 investigational compound : placebo accrual ratio ) frequently is used to compare a smaller group of patients in the placebo group to an investigational product group if determined to be acceptable by a formal statistical assessment of outcome expectations . the selection and evaluation of meaningful and measureable endpoints for each study must be clearly described in the protocol . for example , pharmacologic measurements , targetdrug interactions , and genetic determinants of treatment outcomes frequently are assessed in personalized therapeutic trials . efficacy endpoint evaluations after a clinical study should be conducted on a defined schedule ( eg , thoracic radiographs every 2 months ) and any variance from this schedule should be reported . otherwise , the progressionfree interval may be subject to significant error as tumor progression or metastasis may be only intermittently evaluated creating a large margin of error . multiple , independent reviews of clinical endpoint assessments by qualified specialists should be considered best practice for clinical trials.29 , 30 overall survival is a relatively poor efficacy endpoint because of multiple , potentially competing , and confounding issues . owner acceptance of palliative or hospice care of their pets is variable , multiple interventions that are challenging to account for may be attempted to achieve responses after tumor progression and censoring rules for death may not be accurately interpreted . in addition , euthanasia is a wellknown confounder for determination of overall survival in animal patients enrolled in clinical trials.31 elective euthanasia , when a patient in a selfsupportive , clinically stable status is euthanized at the owner 's request , must be considered carefully when examining outcome data from clinical trials . when clinical trials are conducted for purposes of registration of results for approval of a new therapeutic entity with the center for veterinary medicine of the federal drug administrtion ( fda ) , a separate standard of oversight and documentation are required . multiple data recorders and monitors , data audit requirements , sample custody issues , and secondary biorepository sites to insure sample security are some of the additional responsibilities . the specific requirements and execution for these trials a recent review assessed the coordination of the approval of clinical trials in companion animals between the institutional iacuc committee and an advisory committee that is comprised of veterinary clinical specialists.32 several institutions have adopted the model of a clinical review board ( crb ) to evaluate the merit , feasibility , and compliance with ethical standards for clinical trials in clientowned animals . the iacuc may support the review from the clinical review board or choose to implement a full review . the avma also has recently addressed clinical trial management and approved a policy entitled establishment and use of veterinary clinical studies committees.33 according to this policy , the vcsc would evaluate clinical research that conforms to general standards of care but requires additional biomaterials that would be in excess of residual portions of samples taken only for routine health screening under a clientveterinarian relationship . similar to the function of the crb , the vcsc serves to ensure informed consent and to protect animals from conflict of interest issues . the avma recommends that when the vcsc determines that the protocol of a clinical research study will influence the management of the animal patient , the veterinary clinical studies committee should refer the proposed work for iacuc review . broad representation of clinical expertise is needed for a clinical review board and should include at least 1 member of the iacuc committee as a liaison for communication and clarification . specific training for crb members has not been established but should be developed in the future . resources developed to aid clinical irb members for trials involving humans to evaluate the scientific merit of a study , informed consent forms ( icfs ) , and the consenting process can be readily adapted for veterinary clinical trials.34 the usual responsibilities of the vcsc or crb include consideration of study merit , study feasibility , and the informed consent process . a review of the clinical study design by a peer group is particularly useful for those trials that have not undergone a formal scientific review . the crb also may be tasked with other responsibilities such as evaluation of conflict of interest , coordination of postapproval monitoring and insuring adverse event reporting for all study participants . administrative support , educational support for the members of the crb and clinical investigators , and data capture management systems are recommended as best practices . several specific considerations for veterinary cancer clinical trials involving new drugs and devices have been emphasized.32 for example , careful consideration of preliminary data on toxicity in the target animal species is critical before to initiating a clinical study . insufficient information about product safety may result in failure of the clinical trial to be approved under the full review and oversight of the iacuc . oversight of the clinical trial approval process in nonacademic or private veterinary practices also must be addressed and should evolve as such facilities are asked to participate in multisite clinical studies . if a study is being conducted under the assurance of an institution with a formal iacuc , that institution is responsible for completion of all requirements for compliance such as the informed consent process and its documentation , communication of adverse events with all other sites involved , and postapproval monitoring procedures . another option is to contract with a professional iacuc associated with a contract research organization ( cro ) that is charged with ensuring that similar responsibilities are met at the nonacademic site . guidelines have been continually revised.35 , 36 , 37 , 38 the guiding principle stated in table 1 [ the consent process must be honest , thorough , wellcommunicated , and the owner must have adequate time to consider participation without real or perceived coercion ] fulfills the intention of the informed consent process from the owner 's perspective and should drive communications relevant to enrollment of an animal patient in clinical oncology trials . recently , the national cancer institute ( nci ) reviewed and simplified its standard informed consent document for human patients in order to ensure better comprehension , and we recommend that veterinary cancer clinical trials adopt a similar template.39 full disclosure of the study purpose , associated risks and benefits , study design and interventions required , funding source for the study , incentives to the owner , and conflict of interest ( coi ) statements from the study staff and investigators are required elements to address during the consent process . examples of the intended purpose of a study include whether the study seeks to improve clinical signs associated with cancer or its treatment , if a study represents a safety or efficacy study and whether the study is intended to ultimately benefit animal health , human health , or both . animal patients participating in clinical trials without an efficacy intent must receive extraordinary protection all potential scenarios of risk should be explained , particularly for phase i trials that are designed to identify a safe or maximally tolerated dose when direct patient benefit may not be achieved . the source of funding for each study should be disclosed because it often indicates whether or not any potential coi exists.40 , 41 , 42 conflict of interest concerns should be disclosed and , if necessary , managed in a manner consistent with the office of research integrity guidelines from the department of health and human services.43 all financial subsidies or incentives should be carefully and fully described . , a standard of care does not formally exist in veterinary oncology for many types of cancer . therefore , the enrollment of animal patients , when conventional treatment offers little likelihood of response or success , requires very careful discussion regarding study goals and expectations . even more critical is the communication with pet owners who may be unable to afford an effective , conventional treatment option , and are inclined to enroll their pets into a preliminary clinical trial to receive a subsidy for the conventional treatment . the ethical issues of considering the enrollment of animal patients into clinical trials under these circumstances should be clearly understood by the study team and discussed honestly with the owner . a primary advocate for the owner of an animal patient being considered for enrollment into a clinical trial currently , the principal investigator or member of the clinical team such as the study coordinator or study nurse often is considered the primary advocate if no conflict of interest exists in the study ( or if it is disclosed and managed ) . careful oversight of whether study personnel are in a position of conflict is vital to the trust required of owners to enroll their pets in clinical trials . a copy of the informed consent document should be provided for review and consultation with the owner and the identified patient advocate . if needed , independent psychological , social , and grief counseling support should be identified and utilized for discussions related to clinical study advocacy during the consenting process . the informed consent document should describe the aspects of the study in simple , concise , and easytounderstand language . an evaluation of the quality of patient comprehension after a standard informed consent process identified several features that were statistically associated with high patient recall of study goals and requirements.44 these include the use of the nci template , the extent of the patient 's education , high english language proficiency , and involvement of a nurse familiar with the study and thorough reading of the consent form with adequate time to consider the study consent issues outside of the examination room . oversight and surveillance of clinical trials after approval and during conduct of the study often are negligible in companion animals but should be rigorously applied to ensure accurate interpretation of results . a confidential mechanism for communication of concerns identified by anyone engaged in clinical studies including owners , staff or students should be available . the process for clinical study postapproval monitoring also could be managed , as with more routine situations , by the institutional veterinarian or could be the responsibility of the clinical review board , iacuc , or the research integrity and compliance review committee at the institutional level or through a cro . the nci has developed audit guidelines that are readily adaptable to veterinary clinical trials.45 the role of the academic hospital administration and facilities in the conduct of clinical research also is a critical component of a clinical trials program in veterinary medicine . the hospital may be the repository for study documents ( eg , consent forms ) , provide trained personnel and facilities to execute sampling and data gathering , offer financial support for animal care , and provide counseling and emotional support for owners . the use of consolidated standards of reporting trials ( consort ) guidelines offers a wellaccepted and standardized method to assess design for clinical trials in humans.46 , 47 many journals now require such reporting guidelines compliance for publication of clinical trials . the veterinary cancer society has developed a voluntary listing of active clinical trials for pets with cancer.49 however , resource limitations preclude archival data collection , data retrieval , and a mechanism for reporting trial results . effective data sharing is considered vital to the success of multiinstitutional clinical trial conduct and was the subject of an iom workshop.50 the scientific and ethical considerations of data sharing are summarized in the following excerpts from that report:the moral and ethical arguments for data sharing center on fulfilling obligations to research participants , minimizing safety risks , and honoring the nature of medical research as a public good . the practical and scientific arguments for data sharing include improving the accuracy of research , informing risk / benefit analysis of treatment options , strengthening collaborations , accelerating biomedical research , and restoring trust in the clinical research enterprise . the moral and ethical arguments for data sharing center on fulfilling obligations to research participants , minimizing safety risks , and honoring the nature of medical research as a public good . the practical and scientific arguments for data sharing include improving the accuracy of research , informing risk / benefit analysis of treatment options , strengthening collaborations , accelerating biomedical research , and restoring trust in the clinical research enterprise . implicit in the concept of data sharing is open access to data , which provides greater transparency of study protocols , study reports , and adverse event documentation . several data management systems have been developed for data sharing , and an effort should be made to develop a usable , costeffective platform that would integrate multiinstitutional trial data , and ideally integrate with data management platforms used in clinical studies in humans . the publication of all trial results , whether associated with a positive or a negative outcome , in a searchable database would address the a recent editorial from the editors of the british journal of pharmacology details a policy of reporting transparency of research involving animals including increased accountability for animal use and open access to all primary data including negative studies.51 such reporting guidelines are likely to be adopted by numerous journals . to fully realize such guidelines , a publication strategy would need to be established for negative study reporting and the release of embargoed data from contractsponsored research after a suitable period . an overarching purpose of the workshop and this document is to encourage continued research and education on clinical trial improvement by outreach to scientists , veterinarians , and the petowning public . veterinary schools need to be informed of the resources available so as to encourage and facilitate the responsible conduct of clinical research and bioethics by their students , faculty , and employees.43 , 52 veterinary specialists need to be aware of research opportunities and how to incorporate them into their practices . owners need to clearly understand how knowledge gained by enrollment in clinical trials might help companion animals and humans in the future even though trials may not be designed to help their individual pet . as with the evolution of clinical trial conduct in human oncology , we suspect that owner participation in data gathering and outcome assessment will become better integrated in trial management . the role of social media methods to support trial completion and to educate the public regarding the critical role of studies to improve animal health inevitably will grow . when properly addressed by adherence to sound scientific principles , ethical conduct of research , and respect for patients and their owners , better health may result in the future for all members of the family animals and people .

motor neuron diseases ( mnd ) are a heterogeneous group of disorders which result in death of motor neurons amyotrophic lateral sclerosis ( als ) is the most common form of mnd in adults , affecting both anterior horn cells and corticospinal tracts . amyotrophic refers to the muscle atrophy , weakness , and fasciculation that signify disease of the motor neurons . fifty percent of patients die within three years of onset of symptoms , and 90% die within five years . the incidence of als is from approximately 2 per 100,000 per year and may be increasing . the majority of als cases which have been reported are sporadic ( sals ) ; 10% are familial ( fals ) , some of which arise from mutations in superoxide dismutase-1 ( sod1 ) , tar dna - binding protein ( tdp43 ) [ 5 , 6 ] and fused in sarcoma / translated in liposarcoma ( fus / tls ) [ 7 , 8 ] , als2 [ 9 , 10 ] , dynactin , and senataxin . genomic studies suggest the existence of at least eleven additional loci for fals , but the genetic defects remain to be identified . using a genome - wide association study ( gwas ) approach , it has been recently reported that a locus on chromosome 9p21 accounted for 40% of familial als and nearly 1 fourth of all als cases in a sample of 405 finnish patients . this association signal had previously been reported by van es et al . as related to als , and a meta - analysis amongst many studies showed that this was indeed the major signal for this disease . similarly , recent gwas for frontotemporal dementia ( ftd ) with tdp-43 pathology had also been identified on this locus . linkage analysis of patients affected with multiple cases of als , ftd , and ftd - als with type 2 tdp-43 pathology had suggested that there was an important locus for the disease on chromosome 9p [ 1821 ] , but it is not clear whether the linkage and association signals related to a single locus or whether the different studies are reporting the same alleles at that locus . some more studies have shown that there is a large hexanucleotide ( ggggcc ) repeat expansion in the first intron of c9orf72 on the affected haplotype , and a common mendelian genetic lesion in c9orf72 is implicated in many cases of sporadic and familial als and ftd . recently one of them is mutations in ubqln2 , which encodes a ubiquitin - like protein , ubiquilin 2 , cause dominantly inherited chromosome x - linked als and als / dementia . ubiquilin 2 is a member of the ubiquilin family ( ubiquilins ) , which regulate the degradation of ubiquitinated proteins and found in both inherited and sporadic form of als . functional analysis showed that mutations in ubqln2 lead to an impairment of protein degradation , abnormal protein aggregation and neurodegeneration . another is mutation in sqstm1 gene , which encodes the ubiquitin - binding protein p62 ( also known as sequestosome-1 ) . these findings provide evidence of a direct genetic role of p62 in als pathogenesis as it is supposed to be involved in protein - recycling system by regulating the protein degradation pathways , and thus , p62 , related to ubqln2 may represent an important therapeutic target in als . the key role these mutations play in als points towards their probable contribution in als metabolomics , which needs to be explored in future research studies . although the root cause of als is not clearly understood , but multiple mechanisms have been found to be associated in the pathogenesis of motor neuron death in als . these include oxidative stress , glutamate - mediated excitotoxicity , protein aggregation , mitochondrial dysfunction , and transition - metal - induced toxicity [ 2628 ] . metabolomics is the comprehensive study of the repertoire of small molecules present in cells , tissues , or other biological samples . the lack of biological tools to detect als together with the nonspecificity and heterogeneity of clinical symptoms leads to difficulty in diagnosing the disease in its early stages . metabolomic analysis is a universally applicable strategy for defining the metabolite composition of cells and tissues . ideally metabolomic analyses are rapid , unbiased , and comprehensive , and , to some extent mass spectrometry , h1 nuclear magnetic resonance ( h1 nmr ) spectroscopy and ir spectroscopy are progressively considered as metabolomic techniques . metabolomic approach evolves to study most or all implicated pathways , thereby revealing a biochemical signature for the disease and providing valuable new insights into disease mechanisms . metabolomics should be seen as a complementary technique to genomics , transcriptomics , and proteomics . additionally , it will be of great interest to integrate metabolomic , transcriptomic , and proteomic data into a system biology approach to understand global changes in als biological states . the understanding of regulatory metabolic processes of a complex living organism at the system level requires the assessment of spatiotemporal interorgan metabolic crosstalks through the analysis of biofluids . this challenge can be addressed by studying the metabolic correspondence among tissue and biofluid metabolic profiles . when associated with a well - defined physiological condition , metabolic profiles provide a snapshot of a functional phenotype , or metabotypes , as a result of multiple interactions between metabolic pathways under the influence of environment , lifestyle , genetics , and microbial factors [ 3032 ] . a specific metabolic profile of a systemic biofluid , such as blood or serum / plasma , reflects the overall metabolic status of an individual as the result of highly complex metabolic exchanges between diverse biological compartments , including organs , biofluids , and microbial symbionts . although h1-nmr is a well - established approach for multicompartmental metabolic profiling of intact tissue and biofluid samples , gc / tofms has the advantage to discover changes in metabolites present at lower concentrations due to the increased sensitivity ; hence , nmr and mass spectrometry should be regarded as complementary techniques . the potential use of blood ( serum or plasma ) and cerebrospinal fluid ( csf ) biomarkers associated with als have been studied by many authors , and these target molecules are supposed to be linked to als pathogenesis [ 3436 ] . recently , high - throughput techniques have been used to evaluate a combination of markers in patients with neurological diseases which is performed via different analytical methods such as high - performance liquid chromatography followed by electrochemical detection . nmr spectroscopy is a noninvasive technique , needs little sample preparation , and gives an overview of the principal metabolic pathways . recently , kumar et al . have analyzed blood ( serum ) metabolic profile to study 13 metabolites by h1 nmr ( figure 1 ) and found a significant elevation of the metabolite glutamate in serum sample of als patients . these higher glutamate signals were consistent with the hypothesis of glutamate excitotoxicity in als pathogenesis . plaitakis proposed the hypothesis of glutamatergic dysfunction in als , that is , imbalance between brain and blood glutamate levels . babu et al . also observed that blood glutamate levels were significantly higher in als patients . furthermore , kumar et al . found that , with increased glutamate concentration , glutamine concentration decreased in als , which might represent the imbalance between glutamate - glutamine conversion cycle that occurs in postsynaptic buttons and astrocytes during excitotoxicity . earlier study of pioro also showed in vivo evidence of abnormal glutamate metabolism in the cns parenchyma of als patients . there were previous studies by authors , who measured glutamate and found decreased glutamate concentrations in cns tissue and increased concentrations in the serum and csf of als patients , and hence , proposed a hypothesis suggesting an imbalance in the intracellular versus extracellular glutamatergic neurotransmitter system [ 43 , 44 ] . further some more important metabolites related to energy and oxidant and antioxidants have been studied , and it has been hypothesized that glutamate excitotoxicity exacerbates the formation of ros , which may be responsible for the oxidant - antioxidant imbalance in serum of als patients . for example , histidine metabolite , which is considered to be powerful antioxidant , and n - acetyl derivative ( n - acetyl - x ) concentrations significantly decreased in serum of als patients with increase concentration of bhbt , acetone , and glutamate ( figure 1 ) . unfortunately due to space limitations only a small number of promising article findings related to blood metabolites can be discussed in this section , and readers are directed to other excellent research articles for some other changes in metabolic profile in blood of als patients [ 4547 ] . csf is known as good source for the study of biomarkers because of its direct contact with the brain , its accessibility , and its dynamic changes with the cerebral environment . reduced levels of cystatin c and increased levels of neurofilaments have been proposed as csf biomarkers for als [ 48 , 49 ] . a recent study has found a relationship between the levels of galectin-3 to be indicative of the onset of als symptoms in mice , and the result was found transferrable to als . measured 17 metabolites in als csf and compared csf metabolic profiles between als and non - als patients . his univariate analysis showed higher concentrations of csf acetone , pyruvate , and ascorbate in als patients , while acetate was found decreased in als compared to controls . in this interesting study , the authors concluded that perturbations in energy metabolism and ketone metabolism were associated with als . however , other earlier studies of ascorbate metabolism have shown contradictory results [ 52 , 53 ] . the elevated levels of this antioxidant molecule ( ascorbate ) are compatible with oxidative stress previously described in als and could also be linked to ascorbic acid release from astrocytes after glutamate stimulation [ 54 , 55 ] . in earlier studies performed over the past twenty years and using different methodologies and usually only a small number of patient samples , glutamic acid has been reported as being elevated , normal , or reduced in the csf of als patients [ 5658 ] . the studies reporting an increase in csf glutamic acid content have been taken as support for the theory of glutamic acid excitotoxicity as a cause of als and could result in elevated levels of ascorbic acid . ascorbic acid might , by itself , modulate neuronal metabolism through the inhibition of glucose consumption during episodes of glutamatergic synaptic activity and by stimulating lactate uptake in neurons . in contrast to the interesting study by blasco et al . , wuolikainen et al . found that the content of ascorbic acid to be nonsignificantly different between als and controls . the sources of the compound , creatine , and creatine phosphate , are important for the energy metabolism and are present at high levels in the cns . the major formation of creatinine in the body , however , takes place in the skeletal muscles . this suggests that amyotrophy is the most likely explanation to the lower csf creatinine levels in als . studied the csf metabolome by using gc - tofms platform in als patients with six different mutations in the sod1 gene and compared it with als - patients without such mutations and found that patients with a sod1 mutation have a distinct metabolic profile in the csf . in particular , the patients who are homozygous for the d90a sod1 mutation showed a distinctively different signature when modeled against als patients with other sod1 mutations and sporadic and familial als patients without a sod1 gene mutation . among the metabolites that contributed most to the csf signature were arginine , lysine , ornithine , serine , threonine , and pyroglutamic acid , all found to be reduced in patients carrying a d90a sod1 mutation . analysis of the neurofilament light chain in the csf suggests that patients with sod1 gene mutations constitute a distinct subgroup within the als syndrome , and more so patients homozygous for the d90a sod1 mutation . wuolikainen et al . also reported that patients with sals have a heterogeneous metabolite signature in the csf . the metabolome of the csf of als patients with a sod1 gene mutation was found to form a separate homogeneous group . analysis of metabolites revealed that glutamate and glutamine were reduced , particularly in patients with a familial predisposition . there are significant differences in the metabolite profile and composition among patients with fals , sals , and patients carrying a mutation in the sod1 gene suggesting that the neurodegenerative process in different subtypes of als may be partially dissimilar . kynurenine pathway ( kp ) plays a very important role in the pathogenesis of als . the kn is a major route for the conversion of l - tryptophan ( trp ) to nicotinamide adenine dinucleotide ( nad ) [ 62 , 63 ] , initiated by either tryptophan 2,3-dioxygenase ( tdo ) or indoleamine 2,3-dioxygenase ( ido ) [ 64 , 65 ] . along the kynurenine ( kn ) , they include the free radical generator 3-hydroxykynurenine ( 3-hk ) , the excitotoxic n - methyl - d - aspartate ( nmda ) receptor agonist , quinolinic acid ( quin ) , the neuroprotective nmda receptor antagonist , kynurenic acid ( kyna ) [ 68 , 69 ] , and the neuroprotective picolinic acid ( pic ) ( figure 2 ) [ 70 , 71 ] . with respect to the pathogenesis of als , there is increasing evidence suggesting the involvement of the kp , especially that of the neurotoxin quin . in familial sod1 als , the formation of covalent aggregates of sod1 is tryptophan dependent . kynurenic acid ( kyna ) , produced from l - kynurenine , is an endogenous antagonist of ionotropic excitatory amino acid receptors acting preferentially at the glycine site associated with the nmda receptor complex . demonstrated the highest kyna levels in the caudate nucleus , lower levels in the thalamus , globus pallidus , hippocampus , parietal cortex and frontal cortex , and the lowest level in the cerebellum . it has been suggested that the reduced concentration of kyna may cause neurodegeneration . on the other hand , this endogenous neuroprotectant could have beneficial effect on several animal models of neurological diseases [ 62 , 63 ] . . demonstrated that kyna concentration was significantly higher in the csf of als patients with bulbar onset of als compared to control group , whereas serum kyna concentration was significantly lower in als patients with severe clinical status than in controls . these findings suggest that in als patients csf kyna concentration does not depend on its production in the periphery . but recent study by chen et al . in als patients has shown twofold increase in csf trp , serum trp , and serum kyn , a tenfold increase in csf kyn and a fourfold increase in intracellular csf ido activity in als compared to controls . although kyn can be transported across the blood brain barrier , the tenfold increase in csf kyn , compared to only a twofold increase in serum kyn , the elevated levels of serum and csf trp are speculated to reflect a dysfunction in protein - binding and l - neutral amino acid transporters , respectively , or perhaps , an over - compensatory response , whereby the increase in csf ido activity results in a decrease in csf trp , which encourages the dissociation of trp from albumin to facilitate transport of trp across the blood brain barrier ( bbb ) . a recently published study with glutamate has found that abnormal neurons exhibit an upregulation in a particular form of glutamate - aspartate transporter , which may make them more vulnerable to glutamate - mediated excitotoxicity . as such , a similar event is hypothesized to occur between quin and motor neurons . pathological concentrations of quin could result in a myriad of unfavourable consequences that could exacerbate and accelerate the condition of als . as an endogenous nmda agonist the main source of quin is immune cells rather than astrocytes or neurons [ 79 , 80 ] . however , high levels of kyn produced by astrocytes can be taken up by surrounding activated microglia for quin production . once quin exceeds the safety threshold ( < 150 nm ) , its adverse effect is exerted via the generation of reactive oxygen species , augmentation of the excitotoxic impact through disruption in the glutamatergic transport system , apoptosis , mitochondrial dysfunction , and the production of cytokines and chemokines , all of which are putatively thought to be contributory factors in als ( figure 1 ) . chen et al . showed a significantly increased microglial activation in als motor cortex and spinal cord which indicate a heightened immune response in als cns . it has been also shown that synthesis of kyna in the brain takes place predominantly in astrocytes . in als , an intensive astrocytosis has been found . reported reactive astrogliosis in the ventral horns , dorsal horns , and at the transition between gray matter and anterior and lateral funiculi in the spinal cords of als patients . feeney et al . showed that motor neuron loss and reactive astrocytosis were correlated with the progression of the disease in sod 1 transgenic mouse model of als . observed that the loss of glutamate transporter subtype 1 ( glt-1 ) in als transgenic sod1 mice selectively occurs in the areas affected by neurodegeneration and reactive astrocytosis . thus , it seems that astrocytosis occurring in als may cause an increase of kyna production and observed elevation of kyna in csf . als is a neurodegenerative disease characterized by progressive degeneration of both upper motor neurons ( umn ) in the primary motor cortex ( pmc ) and lower motor neurons ( lmn ) in the brain stem and spinal cord anterior horns . despite the identification of pathologies in the pmc and corticospinal tracts ( cst ) of autopsy tissue samples with als [ 8487 ] , there are no biomarkers identified to date that reliably indicate presence of such pathologies in the brain of patients with als . in an effort to find neuroimaging biomarkers indicative of umn degeneration , advanced mr techniques ( e.g. , magnetic resonance spectroscopy , diffusion tensor imaging , and functional mri ) have been explored [ 88 , 89 ] . it is hypothesized that the neuropathologically proven motor neuron and cst degeneration in als might occur gradually with no apparent manifestation of macroscopic tissue structural changes in the early stage which are detectable by conventional mri methods . in contrast , neurochemicals indicative of metabolic processes responsible for degeneration of the motor neurons and cst in patients with als can be accessed from the disease onset stage using proton mr spectroscopic ( 1h mrs ) methods . several previous cross - sectional and longitudinal studies have demonstrated the value of proton mrs for the evaluation of metabolite alterations in the pmc [ 90100 ] and cst [ 95 , 101 ] of patients with als using single voxel svs and 2d mr spectroscopic imaging [ 101103 ] methods . in general , most of these studies have reported significantly altered metabolite concentrations or ratios in the motor pathways of patients with als . 1h mrs of the human brain reveals important information about compounds such as n - acetylaspartate ( naa ) which has been suggested to be a marker for neuron viability and axonal density in the brain , creatine ( cr ) as a putative marker of gliosis [ 105 , 106 ] , and choline ( cho ) which is associated with membrane phospholipids . several 1h mrs studies in patients with definite als and clear signs of umn involvement showed a reduction of naa in the motor cortex [ 90 , 108110 ] . in another study , chemical shift - imaging follow - up measurements in als patients after a three - month period demonstrated a decline in concentrations of naa , cr , and cho in the most affected motor cortex areas of als in comparison with healthy controls . . found significant changes of naa concentrations and the naa/(cr + cho ) ratio of the investigated grey matter areas . it has been observed that the ratio of naa and naa/(cr + cho ) as a biomarker of neuronal loss or neuronal metabolism in als patients is significantly apparent . this was supported by significantly lower naa values in the more affected hemispheres at the time of study inclusion . also reported significantly decreased naa / myo inositol in als patients compared to healthy controls . today als is thought to be to some degree a multisystem disorder and mitochondrial abnormalities that have been observed not just in motor neurons but even in skeletal muscle cells of als patients . recent work in msod1 mice has shown that motor neuron death is not cell autonomous and involves defects in other cell types than neurons [ 112 , 113 ] . consistent with this notion , recent evidence showed that the pathophysiology of als includes widely systemic defects in both patients and animal models . in particular , energy homeostasis is strikingly abnormal in als patients since both sals and fals patients present with increased energy expenditure ( hypermetabolism ) and hyperlipidemia [ 114 , 115 ] . further it is also reported that , a severe deficiency of nicotinamide adenine dinucleotide to coq oxidoreductase ( naoh : coq ) in skeletal muscle biopsies of patients with sals was observed using saponin - permeabilized muscle fibers . this muscular mitochondrial defect had a heterogeneous distribution and was correlated in some patients with multiple mitochondrial dna deletions , decreased mitochondrial dna levels , and low levels of membrane - associated manganese sod . these results suggest that some sals patients may have muscular mitochondrial damage that may contribute to disease pathogenesis . it has been found that metabolism is higher and bodyweight and fat mass are lower in mutant sod1 mice than in wild - type mice . loss of tdp-43 or its overexpression [ 121 , 122 ] both lead to growth retardation and thus impaired energy homoeostasis . furthermore , adult loss of tdp-43 led to massive decreases in adipose tissue , probably through muscle hypermetabolism , and tdp-43 overexpression led to the formation of morphologically abnormal mitochondria [ 121 , 122 ] . tdp-43-als , like mutant sod1-als , therefore , seems to be associated with impaired energy homoeostasis in transgenic animals . abnormalities in muscle energy metabolism have been suggested as the direct cause of energy deficit and hypermetabolism in mutant sod1 mice . cellular levels of atp are decreased [ 122124 ] and expression of mitochondrial uncoupling proteins and concentrations of markers of lipid and carbohydrate use are increased [ 120 , 125 ] . several reports have indicated the existence of mitochondrial defects in the muscle tissue of patients with als that develops as the disease progresses [ 126128 ] . low - level mitochondrial defects might , however , be present earlier , as increased sensitivity of als myoblasts to oxidative stress has been seen in some patients . the extent and origin of such mitochondrial dysfunction remain controversial , however , and the impairment of energy metabolism seen in patients with als and mutant sod1 mice might be due at least partly to dysfunctional regulation of metabolic pathways . numerous studies in als patients have shown collagen abnormalities of skin , such as decreased amount of collagen , alteration of cross - linking of collagen , and increased solubility of collagen . recently , it has been demonstrated that the basement membrane of skin in als patients is weakly positive for type iv collagen compared with controls , and also serum type iv collagen levels in als patients are lower than controls . it showed that the absolute and relative concentrations of two collagen metabolites , glucosylgalactosyl hydroxylysine ( glu - gal hyl ) , and galactosyl hydroxylysine ( gal hyl ) , excreted in urine , indicate the tissue origin of the collagen metabolites and the rate of degradation of collagen [ 135137 ] . glu - gal hyl accounts for 61% of the hydroxylysine glycosides in skin collagen , and gal hyl constitutes approximately 75% of the glycosides in bone collagen [ 135 , 138 ] . measurement of glu - gal hyl and gal hyl may indicate whether bone or skin collagen is preferentially being degraded . the study demonstrated that large increase in degradation of skin collagen produces a large increase in urinary excretion of glu - gal hyl . it has also been reported that , in the case of bone collagen , patients with bone complications tend to have elevated urinary gal hyl excretion . a high ratio of glu - gal hyl to gal hyl increases the probability that the patient will develop skin complications , and a low ratio is associated with bone - related complications . therefore , these two hydroxylysines , that is , glu - gal hyl and gal hyl , offer more reliable parameters of collagen breakdown than hydroxyproline . the present study has indicated that the urinary excretion of glu - gal hyl is markedly decreased in als and is more pronounced with the duration of illness . [ 141 , 142 ] reported markedly decreased contents of collagen and collagen - associated amino acids in the lateral corticospinal tract and the anterior horn in the spinal cord of patients with als , suggesting that these data could be associated with the degeneration of the upper and lower motor neurons in patients with als . riluzole was the first drug approved by the fda ( usa ) in 1995 for the treatment of als , but its mechanisms of action in slowing the progression of this disease remain obscure . in initial study , to evaluate the efficacy and safety of the antiglutamate agent riluzole , bensimon et al . conducted a prospective , double - blind , placebocontrolled trial in 155 als patients and found that the antiglutamate agent riluzole appears to slow the progression of als disease , and it may improve survival in patients with bulbar onset of disease . further , lacomblez et al . also carried out a double - blind , placebocontrolled at multicentre and found that the 100 mg dose of riluzole has the best benefit - to - risk ratio , and it decreased mortality and slowed muscle - strength deterioration in als patients . it has been reported that the drug riluzole induces a strong metabolic signature which seems not to derive chemically from the drug , but may reflect modified metabolic processes . elucidation of the structures of these molecules could identify biochemical pathways that are subject to perturbations by the drug , some of which could contribute to its efficacy and some to its side effects . though the precise mechanism of action for riluzole remains unclear , it appears to interfere with excitatory amino acid signalling , possibly through the inhibition of glutamate release [ 145147 ] , blockade of inactivated sodium channels , and interaction with guanosine triphosphate - binding proteins . it is a tetracycline with anti - inflammatory action coupled with an independent antimicrobial property . minocycline has proved to be a promising neuroprotective agent [ 151154 ] when studied in mouse models of cerebral ischemia , spinal cord injury , and parkinson 's disease ( pd ) . as an anti - inflammatory , minocycline inhibits apoptosis ( programmed cell death ) via the attenuation of tnf- and downregulating proinflammatory cytokine output . this effect is mediated by a direct action of minocycline on the activated t cells and on microglia , which results in the decreased ability of t cells to contact microglia which impairs cytokine production in t - cell - microglia signal transduction . it has been also found that minocycline inhibits mitochondrial permeability - transition - mediated cytochrome - c release . a pilot study on als patients showed that it could be safe to take minocycline together with riluzole with no significant side effects . in a recently completed multicentre randomized placebocontrolled phase iii trial , however , the positive outcome seen in als mouse models was not reproduced in the human study . it has been reported that minocycline , which is known as an antibiotic and inhibitor of microglial activation , riluzole , which works as an inhibitor of glutamate release , and nimodipine , which is voltage - gated calcium channel blocker , is designed to target the different pathways leading to neuronal death . formerly , minocycline and riluzole have each alone been effective in delaying progression of als [ 143 , 144 , 156 , 160 , 161 ] . when the three drugs were tested in combination on sod1 mice , it resulted in strikingly improved conditions , delaying disease onset by 4 weeks , increasing survival by 6 weeks , and attenuating neurodegeneration by reducing cyclin - dependent kinase 5 mislocalization , caspase-3 activation , astrocytosis , and microgliosis . it has been found that in rat model it has the capability to increase glt1 expression by threefold . it has been also reported that ceftriaxone considerably reduced motor neuron loss and hypercellular gliosis , delaying muscle strength and body weight loss and improving longevity in sod1g93a mice . various antioxidants have been used as potential therapeutic agents in transgenic mice expressing the mutated human sod-1 enzyme . polyamine or putrescine - modified catalase , an antioxidant enzyme that removes hydrogen peroxide and has good permeability at the bbb , increases the survival of transgenic mice bearing the human msod-1g93a [ 147 , 148 , 163 , 164 ] . moreover , the copper chelator and thiol compound penicillamine , the copper chelator trientine , carboxyfullerenes , vitamin e , and n - acetylcysteine have been reported to increase the survival time in this mouse model and/or delay the onset of the disease to a small extent [ 165 , 166 ] . in als , weight loss is frequently observed and can occur early or later during the course of disease . all patients lose some weight due to the unavoidable reduction in skeletal muscle mass that results from denervation and decreased physical activity . in many cases , however , weight loss also has a nutritional component , with loss of fat mass and fat - free mass ( ffm ) attributable to malnutrition , the principal cause of which is decreased dietary intake [ 168 , 169 ] . swallowing disorders and a reduction of energy intake is associated with increased weight loss and the degree of dysphagia . anorexia , digestive disorders , and upper extremity motor difficulties also contribute to low intake . reports that resting energy expenditure ( ree ) may be increased in this context [ 171 , 172 ] appear paradoxical because ffm , the main determinant of ree , decreases in als . many research studies point towards significant role of mitochondria in als pathogenesis and thus may provide a cause of malnutrition in als patients . it has been found that prevalence of substantial mitochondrial degeneration in motor neurons of g93a sod1 mutant mice at the onset of the disease , when electron transfer chain activity and atp synthesis appear severely dysfunctional . furthermore , reduction in the activity of cytochrome - c oxidase , encoded by the mitochondrial genome , in motor neurons of sals patients has also been reported . there is a direct correlation between the amount of mitochondrial dna in the spinal cord of als patients with decrease in the activities of citrate synthase and the respiratory chain complexes i , ii , iii , and iv . not only nervous system but other tissues , including liver , lymphocytes , and muscle , are also affected by mitochondrial dysfunction . found a deficiency of nadh : coq oxidoreductase in muscles of sals and also noticed that decreased activities of nadh : coq oxidoreductase and cytochrome c oxidase are associated with dna abnormalities and reduced levels of the mitochondrial mn - superoxide dismutase . reported the early increase in the mrna levels of the mitochondrial uncoupling protein-3 in skeletal muscles of als - linked g86r sod1 mutant mice . in addition , isolated mitochondria from g86r muscle tissue exhibited a reduced respiratory control ratio , which is in line with the existence of mitochondrial uncoupling . supporting the mitochondrial dysfunction hypothesis , and administration of creatine , an intracellular energy shuttle between mitochondria and sites of energy consumption that is known to ameliorate muscle function , increased the life expectancy of g93a mice . although clinical trials failed to show a beneficial effect of creatine in als patients , the findings in g93a mice invoke the existence of a characteristic energetic imbalance . currently , hypermetabolism in als remains unexplained . the suggestion that hypermetabolism can be explained by an increase in the work of respiratory muscles lacks credibility because there is no correlation between ree and pulmonary function . the evaluation of a patient 's nutritional status and elucidation of the causes of any malnutrition are essential to provision of adequate nutritional care . in fact , malnutrition is common in als , with a frequency reported to be between 15 and 55% , depending on the stage of disease and the definition of malnutrition adopted . moreover , malnutrition is an independent prognostic factor for survival [ 171 , 172 ] . . indicated a chronic hypermetabolism in a subset of sporadic als patients , which complements a decrease in fat - free mass frequently observed in these patients . reported that fat - enriched high - energy diet to g86r mice resulted in an increase in body mass and adipose tissue accumulation , thus showing that increasing energy intake is sufficient to reduce the energetic deficit . . also showed that the number of cells in the ventral horns of the lumbar spinal cord , which is significant lost in large sized cells , most probably representing motor neurons , in symptomatic g86r , mice was prevented in high - fat diet - fed animals of the same age . it is worth emphasizing , however , that at least a subset of als patients showed characteristic hypermetabolic phenotype reminiscent of that observed in mice . from clinical perspective , the nutritional status is a prognostic factor for survival in als , and more evidence suggests that the proper customized nutritional management of patients may constitute a primary symptomatic treatment for the disease . nutritional intervention may prove to be a very significant aspect in als and can be explored further . the kp is a major route for the conversion of trp to nicotinamide adenine dinucleotide [ 62 , 63 ] generating neuroactive intermediates in the process . targeting the kp could offer a new therapeutic option to improve als treatment . in order to proceed further in this regard , two possible approaches can be taken , either to develop analogues of the neuroprotective kynurenines or to inhibit the synthesis of the neurotoxic quin . some of the below - mentioned drugs may show some promising therapeutic approach towards als . ( a ) intracerebral and intraperitoneal administration of 4-chlorokynurenine , which is a precursor of 7-chlorokynurenate , with quin , showed successful enzymatic transamination into the active 7-chlorokynurenate , conferring neuroprotection [ 188 , 189 ] by preventing quin neurotoxicity . ( b ) laquinimod , which is a novel synthetic quinoline , inhibited disease progression and infiltration of cd4 + t - cells and macrophages into the central nervous system ( cns ) . ( c ) an immunosuppressive and anti - inflammatory prodrug , leflunomide , gets converted to its active open - ring metabolite , teriflunomide , an inhibitor of mitochondrial dihydroorotate dehydrogenase , and known to successfully reduce the development of active lesions in patients with relapsing multiple sclerosis . ( d ) the synthesis of quin can also be blocked by inhibiting either kynureninase or kynurenine hydroxylase activity , thus , diverting the kp towards the synthesis of kyna . in immune activated mice , meta - nitronemzoylalanine also significantly reduced the formation of quin in the blood and brain . ( e ) ro61 - 8048 is another potent kynurenine-3-mono - oxygenase ( kmo ) inhibitor . it also reduces glutamate concentrations in the extracellular spaces of the basal ganglia in rats without impairing the learning or memory process typically associated with glutamate receptor antagonists and significantly reduces the neurotoxic levels of 3-hk and quin in the cns . ( f ) clioquinol is a quinoline metal chelator that binds selectively to zinc and copper ions with a hydrophobic nature that allows it to pass easily across the bbb . recent research with clioquinol in neurological disorders involving an imbalance in metal ions has led to promising results , presenting the possibility of a new therapeutic strategy . the wide range of survival time in als patients suggests that multiple prognostic factors are involved ; only some have been clearly identified . a specific biochemical marker for early diagnosing and for monitoring disease progression in als will have important clinical applications . a minority of patients carries mutations in the cu / zn - superoxide dismutase ( sod1 ) gene , but the disease mechanism remains unknown for all types of als . as the glutamate modulator , riluzole , is the only drug currently approved for als treatment , however , combination therapies that target other pathogenic mechanisms may be more effective in slowing disease progression and prolonging survival . the most recent finding in the als genetics suggests a very important role of mutation in sqstm1 and ubqln2 genes , and it is suggested that als patients should be monitored in a broad spectrum to study the altered effect on metabolic pathways [ 24 , 25 ] . the d90a sod1 mutation findings suggest that metabolomic profiling using gc - tofms and multivariate data analysis may be a future tool for diagnosing and monitoring disease progression and may cast light on the disease mechanisms in als . for this , the use of metabolomics as the link to pathway information in genome - wide association studies could be of great interest for mapping and interpreting the effects of different mutations and even combinations of mutations in relation to als subtypes . global metabolomics can be used for detecting changes of metabolite concentrations in samples of fluids such as csf . exploration of metabolomics by the use of small molecules derived from biofluids provides a strong platform to understand the metabolic characters of the living system and plays a significant role in the detection of diagnostic biomarkers . future clinical and experimental studies , therefore , need to concentrate on the complex relations between metabolism and als , and in this way may answer the question of whether targeting defective metabolism in als is an efficient way to alter disease progression . though a single technique is not sufficient to study the entire metabolomic profile , there is a need to converge some state of the art techniques namely , gc / ms , lc / ms , and nmr to realize the full worth of metabolomics . there is definite need to further elaborate on the sensitive and specific metabolic therapeutic signatures by advance analytical means and bioinformatics application which will help us to elucidate the structures of more signature molecules in als disease and provide us insight into aberrant biochemical pathways and may prove to be helpful in building up diagnostic markers and targets for drug design .

amyotrophic lateral sclerosis ( als ) is one of the most common motor neurodegenerative disorders , primarily affecting upper and lower motor neurons in the brain , brainstem , and spinal cord , resulting in paralysis due to muscle weakness and atrophy . the majority of patients die within 35 years of symptom onset as a consequence of respiratory failure . due to relatively fast progression of the disease , early diagnosis is essential . metabolomics offer a unique opportunity to understand the spatiotemporal metabolic crosstalks through the assessment of body fluids and tissue . so far , one of the most challenging issues related to als is to understand the variation of metabolites in body fluids and cns with the progression of disease . in this paper we will review the changes in metabolic profile in response to disease progression condition and also see the therapeutic implication of various drugs in als patients .

1. Introduction 2. Metabolomics 3. ALS and Peripheral Biomarkers 4. Brain Metabolites 5. Muscle Metabolite 6. Urinary Collagen Metabolite 7. Therapeutic Consequences 8. Conclusion

motor neuron diseases ( mnd ) are a heterogeneous group of disorders which result in death of motor neurons amyotrophic lateral sclerosis ( als ) is the most common form of mnd in adults , affecting both anterior horn cells and corticospinal tracts . amyotrophic refers to the muscle atrophy , weakness , and fasciculation that signify disease of the motor neurons . fifty percent of patients die within three years of onset of symptoms , and 90% die within five years . the majority of als cases which have been reported are sporadic ( sals ) ; 10% are familial ( fals ) , some of which arise from mutations in superoxide dismutase-1 ( sod1 ) , tar dna - binding protein ( tdp43 ) [ 5 , 6 ] and fused in sarcoma / translated in liposarcoma ( fus / tls ) [ 7 , 8 ] , als2 [ 9 , 10 ] , dynactin , and senataxin . as related to als , and a meta - analysis amongst many studies showed that this was indeed the major signal for this disease . linkage analysis of patients affected with multiple cases of als , ftd , and ftd - als with type 2 tdp-43 pathology had suggested that there was an important locus for the disease on chromosome 9p [ 1821 ] , but it is not clear whether the linkage and association signals related to a single locus or whether the different studies are reporting the same alleles at that locus . some more studies have shown that there is a large hexanucleotide ( ggggcc ) repeat expansion in the first intron of c9orf72 on the affected haplotype , and a common mendelian genetic lesion in c9orf72 is implicated in many cases of sporadic and familial als and ftd . these findings provide evidence of a direct genetic role of p62 in als pathogenesis as it is supposed to be involved in protein - recycling system by regulating the protein degradation pathways , and thus , p62 , related to ubqln2 may represent an important therapeutic target in als . although the root cause of als is not clearly understood , but multiple mechanisms have been found to be associated in the pathogenesis of motor neuron death in als . the lack of biological tools to detect als together with the nonspecificity and heterogeneity of clinical symptoms leads to difficulty in diagnosing the disease in its early stages . additionally , it will be of great interest to integrate metabolomic , transcriptomic , and proteomic data into a system biology approach to understand global changes in als biological states . the understanding of regulatory metabolic processes of a complex living organism at the system level requires the assessment of spatiotemporal interorgan metabolic crosstalks through the analysis of biofluids . when associated with a well - defined physiological condition , metabolic profiles provide a snapshot of a functional phenotype , or metabotypes , as a result of multiple interactions between metabolic pathways under the influence of environment , lifestyle , genetics , and microbial factors [ 3032 ] . a specific metabolic profile of a systemic biofluid , such as blood or serum / plasma , reflects the overall metabolic status of an individual as the result of highly complex metabolic exchanges between diverse biological compartments , including organs , biofluids , and microbial symbionts . although h1-nmr is a well - established approach for multicompartmental metabolic profiling of intact tissue and biofluid samples , gc / tofms has the advantage to discover changes in metabolites present at lower concentrations due to the increased sensitivity ; hence , nmr and mass spectrometry should be regarded as complementary techniques . the potential use of blood ( serum or plasma ) and cerebrospinal fluid ( csf ) biomarkers associated with als have been studied by many authors , and these target molecules are supposed to be linked to als pathogenesis [ 3436 ] . nmr spectroscopy is a noninvasive technique , needs little sample preparation , and gives an overview of the principal metabolic pathways . have analyzed blood ( serum ) metabolic profile to study 13 metabolites by h1 nmr ( figure 1 ) and found a significant elevation of the metabolite glutamate in serum sample of als patients . these higher glutamate signals were consistent with the hypothesis of glutamate excitotoxicity in als pathogenesis . also observed that blood glutamate levels were significantly higher in als patients . earlier study of pioro also showed in vivo evidence of abnormal glutamate metabolism in the cns parenchyma of als patients . there were previous studies by authors , who measured glutamate and found decreased glutamate concentrations in cns tissue and increased concentrations in the serum and csf of als patients , and hence , proposed a hypothesis suggesting an imbalance in the intracellular versus extracellular glutamatergic neurotransmitter system [ 43 , 44 ] . further some more important metabolites related to energy and oxidant and antioxidants have been studied , and it has been hypothesized that glutamate excitotoxicity exacerbates the formation of ros , which may be responsible for the oxidant - antioxidant imbalance in serum of als patients . for example , histidine metabolite , which is considered to be powerful antioxidant , and n - acetyl derivative ( n - acetyl - x ) concentrations significantly decreased in serum of als patients with increase concentration of bhbt , acetone , and glutamate ( figure 1 ) . unfortunately due to space limitations only a small number of promising article findings related to blood metabolites can be discussed in this section , and readers are directed to other excellent research articles for some other changes in metabolic profile in blood of als patients [ 4547 ] . csf is known as good source for the study of biomarkers because of its direct contact with the brain , its accessibility , and its dynamic changes with the cerebral environment . a recent study has found a relationship between the levels of galectin-3 to be indicative of the onset of als symptoms in mice , and the result was found transferrable to als . measured 17 metabolites in als csf and compared csf metabolic profiles between als and non - als patients . his univariate analysis showed higher concentrations of csf acetone , pyruvate , and ascorbate in als patients , while acetate was found decreased in als compared to controls . in earlier studies performed over the past twenty years and using different methodologies and usually only a small number of patient samples , glutamic acid has been reported as being elevated , normal , or reduced in the csf of als patients [ 5658 ] . the sources of the compound , creatine , and creatine phosphate , are important for the energy metabolism and are present at high levels in the cns . this suggests that amyotrophy is the most likely explanation to the lower csf creatinine levels in als . studied the csf metabolome by using gc - tofms platform in als patients with six different mutations in the sod1 gene and compared it with als - patients without such mutations and found that patients with a sod1 mutation have a distinct metabolic profile in the csf . analysis of the neurofilament light chain in the csf suggests that patients with sod1 gene mutations constitute a distinct subgroup within the als syndrome , and more so patients homozygous for the d90a sod1 mutation . the metabolome of the csf of als patients with a sod1 gene mutation was found to form a separate homogeneous group . there are significant differences in the metabolite profile and composition among patients with fals , sals , and patients carrying a mutation in the sod1 gene suggesting that the neurodegenerative process in different subtypes of als may be partially dissimilar . demonstrated the highest kyna levels in the caudate nucleus , lower levels in the thalamus , globus pallidus , hippocampus , parietal cortex and frontal cortex , and the lowest level in the cerebellum . demonstrated that kyna concentration was significantly higher in the csf of als patients with bulbar onset of als compared to control group , whereas serum kyna concentration was significantly lower in als patients with severe clinical status than in controls . these findings suggest that in als patients csf kyna concentration does not depend on its production in the periphery . in als patients has shown twofold increase in csf trp , serum trp , and serum kyn , a tenfold increase in csf kyn and a fourfold increase in intracellular csf ido activity in als compared to controls . once quin exceeds the safety threshold ( < 150 nm ) , its adverse effect is exerted via the generation of reactive oxygen species , augmentation of the excitotoxic impact through disruption in the glutamatergic transport system , apoptosis , mitochondrial dysfunction , and the production of cytokines and chemokines , all of which are putatively thought to be contributory factors in als ( figure 1 ) . showed a significantly increased microglial activation in als motor cortex and spinal cord which indicate a heightened immune response in als cns . it has been also shown that synthesis of kyna in the brain takes place predominantly in astrocytes . reported reactive astrogliosis in the ventral horns , dorsal horns , and at the transition between gray matter and anterior and lateral funiculi in the spinal cords of als patients . showed that motor neuron loss and reactive astrocytosis were correlated with the progression of the disease in sod 1 transgenic mouse model of als . als is a neurodegenerative disease characterized by progressive degeneration of both upper motor neurons ( umn ) in the primary motor cortex ( pmc ) and lower motor neurons ( lmn ) in the brain stem and spinal cord anterior horns . despite the identification of pathologies in the pmc and corticospinal tracts ( cst ) of autopsy tissue samples with als [ 8487 ] , there are no biomarkers identified to date that reliably indicate presence of such pathologies in the brain of patients with als . it is hypothesized that the neuropathologically proven motor neuron and cst degeneration in als might occur gradually with no apparent manifestation of macroscopic tissue structural changes in the early stage which are detectable by conventional mri methods . in contrast , neurochemicals indicative of metabolic processes responsible for degeneration of the motor neurons and cst in patients with als can be accessed from the disease onset stage using proton mr spectroscopic ( 1h mrs ) methods . several previous cross - sectional and longitudinal studies have demonstrated the value of proton mrs for the evaluation of metabolite alterations in the pmc [ 90100 ] and cst [ 95 , 101 ] of patients with als using single voxel svs and 2d mr spectroscopic imaging [ 101103 ] methods . 1h mrs of the human brain reveals important information about compounds such as n - acetylaspartate ( naa ) which has been suggested to be a marker for neuron viability and axonal density in the brain , creatine ( cr ) as a putative marker of gliosis [ 105 , 106 ] , and choline ( cho ) which is associated with membrane phospholipids . in another study , chemical shift - imaging follow - up measurements in als patients after a three - month period demonstrated a decline in concentrations of naa , cr , and cho in the most affected motor cortex areas of als in comparison with healthy controls . it has been observed that the ratio of naa and naa/(cr + cho ) as a biomarker of neuronal loss or neuronal metabolism in als patients is significantly apparent . also reported significantly decreased naa / myo inositol in als patients compared to healthy controls . today als is thought to be to some degree a multisystem disorder and mitochondrial abnormalities that have been observed not just in motor neurons but even in skeletal muscle cells of als patients . in particular , energy homeostasis is strikingly abnormal in als patients since both sals and fals patients present with increased energy expenditure ( hypermetabolism ) and hyperlipidemia [ 114 , 115 ] . several reports have indicated the existence of mitochondrial defects in the muscle tissue of patients with als that develops as the disease progresses [ 126128 ] . numerous studies in als patients have shown collagen abnormalities of skin , such as decreased amount of collagen , alteration of cross - linking of collagen , and increased solubility of collagen . recently , it has been demonstrated that the basement membrane of skin in als patients is weakly positive for type iv collagen compared with controls , and also serum type iv collagen levels in als patients are lower than controls . it showed that the absolute and relative concentrations of two collagen metabolites , glucosylgalactosyl hydroxylysine ( glu - gal hyl ) , and galactosyl hydroxylysine ( gal hyl ) , excreted in urine , indicate the tissue origin of the collagen metabolites and the rate of degradation of collagen [ 135137 ] . glu - gal hyl accounts for 61% of the hydroxylysine glycosides in skin collagen , and gal hyl constitutes approximately 75% of the glycosides in bone collagen [ 135 , 138 ] . the present study has indicated that the urinary excretion of glu - gal hyl is markedly decreased in als and is more pronounced with the duration of illness . [ 141 , 142 ] reported markedly decreased contents of collagen and collagen - associated amino acids in the lateral corticospinal tract and the anterior horn in the spinal cord of patients with als , suggesting that these data could be associated with the degeneration of the upper and lower motor neurons in patients with als . riluzole was the first drug approved by the fda ( usa ) in 1995 for the treatment of als , but its mechanisms of action in slowing the progression of this disease remain obscure . conducted a prospective , double - blind , placebocontrolled trial in 155 als patients and found that the antiglutamate agent riluzole appears to slow the progression of als disease , and it may improve survival in patients with bulbar onset of disease . also carried out a double - blind , placebocontrolled at multicentre and found that the 100 mg dose of riluzole has the best benefit - to - risk ratio , and it decreased mortality and slowed muscle - strength deterioration in als patients . though the precise mechanism of action for riluzole remains unclear , it appears to interfere with excitatory amino acid signalling , possibly through the inhibition of glutamate release [ 145147 ] , blockade of inactivated sodium channels , and interaction with guanosine triphosphate - binding proteins . minocycline has proved to be a promising neuroprotective agent [ 151154 ] when studied in mouse models of cerebral ischemia , spinal cord injury , and parkinson 's disease ( pd ) . in a recently completed multicentre randomized placebocontrolled phase iii trial , however , the positive outcome seen in als mouse models was not reproduced in the human study . moreover , the copper chelator and thiol compound penicillamine , the copper chelator trientine , carboxyfullerenes , vitamin e , and n - acetylcysteine have been reported to increase the survival time in this mouse model and/or delay the onset of the disease to a small extent [ 165 , 166 ] . in als , weight loss is frequently observed and can occur early or later during the course of disease . anorexia , digestive disorders , and upper extremity motor difficulties also contribute to low intake . many research studies point towards significant role of mitochondria in als pathogenesis and thus may provide a cause of malnutrition in als patients . it has been found that prevalence of substantial mitochondrial degeneration in motor neurons of g93a sod1 mutant mice at the onset of the disease , when electron transfer chain activity and atp synthesis appear severely dysfunctional . furthermore , reduction in the activity of cytochrome - c oxidase , encoded by the mitochondrial genome , in motor neurons of sals patients has also been reported . there is a direct correlation between the amount of mitochondrial dna in the spinal cord of als patients with decrease in the activities of citrate synthase and the respiratory chain complexes i , ii , iii , and iv . found a deficiency of nadh : coq oxidoreductase in muscles of sals and also noticed that decreased activities of nadh : coq oxidoreductase and cytochrome c oxidase are associated with dna abnormalities and reduced levels of the mitochondrial mn - superoxide dismutase . reported the early increase in the mrna levels of the mitochondrial uncoupling protein-3 in skeletal muscles of als - linked g86r sod1 mutant mice . although clinical trials failed to show a beneficial effect of creatine in als patients , the findings in g93a mice invoke the existence of a characteristic energetic imbalance . the suggestion that hypermetabolism can be explained by an increase in the work of respiratory muscles lacks credibility because there is no correlation between ree and pulmonary function . in fact , malnutrition is common in als , with a frequency reported to be between 15 and 55% , depending on the stage of disease and the definition of malnutrition adopted . also showed that the number of cells in the ventral horns of the lumbar spinal cord , which is significant lost in large sized cells , most probably representing motor neurons , in symptomatic g86r , mice was prevented in high - fat diet - fed animals of the same age . from clinical perspective , the nutritional status is a prognostic factor for survival in als , and more evidence suggests that the proper customized nutritional management of patients may constitute a primary symptomatic treatment for the disease . in order to proceed further in this regard , two possible approaches can be taken , either to develop analogues of the neuroprotective kynurenines or to inhibit the synthesis of the neurotoxic quin . it also reduces glutamate concentrations in the extracellular spaces of the basal ganglia in rats without impairing the learning or memory process typically associated with glutamate receptor antagonists and significantly reduces the neurotoxic levels of 3-hk and quin in the cns . the wide range of survival time in als patients suggests that multiple prognostic factors are involved ; only some have been clearly identified . a specific biochemical marker for early diagnosing and for monitoring disease progression in als will have important clinical applications . a minority of patients carries mutations in the cu / zn - superoxide dismutase ( sod1 ) gene , but the disease mechanism remains unknown for all types of als . the most recent finding in the als genetics suggests a very important role of mutation in sqstm1 and ubqln2 genes , and it is suggested that als patients should be monitored in a broad spectrum to study the altered effect on metabolic pathways [ 24 , 25 ] . the d90a sod1 mutation findings suggest that metabolomic profiling using gc - tofms and multivariate data analysis may be a future tool for diagnosing and monitoring disease progression and may cast light on the disease mechanisms in als . exploration of metabolomics by the use of small molecules derived from biofluids provides a strong platform to understand the metabolic characters of the living system and plays a significant role in the detection of diagnostic biomarkers . future clinical and experimental studies , therefore , need to concentrate on the complex relations between metabolism and als , and in this way may answer the question of whether targeting defective metabolism in als is an efficient way to alter disease progression . though a single technique is not sufficient to study the entire metabolomic profile , there is a need to converge some state of the art techniques namely , gc / ms , lc / ms , and nmr to realize the full worth of metabolomics .

motor neuron diseases ( mnd ) are a heterogeneous group of disorders which result in death of motor neurons amyotrophic lateral sclerosis ( als ) is the most common form of mnd in adults , affecting both anterior horn cells and corticospinal tracts . the majority of als cases which have been reported are sporadic ( sals ) ; 10% are familial ( fals ) , some of which arise from mutations in superoxide dismutase-1 ( sod1 ) , tar dna - binding protein ( tdp43 ) [ 5 , 6 ] and fused in sarcoma / translated in liposarcoma ( fus / tls ) [ 7 , 8 ] , als2 [ 9 , 10 ] , dynactin , and senataxin . using a genome - wide association study ( gwas ) approach , it has been recently reported that a locus on chromosome 9p21 accounted for 40% of familial als and nearly 1 fourth of all als cases in a sample of 405 finnish patients . linkage analysis of patients affected with multiple cases of als , ftd , and ftd - als with type 2 tdp-43 pathology had suggested that there was an important locus for the disease on chromosome 9p [ 1821 ] , but it is not clear whether the linkage and association signals related to a single locus or whether the different studies are reporting the same alleles at that locus . some more studies have shown that there is a large hexanucleotide ( ggggcc ) repeat expansion in the first intron of c9orf72 on the affected haplotype , and a common mendelian genetic lesion in c9orf72 is implicated in many cases of sporadic and familial als and ftd . these findings provide evidence of a direct genetic role of p62 in als pathogenesis as it is supposed to be involved in protein - recycling system by regulating the protein degradation pathways , and thus , p62 , related to ubqln2 may represent an important therapeutic target in als . although the root cause of als is not clearly understood , but multiple mechanisms have been found to be associated in the pathogenesis of motor neuron death in als . ideally metabolomic analyses are rapid , unbiased , and comprehensive , and , to some extent mass spectrometry , h1 nuclear magnetic resonance ( h1 nmr ) spectroscopy and ir spectroscopy are progressively considered as metabolomic techniques . the understanding of regulatory metabolic processes of a complex living organism at the system level requires the assessment of spatiotemporal interorgan metabolic crosstalks through the analysis of biofluids . when associated with a well - defined physiological condition , metabolic profiles provide a snapshot of a functional phenotype , or metabotypes , as a result of multiple interactions between metabolic pathways under the influence of environment , lifestyle , genetics , and microbial factors [ 3032 ] . a specific metabolic profile of a systemic biofluid , such as blood or serum / plasma , reflects the overall metabolic status of an individual as the result of highly complex metabolic exchanges between diverse biological compartments , including organs , biofluids , and microbial symbionts . although h1-nmr is a well - established approach for multicompartmental metabolic profiling of intact tissue and biofluid samples , gc / tofms has the advantage to discover changes in metabolites present at lower concentrations due to the increased sensitivity ; hence , nmr and mass spectrometry should be regarded as complementary techniques . the potential use of blood ( serum or plasma ) and cerebrospinal fluid ( csf ) biomarkers associated with als have been studied by many authors , and these target molecules are supposed to be linked to als pathogenesis [ 3436 ] . recently , high - throughput techniques have been used to evaluate a combination of markers in patients with neurological diseases which is performed via different analytical methods such as high - performance liquid chromatography followed by electrochemical detection . there were previous studies by authors , who measured glutamate and found decreased glutamate concentrations in cns tissue and increased concentrations in the serum and csf of als patients , and hence , proposed a hypothesis suggesting an imbalance in the intracellular versus extracellular glutamatergic neurotransmitter system [ 43 , 44 ] . further some more important metabolites related to energy and oxidant and antioxidants have been studied , and it has been hypothesized that glutamate excitotoxicity exacerbates the formation of ros , which may be responsible for the oxidant - antioxidant imbalance in serum of als patients . for example , histidine metabolite , which is considered to be powerful antioxidant , and n - acetyl derivative ( n - acetyl - x ) concentrations significantly decreased in serum of als patients with increase concentration of bhbt , acetone , and glutamate ( figure 1 ) . unfortunately due to space limitations only a small number of promising article findings related to blood metabolites can be discussed in this section , and readers are directed to other excellent research articles for some other changes in metabolic profile in blood of als patients [ 4547 ] . csf is known as good source for the study of biomarkers because of its direct contact with the brain , its accessibility , and its dynamic changes with the cerebral environment . a recent study has found a relationship between the levels of galectin-3 to be indicative of the onset of als symptoms in mice , and the result was found transferrable to als . the elevated levels of this antioxidant molecule ( ascorbate ) are compatible with oxidative stress previously described in als and could also be linked to ascorbic acid release from astrocytes after glutamate stimulation [ 54 , 55 ] . studied the csf metabolome by using gc - tofms platform in als patients with six different mutations in the sod1 gene and compared it with als - patients without such mutations and found that patients with a sod1 mutation have a distinct metabolic profile in the csf . in particular , the patients who are homozygous for the d90a sod1 mutation showed a distinctively different signature when modeled against als patients with other sod1 mutations and sporadic and familial als patients without a sod1 gene mutation . among the metabolites that contributed most to the csf signature were arginine , lysine , ornithine , serine , threonine , and pyroglutamic acid , all found to be reduced in patients carrying a d90a sod1 mutation . analysis of the neurofilament light chain in the csf suggests that patients with sod1 gene mutations constitute a distinct subgroup within the als syndrome , and more so patients homozygous for the d90a sod1 mutation . there are significant differences in the metabolite profile and composition among patients with fals , sals , and patients carrying a mutation in the sod1 gene suggesting that the neurodegenerative process in different subtypes of als may be partially dissimilar . the kn is a major route for the conversion of l - tryptophan ( trp ) to nicotinamide adenine dinucleotide ( nad ) [ 62 , 63 ] , initiated by either tryptophan 2,3-dioxygenase ( tdo ) or indoleamine 2,3-dioxygenase ( ido ) [ 64 , 65 ] . along the kynurenine ( kn ) , they include the free radical generator 3-hydroxykynurenine ( 3-hk ) , the excitotoxic n - methyl - d - aspartate ( nmda ) receptor agonist , quinolinic acid ( quin ) , the neuroprotective nmda receptor antagonist , kynurenic acid ( kyna ) [ 68 , 69 ] , and the neuroprotective picolinic acid ( pic ) ( figure 2 ) [ 70 , 71 ] . with respect to the pathogenesis of als , there is increasing evidence suggesting the involvement of the kp , especially that of the neurotoxin quin . demonstrated the highest kyna levels in the caudate nucleus , lower levels in the thalamus , globus pallidus , hippocampus , parietal cortex and frontal cortex , and the lowest level in the cerebellum . demonstrated that kyna concentration was significantly higher in the csf of als patients with bulbar onset of als compared to control group , whereas serum kyna concentration was significantly lower in als patients with severe clinical status than in controls . in als patients has shown twofold increase in csf trp , serum trp , and serum kyn , a tenfold increase in csf kyn and a fourfold increase in intracellular csf ido activity in als compared to controls . although kyn can be transported across the blood brain barrier , the tenfold increase in csf kyn , compared to only a twofold increase in serum kyn , the elevated levels of serum and csf trp are speculated to reflect a dysfunction in protein - binding and l - neutral amino acid transporters , respectively , or perhaps , an over - compensatory response , whereby the increase in csf ido activity results in a decrease in csf trp , which encourages the dissociation of trp from albumin to facilitate transport of trp across the blood brain barrier ( bbb ) . once quin exceeds the safety threshold ( < 150 nm ) , its adverse effect is exerted via the generation of reactive oxygen species , augmentation of the excitotoxic impact through disruption in the glutamatergic transport system , apoptosis , mitochondrial dysfunction , and the production of cytokines and chemokines , all of which are putatively thought to be contributory factors in als ( figure 1 ) . als is a neurodegenerative disease characterized by progressive degeneration of both upper motor neurons ( umn ) in the primary motor cortex ( pmc ) and lower motor neurons ( lmn ) in the brain stem and spinal cord anterior horns . despite the identification of pathologies in the pmc and corticospinal tracts ( cst ) of autopsy tissue samples with als [ 8487 ] , there are no biomarkers identified to date that reliably indicate presence of such pathologies in the brain of patients with als . it is hypothesized that the neuropathologically proven motor neuron and cst degeneration in als might occur gradually with no apparent manifestation of macroscopic tissue structural changes in the early stage which are detectable by conventional mri methods . in contrast , neurochemicals indicative of metabolic processes responsible for degeneration of the motor neurons and cst in patients with als can be accessed from the disease onset stage using proton mr spectroscopic ( 1h mrs ) methods . several previous cross - sectional and longitudinal studies have demonstrated the value of proton mrs for the evaluation of metabolite alterations in the pmc [ 90100 ] and cst [ 95 , 101 ] of patients with als using single voxel svs and 2d mr spectroscopic imaging [ 101103 ] methods . 1h mrs of the human brain reveals important information about compounds such as n - acetylaspartate ( naa ) which has been suggested to be a marker for neuron viability and axonal density in the brain , creatine ( cr ) as a putative marker of gliosis [ 105 , 106 ] , and choline ( cho ) which is associated with membrane phospholipids . in another study , chemical shift - imaging follow - up measurements in als patients after a three - month period demonstrated a decline in concentrations of naa , cr , and cho in the most affected motor cortex areas of als in comparison with healthy controls . this muscular mitochondrial defect had a heterogeneous distribution and was correlated in some patients with multiple mitochondrial dna deletions , decreased mitochondrial dna levels , and low levels of membrane - associated manganese sod . furthermore , adult loss of tdp-43 led to massive decreases in adipose tissue , probably through muscle hypermetabolism , and tdp-43 overexpression led to the formation of morphologically abnormal mitochondria [ 121 , 122 ] . the extent and origin of such mitochondrial dysfunction remain controversial , however , and the impairment of energy metabolism seen in patients with als and mutant sod1 mice might be due at least partly to dysfunctional regulation of metabolic pathways . recently , it has been demonstrated that the basement membrane of skin in als patients is weakly positive for type iv collagen compared with controls , and also serum type iv collagen levels in als patients are lower than controls . it showed that the absolute and relative concentrations of two collagen metabolites , glucosylgalactosyl hydroxylysine ( glu - gal hyl ) , and galactosyl hydroxylysine ( gal hyl ) , excreted in urine , indicate the tissue origin of the collagen metabolites and the rate of degradation of collagen [ 135137 ] . [ 141 , 142 ] reported markedly decreased contents of collagen and collagen - associated amino acids in the lateral corticospinal tract and the anterior horn in the spinal cord of patients with als , suggesting that these data could be associated with the degeneration of the upper and lower motor neurons in patients with als . riluzole was the first drug approved by the fda ( usa ) in 1995 for the treatment of als , but its mechanisms of action in slowing the progression of this disease remain obscure . conducted a prospective , double - blind , placebocontrolled trial in 155 als patients and found that the antiglutamate agent riluzole appears to slow the progression of als disease , and it may improve survival in patients with bulbar onset of disease . also carried out a double - blind , placebocontrolled at multicentre and found that the 100 mg dose of riluzole has the best benefit - to - risk ratio , and it decreased mortality and slowed muscle - strength deterioration in als patients . though the precise mechanism of action for riluzole remains unclear , it appears to interfere with excitatory amino acid signalling , possibly through the inhibition of glutamate release [ 145147 ] , blockade of inactivated sodium channels , and interaction with guanosine triphosphate - binding proteins . this effect is mediated by a direct action of minocycline on the activated t cells and on microglia , which results in the decreased ability of t cells to contact microglia which impairs cytokine production in t - cell - microglia signal transduction . it has been reported that minocycline , which is known as an antibiotic and inhibitor of microglial activation , riluzole , which works as an inhibitor of glutamate release , and nimodipine , which is voltage - gated calcium channel blocker , is designed to target the different pathways leading to neuronal death . when the three drugs were tested in combination on sod1 mice , it resulted in strikingly improved conditions , delaying disease onset by 4 weeks , increasing survival by 6 weeks , and attenuating neurodegeneration by reducing cyclin - dependent kinase 5 mislocalization , caspase-3 activation , astrocytosis , and microgliosis . moreover , the copper chelator and thiol compound penicillamine , the copper chelator trientine , carboxyfullerenes , vitamin e , and n - acetylcysteine have been reported to increase the survival time in this mouse model and/or delay the onset of the disease to a small extent [ 165 , 166 ] . in many cases , however , weight loss also has a nutritional component , with loss of fat mass and fat - free mass ( ffm ) attributable to malnutrition , the principal cause of which is decreased dietary intake [ 168 , 169 ] . it has been found that prevalence of substantial mitochondrial degeneration in motor neurons of g93a sod1 mutant mice at the onset of the disease , when electron transfer chain activity and atp synthesis appear severely dysfunctional . there is a direct correlation between the amount of mitochondrial dna in the spinal cord of als patients with decrease in the activities of citrate synthase and the respiratory chain complexes i , ii , iii , and iv . found a deficiency of nadh : coq oxidoreductase in muscles of sals and also noticed that decreased activities of nadh : coq oxidoreductase and cytochrome c oxidase are associated with dna abnormalities and reduced levels of the mitochondrial mn - superoxide dismutase . supporting the mitochondrial dysfunction hypothesis , and administration of creatine , an intracellular energy shuttle between mitochondria and sites of energy consumption that is known to ameliorate muscle function , increased the life expectancy of g93a mice . also showed that the number of cells in the ventral horns of the lumbar spinal cord , which is significant lost in large sized cells , most probably representing motor neurons , in symptomatic g86r , mice was prevented in high - fat diet - fed animals of the same age . from clinical perspective , the nutritional status is a prognostic factor for survival in als , and more evidence suggests that the proper customized nutritional management of patients may constitute a primary symptomatic treatment for the disease . in order to proceed further in this regard , two possible approaches can be taken , either to develop analogues of the neuroprotective kynurenines or to inhibit the synthesis of the neurotoxic quin . ( c ) an immunosuppressive and anti - inflammatory prodrug , leflunomide , gets converted to its active open - ring metabolite , teriflunomide , an inhibitor of mitochondrial dihydroorotate dehydrogenase , and known to successfully reduce the development of active lesions in patients with relapsing multiple sclerosis . it also reduces glutamate concentrations in the extracellular spaces of the basal ganglia in rats without impairing the learning or memory process typically associated with glutamate receptor antagonists and significantly reduces the neurotoxic levels of 3-hk and quin in the cns . a minority of patients carries mutations in the cu / zn - superoxide dismutase ( sod1 ) gene , but the disease mechanism remains unknown for all types of als . the most recent finding in the als genetics suggests a very important role of mutation in sqstm1 and ubqln2 genes , and it is suggested that als patients should be monitored in a broad spectrum to study the altered effect on metabolic pathways [ 24 , 25 ] . the d90a sod1 mutation findings suggest that metabolomic profiling using gc - tofms and multivariate data analysis may be a future tool for diagnosing and monitoring disease progression and may cast light on the disease mechanisms in als . for this , the use of metabolomics as the link to pathway information in genome - wide association studies could be of great interest for mapping and interpreting the effects of different mutations and even combinations of mutations in relation to als subtypes . exploration of metabolomics by the use of small molecules derived from biofluids provides a strong platform to understand the metabolic characters of the living system and plays a significant role in the detection of diagnostic biomarkers . future clinical and experimental studies , therefore , need to concentrate on the complex relations between metabolism and als , and in this way may answer the question of whether targeting defective metabolism in als is an efficient way to alter disease progression . though a single technique is not sufficient to study the entire metabolomic profile , there is a need to converge some state of the art techniques namely , gc / ms , lc / ms , and nmr to realize the full worth of metabolomics . there is definite need to further elaborate on the sensitive and specific metabolic therapeutic signatures by advance analytical means and bioinformatics application which will help us to elucidate the structures of more signature molecules in als disease and provide us insight into aberrant biochemical pathways and may prove to be helpful in building up diagnostic markers and targets for drug design .

dental implant - supported prostheses have become one of the significant treatment modalities for replacement of teeth , with reported success rates of over 98.2% . the success of dental implants is highly dependent upon the integration between the implant and the intraoral hard / soft tissues . the long term success or survival of dental implants is determined by the transmission of occlusal load and resultant stress distribution in the surrounding bone . load transfer at the bone - implant interface depends on : ( 1 ) the implant geometry and the design of implant abutment connection ; ( 2 ) the loading protocol and the type of occlusion ; ( 3 ) the number of implants and position ; ( 4 ) the quality and quantity of the surrounding bone . it has been demonstrated that vertical and transverse masticatory loads induce axial forces and bending moments that results in stress gradients in the implant , as well as in the bone . rieger et al . reported that stresses in the range 1.4 - 5.0 mpa may be required for healthy maintenance of bone , stresses outside this range have been reported to cause bone resorption . according to frost 's mechanostat concept , bone fractures at 10,00020,000 microstrains . however , just 20% to 40% of the amount of strain required for fracture ( i.e. , 4,000 microstrains ) may trigger cytokine to activate a resorptive response . a persistent load increases the stress and may provoke micro - fractures and osteoclastic activity in the bone . there has been a continuous evolution of the implant abutment connection design with the intensions of reducing these stress concentrations . based on the various prosthetic and biological complications encountered in the clinical scenario and the results of various studies , the initial external hex design which had the interface above the implant and the osseous crest , has evolved into the internal hex with the implant abutment interface being placed more apically and away from the osseous crest . the development of these implant abutment interfaces have reduced the amount of stress that is transmitted to the implant or the surrounding bone and therefore considerably reducing the crestal bone loss , although not entirely eliminated indicating a multi - factorial cause . the structural complexity of implant abutment interface design due to the continuous improvement in the geometry has made it difficult to evaluate occlusal forces in the bone around the dental implant and the stresses within the implant . finite element analysis ( fea ) is a useful tool for the prediction of the effects of stress on the implant and its surrounding bone . the use of the finite element method to analyze stress concentrations was initially introduced into implant dentistry by weinstein et al . in 1976 . in fea , the mechanical performance of the implant abutment interface could be evaluated by von misses stresses . von misses stress criterion is important to interpret the stresses within the ductile material , such as the implant material ; as deformation occurs when the von misses stress value exceeds the yield strength . finite element studies comparing different connections and the effect of the change in occlusal table width dimensions on stress distribution pattern in and around the implant abutment interface are limited . in this study , an attempt is made to compare the stress distribution pattern of a passive fit and friction fit implant abutment interface in different areas of the implant and bone and the influence of occlusal table width on stress distribution . finite element analysis is a computerized numerical technique used to determine the stress and displacements through a predetermined model . fea solves a complex problem by dividing it into a series of interrelated simple problems . a mesh is needed in fea to divide the complex geometry into smaller elements in which the field variables can be interpolated with the use of shape functions . the process of creating the mesh , elements , their respective nodes and defining boundary conditions is referred to as the study models were constructed using reverse engineering technique in pro - engineer 05 through three - dimensional ( 3d ) optical scanning and point cloud data extraction . the reverse - engineering process involves measuring an object and then reconstructing it as a 3d model . two cad models of implants were constructed with two different types of implant abutment connections currently available in the market , the passive fit or the slip - fit represented by the nobel replace tri - lobe connection ( nobel replace , tapered groovy , rp 4.3 mm 13 mm ) and the friction fit or active fit represented by the nobel active conical connection ( nobel active , internal rp 4.3 mm 13 mm ) along with their respective snappy abutments . the bone structure was modeled through a computed tomography ( ct ) scan that can provide results closer to a real scenario , because there is a difference in the behavior of stresses in work conducted with elliptical models , cobblestones , and ct scan data . the thickness of the cortical bone was kept 2 mm , and a uniform layer of cortical bone was modeled on the outer surface of the cancellous core . a bone block model was constructed based on a cross - sectional image of the human mandible in the premolar region , 25 mm high , 12 mm wide , and 10 mm thick consisting of a spongy center surrounded by a 2-mm cortical bone . three crowns with different occlusal table dimensions were constructed by changing the buccolingual dimensions and keeping mesiodistal and the cervicoocclusal length constant . the dimensions of a mandibular premolar are 7.5 mm buccolingually , 9 mm mesiodistally , and 8 mm cervicoocclusally . the buccolingual dimension with 7.5 mm was considered as ideal , and then crowns with narrow and wider occlusal tables were constructed with 6 mm and 10 mm buccolingual dimension , respectively . they were then placed over the passive connection and the friction connection making a total of six models . the implant abutment complex thus constructed using reverse - engineering technique was then positioned in the cortical and cancellous bone block . the 3d models corresponding to the geometric model was meshed using hypermesh 10 and then imported into ansys 13 software to perform the numerical simulation . it is a 2 order tetra element which has a quadratic displacement behavior and is well suited to model irregular meshes . the element is defined by 10 nodes having 3 of freedom at each node and 3 translations in nodal x , y , and z directions . the numbers of nodes in friction connection for ideal , narrow , and wider occlusal table are 80,786 , 82,047 , and 83,972 ; and the number elements are 57,082 , 57,737 , and 58,931 , respectively . the numbers of nodes in passive connection for ideal , narrow , and wider occlusal table are 76,330 , 77,469 , and 79,527 ; and the number elements are 53,569 , 54,137 , and 55,414 , respectively . meshed models of passive - fit connection and friction fit connection are shown in figure 1a . ( b ) the vertical and oblique loading directions on a meshed model of a passive fit connection in this study , we assumed the implant , abutment , and screws were homogeneous , linear elastic , and isotropic mechanical properties . material properties for bone and implant components [ table 1 ] were collected from reliable resources and published data . the implant was pure titanium , and other components were titanium alloys , with homogeneous and isotropic elastic properties . it was assumed that there is complete osseointegration between the implant and the surrounding bone . mechanical properties of different materials . a distributed force of 100 n was applied onto the top surface of the crown vertically along the long axis and then obliquely at 45 to the longitudinal axis of the implant . for a direct and systematic comparison , the same loading conditions , boundary conditions , and constraints was applied in all the models . the vertical and oblique loading directions on a meshed model of a passive fit connection as an example are shown in figure 1b . the study models were constructed using reverse engineering technique in pro - engineer 05 through three - dimensional ( 3d ) optical scanning and point cloud data extraction . the reverse - engineering process involves measuring an object and then reconstructing it as a 3d model . two cad models of implants were constructed with two different types of implant abutment connections currently available in the market , the passive fit or the slip - fit represented by the nobel replace tri - lobe connection ( nobel replace , tapered groovy , rp 4.3 mm 13 mm ) and the friction fit or active fit represented by the nobel active conical connection ( nobel active , internal rp 4.3 mm 13 mm ) along with their respective snappy abutments . the bone structure was modeled through a computed tomography ( ct ) scan that can provide results closer to a real scenario , because there is a difference in the behavior of stresses in work conducted with elliptical models , cobblestones , and ct scan data . the thickness of the cortical bone was kept 2 mm , and a uniform layer of cortical bone was modeled on the outer surface of the cancellous core . a bone block model was constructed based on a cross - sectional image of the human mandible in the premolar region , 25 mm high , 12 mm wide , and 10 mm thick consisting of a spongy center surrounded by a 2-mm cortical bone . three crowns with different occlusal table dimensions were constructed by changing the buccolingual dimensions and keeping mesiodistal and the cervicoocclusal length constant . the dimensions of a mandibular premolar are 7.5 mm buccolingually , 9 mm mesiodistally , and 8 mm cervicoocclusally . the buccolingual dimension with 7.5 mm was considered as ideal , and then crowns with narrow and wider occlusal tables were constructed with 6 mm and 10 mm buccolingual dimension , respectively . they were then placed over the passive connection and the friction connection making a total of six models . the implant abutment complex thus constructed using reverse - engineering technique was then positioned in the cortical and cancellous bone block . the 3d models corresponding to the geometric model was meshed using hypermesh 10 and then imported into ansys 13 software to perform the numerical simulation . it is a 2 order tetra element which has a quadratic displacement behavior and is well suited to model irregular meshes . the element is defined by 10 nodes having 3 of freedom at each node and 3 translations in nodal x , y , and z directions . the numbers of nodes in friction connection for ideal , narrow , and wider occlusal table are 80,786 , 82,047 , and 83,972 ; and the number elements are 57,082 , 57,737 , and 58,931 , respectively . the numbers of nodes in passive connection for ideal , narrow , and wider occlusal table are 76,330 , 77,469 , and 79,527 ; and the number elements are 53,569 , 54,137 , and 55,414 , respectively . meshed models of passive - fit connection and friction fit connection are shown in figure 1a . ( ( b ) the vertical and oblique loading directions on a meshed model of a passive fit connection in this study , we assumed the implant , abutment , and screws were homogeneous , linear elastic , and isotropic mechanical properties . material properties for bone and implant components [ table 1 ] were collected from reliable resources and published data . the implant was pure titanium , and other components were titanium alloys , with homogeneous and isotropic elastic properties . it was assumed that there is complete osseointegration between the implant and the surrounding bone . a distributed force of 100 n was applied onto the top surface of the crown vertically along the long axis and then obliquely at 45 to the longitudinal axis of the implant . for a direct and systematic comparison , the same loading conditions , boundary conditions , and constraints was applied in all the models . the vertical and oblique loading directions on a meshed model of a passive fit connection as an example are shown in figure 1b . the data obtained from ansys calculation can be presented in a stress distribution map with a color scale , which makes it possible to compare directly the stress level in various component structures of all models . the amount of stress and the pattern of stress generated after applying a load of 100 n on each model in vertical and oblique direction were recorded on a color scale . the figures 24 shows the von misses stresses during vertical and oblique loading with 100 n on a narrow occlusal table in different regions of friction fit and passive fit implant abutment interface and figures 5 and 6 show graphs of von misses stress ( in mpa ) on narrow , ideal , and wider occlusal tables in vertical and oblique loading on implant , implant abutment interface , and bone in friction fit and passive fit connections . von misses stress ( in mpa ) on the implant abutment complex with prosthesis having narrow , ideal and wider occlusal tables ( a ) von misses stress on an implant with friction fit connection and passive fit connection during vertical loading with a load of 100 n in models with narrow occlusal table . ( b ) von misses stress on an implant with friction fit connection and passive fit connection during oblique loading with a load of 100 n in models with narrow occlusal table ( a ) von misses stress on an implant abutment interface with friction fit connection and passive fit connection during vertical loading with a load of 100 n in models with narrow occlusal table . ( b ) von misses stress on an implant abutment interface with friction fit connection and passive fit connection during oblique loading with a load of 100 n in models with narrow occlusal table ( a ) von misses stress on the bone with friction fit connection and passive fit connection during vertical loading with a load of 100 n in models with narrow occlusal table ( b ) von misses stress on the bone with friction fit connection and passive fit connection during oblique loading with a load of 100 n in models with narrow occlusal table graph showing von misses stress ( in mpa ) on narrow , ideal , and wider occlusal tables in vertical and oblique loading on implant and implant abutment interface in friction fit and passive fit connections graph showing von misses stress ( in mpa ) on narrow , ideal , and wider occlusal tables in vertical and oblique loading on bone in friction fit and passive fit connections from the values given in table 2 the following data and results have been obtained : the overall maximum von misses stress on the implant is more significant for friction fit than the passive fit implant abutment interface in both vertical and oblique loading for all the models testedat the implant abutment interface and at the neck of the implant , the von misses stress was higher for the friction fit . whereas on the outer surface of the abutment and on the internal surface of the fixture ; the passive fit shows lesser stress in both vertical and oblique loading for all the models testedthe overall maximum von misses stress on the bone is significantly less for friction fit than the passive fit in both vertical and oblique loading for all the modelsirrespective of the type of abutment connection used , the maximum von misses stress was seen in the region of the cortical or the marginal bone . it is showed that a significant reduction in von misses stress was observed at the boundary between cortical bone and cancellous bone in both loading conditionsthe narrow occlusal table , irrespective of their connection type has reduced the stress generated . this shows that the width of the occlusal table has got a significant influence on the stress generated on the implant , as well as on the bone . the overall maximum von misses stress on the implant is more significant for friction fit than the passive fit implant abutment interface in both vertical and oblique loading for all the models tested at the implant abutment interface and at the neck of the implant , the von misses stress was higher for the friction fit . whereas on the outer surface of the abutment and on the internal surface of the fixture ; the passive fit shows lesser stress in both vertical and oblique loading for all the models tested the overall maximum von misses stress on the bone is significantly less for friction fit than the passive fit in both vertical and oblique loading for all the models irrespective of the type of abutment connection used , the maximum von misses stress was seen in the region of the cortical or the marginal bone . it is showed that a significant reduction in von misses stress was observed at the boundary between cortical bone and cancellous bone in both loading conditions the narrow occlusal table , irrespective of their connection type has reduced the stress generated . this shows that the width of the occlusal table has got a significant influence on the stress generated on the implant , as well as on the bone . the aim of the study was to analyze the influence of two different types of implant abutment connection on the stress distribution pattern in the implant and the surrounding bone . the implant abutment interface that have been analyzed in the study represent two broad categories of implant abutment connection currently available in the market , the passive fit or the slip - fit represented by the nobel replace tri - lobe connection and the friction fit or active fit represented by the nobel active conical connection . six models were constructed in pro - engineer 05 of the two implant abutment connection for three different occlusal dimensions each . the implant and abutment complex was placed in cortical and cancellous bone modeled using a ct scan . this complex was subjected to a force of 100 n in the axial and oblique direction . the amount of stress and the pattern of stress generated were recorded on a color scale . the mean values of overall stress on the implant with friction fit connection were 88.725 for vertical load and 258.962 for oblique load whereas on passive fit were 49.805 and 136.249 , respectively , indicating that friction fit produced higher overall stress on the implant than the passive fit . the mean values of overall stress on the bone for vertical and oblique loading on friction fit connection were 15.907 and 57.236 whereas on a passive fit connection were 30.680 and 88.983 , respectively . this show that the stress generated by passive fit connection on bone is almost double the stress generated by the friction fit connection . the mean values of the overall stresses on the implant and the bone shows that the friction fit connection absorbs more stress and dissipates less stress to the surrounding bone . the larger contact area and deeper position inside the implant for friction fit connection allowed for better stability and broader stress distribution , as has been observed in several other studies . this frictional fit creates wedging effects to improve the implant abutment joint stability against the lateral force and helps to transfer the loading force along the conical surface to distribute the stress on the implant , ultimately reducing biological and biomechanical complications . the internal conical connections help the abutment screw retain greater preload after repeated loads since the loading stress is not entirely concentrated on the screw as in the external hex butt joint implant systems . the friction - locking mechanics and the solid design of the friction fit connections provided greater resistance to deformation and fracture under oblique compressive loading when compared to the passive fit connection . in passive fit connection , the cold welding does not occur when the abutments are tightened thus an inevitable gap between the implant and abutment may still exist . this can cause micro - motion at the interface during clinical loading , which in turn may contribute to stress on the screw and therefore loss of preload and loosening of abutment thereby leading to bacterial colonization of the micro gap . the threshold of deleterious micro - motion level asserted by various researchers lies within the range of 50150 m . beyond these levels of micro - motion the mean values of stress on the crestal bone for vertical and oblique loading on friction fit connection were 15.391 and 25.449 whereas on a passive fit connection were 19.024 and 29.674 , respectively . the highest stress occurs in the implant 's most cervical region when an occlusal load is applied upon an implant , and the load is partially transferred to the bone . this phenomenon is due to one of the principles of engineering , that is , when two materials are in contact with each other , and one of them is loaded , the stresses will be higher at the materials initial point of contact . this explains why the cervical region of the implant is the site where the greatest micro - deformations occur independently of the type of bone and the design of the implant , the configuration of the prosthesis and the load . the results of the current fea for the osseointegrated model are in accordance with the findings of hansson . using fea , hansson showed that a conical implant abutment interface at the level of the bone crest decreases the peak bone - implant interfacial stress as compared with the flat top interface . for the friction fit implant abutment interface , this peak interfacial shear stress was located at some depth in the marginal bone . the mean values of stress on the apical area of bone for vertical and oblique loading on friction fit connection were 0.019 and 0.021 whereas on a passive fit connection were 0.0215 and 0.0278 , respectively . in this study , significantly larger stress values were seen in the neck area versus the apex area among all models in all conditions , which is consistent with the results of other studies . stresses induced by occlusal load are initially transferred from implant to the cortical bone , and a small amount of remaining stress spreads to cancellous bone . higher stress values are observed in cortical bone because of higher modulus of elasticity and bone density compared to the cancellous bone . richter has reported that the highest stress in the crestal bone is a result of a transverse load and clenching at centric contacts . the width of almost every natural tooth is greater than the width of the implant used to replace the tooth . the greater the width of a transosteal structure , the lesser the magnitude of stress transmitted to the surrounding bone . the cross - sectional shape of the natural tooth at the crest is biomechanically optimized to resist lateral loads , implants , however , are almost round in cross - section , which is less effective in resisting lateral bending loads thereby concentrating loads in the crestal region . the mean values of axial displacement of teeth in the socket are 25 - 100 m , whereas the range of motion of osseointegrated dental implants has been reported approximately 3 - 5 m . the elastic modulus of the tooth is closest to bone compared to the available implant biomaterials . hence , under similar loading conditions implant generates greater stresses and strain at the crest of bone than a natural tooth . from the results of the study , it is shown that the narrow occlusal table , irrespective of their connection type has reduced the stress generated . this shows that the width of the occlusal table has got a significant influence on the stress generated on the implant , as well as on the bone . typically , a 30% to 40% reduction in the occlusal table in a molar region has been suggested because any dimension larger than the implant diameter can cause cantilever effects and eventual bending moments in single - implant prostheses . a narrow occlusal table reduces the chance of offset loading and increases axial loading , which eventually can decrease the bending moment . misch has described how a narrow occlusal table can improve oral hygiene and reduce the risk of porcelain fracture . the proposed key factors to control bend overload in posterior restorations were reduced the inclination of cusps , centrally oriented contacts with a 11.5 mm flat area , a narrowed occlusal table , and elimination of cantilevers . as the wider occlusal table will increase stress on the abutment screws , the occlusal table should be reduced in width compared with natural teeth in nonesthetic regions of the mouth . analysis of finite elements was shown to be a versatile and promising methodology for analyzing stress concentrations in implant dentistry , but it is worth emphasizing that the fea is an approximate virtual simulation of clinical situations , presenting certain limitations . hart et al . demonstrated that fea models with more than 10,420 nodes showed convergent results . therefore , the results derived from this fea may be considered to be reasonably accurate and acceptable . within the limits of the present study , the following conclusions can be derived : the overall maximum von misses stress on the implant is significantly more for friction fit connection than the passive fit connection in both vertical and oblique loading in all the models ; whereas the overall maximum von misses stress on the bone is significantly less for friction fit connection than the passive fit connection in both vertical and oblique loading in all the models . the overall maximum von misses stress values on the implant and the bone show that the friction fit connection absorbs more stress and dissipates less stress to the surrounding bone . further studies on the permissible amount of micro - movement allowed in a passive fit implant abutment interface may need to be conductedirrespective of the type of abutment connection used the maximum von misses stress was seen in the region of the cortical or the crestal bone . it is shown that a significant reduction in von misses stress was observed at the boundaries between cortical bone and cancellous bone in both loading conditions because of relatively low elastic modulus of cancellous boneon comparing the stresses on narrow , ideal and wider occlusal tables for both frictions fit connection and passive fit connection , the results show that the narrow occlusal table has the least stress followed by ideal and the wider occlusal tables . this shows that the width of the occlusal table has got a significant influence on the stress generated on the implant , as well as on the bone . the overall maximum von misses stress on the implant is significantly more for friction fit connection than the passive fit connection in both vertical and oblique loading in all the models ; whereas the overall maximum von misses stress on the bone is significantly less for friction fit connection than the passive fit connection in both vertical and oblique loading in all the models . the overall maximum von misses stress values on the implant and the bone show that the friction fit connection absorbs more stress and dissipates less stress to the surrounding bone . further studies on the permissible amount of micro - movement allowed in a passive fit implant abutment interface may need to be conducted irrespective of the type of abutment connection used the maximum von misses stress was seen in the region of the cortical or the crestal bone . it is shown that a significant reduction in von misses stress was observed at the boundaries between cortical bone and cancellous bone in both loading conditions because of relatively low elastic modulus of cancellous bone on comparing the stresses on narrow , ideal and wider occlusal tables for both frictions fit connection and passive fit connection , the results show that the narrow occlusal table has the least stress followed by ideal and the wider occlusal tables . this shows that the width of the occlusal table has got a significant influence on the stress generated on the implant , as well as on the bone . the friction fit connection is superior to the passive fit connection , as the friction fit creates wedging effects to improve the implant abutment joint stability against the lateral force and helps to transfer the loading force along the conical surface to distribute the stress on the implant , ultimately reducing biological and biomechanical complications . a narrow occlusal table may increase axial loading and decrease nonaxial loading for the implants thereby reducing the stress on the implant , implant abutment interface , and bone . thus , it is recommended that the size of the occlusal table to be 30% to 40% smaller for molars .

aims : the aim of the study was to evaluate the stress distribution pattern in the implant and the surrounding bone for a passive and a friction fit implant abutment interface and to analyze the influence of occlusal table dimension on the stress generated.materials and methods : cad models of two different types of implant abutment connections , the passive fit or the slip - fit represented by the nobel replace tri - lobe connection and the friction fit or active fit represented by the nobel active conical connection were made . the stress distribution pattern was studied at different occlusal dimension . six models were constructed in pro - engineer 05 of the two implant abutment connection for three different occlusal dimensions each . the implant and abutment complex was placed in cortical and cancellous bone modeled using a computed tomography scan . this complex was subjected to a force of 100 n in the axial and oblique direction . the amount of stress and the pattern of stress generated were recorded on a color scale using ansys 13 software.results:the results showed that overall maximum von misses stress on the bone is significantly less for friction fit than the passive fit in any loading conditions stresses on the implant were significantly higher for the friction fit than the passive fit . the narrow occlusal table models generated the least amount of stress on the implant abutment interface.conclusion:it can thus be concluded that the conical connection distributes more stress to the implant body and dissipates less stress to the surrounding bone . a narrow occlusal table considerably reduces the occlusal overload .

INTRODUCTION MATERIALS AND METHODS Construction of geometric model Mesh generation of the model Boundary conditions and constraints Loading conditions RESULTS DISCUSSION CONCLUSION

the long term success or survival of dental implants is determined by the transmission of occlusal load and resultant stress distribution in the surrounding bone . load transfer at the bone - implant interface depends on : ( 1 ) the implant geometry and the design of implant abutment connection ; ( 2 ) the loading protocol and the type of occlusion ; ( 3 ) the number of implants and position ; ( 4 ) the quality and quantity of the surrounding bone . it has been demonstrated that vertical and transverse masticatory loads induce axial forces and bending moments that results in stress gradients in the implant , as well as in the bone . a persistent load increases the stress and may provoke micro - fractures and osteoclastic activity in the bone . there has been a continuous evolution of the implant abutment connection design with the intensions of reducing these stress concentrations . based on the various prosthetic and biological complications encountered in the clinical scenario and the results of various studies , the initial external hex design which had the interface above the implant and the osseous crest , has evolved into the internal hex with the implant abutment interface being placed more apically and away from the osseous crest . the development of these implant abutment interfaces have reduced the amount of stress that is transmitted to the implant or the surrounding bone and therefore considerably reducing the crestal bone loss , although not entirely eliminated indicating a multi - factorial cause . the structural complexity of implant abutment interface design due to the continuous improvement in the geometry has made it difficult to evaluate occlusal forces in the bone around the dental implant and the stresses within the implant . finite element analysis ( fea ) is a useful tool for the prediction of the effects of stress on the implant and its surrounding bone . in fea , the mechanical performance of the implant abutment interface could be evaluated by von misses stresses . finite element studies comparing different connections and the effect of the change in occlusal table width dimensions on stress distribution pattern in and around the implant abutment interface are limited . in this study , an attempt is made to compare the stress distribution pattern of a passive fit and friction fit implant abutment interface in different areas of the implant and bone and the influence of occlusal table width on stress distribution . the process of creating the mesh , elements , their respective nodes and defining boundary conditions is referred to as the study models were constructed using reverse engineering technique in pro - engineer 05 through three - dimensional ( 3d ) optical scanning and point cloud data extraction . two cad models of implants were constructed with two different types of implant abutment connections currently available in the market , the passive fit or the slip - fit represented by the nobel replace tri - lobe connection ( nobel replace , tapered groovy , rp 4.3 mm 13 mm ) and the friction fit or active fit represented by the nobel active conical connection ( nobel active , internal rp 4.3 mm 13 mm ) along with their respective snappy abutments . the bone structure was modeled through a computed tomography ( ct ) scan that can provide results closer to a real scenario , because there is a difference in the behavior of stresses in work conducted with elliptical models , cobblestones , and ct scan data . a bone block model was constructed based on a cross - sectional image of the human mandible in the premolar region , 25 mm high , 12 mm wide , and 10 mm thick consisting of a spongy center surrounded by a 2-mm cortical bone . three crowns with different occlusal table dimensions were constructed by changing the buccolingual dimensions and keeping mesiodistal and the cervicoocclusal length constant . they were then placed over the passive connection and the friction connection making a total of six models . the implant abutment complex thus constructed using reverse - engineering technique was then positioned in the cortical and cancellous bone block . the numbers of nodes in passive connection for ideal , narrow , and wider occlusal table are 76,330 , 77,469 , and 79,527 ; and the number elements are 53,569 , 54,137 , and 55,414 , respectively . meshed models of passive - fit connection and friction fit connection are shown in figure 1a . ( b ) the vertical and oblique loading directions on a meshed model of a passive fit connection in this study , we assumed the implant , abutment , and screws were homogeneous , linear elastic , and isotropic mechanical properties . it was assumed that there is complete osseointegration between the implant and the surrounding bone . a distributed force of 100 n was applied onto the top surface of the crown vertically along the long axis and then obliquely at 45 to the longitudinal axis of the implant . the vertical and oblique loading directions on a meshed model of a passive fit connection as an example are shown in figure 1b . the study models were constructed using reverse engineering technique in pro - engineer 05 through three - dimensional ( 3d ) optical scanning and point cloud data extraction . two cad models of implants were constructed with two different types of implant abutment connections currently available in the market , the passive fit or the slip - fit represented by the nobel replace tri - lobe connection ( nobel replace , tapered groovy , rp 4.3 mm 13 mm ) and the friction fit or active fit represented by the nobel active conical connection ( nobel active , internal rp 4.3 mm 13 mm ) along with their respective snappy abutments . the bone structure was modeled through a computed tomography ( ct ) scan that can provide results closer to a real scenario , because there is a difference in the behavior of stresses in work conducted with elliptical models , cobblestones , and ct scan data . three crowns with different occlusal table dimensions were constructed by changing the buccolingual dimensions and keeping mesiodistal and the cervicoocclusal length constant . they were then placed over the passive connection and the friction connection making a total of six models . the implant abutment complex thus constructed using reverse - engineering technique was then positioned in the cortical and cancellous bone block . the numbers of nodes in friction connection for ideal , narrow , and wider occlusal table are 80,786 , 82,047 , and 83,972 ; and the number elements are 57,082 , 57,737 , and 58,931 , respectively . meshed models of passive - fit connection and friction fit connection are shown in figure 1a . ( ( b ) the vertical and oblique loading directions on a meshed model of a passive fit connection in this study , we assumed the implant , abutment , and screws were homogeneous , linear elastic , and isotropic mechanical properties . it was assumed that there is complete osseointegration between the implant and the surrounding bone . a distributed force of 100 n was applied onto the top surface of the crown vertically along the long axis and then obliquely at 45 to the longitudinal axis of the implant . the vertical and oblique loading directions on a meshed model of a passive fit connection as an example are shown in figure 1b . the data obtained from ansys calculation can be presented in a stress distribution map with a color scale , which makes it possible to compare directly the stress level in various component structures of all models . the amount of stress and the pattern of stress generated after applying a load of 100 n on each model in vertical and oblique direction were recorded on a color scale . the figures 24 shows the von misses stresses during vertical and oblique loading with 100 n on a narrow occlusal table in different regions of friction fit and passive fit implant abutment interface and figures 5 and 6 show graphs of von misses stress ( in mpa ) on narrow , ideal , and wider occlusal tables in vertical and oblique loading on implant , implant abutment interface , and bone in friction fit and passive fit connections . von misses stress ( in mpa ) on the implant abutment complex with prosthesis having narrow , ideal and wider occlusal tables ( a ) von misses stress on an implant with friction fit connection and passive fit connection during vertical loading with a load of 100 n in models with narrow occlusal table . ( b ) von misses stress on an implant with friction fit connection and passive fit connection during oblique loading with a load of 100 n in models with narrow occlusal table ( a ) von misses stress on an implant abutment interface with friction fit connection and passive fit connection during vertical loading with a load of 100 n in models with narrow occlusal table . ( b ) von misses stress on an implant abutment interface with friction fit connection and passive fit connection during oblique loading with a load of 100 n in models with narrow occlusal table ( a ) von misses stress on the bone with friction fit connection and passive fit connection during vertical loading with a load of 100 n in models with narrow occlusal table ( b ) von misses stress on the bone with friction fit connection and passive fit connection during oblique loading with a load of 100 n in models with narrow occlusal table graph showing von misses stress ( in mpa ) on narrow , ideal , and wider occlusal tables in vertical and oblique loading on implant and implant abutment interface in friction fit and passive fit connections graph showing von misses stress ( in mpa ) on narrow , ideal , and wider occlusal tables in vertical and oblique loading on bone in friction fit and passive fit connections from the values given in table 2 the following data and results have been obtained : the overall maximum von misses stress on the implant is more significant for friction fit than the passive fit implant abutment interface in both vertical and oblique loading for all the models testedat the implant abutment interface and at the neck of the implant , the von misses stress was higher for the friction fit . whereas on the outer surface of the abutment and on the internal surface of the fixture ; the passive fit shows lesser stress in both vertical and oblique loading for all the models testedthe overall maximum von misses stress on the bone is significantly less for friction fit than the passive fit in both vertical and oblique loading for all the modelsirrespective of the type of abutment connection used , the maximum von misses stress was seen in the region of the cortical or the marginal bone . it is showed that a significant reduction in von misses stress was observed at the boundary between cortical bone and cancellous bone in both loading conditionsthe narrow occlusal table , irrespective of their connection type has reduced the stress generated . this shows that the width of the occlusal table has got a significant influence on the stress generated on the implant , as well as on the bone . the overall maximum von misses stress on the implant is more significant for friction fit than the passive fit implant abutment interface in both vertical and oblique loading for all the models tested at the implant abutment interface and at the neck of the implant , the von misses stress was higher for the friction fit . whereas on the outer surface of the abutment and on the internal surface of the fixture ; the passive fit shows lesser stress in both vertical and oblique loading for all the models tested the overall maximum von misses stress on the bone is significantly less for friction fit than the passive fit in both vertical and oblique loading for all the models irrespective of the type of abutment connection used , the maximum von misses stress was seen in the region of the cortical or the marginal bone . it is showed that a significant reduction in von misses stress was observed at the boundary between cortical bone and cancellous bone in both loading conditions the narrow occlusal table , irrespective of their connection type has reduced the stress generated . this shows that the width of the occlusal table has got a significant influence on the stress generated on the implant , as well as on the bone . the aim of the study was to analyze the influence of two different types of implant abutment connection on the stress distribution pattern in the implant and the surrounding bone . the implant abutment interface that have been analyzed in the study represent two broad categories of implant abutment connection currently available in the market , the passive fit or the slip - fit represented by the nobel replace tri - lobe connection and the friction fit or active fit represented by the nobel active conical connection . six models were constructed in pro - engineer 05 of the two implant abutment connection for three different occlusal dimensions each . the implant and abutment complex was placed in cortical and cancellous bone modeled using a ct scan . this complex was subjected to a force of 100 n in the axial and oblique direction . the amount of stress and the pattern of stress generated were recorded on a color scale . the mean values of overall stress on the implant with friction fit connection were 88.725 for vertical load and 258.962 for oblique load whereas on passive fit were 49.805 and 136.249 , respectively , indicating that friction fit produced higher overall stress on the implant than the passive fit . the mean values of overall stress on the bone for vertical and oblique loading on friction fit connection were 15.907 and 57.236 whereas on a passive fit connection were 30.680 and 88.983 , respectively . this show that the stress generated by passive fit connection on bone is almost double the stress generated by the friction fit connection . the mean values of the overall stresses on the implant and the bone shows that the friction fit connection absorbs more stress and dissipates less stress to the surrounding bone . the larger contact area and deeper position inside the implant for friction fit connection allowed for better stability and broader stress distribution , as has been observed in several other studies . this frictional fit creates wedging effects to improve the implant abutment joint stability against the lateral force and helps to transfer the loading force along the conical surface to distribute the stress on the implant , ultimately reducing biological and biomechanical complications . the friction - locking mechanics and the solid design of the friction fit connections provided greater resistance to deformation and fracture under oblique compressive loading when compared to the passive fit connection . in passive fit connection , the cold welding does not occur when the abutments are tightened thus an inevitable gap between the implant and abutment may still exist . beyond these levels of micro - motion the mean values of stress on the crestal bone for vertical and oblique loading on friction fit connection were 15.391 and 25.449 whereas on a passive fit connection were 19.024 and 29.674 , respectively . the highest stress occurs in the implant 's most cervical region when an occlusal load is applied upon an implant , and the load is partially transferred to the bone . this explains why the cervical region of the implant is the site where the greatest micro - deformations occur independently of the type of bone and the design of the implant , the configuration of the prosthesis and the load . using fea , hansson showed that a conical implant abutment interface at the level of the bone crest decreases the peak bone - implant interfacial stress as compared with the flat top interface . for the friction fit implant abutment interface , this peak interfacial shear stress was located at some depth in the marginal bone . the mean values of stress on the apical area of bone for vertical and oblique loading on friction fit connection were 0.019 and 0.021 whereas on a passive fit connection were 0.0215 and 0.0278 , respectively . stresses induced by occlusal load are initially transferred from implant to the cortical bone , and a small amount of remaining stress spreads to cancellous bone . the greater the width of a transosteal structure , the lesser the magnitude of stress transmitted to the surrounding bone . from the results of the study , it is shown that the narrow occlusal table , irrespective of their connection type has reduced the stress generated . this shows that the width of the occlusal table has got a significant influence on the stress generated on the implant , as well as on the bone . typically , a 30% to 40% reduction in the occlusal table in a molar region has been suggested because any dimension larger than the implant diameter can cause cantilever effects and eventual bending moments in single - implant prostheses . a narrow occlusal table reduces the chance of offset loading and increases axial loading , which eventually can decrease the bending moment . as the wider occlusal table will increase stress on the abutment screws , the occlusal table should be reduced in width compared with natural teeth in nonesthetic regions of the mouth . within the limits of the present study , the following conclusions can be derived : the overall maximum von misses stress on the implant is significantly more for friction fit connection than the passive fit connection in both vertical and oblique loading in all the models ; whereas the overall maximum von misses stress on the bone is significantly less for friction fit connection than the passive fit connection in both vertical and oblique loading in all the models . the overall maximum von misses stress values on the implant and the bone show that the friction fit connection absorbs more stress and dissipates less stress to the surrounding bone . further studies on the permissible amount of micro - movement allowed in a passive fit implant abutment interface may need to be conductedirrespective of the type of abutment connection used the maximum von misses stress was seen in the region of the cortical or the crestal bone . it is shown that a significant reduction in von misses stress was observed at the boundaries between cortical bone and cancellous bone in both loading conditions because of relatively low elastic modulus of cancellous boneon comparing the stresses on narrow , ideal and wider occlusal tables for both frictions fit connection and passive fit connection , the results show that the narrow occlusal table has the least stress followed by ideal and the wider occlusal tables . this shows that the width of the occlusal table has got a significant influence on the stress generated on the implant , as well as on the bone . the overall maximum von misses stress on the implant is significantly more for friction fit connection than the passive fit connection in both vertical and oblique loading in all the models ; whereas the overall maximum von misses stress on the bone is significantly less for friction fit connection than the passive fit connection in both vertical and oblique loading in all the models . the overall maximum von misses stress values on the implant and the bone show that the friction fit connection absorbs more stress and dissipates less stress to the surrounding bone . further studies on the permissible amount of micro - movement allowed in a passive fit implant abutment interface may need to be conducted irrespective of the type of abutment connection used the maximum von misses stress was seen in the region of the cortical or the crestal bone . it is shown that a significant reduction in von misses stress was observed at the boundaries between cortical bone and cancellous bone in both loading conditions because of relatively low elastic modulus of cancellous bone on comparing the stresses on narrow , ideal and wider occlusal tables for both frictions fit connection and passive fit connection , the results show that the narrow occlusal table has the least stress followed by ideal and the wider occlusal tables . this shows that the width of the occlusal table has got a significant influence on the stress generated on the implant , as well as on the bone . the friction fit connection is superior to the passive fit connection , as the friction fit creates wedging effects to improve the implant abutment joint stability against the lateral force and helps to transfer the loading force along the conical surface to distribute the stress on the implant , ultimately reducing biological and biomechanical complications . a narrow occlusal table may increase axial loading and decrease nonaxial loading for the implants thereby reducing the stress on the implant , implant abutment interface , and bone . thus , it is recommended that the size of the occlusal table to be 30% to 40% smaller for molars .

dental implant - supported prostheses have become one of the significant treatment modalities for replacement of teeth , with reported success rates of over 98.2% . the success of dental implants is highly dependent upon the integration between the implant and the intraoral hard / soft tissues . the long term success or survival of dental implants is determined by the transmission of occlusal load and resultant stress distribution in the surrounding bone . load transfer at the bone - implant interface depends on : ( 1 ) the implant geometry and the design of implant abutment connection ; ( 2 ) the loading protocol and the type of occlusion ; ( 3 ) the number of implants and position ; ( 4 ) the quality and quantity of the surrounding bone . it has been demonstrated that vertical and transverse masticatory loads induce axial forces and bending moments that results in stress gradients in the implant , as well as in the bone . reported that stresses in the range 1.4 - 5.0 mpa may be required for healthy maintenance of bone , stresses outside this range have been reported to cause bone resorption . a persistent load increases the stress and may provoke micro - fractures and osteoclastic activity in the bone . there has been a continuous evolution of the implant abutment connection design with the intensions of reducing these stress concentrations . based on the various prosthetic and biological complications encountered in the clinical scenario and the results of various studies , the initial external hex design which had the interface above the implant and the osseous crest , has evolved into the internal hex with the implant abutment interface being placed more apically and away from the osseous crest . the development of these implant abutment interfaces have reduced the amount of stress that is transmitted to the implant or the surrounding bone and therefore considerably reducing the crestal bone loss , although not entirely eliminated indicating a multi - factorial cause . the structural complexity of implant abutment interface design due to the continuous improvement in the geometry has made it difficult to evaluate occlusal forces in the bone around the dental implant and the stresses within the implant . finite element analysis ( fea ) is a useful tool for the prediction of the effects of stress on the implant and its surrounding bone . the use of the finite element method to analyze stress concentrations was initially introduced into implant dentistry by weinstein et al . in fea , the mechanical performance of the implant abutment interface could be evaluated by von misses stresses . von misses stress criterion is important to interpret the stresses within the ductile material , such as the implant material ; as deformation occurs when the von misses stress value exceeds the yield strength . finite element studies comparing different connections and the effect of the change in occlusal table width dimensions on stress distribution pattern in and around the implant abutment interface are limited . in this study , an attempt is made to compare the stress distribution pattern of a passive fit and friction fit implant abutment interface in different areas of the implant and bone and the influence of occlusal table width on stress distribution . a mesh is needed in fea to divide the complex geometry into smaller elements in which the field variables can be interpolated with the use of shape functions . the process of creating the mesh , elements , their respective nodes and defining boundary conditions is referred to as the study models were constructed using reverse engineering technique in pro - engineer 05 through three - dimensional ( 3d ) optical scanning and point cloud data extraction . two cad models of implants were constructed with two different types of implant abutment connections currently available in the market , the passive fit or the slip - fit represented by the nobel replace tri - lobe connection ( nobel replace , tapered groovy , rp 4.3 mm 13 mm ) and the friction fit or active fit represented by the nobel active conical connection ( nobel active , internal rp 4.3 mm 13 mm ) along with their respective snappy abutments . the bone structure was modeled through a computed tomography ( ct ) scan that can provide results closer to a real scenario , because there is a difference in the behavior of stresses in work conducted with elliptical models , cobblestones , and ct scan data . the thickness of the cortical bone was kept 2 mm , and a uniform layer of cortical bone was modeled on the outer surface of the cancellous core . a bone block model was constructed based on a cross - sectional image of the human mandible in the premolar region , 25 mm high , 12 mm wide , and 10 mm thick consisting of a spongy center surrounded by a 2-mm cortical bone . three crowns with different occlusal table dimensions were constructed by changing the buccolingual dimensions and keeping mesiodistal and the cervicoocclusal length constant . the numbers of nodes in friction connection for ideal , narrow , and wider occlusal table are 80,786 , 82,047 , and 83,972 ; and the number elements are 57,082 , 57,737 , and 58,931 , respectively . the numbers of nodes in passive connection for ideal , narrow , and wider occlusal table are 76,330 , 77,469 , and 79,527 ; and the number elements are 53,569 , 54,137 , and 55,414 , respectively . ( b ) the vertical and oblique loading directions on a meshed model of a passive fit connection in this study , we assumed the implant , abutment , and screws were homogeneous , linear elastic , and isotropic mechanical properties . a distributed force of 100 n was applied onto the top surface of the crown vertically along the long axis and then obliquely at 45 to the longitudinal axis of the implant . two cad models of implants were constructed with two different types of implant abutment connections currently available in the market , the passive fit or the slip - fit represented by the nobel replace tri - lobe connection ( nobel replace , tapered groovy , rp 4.3 mm 13 mm ) and the friction fit or active fit represented by the nobel active conical connection ( nobel active , internal rp 4.3 mm 13 mm ) along with their respective snappy abutments . the bone structure was modeled through a computed tomography ( ct ) scan that can provide results closer to a real scenario , because there is a difference in the behavior of stresses in work conducted with elliptical models , cobblestones , and ct scan data . the thickness of the cortical bone was kept 2 mm , and a uniform layer of cortical bone was modeled on the outer surface of the cancellous core . a bone block model was constructed based on a cross - sectional image of the human mandible in the premolar region , 25 mm high , 12 mm wide , and 10 mm thick consisting of a spongy center surrounded by a 2-mm cortical bone . the numbers of nodes in friction connection for ideal , narrow , and wider occlusal table are 80,786 , 82,047 , and 83,972 ; and the number elements are 57,082 , 57,737 , and 58,931 , respectively . the numbers of nodes in passive connection for ideal , narrow , and wider occlusal table are 76,330 , 77,469 , and 79,527 ; and the number elements are 53,569 , 54,137 , and 55,414 , respectively . ( ( b ) the vertical and oblique loading directions on a meshed model of a passive fit connection in this study , we assumed the implant , abutment , and screws were homogeneous , linear elastic , and isotropic mechanical properties . a distributed force of 100 n was applied onto the top surface of the crown vertically along the long axis and then obliquely at 45 to the longitudinal axis of the implant . the data obtained from ansys calculation can be presented in a stress distribution map with a color scale , which makes it possible to compare directly the stress level in various component structures of all models . the amount of stress and the pattern of stress generated after applying a load of 100 n on each model in vertical and oblique direction were recorded on a color scale . the figures 24 shows the von misses stresses during vertical and oblique loading with 100 n on a narrow occlusal table in different regions of friction fit and passive fit implant abutment interface and figures 5 and 6 show graphs of von misses stress ( in mpa ) on narrow , ideal , and wider occlusal tables in vertical and oblique loading on implant , implant abutment interface , and bone in friction fit and passive fit connections . von misses stress ( in mpa ) on the implant abutment complex with prosthesis having narrow , ideal and wider occlusal tables ( a ) von misses stress on an implant with friction fit connection and passive fit connection during vertical loading with a load of 100 n in models with narrow occlusal table . ( b ) von misses stress on an implant with friction fit connection and passive fit connection during oblique loading with a load of 100 n in models with narrow occlusal table ( a ) von misses stress on an implant abutment interface with friction fit connection and passive fit connection during vertical loading with a load of 100 n in models with narrow occlusal table . ( b ) von misses stress on an implant abutment interface with friction fit connection and passive fit connection during oblique loading with a load of 100 n in models with narrow occlusal table ( a ) von misses stress on the bone with friction fit connection and passive fit connection during vertical loading with a load of 100 n in models with narrow occlusal table ( b ) von misses stress on the bone with friction fit connection and passive fit connection during oblique loading with a load of 100 n in models with narrow occlusal table graph showing von misses stress ( in mpa ) on narrow , ideal , and wider occlusal tables in vertical and oblique loading on implant and implant abutment interface in friction fit and passive fit connections graph showing von misses stress ( in mpa ) on narrow , ideal , and wider occlusal tables in vertical and oblique loading on bone in friction fit and passive fit connections from the values given in table 2 the following data and results have been obtained : the overall maximum von misses stress on the implant is more significant for friction fit than the passive fit implant abutment interface in both vertical and oblique loading for all the models testedat the implant abutment interface and at the neck of the implant , the von misses stress was higher for the friction fit . whereas on the outer surface of the abutment and on the internal surface of the fixture ; the passive fit shows lesser stress in both vertical and oblique loading for all the models testedthe overall maximum von misses stress on the bone is significantly less for friction fit than the passive fit in both vertical and oblique loading for all the modelsirrespective of the type of abutment connection used , the maximum von misses stress was seen in the region of the cortical or the marginal bone . it is showed that a significant reduction in von misses stress was observed at the boundary between cortical bone and cancellous bone in both loading conditionsthe narrow occlusal table , irrespective of their connection type has reduced the stress generated . this shows that the width of the occlusal table has got a significant influence on the stress generated on the implant , as well as on the bone . the overall maximum von misses stress on the implant is more significant for friction fit than the passive fit implant abutment interface in both vertical and oblique loading for all the models tested at the implant abutment interface and at the neck of the implant , the von misses stress was higher for the friction fit . whereas on the outer surface of the abutment and on the internal surface of the fixture ; the passive fit shows lesser stress in both vertical and oblique loading for all the models tested the overall maximum von misses stress on the bone is significantly less for friction fit than the passive fit in both vertical and oblique loading for all the models irrespective of the type of abutment connection used , the maximum von misses stress was seen in the region of the cortical or the marginal bone . it is showed that a significant reduction in von misses stress was observed at the boundary between cortical bone and cancellous bone in both loading conditions the narrow occlusal table , irrespective of their connection type has reduced the stress generated . this shows that the width of the occlusal table has got a significant influence on the stress generated on the implant , as well as on the bone . the aim of the study was to analyze the influence of two different types of implant abutment connection on the stress distribution pattern in the implant and the surrounding bone . the implant abutment interface that have been analyzed in the study represent two broad categories of implant abutment connection currently available in the market , the passive fit or the slip - fit represented by the nobel replace tri - lobe connection and the friction fit or active fit represented by the nobel active conical connection . the mean values of overall stress on the implant with friction fit connection were 88.725 for vertical load and 258.962 for oblique load whereas on passive fit were 49.805 and 136.249 , respectively , indicating that friction fit produced higher overall stress on the implant than the passive fit . the mean values of the overall stresses on the implant and the bone shows that the friction fit connection absorbs more stress and dissipates less stress to the surrounding bone . this frictional fit creates wedging effects to improve the implant abutment joint stability against the lateral force and helps to transfer the loading force along the conical surface to distribute the stress on the implant , ultimately reducing biological and biomechanical complications . the friction - locking mechanics and the solid design of the friction fit connections provided greater resistance to deformation and fracture under oblique compressive loading when compared to the passive fit connection . this can cause micro - motion at the interface during clinical loading , which in turn may contribute to stress on the screw and therefore loss of preload and loosening of abutment thereby leading to bacterial colonization of the micro gap . beyond these levels of micro - motion the mean values of stress on the crestal bone for vertical and oblique loading on friction fit connection were 15.391 and 25.449 whereas on a passive fit connection were 19.024 and 29.674 , respectively . this phenomenon is due to one of the principles of engineering , that is , when two materials are in contact with each other , and one of them is loaded , the stresses will be higher at the materials initial point of contact . this explains why the cervical region of the implant is the site where the greatest micro - deformations occur independently of the type of bone and the design of the implant , the configuration of the prosthesis and the load . using fea , hansson showed that a conical implant abutment interface at the level of the bone crest decreases the peak bone - implant interfacial stress as compared with the flat top interface . the mean values of stress on the apical area of bone for vertical and oblique loading on friction fit connection were 0.019 and 0.021 whereas on a passive fit connection were 0.0215 and 0.0278 , respectively . in this study , significantly larger stress values were seen in the neck area versus the apex area among all models in all conditions , which is consistent with the results of other studies . the cross - sectional shape of the natural tooth at the crest is biomechanically optimized to resist lateral loads , implants , however , are almost round in cross - section , which is less effective in resisting lateral bending loads thereby concentrating loads in the crestal region . the mean values of axial displacement of teeth in the socket are 25 - 100 m , whereas the range of motion of osseointegrated dental implants has been reported approximately 3 - 5 m . from the results of the study , it is shown that the narrow occlusal table , irrespective of their connection type has reduced the stress generated . this shows that the width of the occlusal table has got a significant influence on the stress generated on the implant , as well as on the bone . typically , a 30% to 40% reduction in the occlusal table in a molar region has been suggested because any dimension larger than the implant diameter can cause cantilever effects and eventual bending moments in single - implant prostheses . the proposed key factors to control bend overload in posterior restorations were reduced the inclination of cusps , centrally oriented contacts with a 11.5 mm flat area , a narrowed occlusal table , and elimination of cantilevers . as the wider occlusal table will increase stress on the abutment screws , the occlusal table should be reduced in width compared with natural teeth in nonesthetic regions of the mouth . analysis of finite elements was shown to be a versatile and promising methodology for analyzing stress concentrations in implant dentistry , but it is worth emphasizing that the fea is an approximate virtual simulation of clinical situations , presenting certain limitations . within the limits of the present study , the following conclusions can be derived : the overall maximum von misses stress on the implant is significantly more for friction fit connection than the passive fit connection in both vertical and oblique loading in all the models ; whereas the overall maximum von misses stress on the bone is significantly less for friction fit connection than the passive fit connection in both vertical and oblique loading in all the models . the overall maximum von misses stress values on the implant and the bone show that the friction fit connection absorbs more stress and dissipates less stress to the surrounding bone . further studies on the permissible amount of micro - movement allowed in a passive fit implant abutment interface may need to be conductedirrespective of the type of abutment connection used the maximum von misses stress was seen in the region of the cortical or the crestal bone . it is shown that a significant reduction in von misses stress was observed at the boundaries between cortical bone and cancellous bone in both loading conditions because of relatively low elastic modulus of cancellous boneon comparing the stresses on narrow , ideal and wider occlusal tables for both frictions fit connection and passive fit connection , the results show that the narrow occlusal table has the least stress followed by ideal and the wider occlusal tables . this shows that the width of the occlusal table has got a significant influence on the stress generated on the implant , as well as on the bone . the overall maximum von misses stress on the implant is significantly more for friction fit connection than the passive fit connection in both vertical and oblique loading in all the models ; whereas the overall maximum von misses stress on the bone is significantly less for friction fit connection than the passive fit connection in both vertical and oblique loading in all the models . further studies on the permissible amount of micro - movement allowed in a passive fit implant abutment interface may need to be conducted irrespective of the type of abutment connection used the maximum von misses stress was seen in the region of the cortical or the crestal bone . it is shown that a significant reduction in von misses stress was observed at the boundaries between cortical bone and cancellous bone in both loading conditions because of relatively low elastic modulus of cancellous bone on comparing the stresses on narrow , ideal and wider occlusal tables for both frictions fit connection and passive fit connection , the results show that the narrow occlusal table has the least stress followed by ideal and the wider occlusal tables . this shows that the width of the occlusal table has got a significant influence on the stress generated on the implant , as well as on the bone . the friction fit connection is superior to the passive fit connection , as the friction fit creates wedging effects to improve the implant abutment joint stability against the lateral force and helps to transfer the loading force along the conical surface to distribute the stress on the implant , ultimately reducing biological and biomechanical complications .

mimicry of interfacial protein segments has led to new classes of rationally designed inhibitors of protein protein interactions ( ppis ) . the identification and analysis of protein complexes mediated by protein secondary structures provide a platform for these explorations . we have recently examined the full set of protein complexes in the protein data bank mediated by -helices and -strands . our work , along with efforts by kritzer et al . to define loop motifs at protein interfaces , aims both to describe the interactions present in the protein data bank and to prescribe effective starting points for the design of ppi inhibitors . individual secondary structures are critical elements of protein interfaces ; however , many ppis feature more complex modes of binding , suggesting a potential role for synthetic tertiary structure mimetics or miniproteins as attractive candidates for the design of new classes of ppi inhibitors . miniproteins consisting of helical bundles , -sheet barrels , and loops , along with synthetic antibodies , are now routinely used to enrich ligands for protein targets , especially for extracellular receptors . in an effort to expand our atomic analysis of protein structural data beyond interactions that can be mediated by a single secondary structure element alone , we chose to begin our survey of protein tertiary interactions by focusing on helix dimers because the dimer is the simplest all - helical tertiary structure stoichiometry . coiled coils and helical bundles are well understood and have been extensively studied in diverse biochemical and biophysical contexts . dimeric coiled coils or similarly structured motifs such as bundles play essential roles in mediating biological processes , iconically driving the multimerization and stabilization of proteins involved in transcription factor complexes and vesicular trafficking , among other critical functions . several computational approaches have been implemented to predict coiled coil - mediated interactions by their pairwise and multimeric residue correlations . seminal studies have produced a comprehensive dataset of the coiled coil interactome . however , computational and experimental methods for the analysis of coiled coils described thus far are largely devoted to characterization of forces that lead to coiled coil formation . to complement these studies , we sought to analyze interactions of helical dimers with globular proteins as a step toward the rational design of coiled coil mimetics as ppi inhibitors . though canonical coiled coils possess supercoiling and particular packing properties , we did not impose these requirements , stipulating only that the helices be proximal and well - oriented . since our motivation for developing this dataset is to identify interactions that may not be inhibited by secondary structure mimics , we also required that critical binding residues be located on both helices . these criteria retain structures of high structural similarity to a coiled coil but eliminate canonical all - alpha tertiary structure motifs like the helix - loop - helix and helix - turn - helix dna binding domains , whose interhelical angles are far from parallel or antiparallel . examination of the helix dimer dataset suggests that coiled coil interfaces can be divided into three broad categories ( figure 1 ) according to their interaction stoichiometry . case 1 features a helical dimer from one protein interacting with a single partner protein . in case 2 , a helical dimer from one protein interacts with two different protein partners . in case 3 , a single helical dimer motif forms at the interface between partner proteins . we anticipate that helical dimers in case 3 would favor different interacting residues from examples in cases 1 and 2 , because in case 3 high - affinity interactions must form between two individual helices rather than a helix dimer and a globular protein . this taxonomy reflects the different properties demanded of potential designed inhibitors : case 1 features interactions on predominantly one dimer face ; case 2 generally interacts with two faces ; case 3 dimer interfaces may be interrupted by a single helix . models depicting three families of coiled coil - like structures at protein protein interfaces . ( a ) in the first family ( case 1 ) , a coiled coil entirely from chain a forms an interaction with protein b. ( b ) in the second family ( case 2 ) , a coiled coil , which may come from one or two proteins , interacts with two different proteins partners . ( c ) in the third family ( case 3 ) , a coiled coil forms across a protein interface . schematic for identification of protein interfaces in the protein data bank ( pdb ) where a helix dimer contributes significantly to complex formation . interfacial helices from the previously described hippdb dataset were culled to produce a set of structures for detailed analysis via stringent distance , orientation , and energetic criteria . on the basis of our evaluations , we classified the interactions among three classes ( cases 13 ) of helical tertiary structure - mediated ppis . the energetic contribution of each interface helix dimer and individual residues was approximated using rosetta . we examined the biophysical properties of each class in relation to each other , to typical interface helices in general , and to canonical coiled coils . coiled coils are defined as two or more -helices that wind around each other to form supercoils . classical coiled coils are characterized by a heptad repeat , ( abcdefg)n , where buried a and d positions form the interface between partner helices . we sought to identify all helix dimers that are in contact with a globular protein irrespective of whether such helices would meet the strict definition . this study has revealed the existence of a set of biologically relevant complexes as potential targets for inhibitor design . we also analyzed the biophysical properties of dimer interfaces , such as the composition of hot spot residues and the degree to which helical dimers differ from coiled coils and protein helices in general . this analysis shows that hot spot residues are concentrated over compact areas , potentially allowing the mimicry of these dimers by small or medium - sized molecules . hippdb was developed to comprise all interface helices of four or more residues that contain two or more hot spot residues , where hot spot residues are defined via computational alanine scanning , performed with rosetta , as those that result in a loss of binding energy ( g ) of at least 1.0 rosetta energy unit ( reu , which scales approximately as 1 kcal / mol ) . in this work , we expanded the hippdb dataset to include over 37 000 high - affinity interfaces and imposed stringent geometric and energetic criteria to obtain a set of over 1000 high - affinity helical dimers . we made modifications to our prior methodology to tailor it to the coiled coil motif ( figure 2 ) . in hippdb , we were interested in identifying minimal inhibitory motifs to aid the design of synthetic inhibitors . this goal requires identification of helical segments of a protein present at an interface without the sequences not in contact . in the context of helical dimers , the challenge is to capture the defining geometric parameters as accurately as possible so any energetically irrelevant residues far from the interface can be discarded separately . we altered our analysis such that any continuous stretch of helical residues counts as a helical element as long as part of the helix is present at the interface . this modified method identifies as a single element of interest helix dimers in which one helix makes contact with a partner protein at multiple separate points along its length . we imposed both geometric and energetic criteria to obtain a dataset of interfaces where coiled coil - like structures play an important role . we stipulated that each helix must contribute at least 6.0 rosetta energy units ( reu ) of g in its interaction with its partner , that the angle between their helical axes must be within 30 of parallel or antiparallel , and that the two helices are within 17 of each other . these conditions ensured coiled coil - like geometry and a substantial energetic contribution , equivalent to at least three strong hot spot residues , from each helix . the 6 reu threshold ensures that the selected coiled coil interfaces contain a sufficient number of hot spot residues to merit their mimicry by a potential synthetic inhibitor . lower energetic thresholds yield additional compelling complexes , but far too many to investigate individually . we also imposed conditions on the percentage of the interaction s overall g contributed by each helix . when both helices come from the same protein chain , we required each helix to contribute at least 20% of the chain s g . this requirement excluded any helical dimers that did not make a substantial total contribution to the binding interface , and ensured that the complexes in the database will truly require a dimer and are not amenable to disruption by mimic of a single helix . finally , we aimed to ensure that the dataset was not dominated by pairs of chains from multimeric helix bundles , as our interest lies in complexes where at least one partner is a globular protein . we observed that in structures of conventional helix bundles , almost every residue of each chain is present at the protein , we required that at least one partner in every complex must have at minimum 30% of its residues distant from the interface . dimers may be queried using pdb identification , total g and sasa , interhelical distances , and angles ( table 1 ) . . each field may be searched and sorted , and data may be downloaded in csv , pdf , or xls format . complexes of weaker affinity ( g cutoff of 4 reu ) are included for comparison in the website dataset . a cursory analysis would suggest that the hot spot residues may be distributed evenly over many heptads , requiring the design of large molecules or biologics for inhibition , as has been true for the design of coiled coil assembly inhibitors . we were surprised to find that a plurality of complexes in dippdb possessed hot spot residues over a relatively small region of the interface ( figure 3 ) . the average length of case 1 , 2 , and 3 helical dimers is 19 , 27 , and 22 residues , respectively , while their average hot spot spans are 13 , 17 , and 16.5 residues . the critical residues are limited to a single heptad in 15% of case 1 dimers and to two heptads in two - thirds of examples . the trend of compact hot segments in helical dimers is observed in each case with two - thirds of critical contact residues averaging fewer than three heptads . dimers that span three heptads or less ( roughly 30% of the dataset ) average a hot spot per 4.7 residues . this signature is strongly suggestive of an interface amenable to inhibition by designed peptidomimetics . however , as peptidic coiled coils of these length scales are not generally stable , we have undertaken an experimental effort to develop cross - linked helix dimers ( chds ) as minimal inhibitors of coiled coils and other helical ppis . histogram of the distance between the first and final hot spot residue in case 1 ( black ) , case 2 ( light gray ) , and case 3 ( dark gray ) helical dimers . further examination of dippdb interfaces suggests that helical dimers at protein protein interfaces have comparable amino acid composition to other high - affinity interface helices but significantly differ from canonical coiled coils . in general , classical coiled coil motifs possess a distribution of amino acids similar to the -helix but with considerable additional bias toward aliphatic residues , owing to their obligate interior packing interactions . we wished to understand whether helical dimers more closely reflected the amino acid distribution on high - affinity helices or classical coiled coils . in examining the distribution of amino acids in these structures , we found it largely consistent with the distribution on helices in general ( supporting information , figure s1 ) . though there is considerable selective pressure for coiled coil motifs to possess high proportions of aliphatic amino acids , especially leucine and isoleucine , for optimal knobs - into - holes packing arrangements , these residue types are not overwhelmingly enriched in the -helices that are part of dippdb . we also examined the distribution of hot spot residues on each family of helix dimers . a summary of these data is shown in figure 4 , and details for all three cases by residue types are included in the supporting information , figure s2 . aliphatic and charged residues are moderately enriched as hot spot residues , though generally consistent with the helical baseline . in contrast , polar residues were greatly depleted , and more so than the general helical case ; conversely , aromatic residues were more enriched in helical dimers . frequency of hot spot residues , normalized to natural abundance , in helix dimers and interfacial single helices , respectively . the plot shows distribution of aliphatic residues ( leu , ile , val ) , polar residues ( gln , asn , ser ) , aromatic residues ( phe , trp , tyr ) , and charged residues ( arg , asp , glu , lys ) in the three contexts . separate from our analysis of amino acid composition in general , we examined the inter - helix contacts made by case 1/2 and case 3 helical dimers and conventional coiled coils ( figure 5a ) . helix dimers feature a larger proportion of interstrand contacts between polar residues than typical coiled coils . in the classical coiled coil motif , core polar mutations may be tolerated via changes in stoichiometry , local distortions in geometry , or an increased inner void volume . the presence of polar contacts at the interior of helical dimers emphasizes the importance of their specific interactions . the leucine zipper coiled coil ( figure 5b ) contains four paired aliphatic knob - into - hole packing interactions , which dominate the interaction . in the particular case 1 dimer example depicted in figure 5c , aliphatic packing interactions are limited and energetically insignificant ; one helix only possesses one aliphatic residue ( a threonine ) facing its partner over five entire turns ; in contrast , the nonpolar residues from the other helix pack into non - canonical holes . the case 3 dimer example ( figure 5d ) possesses energetically important nonpolar residues , but they are not organized into the classic interior groove of a coiled coil , and the phenylalanines are more than twice as energetically important as the leucines by g . visual inspection of these examples and others inspired us to quantify the degree to which the helical dimer forms non - canonical packing interactions . ( a ) amino acid composition of inter - helical contact residues in helix dimers as opposed to true coiled coils . ( b ) the packing of a classic leucine zipper , gcn4 , features an aliphatic a / d groove with each residue packing into a complementary hole . ( c ) this case 1 helical dimer from 1,2-hydroquinol dehydrogenase homodimer illustrates highly non - canonical packing interactions . ( d ) the orphan nuclear receptor nur77 contains an aliphatic core but lacks any heptad repeat structure and knob / hole packing orientation . we found the examples of non - canonical inner grooves compelling and performed a more comprehensive analysis to determine how non - canonical these motifs are as compared to the classical coiled coil . we employed woolfson s socket analysis to explore the dataset ( supporting information , figure s4 ) . the socket algorithm identifies knobs - into - holes packing of coiled coils to distinguish them from helix dimers . of the 262 case 1 dimers , only 24 dimers were identified as being coiled coils by socket ( 9.2% ) , of which 22 were antiparallel and 2 were parallel . an additional 17 dimers contained only one complementary knob - into - hole packing interaction . might have four such interactions per heptad ( two per partner ) . moreover , more than half of those 24 coiled coils identified were fewer than three heptads in length . this analysis implies that while helical dimers may occasionally exhibit packing characteristic of coiled coil motifs , they are too short to be stable on their own . 27/261 case 2 dimers contained significant coiled coil structure ( 10.3% ) and 21 contained one complementary interaction . of case 3 dimers , 133/919 were identified as coiled coils by socket ( 14.4% ) and an additional 50 contained a single knob - in - hole interaction . to follow up on the disparities in inner groove composition demonstrated in figure 5 , we specifically studied the frequency of hydrophilic inner grooves . we found hydrophilic inner groove residues quite common : each case averaged at least three , and 40% of complexes overall had at least four such residues . we profile two complexes whose interfacial g results almost exclusively ( > 90% ) from hydrophilic contacts in the supporting information , figure s5 . such features are uncommon in canonical coiled coils . polar residues may be found at the interior of inside - out coiled coil motifs found in membrane proteins , though there is debate regarding the extent of polarity on the inside . because the vast majority of the proteins in dippdb are cytosolic , including these unusual examples , we hypothesized that a similar environment may make this inside - out geometry possible : the helical dimer is surrounded by hydrophobic residues presented by the protein in which it is found and by its binding partner , serving an analogous role to membrane lipids . the networks of buried hydrogen bonds that may form at the interior of a coiled coil are well studied , although the partially polar interiors observed here present an extreme case . we obtained the set of hydrophilic contact residues in case 1 helical dimers . a total of 79.4% of hydrophilic contact residues were flanked by at least one nonpolar residue . buried polar residues averaged under 30% relative sasa ( supporting information , figure s6 ) . this degree of burial is highly destabilizing if hydrogen bonds are left buried but unsatisfied as a result , but it concomitantly increases the value of satisfied hydrogen bonds , contributing to the strength of these interfaces . overall , though it is possible to find recognizably canonical packing at the interior of helical dimers , the majority do not succumb to the same generalizations as the coiled coil motif . as discussed above , the knobs - into - holes packing of coiled coils distinguishes them from helical dimers . because the two helices of a case 3 helical dimers come from different chains , we were able to compare knob hole packing interactions to the g of the knob residue by identifying the nearest three - residue hole on the partner helix ( i , i+1 , i+4 or i , i+3 , i+4 ) to each knob . we restricted this analysis to the inner groove of each helix and furthermore recomputed the knob g values on complexes only including the dimer helices so as to omit any interactions with other components of the protein . though the packing is not conventional enough to identify the dimers as coiled coils by socket , we anticipated that we would still be able to identify some trends . in contrast to the classical knobs - into - holes aliphatic model , residues of all types could form inner - groove contacts of considerable g . instead , we found the key feature , tightly correlated to g , was that knob residues made contact with chemically complementary holes . although aliphatic contact residues of low to moderate g do frequently pack into polar holes and vice versa , every aliphatic and aromatic residue type had higher average g when packing in a mostly aliphatic or aromatic hole than in a mostly polar one ( supporting information , figure s7 ) . in the creation of this dataset , we made no stipulation about the relative position of these helix bundles in the protein(s ) in question . we conjectured that helix - turn - helix motifs , or helical hairpins , which are common sources of ideal antiparallel coiled coils , might be particularly prominent interface elements . of the 262 case 1 helical dimers , 81 are separated by two to eight nonhelical residues . this substantial proportion of helix - turn - helix motifs is encouraging , as it suggests that the tertiary structure present at the interface is largely governed by local forces that may be mimicked by a designed inhibitor . overall , there are 133 parallel and 129 antiparallel dimers ; thus , development of scaffolds appropriate for both motifs is critical . in contrast , of the 115 case 2 helical dimers in which both dimer helices come from the same chain , only 25 exhibit helix - turn - helix motifs . the possible interplay between interface tertiary structure geometry and the stoichiometry of formed complexes merits further study , as it suggests a difference in folding cooperativity . even though the n and c termini of parallel dimers on the same chain are distant , the majority of parallel dimers are connected by a small number of motifs . of the 133 parallel case 1 dimers , 55 are connected by a two to four residue loop , a strand whose length varies with that of the dimer , a single residue turn , a short 310 or -helix , followed by another short loop ( figure 7 ) . the second most prevalent motif includes 16 examples of a strand bracketed by two 45-residue loops . the two helices are linked by a loop - helix - loop in only four instances . five possess the loop - strand - turn - helix - loop linker , five are connected by a loop - helix - loop , eight have a loop - strand - loop , but 11 contain a loop - helix - loop - helix - loop - helix - loop . ( specific pdbs for each motif are listed in the supporting information . ) to our knowledge , this is the first effort to establish common protein folds connecting helices of defined orientation outside of canonical coiled coils . redesigned and optimized derivatives of these motifs particularly the loop - strand - turn - helix - loop motif , as it is by far the most common and the most structurally interesting may serve as miniprotein scaffolds . common antiparallel ( a ) and parallel ( b ) helical dimer motifs feature linkers of a simple turn , a loop - strand - helix - loop , a loop - strand - loop , and a loop - helix - loop . given the importance of salt bridge interactions holding coiled coils together , we investigated the distribution of charge across helix monomers in each case . in case 1 , the average helix dimer has helices with net charges of 1.19 and 1.05 , and 61% are net neutral or positive in total . in contrast , though case 2 features dimers with net charges per helix of 0.89 and 0.33 , and thus a higher average charge , 66% are net neutral or positive . case 3 dimers are 70% net neutral or positive and have net charges for each helix of 0.88 and 0.08 . these percentages suggest that the surface bound by such helical dimers may also be frequently negative or neutral . we calculated the surface charge on proteins bound by case 1 dimers to be 70% net neutral or negative , with an average net charge of 0.75 . case 2 dimers bind surfaces that are 80% net neutral or negative with an average net charge of 0.68 . case 3 dimers bind surfaces that are 68% neutral or negative with an average charge of 0.61 . the net positive charge in dimers is consistent with the higher number of positively charged protein helices in general . ( a ) interfaces mediated by case 1 and case 2 helical dimers possess diverse functions and are dominated by enzyme complexes . ( b ) t. brucei s udp - galactose 4-epimerase features a mutation in the active site relative to the human enzyme , potentially permitting specific targeting . galactose metabolism is essential to the parasite s ability to cause african sleeping sickness , a neglected tropical disease . the enzyme is active in dimeric and tetrameric forms ; the target monomer is shown as gray surface . ( c ) pcsk9 propeptide ( surface ) binds with high affinity to pcsk9 ( cartoon ) and inhibits its proteolytic activity . this enzyme is linked to atherosclerosis and cardiovascular disease . in both figures , the nearby active site from the dimer chain the interfaces collated in dippdb have the traits of prime targets for drug design . we categorized the functions of ppis as defined in the pdb ( figure 8a ) and observed that they are implicated in biological processes from enzymatic function to transcription to the immune response . we identified a total of 523 interactions ( cases 1 and 2 ) that would require a helix dimer mimetic or miniprotein for inhibition . case 3 dimers , in contrast , feature a pair of single helices interacting with each other across an interface ; thus , mimics of a single helix can disrupt these interactions . intracellular ppis dominate the dataset ; thus , inhibition of these complexes will require development of synthetic analogues that can permeate the cell membrane . some of these newly identified targets will potentially aid efforts in drug discovery . in particular , it is interesting to note that the largest category , various enzymes , accounts for 63% of dipp interactions . this category contains many hydrolases , oxidoreductases , and transferases , among other enzymes . although enzyme function has typically been controlled using substrate or transition state analogues , helix dimers offer a potentially attractive alternative scaffold . figure 8b , c highlights two examples of interactions involving udp - galactose 4-epimerase and pcsk9 where helical dimers at dimeric interfaces in enzyme biological assemblies of considerable medical relevance bind near enzyme active sites . several targets in dippdb are critically relevant to cancer phenotypes . in particular , we explored classic cases leading to hallmarks of cancer and discovered a set of possible targets mediated by helical dimers . for example , three complexes mhf histone tetramer / fancm helicase ( pdb code 4e45 ) , mre11 nuclease / rad50 abc atpase ( pdb code 3qf7 ) , and the n- and c - terminal domains of the mms21 subunit of smc5 ( pdb code 3htk)have a role in dna repair . the interaction between epo and its receptor ( pdb code 1eer ) is implicated in the hypoxic response ; the complex between murine ifnar1 and interferon - beta ( pdb code 3wcy ) is implicated in the regulation of apoptosis , and the inhibition of the catalytic subunit p110 by the sh2 domain of the regulatory subunit p85 of phosphoinositide 3-kinase ( pdb code 2y3a ) is implicated in angiogenesis and invasive cell growth . in each case , mimicry of both helices is predicted to be essential to optimize binding affinity . beyond these targets , helical dimer interfaces include bacterial transcription and metabolism , quorum sensing , cell signaling , and more . a list of targets implicated in transcription is included in the supporting information , table s1 . we examined the 354 pdb structures that contain at least one case 1 or case 2 helical dimer and explored the gene ontology ( go ) terms annotating each complex . for example , two structures ( pdb codes 2p5 t and 1gvn ) were toxins annotated with nucleic acid binding transcription factor activity , and seven are implicated in organismal development ; two are involved in immune responses . we anticipate that these and more targets will become tractable for modulation by designed tertiary structure mimetics ; in figure 9 , we depict several interactions of particular pharmacological interest whose tertiary structure binding sites have not been drugged . these complexes , or the pathways they modulate , are known to be of therapeutic interest . we illustrate them here to highlight the importance of helix dimer domains in coaxing the formation of these complexes and the potential of dimer mimics as inhibitors . ( a ) lsd1-mediated nucleosome demethylation requires complexation with corest ; existing inhibitors of lsd1 bind its substrate pocket , and predicted binding sites have thus far omitted the helical dimer interface . ( b ) sam68 rna - binding protein is implicated in pro - oncogenic activity and modulates alternative splicing of cd44 and bcl - xl . ( c ) the kaposi s sarcoma protein vflip forms an a2b2 heterotetramer with nf - kappa - b essential modulator ( nemo ) coiled coil ; thus far , inhibition of this complex s function has only been achieved through geldanamycin inhibition of hsp90 . the aim of any systematic study of protein structures is both to uncover general principles governing protein geometry and to develop new insight into how to practically modulate protein function . topologically defined segments often mediate protein protein interactions , and mimicry of these regions has emerged as a successful strategy for inhibitor design . several examples of ppi inhibitors derived from mimicry of interfacial -helical and -strand domains have been described . emerging examples of tertiary mimetics as ppi inhibitors illustrate the broad potential of moving beyond secondary structure mimicry . we were motivated to analyze protein complexes featuring helical dimers to create a list of potential targets where mimics of a single helix may not be sufficient . modification of the protocol used to develop a database of interface helices ( hippdb ) provided a set of protein protein interactions where the critical binding residues reside on two helices oriented in parallel or antiparallel configurations . the dataset includes some helical dimers that would be classified as true coiled coils because of their heptad repeats and supercoiling as well as helical dimers that find other means of making contacts with each other . some helical dimers violate the typical expectations for coiled coil interiors and are held together largely by salt bridges and hydrogen bonds , while others violate expectations for typical protein interfaces , which contain mostly large , aliphatic hot spot residues . the length of dimers in contact with the protein partners spans one to three heptads , suggesting that medium - sized molecules or miniproteins will be able to disrupt these complexes . the online database provides a list of all entries in the dataset along with their pdb identifiers and the energetic contributions of the hot spot residues .

the modulation of protein protein interactions ( ppis ) by means of creating or stabilizing secondary structure conformations is a rapidly growing area of research . recent success in the inhibition of difficult ppis by secondary structure mimetics also points to potential limitations , because often , specific cases require tertiary structure mimetics . to streamline protein structure - based inhibitor design , we have previously described the examination of protein complexes in the protein data bank where -helices or -strands form critical contacts . here , we examined coiled coils and helix bundles that mediate complex formation to create a platform for the discovery of potential tertiary structure mimetics . though there has been extensive analysis of coiled coil motifs , the interactions between pre - formed coiled coils and globular proteins have not been systematically analyzed . this article identifies critical features of these helical interfaces with respect to coiled coil and other helical ppis . we expect the analysis to prove useful for the rational design of modulators of this fundamental class of protein assemblies .

Introduction Results and Discussion Conclusion

mimicry of interfacial protein segments has led to new classes of rationally designed inhibitors of protein protein interactions ( ppis ) . the identification and analysis of protein complexes mediated by protein secondary structures provide a platform for these explorations . we have recently examined the full set of protein complexes in the protein data bank mediated by -helices and -strands . to define loop motifs at protein interfaces , aims both to describe the interactions present in the protein data bank and to prescribe effective starting points for the design of ppi inhibitors . individual secondary structures are critical elements of protein interfaces ; however , many ppis feature more complex modes of binding , suggesting a potential role for synthetic tertiary structure mimetics or miniproteins as attractive candidates for the design of new classes of ppi inhibitors . in an effort to expand our atomic analysis of protein structural data beyond interactions that can be mediated by a single secondary structure element alone , we chose to begin our survey of protein tertiary interactions by focusing on helix dimers because the dimer is the simplest all - helical tertiary structure stoichiometry . coiled coils and helical bundles are well understood and have been extensively studied in diverse biochemical and biophysical contexts . dimeric coiled coils or similarly structured motifs such as bundles play essential roles in mediating biological processes , iconically driving the multimerization and stabilization of proteins involved in transcription factor complexes and vesicular trafficking , among other critical functions . however , computational and experimental methods for the analysis of coiled coils described thus far are largely devoted to characterization of forces that lead to coiled coil formation . to complement these studies , we sought to analyze interactions of helical dimers with globular proteins as a step toward the rational design of coiled coil mimetics as ppi inhibitors . though canonical coiled coils possess supercoiling and particular packing properties , we did not impose these requirements , stipulating only that the helices be proximal and well - oriented . since our motivation for developing this dataset is to identify interactions that may not be inhibited by secondary structure mimics , we also required that critical binding residues be located on both helices . these criteria retain structures of high structural similarity to a coiled coil but eliminate canonical all - alpha tertiary structure motifs like the helix - loop - helix and helix - turn - helix dna binding domains , whose interhelical angles are far from parallel or antiparallel . examination of the helix dimer dataset suggests that coiled coil interfaces can be divided into three broad categories ( figure 1 ) according to their interaction stoichiometry . we anticipate that helical dimers in case 3 would favor different interacting residues from examples in cases 1 and 2 , because in case 3 high - affinity interactions must form between two individual helices rather than a helix dimer and a globular protein . this taxonomy reflects the different properties demanded of potential designed inhibitors : case 1 features interactions on predominantly one dimer face ; case 2 generally interacts with two faces ; case 3 dimer interfaces may be interrupted by a single helix . models depicting three families of coiled coil - like structures at protein protein interfaces . ( a ) in the first family ( case 1 ) , a coiled coil entirely from chain a forms an interaction with protein b. ( b ) in the second family ( case 2 ) , a coiled coil , which may come from one or two proteins , interacts with two different proteins partners . ( c ) in the third family ( case 3 ) , a coiled coil forms across a protein interface . schematic for identification of protein interfaces in the protein data bank ( pdb ) where a helix dimer contributes significantly to complex formation . interfacial helices from the previously described hippdb dataset were culled to produce a set of structures for detailed analysis via stringent distance , orientation , and energetic criteria . on the basis of our evaluations , we classified the interactions among three classes ( cases 13 ) of helical tertiary structure - mediated ppis . we examined the biophysical properties of each class in relation to each other , to typical interface helices in general , and to canonical coiled coils . coiled coils are defined as two or more -helices that wind around each other to form supercoils . classical coiled coils are characterized by a heptad repeat , ( abcdefg)n , where buried a and d positions form the interface between partner helices . we also analyzed the biophysical properties of dimer interfaces , such as the composition of hot spot residues and the degree to which helical dimers differ from coiled coils and protein helices in general . in this work , we expanded the hippdb dataset to include over 37 000 high - affinity interfaces and imposed stringent geometric and energetic criteria to obtain a set of over 1000 high - affinity helical dimers . we made modifications to our prior methodology to tailor it to the coiled coil motif ( figure 2 ) . in hippdb , we were interested in identifying minimal inhibitory motifs to aid the design of synthetic inhibitors . in the context of helical dimers , the challenge is to capture the defining geometric parameters as accurately as possible so any energetically irrelevant residues far from the interface can be discarded separately . we imposed both geometric and energetic criteria to obtain a dataset of interfaces where coiled coil - like structures play an important role . the 6 reu threshold ensures that the selected coiled coil interfaces contain a sufficient number of hot spot residues to merit their mimicry by a potential synthetic inhibitor . this requirement excluded any helical dimers that did not make a substantial total contribution to the binding interface , and ensured that the complexes in the database will truly require a dimer and are not amenable to disruption by mimic of a single helix . finally , we aimed to ensure that the dataset was not dominated by pairs of chains from multimeric helix bundles , as our interest lies in complexes where at least one partner is a globular protein . we observed that in structures of conventional helix bundles , almost every residue of each chain is present at the protein , we required that at least one partner in every complex must have at minimum 30% of its residues distant from the interface . complexes of weaker affinity ( g cutoff of 4 reu ) are included for comparison in the website dataset . a cursory analysis would suggest that the hot spot residues may be distributed evenly over many heptads , requiring the design of large molecules or biologics for inhibition , as has been true for the design of coiled coil assembly inhibitors . we were surprised to find that a plurality of complexes in dippdb possessed hot spot residues over a relatively small region of the interface ( figure 3 ) . however , as peptidic coiled coils of these length scales are not generally stable , we have undertaken an experimental effort to develop cross - linked helix dimers ( chds ) as minimal inhibitors of coiled coils and other helical ppis . further examination of dippdb interfaces suggests that helical dimers at protein protein interfaces have comparable amino acid composition to other high - affinity interface helices but significantly differ from canonical coiled coils . in general , classical coiled coil motifs possess a distribution of amino acids similar to the -helix but with considerable additional bias toward aliphatic residues , owing to their obligate interior packing interactions . we wished to understand whether helical dimers more closely reflected the amino acid distribution on high - affinity helices or classical coiled coils . though there is considerable selective pressure for coiled coil motifs to possess high proportions of aliphatic amino acids , especially leucine and isoleucine , for optimal knobs - into - holes packing arrangements , these residue types are not overwhelmingly enriched in the -helices that are part of dippdb . a summary of these data is shown in figure 4 , and details for all three cases by residue types are included in the supporting information , figure s2 . the plot shows distribution of aliphatic residues ( leu , ile , val ) , polar residues ( gln , asn , ser ) , aromatic residues ( phe , trp , tyr ) , and charged residues ( arg , asp , glu , lys ) in the three contexts . separate from our analysis of amino acid composition in general , we examined the inter - helix contacts made by case 1/2 and case 3 helical dimers and conventional coiled coils ( figure 5a ) . helix dimers feature a larger proportion of interstrand contacts between polar residues than typical coiled coils . in the classical coiled coil motif , core polar mutations may be tolerated via changes in stoichiometry , local distortions in geometry , or an increased inner void volume . in the particular case 1 dimer example depicted in figure 5c , aliphatic packing interactions are limited and energetically insignificant ; one helix only possesses one aliphatic residue ( a threonine ) facing its partner over five entire turns ; in contrast , the nonpolar residues from the other helix pack into non - canonical holes . ( a ) amino acid composition of inter - helical contact residues in helix dimers as opposed to true coiled coils . we found the examples of non - canonical inner grooves compelling and performed a more comprehensive analysis to determine how non - canonical these motifs are as compared to the classical coiled coil . we employed woolfson s socket analysis to explore the dataset ( supporting information , figure s4 ) . the socket algorithm identifies knobs - into - holes packing of coiled coils to distinguish them from helix dimers . of the 262 case 1 dimers , only 24 dimers were identified as being coiled coils by socket ( 9.2% ) , of which 22 were antiparallel and 2 were parallel . moreover , more than half of those 24 coiled coils identified were fewer than three heptads in length . this analysis implies that while helical dimers may occasionally exhibit packing characteristic of coiled coil motifs , they are too short to be stable on their own . 27/261 case 2 dimers contained significant coiled coil structure ( 10.3% ) and 21 contained one complementary interaction . of case 3 dimers , 133/919 were identified as coiled coils by socket ( 14.4% ) and an additional 50 contained a single knob - in - hole interaction . to follow up on the disparities in inner groove composition demonstrated in figure 5 , we specifically studied the frequency of hydrophilic inner grooves . we profile two complexes whose interfacial g results almost exclusively ( > 90% ) from hydrophilic contacts in the supporting information , figure s5 . such features are uncommon in canonical coiled coils . polar residues may be found at the interior of inside - out coiled coil motifs found in membrane proteins , though there is debate regarding the extent of polarity on the inside . because the vast majority of the proteins in dippdb are cytosolic , including these unusual examples , we hypothesized that a similar environment may make this inside - out geometry possible : the helical dimer is surrounded by hydrophobic residues presented by the protein in which it is found and by its binding partner , serving an analogous role to membrane lipids . this degree of burial is highly destabilizing if hydrogen bonds are left buried but unsatisfied as a result , but it concomitantly increases the value of satisfied hydrogen bonds , contributing to the strength of these interfaces . overall , though it is possible to find recognizably canonical packing at the interior of helical dimers , the majority do not succumb to the same generalizations as the coiled coil motif . as discussed above , the knobs - into - holes packing of coiled coils distinguishes them from helical dimers . because the two helices of a case 3 helical dimers come from different chains , we were able to compare knob hole packing interactions to the g of the knob residue by identifying the nearest three - residue hole on the partner helix ( i , i+1 , i+4 or i , i+3 , i+4 ) to each knob . we restricted this analysis to the inner groove of each helix and furthermore recomputed the knob g values on complexes only including the dimer helices so as to omit any interactions with other components of the protein . though the packing is not conventional enough to identify the dimers as coiled coils by socket , we anticipated that we would still be able to identify some trends . instead , we found the key feature , tightly correlated to g , was that knob residues made contact with chemically complementary holes . in the creation of this dataset , we made no stipulation about the relative position of these helix bundles in the protein(s ) in question . we conjectured that helix - turn - helix motifs , or helical hairpins , which are common sources of ideal antiparallel coiled coils , might be particularly prominent interface elements . this substantial proportion of helix - turn - helix motifs is encouraging , as it suggests that the tertiary structure present at the interface is largely governed by local forces that may be mimicked by a designed inhibitor . the possible interplay between interface tertiary structure geometry and the stoichiometry of formed complexes merits further study , as it suggests a difference in folding cooperativity . even though the n and c termini of parallel dimers on the same chain are distant , the majority of parallel dimers are connected by a small number of motifs . ( specific pdbs for each motif are listed in the supporting information . ) to our knowledge , this is the first effort to establish common protein folds connecting helices of defined orientation outside of canonical coiled coils . redesigned and optimized derivatives of these motifs particularly the loop - strand - turn - helix - loop motif , as it is by far the most common and the most structurally interesting may serve as miniprotein scaffolds . given the importance of salt bridge interactions holding coiled coils together , we investigated the distribution of charge across helix monomers in each case . ( b ) t. brucei s udp - galactose 4-epimerase features a mutation in the active site relative to the human enzyme , potentially permitting specific targeting . in both figures , the nearby active site from the dimer chain the interfaces collated in dippdb have the traits of prime targets for drug design . we identified a total of 523 interactions ( cases 1 and 2 ) that would require a helix dimer mimetic or miniprotein for inhibition . intracellular ppis dominate the dataset ; thus , inhibition of these complexes will require development of synthetic analogues that can permeate the cell membrane . some of these newly identified targets will potentially aid efforts in drug discovery . in particular , we explored classic cases leading to hallmarks of cancer and discovered a set of possible targets mediated by helical dimers . the interaction between epo and its receptor ( pdb code 1eer ) is implicated in the hypoxic response ; the complex between murine ifnar1 and interferon - beta ( pdb code 3wcy ) is implicated in the regulation of apoptosis , and the inhibition of the catalytic subunit p110 by the sh2 domain of the regulatory subunit p85 of phosphoinositide 3-kinase ( pdb code 2y3a ) is implicated in angiogenesis and invasive cell growth . a list of targets implicated in transcription is included in the supporting information , table s1 . we anticipate that these and more targets will become tractable for modulation by designed tertiary structure mimetics ; in figure 9 , we depict several interactions of particular pharmacological interest whose tertiary structure binding sites have not been drugged . ( c ) the kaposi s sarcoma protein vflip forms an a2b2 heterotetramer with nf - kappa - b essential modulator ( nemo ) coiled coil ; thus far , inhibition of this complex s function has only been achieved through geldanamycin inhibition of hsp90 . the aim of any systematic study of protein structures is both to uncover general principles governing protein geometry and to develop new insight into how to practically modulate protein function . topologically defined segments often mediate protein protein interactions , and mimicry of these regions has emerged as a successful strategy for inhibitor design . emerging examples of tertiary mimetics as ppi inhibitors illustrate the broad potential of moving beyond secondary structure mimicry . we were motivated to analyze protein complexes featuring helical dimers to create a list of potential targets where mimics of a single helix may not be sufficient . modification of the protocol used to develop a database of interface helices ( hippdb ) provided a set of protein protein interactions where the critical binding residues reside on two helices oriented in parallel or antiparallel configurations . the dataset includes some helical dimers that would be classified as true coiled coils because of their heptad repeats and supercoiling as well as helical dimers that find other means of making contacts with each other . some helical dimers violate the typical expectations for coiled coil interiors and are held together largely by salt bridges and hydrogen bonds , while others violate expectations for typical protein interfaces , which contain mostly large , aliphatic hot spot residues . the length of dimers in contact with the protein partners spans one to three heptads , suggesting that medium - sized molecules or miniproteins will be able to disrupt these complexes . the online database provides a list of all entries in the dataset along with their pdb identifiers and the energetic contributions of the hot spot residues .

mimicry of interfacial protein segments has led to new classes of rationally designed inhibitors of protein protein interactions ( ppis ) . we have recently examined the full set of protein complexes in the protein data bank mediated by -helices and -strands . to define loop motifs at protein interfaces , aims both to describe the interactions present in the protein data bank and to prescribe effective starting points for the design of ppi inhibitors . individual secondary structures are critical elements of protein interfaces ; however , many ppis feature more complex modes of binding , suggesting a potential role for synthetic tertiary structure mimetics or miniproteins as attractive candidates for the design of new classes of ppi inhibitors . miniproteins consisting of helical bundles , -sheet barrels , and loops , along with synthetic antibodies , are now routinely used to enrich ligands for protein targets , especially for extracellular receptors . in an effort to expand our atomic analysis of protein structural data beyond interactions that can be mediated by a single secondary structure element alone , we chose to begin our survey of protein tertiary interactions by focusing on helix dimers because the dimer is the simplest all - helical tertiary structure stoichiometry . dimeric coiled coils or similarly structured motifs such as bundles play essential roles in mediating biological processes , iconically driving the multimerization and stabilization of proteins involved in transcription factor complexes and vesicular trafficking , among other critical functions . several computational approaches have been implemented to predict coiled coil - mediated interactions by their pairwise and multimeric residue correlations . however , computational and experimental methods for the analysis of coiled coils described thus far are largely devoted to characterization of forces that lead to coiled coil formation . to complement these studies , we sought to analyze interactions of helical dimers with globular proteins as a step toward the rational design of coiled coil mimetics as ppi inhibitors . though canonical coiled coils possess supercoiling and particular packing properties , we did not impose these requirements , stipulating only that the helices be proximal and well - oriented . since our motivation for developing this dataset is to identify interactions that may not be inhibited by secondary structure mimics , we also required that critical binding residues be located on both helices . these criteria retain structures of high structural similarity to a coiled coil but eliminate canonical all - alpha tertiary structure motifs like the helix - loop - helix and helix - turn - helix dna binding domains , whose interhelical angles are far from parallel or antiparallel . examination of the helix dimer dataset suggests that coiled coil interfaces can be divided into three broad categories ( figure 1 ) according to their interaction stoichiometry . we anticipate that helical dimers in case 3 would favor different interacting residues from examples in cases 1 and 2 , because in case 3 high - affinity interactions must form between two individual helices rather than a helix dimer and a globular protein . this taxonomy reflects the different properties demanded of potential designed inhibitors : case 1 features interactions on predominantly one dimer face ; case 2 generally interacts with two faces ; case 3 dimer interfaces may be interrupted by a single helix . ( a ) in the first family ( case 1 ) , a coiled coil entirely from chain a forms an interaction with protein b. ( b ) in the second family ( case 2 ) , a coiled coil , which may come from one or two proteins , interacts with two different proteins partners . schematic for identification of protein interfaces in the protein data bank ( pdb ) where a helix dimer contributes significantly to complex formation . on the basis of our evaluations , we classified the interactions among three classes ( cases 13 ) of helical tertiary structure - mediated ppis . we examined the biophysical properties of each class in relation to each other , to typical interface helices in general , and to canonical coiled coils . we sought to identify all helix dimers that are in contact with a globular protein irrespective of whether such helices would meet the strict definition . we also analyzed the biophysical properties of dimer interfaces , such as the composition of hot spot residues and the degree to which helical dimers differ from coiled coils and protein helices in general . hippdb was developed to comprise all interface helices of four or more residues that contain two or more hot spot residues , where hot spot residues are defined via computational alanine scanning , performed with rosetta , as those that result in a loss of binding energy ( g ) of at least 1.0 rosetta energy unit ( reu , which scales approximately as 1 kcal / mol ) . in this work , we expanded the hippdb dataset to include over 37 000 high - affinity interfaces and imposed stringent geometric and energetic criteria to obtain a set of over 1000 high - affinity helical dimers . in hippdb , we were interested in identifying minimal inhibitory motifs to aid the design of synthetic inhibitors . this goal requires identification of helical segments of a protein present at an interface without the sequences not in contact . in the context of helical dimers , the challenge is to capture the defining geometric parameters as accurately as possible so any energetically irrelevant residues far from the interface can be discarded separately . we stipulated that each helix must contribute at least 6.0 rosetta energy units ( reu ) of g in its interaction with its partner , that the angle between their helical axes must be within 30 of parallel or antiparallel , and that the two helices are within 17 of each other . this requirement excluded any helical dimers that did not make a substantial total contribution to the binding interface , and ensured that the complexes in the database will truly require a dimer and are not amenable to disruption by mimic of a single helix . finally , we aimed to ensure that the dataset was not dominated by pairs of chains from multimeric helix bundles , as our interest lies in complexes where at least one partner is a globular protein . we observed that in structures of conventional helix bundles , almost every residue of each chain is present at the protein , we required that at least one partner in every complex must have at minimum 30% of its residues distant from the interface . a cursory analysis would suggest that the hot spot residues may be distributed evenly over many heptads , requiring the design of large molecules or biologics for inhibition , as has been true for the design of coiled coil assembly inhibitors . we were surprised to find that a plurality of complexes in dippdb possessed hot spot residues over a relatively small region of the interface ( figure 3 ) . the trend of compact hot segments in helical dimers is observed in each case with two - thirds of critical contact residues averaging fewer than three heptads . however , as peptidic coiled coils of these length scales are not generally stable , we have undertaken an experimental effort to develop cross - linked helix dimers ( chds ) as minimal inhibitors of coiled coils and other helical ppis . histogram of the distance between the first and final hot spot residue in case 1 ( black ) , case 2 ( light gray ) , and case 3 ( dark gray ) helical dimers . further examination of dippdb interfaces suggests that helical dimers at protein protein interfaces have comparable amino acid composition to other high - affinity interface helices but significantly differ from canonical coiled coils . in general , classical coiled coil motifs possess a distribution of amino acids similar to the -helix but with considerable additional bias toward aliphatic residues , owing to their obligate interior packing interactions . in examining the distribution of amino acids in these structures , we found it largely consistent with the distribution on helices in general ( supporting information , figure s1 ) . though there is considerable selective pressure for coiled coil motifs to possess high proportions of aliphatic amino acids , especially leucine and isoleucine , for optimal knobs - into - holes packing arrangements , these residue types are not overwhelmingly enriched in the -helices that are part of dippdb . a summary of these data is shown in figure 4 , and details for all three cases by residue types are included in the supporting information , figure s2 . the plot shows distribution of aliphatic residues ( leu , ile , val ) , polar residues ( gln , asn , ser ) , aromatic residues ( phe , trp , tyr ) , and charged residues ( arg , asp , glu , lys ) in the three contexts . separate from our analysis of amino acid composition in general , we examined the inter - helix contacts made by case 1/2 and case 3 helical dimers and conventional coiled coils ( figure 5a ) . in the classical coiled coil motif , core polar mutations may be tolerated via changes in stoichiometry , local distortions in geometry , or an increased inner void volume . in the particular case 1 dimer example depicted in figure 5c , aliphatic packing interactions are limited and energetically insignificant ; one helix only possesses one aliphatic residue ( a threonine ) facing its partner over five entire turns ; in contrast , the nonpolar residues from the other helix pack into non - canonical holes . the case 3 dimer example ( figure 5d ) possesses energetically important nonpolar residues , but they are not organized into the classic interior groove of a coiled coil , and the phenylalanines are more than twice as energetically important as the leucines by g . we found the examples of non - canonical inner grooves compelling and performed a more comprehensive analysis to determine how non - canonical these motifs are as compared to the classical coiled coil . of the 262 case 1 dimers , only 24 dimers were identified as being coiled coils by socket ( 9.2% ) , of which 22 were antiparallel and 2 were parallel . to follow up on the disparities in inner groove composition demonstrated in figure 5 , we specifically studied the frequency of hydrophilic inner grooves . polar residues may be found at the interior of inside - out coiled coil motifs found in membrane proteins , though there is debate regarding the extent of polarity on the inside . because the vast majority of the proteins in dippdb are cytosolic , including these unusual examples , we hypothesized that a similar environment may make this inside - out geometry possible : the helical dimer is surrounded by hydrophobic residues presented by the protein in which it is found and by its binding partner , serving an analogous role to membrane lipids . the networks of buried hydrogen bonds that may form at the interior of a coiled coil are well studied , although the partially polar interiors observed here present an extreme case . overall , though it is possible to find recognizably canonical packing at the interior of helical dimers , the majority do not succumb to the same generalizations as the coiled coil motif . because the two helices of a case 3 helical dimers come from different chains , we were able to compare knob hole packing interactions to the g of the knob residue by identifying the nearest three - residue hole on the partner helix ( i , i+1 , i+4 or i , i+3 , i+4 ) to each knob . we restricted this analysis to the inner groove of each helix and furthermore recomputed the knob g values on complexes only including the dimer helices so as to omit any interactions with other components of the protein . though the packing is not conventional enough to identify the dimers as coiled coils by socket , we anticipated that we would still be able to identify some trends . in contrast to the classical knobs - into - holes aliphatic model , residues of all types could form inner - groove contacts of considerable g . instead , we found the key feature , tightly correlated to g , was that knob residues made contact with chemically complementary holes . although aliphatic contact residues of low to moderate g do frequently pack into polar holes and vice versa , every aliphatic and aromatic residue type had higher average g when packing in a mostly aliphatic or aromatic hole than in a mostly polar one ( supporting information , figure s7 ) . in the creation of this dataset , we made no stipulation about the relative position of these helix bundles in the protein(s ) in question . this substantial proportion of helix - turn - helix motifs is encouraging , as it suggests that the tertiary structure present at the interface is largely governed by local forces that may be mimicked by a designed inhibitor . the possible interplay between interface tertiary structure geometry and the stoichiometry of formed complexes merits further study , as it suggests a difference in folding cooperativity . even though the n and c termini of parallel dimers on the same chain are distant , the majority of parallel dimers are connected by a small number of motifs . of the 133 parallel case 1 dimers , 55 are connected by a two to four residue loop , a strand whose length varies with that of the dimer , a single residue turn , a short 310 or -helix , followed by another short loop ( figure 7 ) . five possess the loop - strand - turn - helix - loop linker , five are connected by a loop - helix - loop , eight have a loop - strand - loop , but 11 contain a loop - helix - loop - helix - loop - helix - loop . redesigned and optimized derivatives of these motifs particularly the loop - strand - turn - helix - loop motif , as it is by far the most common and the most structurally interesting may serve as miniprotein scaffolds . common antiparallel ( a ) and parallel ( b ) helical dimer motifs feature linkers of a simple turn , a loop - strand - helix - loop , a loop - strand - loop , and a loop - helix - loop . given the importance of salt bridge interactions holding coiled coils together , we investigated the distribution of charge across helix monomers in each case . in both figures , the nearby active site from the dimer chain the interfaces collated in dippdb have the traits of prime targets for drug design . we categorized the functions of ppis as defined in the pdb ( figure 8a ) and observed that they are implicated in biological processes from enzymatic function to transcription to the immune response . figure 8b , c highlights two examples of interactions involving udp - galactose 4-epimerase and pcsk9 where helical dimers at dimeric interfaces in enzyme biological assemblies of considerable medical relevance bind near enzyme active sites . in particular , we explored classic cases leading to hallmarks of cancer and discovered a set of possible targets mediated by helical dimers . for example , three complexes mhf histone tetramer / fancm helicase ( pdb code 4e45 ) , mre11 nuclease / rad50 abc atpase ( pdb code 3qf7 ) , and the n- and c - terminal domains of the mms21 subunit of smc5 ( pdb code 3htk)have a role in dna repair . the interaction between epo and its receptor ( pdb code 1eer ) is implicated in the hypoxic response ; the complex between murine ifnar1 and interferon - beta ( pdb code 3wcy ) is implicated in the regulation of apoptosis , and the inhibition of the catalytic subunit p110 by the sh2 domain of the regulatory subunit p85 of phosphoinositide 3-kinase ( pdb code 2y3a ) is implicated in angiogenesis and invasive cell growth . for example , two structures ( pdb codes 2p5 t and 1gvn ) were toxins annotated with nucleic acid binding transcription factor activity , and seven are implicated in organismal development ; two are involved in immune responses . we anticipate that these and more targets will become tractable for modulation by designed tertiary structure mimetics ; in figure 9 , we depict several interactions of particular pharmacological interest whose tertiary structure binding sites have not been drugged . we illustrate them here to highlight the importance of helix dimer domains in coaxing the formation of these complexes and the potential of dimer mimics as inhibitors . ( a ) lsd1-mediated nucleosome demethylation requires complexation with corest ; existing inhibitors of lsd1 bind its substrate pocket , and predicted binding sites have thus far omitted the helical dimer interface . ( b ) sam68 rna - binding protein is implicated in pro - oncogenic activity and modulates alternative splicing of cd44 and bcl - xl . ( c ) the kaposi s sarcoma protein vflip forms an a2b2 heterotetramer with nf - kappa - b essential modulator ( nemo ) coiled coil ; thus far , inhibition of this complex s function has only been achieved through geldanamycin inhibition of hsp90 . the aim of any systematic study of protein structures is both to uncover general principles governing protein geometry and to develop new insight into how to practically modulate protein function . modification of the protocol used to develop a database of interface helices ( hippdb ) provided a set of protein protein interactions where the critical binding residues reside on two helices oriented in parallel or antiparallel configurations .

we studied 3,811 pregnant women of european ancestry ( manchester and belfast , u.k . , and newcastle and brisbane , australia ) and 1,706 pregnant women of south east asian ancestry ( bangkok , thailand ) . the protocol was approved by the institutional review board at each field center , and all participants gave written , informed consent . briefly , eligible women ( 30 ) underwent a 75-g ogtt at 2432 weeks ' gestation ( as close to 28 weeks as possible ) . height , weight , and blood pressure were also measured using standardized procedures and calibrated equipment . a sample for random plasma glucose was collected at 3437 weeks ' gestation as a safety measure to identify cases with hyperglycemia above a predefined threshold . demographic and lifestyle characteristics , age , and parity were collected via questionnaire . race / ethnicity was self - identified . participants , caregivers , and hapo study staff ( except laboratory personnel ) remained blinded to glucose values unless fasting plasma glucose was > 5.8 mmol / l , 2-h ogtt plasma glucose was > 11.1 mmol / l , or any plasma glucose value was < 2.5 mmol / l . although unblinded participants were excluded from the original hapo study of pregnancy outcome ( 1 ) , we did not exclude them from maternal glucose analyses in the current study because there was no intervention to alter maternal glucose levels before the ogtt . cord blood samples for dna , plasma glucose , and serum c - peptide were collected from the offspring at delivery . these have been described in detail previously ( 31 ) . in the current study , we analyzed weight , length , head circumference , triceps , and flank and subscapular skinfold thicknesses . birth weight , length , and head circumference were available from medical records in addition to the measurements taken by hapo study personnel . to maximize sample size , we used the medical record birth weight because it was more widely available and highly correlated with the hapo - measured birth weight ( r = 0.98 ) . we used the hapo study measures for length and head circumference and added medical record values when these were unavailable . fat mass at birth ( grams ) was derived using the following formula ( 32 ) : where bw is birth weight in grams , fls is flank skinfold thickness in millimeters , and bl is birth length in centimeters ( all measured by hapo study personnel ) . from this , percent body fat was derived as follows : 100 fat mass / birth weight . we selected the rs1799884 ( gck ) and rs7903146 ( tcf7l2 ) single nucleotide polymorphisms ( snps ) for association analysis . as well as being robustly associated with higher fasting glucose and type 2 diabetes in europeans ( 4,10,19 ) , these snps , or perfectly correlated ( r = 1 ) proxies , show similar associations in east asian and south asian populations ( 3337 ) . however , we also selected tcf7l2 rs290487 and rs11196218 for analysis in our thai samples because they have shown independent evidence of association with type 2 diabetes in east asians ( r < 0.1 with rs7903146 and r = 0.01 with each other in the hapmap han chinese from beijing and japanese from tokyo reference samples ) ( 35,3840 ) and are more common than rs7903146 in these samples , giving greater power to detect associations . genotyping was carried out as part of a larger candidate panel of 1,536 snps using the illumina golden gate platform in the northwestern university genomics core . we included dna samples in our study that were successfully genotyped for over 94% of snps . call rates for the gck and tcf7l2 snps exceeded 99% in these samples , and there was not deviation from hardy - weinberg equilibrium ( p > 0.01 for the four snps in each of the five field centers ) . the frequency of the t - allele of gck rs1799884 ( associated with higher glucose levels ) ranged from 17.1 to 18.7% in the european samples and was 10.3% in the thai sample . the type 2 diabetes risk allele frequency of tcf7l2 rs7903146 ranged from 29.2 to 30.6% in the europeans and was 4.7% in the thai . the rs290487 and rs11196218 risk allele frequencies ( risk alleles defined by previous studies ) ( 35,3840 ) were 47.4 and 72.4% , respectively , in the thai sample . all analyses were carried out using stata ( version 10 ; stata , college station , tx ) . we analyzed the association between each of the three primary outcome measures ( fasting plasma glucose , 1-h ogtt glucose , and 2-h ogtt glucose ) and genotype using linear regression under an additive genetic model . we included maternal age , bmi , and mean arterial pressure as covariates in the model . we repeated the analysis including additional covariates ( bmi squared , gestational age at ogtt , parity , sex of the baby , and maternal height ) to check that they did not change the results . we analyzed the european and thai samples separately and included field center as a covariate in all analyses of europeans . we also sought to investigate the association of each snp with maternal glucose levels that carry a substantially higher risk of adverse pregnancy outcome . we created a high glucose variable with a value of 1 if the participant had one or more high values in the ogtt ( fasting plasma glucose 5.1 mmol / l , 1-h glucose 10.0 mmol / l ) and a value of 0 if the glucose was below these thresholds . the high glucose threshold corresponds to an odds ratio ( or ) of 1.75 , relative to the mean glucose level , averaged across three outcomes in the hapo study : offspring birth weight > 90th percentile , cord c - peptide > 90th percentile , offspring percent body fat > 90th percentile , and is the iadpsg - recommended threshold for gestational diabetes mellitus ( 28 ) . we analyzed the association between each snp and the odds of high glucose using logistic regression ( log - additive genetic model ) , adjusting for field center ( european ancestry samples only ) maternal age , bmi , and mean arterial pressure . using all 5,515 study subjects with both gck and tcf7l2 genotype available ( and adjusting for field center ) , we then analyzed the association between the odds of high glucose and the combined number of t - alleles at both loci ( 0 , 1 , 2 , and 3 or 4 ) , with the same covariates as before . we combined individuals with three or four t - alleles into one group because of low numbers in the final category . to test for deviation from a multiplicative trend across the four groups , we compared the results with a full model ( including the allele score as indicator variables ) using a likelihood ratio test . to guard against possible population stratification , we generated principal components of ancestry using smartpca from the eigensoft software package ( 41 ) based on 141 ancestry informative markers that were genotyped in the same panel of 1,536 snps as the gck and tcf7l2 snps . we analyzed the association between each neonatal outcome and maternal genotype using linear regression ( additive genetic model ) . we performed the analysis twice : 1 ) a minimally adjusted model with field center ( european ancestry only ) , sex of the baby , and gestational age at delivery as covariates and 2 ) a fully adjusted model including maternal age at ogtt , maternal bmi at ogtt , maternal bmi at ogtt squared , parity , maternal smoking ( yes / no ) , mean arterial pressure , and maternal height as additional covariates . the first analysis was performed to enable comparison with previously published studies ( 4,20,22 ) , and the second was performed for comparison with the first to verify that the additional covariates did not change the results . all analyses of neonatal outcomes excluded babies born preterm ( before 37 full weeks of gestation ) and pregnancies in which caregivers were unblinded to maternal glucose levels . we performed inverse variance meta - analysis ( fixed effects ) of the association between each snp and birth weight to combine the hapo study data with data from previously published studies ( 4,20,22 ) . within each study sample , the association between genotype and birth weight was analyzed using linear regression under an additive genetic model , with sex and gestational age at delivery as covariates . for the meta - analysis , we used the metan module developed for stata ( 42 ) . heterogeneity between studies was estimated using cochran 's q test and the i statistic ( 43 ) . we also used cochran 's q test to assess evidence of heterogeneity between the analyses of maternal glucose outcomes in the european and thai samples . we analyzed the association between birth weight and fetal genotype using linear regression ( additive model ) , adjusting for field center ( european ancestry only ) , sex , and gestational age and again excluding preterm births and unblinded pregnancies . since maternal and fetal genotypes are correlated , we then repeated the analysis stratifying by maternal genotype and tested for evidence of a fetal genotype birth weight association within each stratum . to address whether fetal genotype alters the impact of maternal glucose levels on offspring birth weight , fat mass , or skinfold thickness , we additionally tested for evidence of interaction between fetal genotype and maternal glucose using a likelihood ratio test . our sample of 3,811 europeans gave us 80% power to detect per - allele differences in an outcome of 0.09 sd and 0.07 sd for the gck and tcf7l2 snps , respectively , at p < 0.05 . due to the lower sample size and allele frequencies in the thai sample , power to detect these associations was reduced ( 35 and 14% , respectively ) . power calculations were performed using quanto , version 1.2 ( 44 ) . to address whether maternal gck and tcf7l2 genotypes improve prediction of birth weight or neonatal adiposity in the presence of different combinations of other known variables , we used logistic regression to model the odds of birth weight , skinfold sum , and percent body fat > 90th percentile against various explanatory variables , including both maternal gck and tcf7l2 genotypes ( included as indicator variables ) . we constructed receiver operator characteristic ( roc ) curves and calculated the area under the curve ( auc ) to estimate the discriminatory power of the model . briefly , eligible women ( 30 ) underwent a 75-g ogtt at 2432 weeks ' gestation ( as close to 28 weeks as possible ) . height , weight , and blood pressure were also measured using standardized procedures and calibrated equipment . a sample for random plasma glucose was collected at 3437 weeks ' gestation as a safety measure to identify cases with hyperglycemia above a predefined threshold . demographic and lifestyle characteristics , age , and parity were collected via questionnaire . race / ethnicity was self - identified . participants , caregivers , and hapo study staff ( except laboratory personnel ) remained blinded to glucose values unless fasting plasma glucose was > 5.8 mmol / l , 2-h ogtt plasma glucose was > 11.1 mmol / l , or any plasma glucose value was < 2.5 mmol / l . although unblinded participants were excluded from the original hapo study of pregnancy outcome ( 1 ) , we did not exclude them from maternal glucose analyses in the current study because there was no intervention to alter maternal glucose levels before the ogtt . cord blood samples for dna , plasma glucose , and serum c - peptide were collected from the offspring at delivery . these have been described in detail previously ( 31 ) . in the current study , we analyzed weight , length , head circumference , triceps , and flank and subscapular skinfold thicknesses . birth weight , length , and head circumference were available from medical records in addition to the measurements taken by hapo study personnel . to maximize sample size , we used the medical record birth weight because it was more widely available and highly correlated with the hapo - measured birth weight ( r = 0.98 ) . we used the hapo study measures for length and head circumference and added medical record values when these were unavailable . fat mass at birth ( grams ) was derived using the following formula ( 32 ) : where bw is birth weight in grams , fls is flank skinfold thickness in millimeters , and bl is birth length in centimeters ( all measured by hapo study personnel ) . from this , percent body fat was derived as follows : 100 fat mass / birth weight . we selected the rs1799884 ( gck ) and rs7903146 ( tcf7l2 ) single nucleotide polymorphisms ( snps ) for association analysis . as well as being robustly associated with higher fasting glucose and type 2 diabetes in europeans ( 4,10,19 ) , these snps , or perfectly correlated ( r = 1 ) proxies , show similar associations in east asian and south asian populations ( 3337 ) . however , we also selected tcf7l2 rs290487 and rs11196218 for analysis in our thai samples because they have shown independent evidence of association with type 2 diabetes in east asians ( r < 0.1 with rs7903146 and r = 0.01 with each other in the hapmap han chinese from beijing and japanese from tokyo reference samples ) ( 35,3840 ) and are more common than rs7903146 in these samples , giving greater power to detect associations . genotyping was carried out as part of a larger candidate panel of 1,536 snps using the illumina golden gate platform in the northwestern university genomics core . we included dna samples in our study that were successfully genotyped for over 94% of snps . call rates for the gck and tcf7l2 snps exceeded 99% in these samples , and there was not deviation from hardy - weinberg equilibrium ( p > 0.01 for the four snps in each of the five field centers ) . the frequency of the t - allele of gck rs1799884 ( associated with higher glucose levels ) ranged from 17.1 to 18.7% in the european samples and was 10.3% in the thai sample . the type 2 diabetes risk allele frequency of tcf7l2 rs7903146 ranged from 29.2 to 30.6% in the europeans and was 4.7% in the thai . the rs290487 and rs11196218 risk allele frequencies ( risk alleles defined by previous studies ) ( 35,3840 ) were 47.4 and 72.4% , respectively , in the thai sample . all analyses were carried out using stata ( version 10 ; stata , college station , tx ) . we analyzed the association between each of the three primary outcome measures ( fasting plasma glucose , 1-h ogtt glucose , and 2-h ogtt glucose ) and genotype using linear regression under an additive genetic model . we included maternal age , bmi , and mean arterial pressure as covariates in the model . we repeated the analysis including additional covariates ( bmi squared , gestational age at ogtt , parity , sex of the baby , and maternal height ) to check that they did not change the results . we analyzed the european and thai samples separately and included field center as a covariate in all analyses of europeans . we also sought to investigate the association of each snp with maternal glucose levels that carry a substantially higher risk of adverse pregnancy outcome . we created a high glucose variable with a value of 1 if the participant had one or more high values in the ogtt ( fasting plasma glucose 5.1 mmol / l , 1-h glucose 10.0 mmol / l ) and a value of 0 if the glucose was below these thresholds . the high glucose threshold corresponds to an odds ratio ( or ) of 1.75 , relative to the mean glucose level , averaged across three outcomes in the hapo study : offspring birth weight > 90th percentile , cord c - peptide > 90th percentile , offspring percent body fat > 90th percentile , and is the iadpsg - recommended threshold for gestational diabetes mellitus ( 28 ) . we analyzed the association between each snp and the odds of high glucose using logistic regression ( log - additive genetic model ) , adjusting for field center ( european ancestry samples only ) maternal age , bmi , and mean arterial pressure . using all 5,515 study subjects with both gck and tcf7l2 genotype available ( and adjusting for field center ) , we then analyzed the association between the odds of high glucose and the combined number of t - alleles at both loci ( 0 , 1 , 2 , and 3 or 4 ) , with the same covariates as before . we combined individuals with three or four t - alleles into one group because of low numbers in the final category . to test for deviation from a multiplicative trend across the four groups , we compared the results with a full model ( including the allele score as indicator variables ) using a likelihood ratio test . to guard against possible population stratification , we generated principal components of ancestry using smartpca from the eigensoft software package ( 41 ) based on 141 ancestry informative markers that were genotyped in the same panel of 1,536 snps as the gck and tcf7l2 snps . we analyzed the association between each neonatal outcome and maternal genotype using linear regression ( additive genetic model ) . we performed the analysis twice : 1 ) a minimally adjusted model with field center ( european ancestry only ) , sex of the baby , and gestational age at delivery as covariates and 2 ) a fully adjusted model including maternal age at ogtt , maternal bmi at ogtt , maternal bmi at ogtt squared , parity , maternal smoking ( yes / no ) , mean arterial pressure , and maternal height as additional covariates . the first analysis was performed to enable comparison with previously published studies ( 4,20,22 ) , and the second was performed for comparison with the first to verify that the additional covariates did not change the results . all analyses of neonatal outcomes excluded babies born preterm ( before 37 full weeks of gestation ) and pregnancies in which caregivers were unblinded to maternal glucose levels . we performed inverse variance meta - analysis ( fixed effects ) of the association between each snp and birth weight to combine the hapo study data with data from previously published studies ( 4,20,22 ) . within each study sample , the association between genotype and birth weight was analyzed using linear regression under an additive genetic model , with sex and gestational age at delivery as covariates . for the meta - analysis , we used the metan module developed for stata ( 42 ) . heterogeneity between studies was estimated using cochran 's q test and the i statistic ( 43 ) . we also used cochran 's q test to assess evidence of heterogeneity between the analyses of maternal glucose outcomes in the european and thai samples . we analyzed the association between birth weight and fetal genotype using linear regression ( additive model ) , adjusting for field center ( european ancestry only ) , sex , and gestational age and again excluding preterm births and unblinded pregnancies . since maternal and fetal genotypes are correlated , we then repeated the analysis stratifying by maternal genotype and tested for evidence of a fetal genotype birth weight association within each stratum . to address whether fetal genotype alters the impact of maternal glucose levels on offspring birth weight , fat mass , or skinfold thickness , we additionally tested for evidence of interaction between fetal genotype and maternal glucose using a likelihood ratio test . our sample of 3,811 europeans gave us 80% power to detect per - allele differences in an outcome of 0.09 sd and 0.07 sd for the gck and tcf7l2 snps , respectively , at p < 0.05 . due to the lower sample size and allele frequencies in the thai sample , power to detect these associations was reduced ( 35 and 14% , respectively ) . to address whether maternal gck and tcf7l2 genotypes improve prediction of birth weight or neonatal adiposity in the presence of different combinations of other known variables , we used logistic regression to model the odds of birth weight , skinfold sum , and percent body fat > 90th percentile against various explanatory variables , including both maternal gck and tcf7l2 genotypes ( included as indicator variables ) . we constructed receiver operator characteristic ( roc ) curves and calculated the area under the curve ( auc ) to estimate the discriminatory power of the model . the associations between continuous measures of maternal glucose , as determined during an ogtt , and the gck and tcf7l2 variants are presented in table 2 and table 3 , respectively . summary data from each of the field centers with european ancestry women are presented in supplementary tables 1 and 2 , available in an online appendix ( http://diabetes.diabetesjournals.org/cgi/content/full/db10-0177/dc1 ) . number of women with fasting glucose , gck rs1779984 genotype , age , bmi , and mean arterial pressure available . excluding births before 37 completed weeks of gestation and pregnancies in which caregivers were not blinded to maternal glucose levels . association between maternal gck rs1799884 genotype and maternal glucose levels in pregnancy the european plasma glucose levels by genotype are the mean values across all four field centers . the t - allele of gck rs1799884 is associated with raised fasting glucose in nondiabetic , nonpregnant populations . the t - allele frequency ranged from 17.1 to 18.7% in the european ancestry samples and was 10.3% in the thai samples . the effect sizes and p values are from linear regression of maternal glucose level against genotype ( coded 0 , 1 , or 2 t - alleles ) , with field center ( european ancestry only ) , age , bmi , and mean arterial pressure as covariates . age , bmi , and mean arterial pressure were measured at a median of 2829 weeks ' gestation , depending on the field center . association between maternal tcf7l2 rs7903146 genotype and maternal glucose levels in pregnancy the european plasma glucose levels by genotype are the mean values across all four field centers . the t - allele of tcf7l2 rs7903146 is associated with an increased risk of type 2 diabetes . the t - allele frequency ranged from 29.2 to 30.6% in the european ancestry samples and was 4.7% in the thai samples . the effect sizes and p values are from linear regression of maternal glucose level against genotype ( coded 0 , 1 , or 2 t - alleles ) , with field center ( european ancestry only ) , age , bmi , and mean arterial pressure as covariates . age , bmi , and mean arterial pressure were measured at a median of 2829 weeks ' gestation , depending on the field center . we observed associations between the gck rs1799884 variant and fasting glucose in both the european ( 0.03 mmol / l higher per t - allele [ 95% ci 0.010.05 ] ; p = 0.001 ) and thai samples ( 0.08 mmol / l higher per t - allele [ 0.040.12 ] ; p < 0.0001 ) . we also observed an association with 1-h glucose in the europeans ( p = 0.001 ) but not in the thais ( p = 0.24 ) . however , the estimated differences were similar ( per t - allele increase 0.15 mmol / l [ 0.060.25 ] in the europeans and 0.11 mmol / l [ 0.07 to 0.29 ] in the thais ) . there was evidence of association with 2-h glucose in the thai sample ( 0.21 mmol / l higher per t - allele [ 95% ci 0.060.35 ] ; p = 0.005 ) , but not in the europeans ( 0.02 mmol / l per t - allele [ 0.050.09 ] ; p = 0.56 ) . there was evidence of heterogeneity between the european and thai samples for the fasting ( p = 0.03 ) and 2-h ( p = 0.02 ) glucose measures but not the 1-h measure ( p = 0.70 ) . when we repeated these analyses including additional covariates , we observed similar results ( supplementary table 3 ) . in the european samples , we observed associations between the tcf7l2 rs7903146 variant and both 1-h ( 0.16 mmol / l higher per t - allele [ 95% ci 0.080.24 ] ; p < 0.0001 ) and 2-h ( 0.13 mmol / l higher per t - allele [ 0.070.19 ] ; p < 0.0001 ) glucose levels . there was also some evidence for association with fasting glucose ( 0.02 mmol / l higher per t - allele [ 0.0020.03 ] ; p = 0.03 ) . we observed no evidence for association at p < 0.05 between the tcf7l2 variant and the maternal glucose measures in the thai samples , reflecting either no association in this population or reduced power due to the lower minor allele frequency . the estimated differences were similar to those in the europeans ( all heterogeneity p values > 0.6 ) . when we repeated these analyses including additional covariates , we observed similar results ( supplementary table 4 ) . there were also no associations between any of the maternal glucose outcomes and the tcf7l2 rs290487 or rs11196218 snps in the thai sample ( p > 0.05 ; data not shown ) . consistent with the results for continuous glucose measures , we observed associations between both the gck and tcf7l2 variant and the odds of high glucose , defined by the iadpsg ( 28 ) as having one or more of the following : fasting plasma glucose 5.1 26% of women with the gck rs1799884 tt genotype had high glucose , compared with 15% with the cc genotype , and the per - allele or was 1.29 ( 95% ci 1.091.50 ) . the corresponding or in the thai sample was 1.42 ( 1.061.77 ) . for tcf7l2 rs7903146 , 20% of women of european ancestry with the tt genotype had high glucose , compared with 16% with the cc genotype . the per - allele ors were 1.15 ( 1.001.31 ) in the europeans and 1.39 ( 0.881.90 ) in the thais . analysis of the complete dataset , combining information from both gck and tcf7l2 , showed strong evidence for a trend to increased odds of high glucose with an increasing number of t - alleles ( or 1.25 [ 95% ci 1.141.37 ] ; p < 0.00001 ) ( supplementary fig . 1 ) . there was no evidence of deviation from a multiplicative model ( p = 0.28 ) . association between gck or tcf7l2 genotype and high * maternal glucose levels * defined as fasting glucose 5.1 mmol / l or 1-h glucose 10.0 mmol / l or 2-h glucose 8.5 ors and p values are from logistic regression of high glucose status ( 1 or 0 ) against genotype ( coded 0 , 1 , or 2 t - alleles ) , with field center ( european ancestry only ) , age , bmi , and mean arterial pressure as covariates . age , bmi , and mean arterial pressure were measured at a median of 2829 weeks ' gestation , depending on the field center . categories are combined because of the small number of tt homozygotes ( n = 3 ) . inclusion of the first two ancestry principal components as covariates did not change the associations between the gck and tcf7l2 variant and maternal glucose outcomes ( data not shown ) . a sensitivity analysis including only participants whose caregivers meta - analysis of the associations between the maternal gck and tcf7l2 variants and offspring birth weight , using both thai and european study samples , showed that those in the hapo study were consistent in size and direction with previously published studies of europeans ( heterogeneity p values > 0.05 ; i < 20% ) ( fig . the overall p values for association with birth weight were p < 0.0001 for maternal gck rs1799884 ( n = 12,643 ) and p < 0.001 for maternal tcf7l2 rs7903146 ( n = 13,406 ) . meta - analysis of the association between offspring birth weight and maternal gck rs1799884 genotype ( overall p < 0.0001 ) ( a ) or maternal tcf7l2 rs7903146 genotype ( overall p < 0.001 ) ( b ) in the hapo study and previously published studies ( 4,20,22 ) . alspac , avon longitudinal study of parents and children ; efsoch , exeter family study of childhood health ; nccgp , north cumbria community genetics project . we analyzed the associations between the maternal gck and tcf7l2 variants and various neonatal traits ( supplementary tables 5 and 6 ) . we did not observe an association with cord c - peptide at either locus ( p > 0.05 ) . after bonferroni adjustment for 10 tests , there was no association at p < 0.05 with neonatal anthropometric traits at either locus . however , in the europeans , the gck rs1799884 effect size estimates for all traits were in the direction expected , given the association with maternal glucose , and the unadjusted p values for association with birth weight , fat mass , percent body fat , and skinfold thickness were all < 0.05 . we observed no overall association between birth weight and the fetal genotype at either locus ( p > 0.05 ; data not shown ) . to address the correlation between maternal and fetal genotype , we stratified by maternal genotype , but there was no evidence of association ( p > 0.05 ) after multiple testing correction ( supplementary fig . we observed no evidence of interaction between fetal genotype and maternal glucose levels on offspring birth weight , fat mass , or skinfold thickness ( p > 0.05 ; data not shown ) . we next addressed 1 ) whether maternal gck and tcf7l2 genotypes improve prediction of birth weight or neonatal adiposity when maternal fasting glucose is not known and 2 ) whether knowledge of maternal genotypes improves prediction of birth weight or neonatal adiposity when maternal fasting glucose is known but maternal 1- and 2-h glucose values are not known . when maternal fasting glucose was added to a model containing various covariates , the observed area under the roc curves for birth weight , skinfold sum , and percent body fat > 90th percentile increased ( p < 0.05 ) . in contrast , the addition of maternal genotype at gck and tcf7l2 did not result in increased discriminatory ability ( p > 0.05 ) . similarly , genotype did not improve the aucs when considered alongside fasting glucose , whereas measured 1- and 2-h glucose values did result in improvement ( p<0.05 ; supplementary table 7 ) . the associations between continuous measures of maternal glucose , as determined during an ogtt , and the gck and tcf7l2 variants are presented in table 2 and table 3 , respectively . summary data from each of the field centers with european ancestry women are presented in supplementary tables 1 and 2 , available in an online appendix ( http://diabetes.diabetesjournals.org/cgi/content/full/db10-0177/dc1 ) . number of women with fasting glucose , gck rs1779984 genotype , age , bmi , and mean arterial pressure available . excluding births before 37 completed weeks of gestation and pregnancies in which caregivers were not blinded to maternal glucose levels . association between maternal gck rs1799884 genotype and maternal glucose levels in pregnancy the european plasma glucose levels by genotype are the mean values across all four field centers . the t - allele of gck rs1799884 is associated with raised fasting glucose in nondiabetic , nonpregnant populations . the t - allele frequency ranged from 17.1 to 18.7% in the european ancestry samples and was 10.3% in the thai samples . the effect sizes and p values are from linear regression of maternal glucose level against genotype ( coded 0 , 1 , or 2 t - alleles ) , with field center ( european ancestry only ) , age , bmi , and mean arterial pressure as covariates . age , bmi , and mean arterial pressure were measured at a median of 2829 weeks ' gestation , depending on the field center . association between maternal tcf7l2 rs7903146 genotype and maternal glucose levels in pregnancy the european plasma glucose levels by genotype are the mean values across all four field centers . the t - allele of tcf7l2 rs7903146 is associated with an increased risk of type 2 diabetes . the t - allele frequency ranged from 29.2 to 30.6% in the european ancestry samples and was 4.7% in the thai samples . the effect sizes and p values are from linear regression of maternal glucose level against genotype ( coded 0 , 1 , or 2 t - alleles ) , with field center ( european ancestry only ) , age , bmi , and mean arterial pressure as covariates . age , bmi , and mean arterial pressure were measured at a median of 2829 weeks ' gestation , depending on the field center . we observed associations between the gck rs1799884 variant and fasting glucose in both the european ( 0.03 mmol / l higher per t - allele [ 95% ci 0.010.05 ] ; p = 0.001 ) and thai samples ( 0.08 mmol / l higher per t - allele [ 0.040.12 ] ; p < 0.0001 ) . we also observed an association with 1-h glucose in the europeans ( p = 0.001 ) but not in the thais ( p = 0.24 ) . however , the estimated differences were similar ( per t - allele increase 0.15 mmol / l [ 0.060.25 ] in the europeans and 0.11 mmol / l [ 0.07 to 0.29 ] in the thais ) . there was evidence of association with 2-h glucose in the thai sample ( 0.21 mmol / l higher per t - allele [ 95% ci 0.060.35 ] ; p = 0.005 ) , but not in the europeans ( 0.02 mmol / l per t - allele [ 0.050.09 ] ; p = 0.56 ) . there was evidence of heterogeneity between the european and thai samples for the fasting ( p = 0.03 ) and 2-h ( p = 0.02 ) glucose measures but not the 1-h measure ( p = 0.70 ) . when we repeated these analyses including additional covariates , we observed similar results ( supplementary table 3 ) . in the european samples , we observed associations between the tcf7l2 rs7903146 variant and both 1-h ( 0.16 mmol / l higher per t - allele [ 95% ci 0.080.24 ] ; p < 0.0001 ) and 2-h ( 0.13 mmol / l higher per t - allele [ 0.070.19 ] ; p < 0.0001 ) glucose levels . there was also some evidence for association with fasting glucose ( 0.02 mmol / l higher per t - allele [ 0.0020.03 ] ; p = 0.03 ) . we observed no evidence for association at p < 0.05 between the tcf7l2 variant and the maternal glucose measures in the thai samples , reflecting either no association in this population or reduced power due to the lower minor allele frequency . the estimated differences were similar to those in the europeans ( all heterogeneity p values > 0.6 ) . when we repeated these analyses including additional covariates , we observed similar results ( supplementary table 4 ) . there were also no associations between any of the maternal glucose outcomes and the tcf7l2 rs290487 or rs11196218 snps in the thai sample ( p > 0.05 ; data not shown ) . consistent with the results for continuous glucose measures , we observed associations between both the gck and tcf7l2 variant and the odds of high glucose , defined by the iadpsg ( 28 ) as having one or more of the following : fasting plasma glucose 5.1 26% of women with the gck rs1799884 tt genotype had high glucose , compared with 15% with the cc genotype , and the per - allele or was 1.29 ( 95% ci 1.091.50 ) . the corresponding or in the thai sample was 1.42 ( 1.061.77 ) . for tcf7l2 rs7903146 , 20% of women of european ancestry with the tt genotype had high glucose , compared with 16% with the cc genotype . the per - allele ors were 1.15 ( 1.001.31 ) in the europeans and 1.39 ( 0.881.90 ) in the thais . analysis of the complete dataset , combining information from both gck and tcf7l2 , showed strong evidence for a trend to increased odds of high glucose with an increasing number of t - alleles ( or 1.25 [ 95% ci 1.141.37 ] ; p < 0.00001 ) ( supplementary fig . 1 ) . there was no evidence of deviation from a multiplicative model ( p = 0.28 ) . association between gck or tcf7l2 genotype and high * maternal glucose levels * defined as fasting glucose 5.1 mmol / l or 1-h glucose 10.0 mmol / l or 2-h glucose 8.5 ors and p values are from logistic regression of high glucose status ( 1 or 0 ) against genotype ( coded 0 , 1 , or 2 t - alleles ) , with field center ( european ancestry only ) , age , bmi , and mean arterial pressure as covariates . age , bmi , and mean arterial pressure were measured at a median of 2829 weeks ' gestation , depending on the field center . categories are combined because of the small number of tt homozygotes ( n = 3 ) . inclusion of the first two ancestry principal components as covariates did not change the associations between the gck and tcf7l2 variant and maternal glucose outcomes ( data not shown ) . a sensitivity analysis including only participants whose caregivers meta - analysis of the associations between the maternal gck and tcf7l2 variants and offspring birth weight , using both thai and european study samples , showed that those in the hapo study were consistent in size and direction with previously published studies of europeans ( heterogeneity p values > 0.05 ; i < 20% ) ( fig . the overall p values for association with birth weight were p < 0.0001 for maternal gck rs1799884 ( n = 12,643 ) and p < 0.001 for maternal tcf7l2 rs7903146 ( n = 13,406 ) . meta - analysis of the association between offspring birth weight and maternal gck rs1799884 genotype ( overall p < 0.0001 ) ( a ) or maternal tcf7l2 rs7903146 genotype ( overall p < 0.001 ) ( b ) in the hapo study and previously published studies ( 4,20,22 ) . alspac , avon longitudinal study of parents and children ; efsoch , exeter family study of childhood health ; nccgp , north cumbria community genetics project . we analyzed the associations between the maternal gck and tcf7l2 variants and various neonatal traits ( supplementary tables 5 and 6 ) . we did not observe an association with cord c - peptide at either locus ( p > 0.05 ) . after bonferroni adjustment for 10 tests , there was no association at p < 0.05 with neonatal anthropometric traits at either locus . however , in the europeans , the gck rs1799884 effect size estimates for all traits were in the direction expected , given the association with maternal glucose , and the unadjusted p values for association with birth weight , fat mass , percent body fat , and skinfold thickness were all < 0.05 . we observed no overall association between birth weight and the fetal genotype at either locus ( p > 0.05 ; data not shown ) . to address the correlation between maternal and fetal genotype , we stratified by maternal genotype , but there was no evidence of association ( p > 0.05 ) after multiple testing correction ( supplementary fig . we observed no evidence of interaction between fetal genotype and maternal glucose levels on offspring birth weight , fat mass , or skinfold thickness ( p > 0.05 ; data not shown ) . we next addressed 1 ) whether maternal gck and tcf7l2 genotypes improve prediction of birth weight or neonatal adiposity when maternal fasting glucose is not known and 2 ) whether knowledge of maternal genotypes improves prediction of birth weight or neonatal adiposity when maternal fasting glucose is known but maternal 1- and 2-h glucose values are not known . when maternal fasting glucose was added to a model containing various covariates , the observed area under the roc curves for birth weight , skinfold sum , and percent body fat > 90th percentile increased ( p < 0.05 ) . in contrast , the addition of maternal genotype at gck and tcf7l2 did not result in increased discriminatory ability ( p > 0.05 ) . similarly , genotype did not improve the aucs when considered alongside fasting glucose , whereas measured 1- and 2-h glucose values did result in improvement ( p<0.05 ; supplementary table 7 ) . in our study of 5,517 women of european and thai ancestry , we have shown for the first time associations between variants at the gck and tcf7l2 loci and continuous ogtt measures of maternal glucose in pregnant women without overt diabetes . an additional new finding in this study is association of these variants with gestational diabetes mellitus , as defined by the new iadpsg consensus recommendation ( 28 ) , itself associated with higher risk of adverse birth outcomes . the gck rs1799884 variant was associated with fasting glucose in both of our study populations . the association in the thai sample was similar to associations previously observed in nonpregnant east asian subjects ( 37 ) , while the estimated fasting glucose difference per allele in the europeans was smaller than previously observed ( p = 0.02 ) ( 7 ) . we observed some evidence of heterogeneity , with the thai sample showing a tendency to larger per - allele differences than the european ancestry sample ( p = 0.03 ) . this is consistent with previous observations from east asian , but not south asian , versus european analyses ( 36,37 ) . changes in glucokinase activity or content in pancreatic -cells are known to impact primarily on fasting glucose levels ( 45 ) . however , the rate of -cell glucose metabolism and , hence , insulin secretion is controlled by glucokinase across the full range of glucose levels ( 46,47 ) , and mutations of the glucokinase gene result in a raising of glucose values throughout the glucose tolerance test compared with non mutation - carrying family members ( 48 ) . in keeping with this , gck rs1799884 was associated with higher 1-h glucose levels in the european population in our study and higher 2-h glucose levels in the thais . association of gck rs1799884 with 2-h glucose was demonstrated previously in a european study of nonpregnant individuals ( 49 ) . the tcf7l2 rs7903146 variant showed associations at p < 0.05 with all measures of maternal glucose , but these were largest for 1- and 2-h glucose in keeping with the known association of this locus with glucose - stimulated insulin secretion and incretin signaling in the islet ( 13 ) . despite the lower minor allele frequency in the thai sample , the glucose differences per allele for all three measures were similar to those observed in the europeans . previous studies of both gck and tcf7l2 have shown associations with gestational diabetes mellitus ( 23,24,26,27 ) . in keeping with these , we have shown associations with the new consensus definition of gestational diabetes mellitus ( 28 ) in both the european and thai samples . we observed strong evidence for a trend to higher odds of high glucose with increasing numbers of t - alleles at gck and tcf7l2 ( p < 0.00001 ) but found no evidence for statistical interaction between these loci ( p > 0.2 ) . it is important that we now assess the impact of all confirmed fasting glucose and type 2 diabetes susceptibility loci on maternal glycemia because we are likely to identify extreme groups within the population who are at greatly increased genetic risk of high glucose levels in pregnancy . due to the adverse impact of glucose levels per se on offspring phenotype , genetic variants that are only associated with steady - state glucose regulation may be associated with potentially harmful outcomes in pregnancy even if they are not associated with disease risk in nonpregnant adults . the associations that we observed between the maternal risk allele at both loci and higher offspring birth weight were similar to those previously published ( 4,20 ) . our finding that the gck variant showed adiposity associations consistent with the continuous relationship between glucose and neonatal adiposity ( 31 ) is a novel observation that extends the previously demonstrated association of this variant with birth weight . larger studies or meta - analyses will be necessary to provide enough statistical power with which to investigate thoroughly the associations between maternal genotype and neonatal anthropometrics . consistent with previous studies ( 4,20,22 ) , we observed no association between birth weight and fetal genotype , suggesting that , unlike rare fetal mutations in gck ( 50 ) , the common variants at gck and tcf7l2 do not influence fetal growth directly . measurement of maternal glucose can help identify women without overt diabetes who have a higher risk of neonatal adiposity > 90th percentile ( 31 ) . we hypothesized that maternal gck and tcf7l2 genotypes might add useful information for predicting neonatal birth weight , skinfold sum , and percent body fat > 90th percentile when maternal glucose is not known . using roc curves , we found that maternal genotypes did not improve the discriminatory ability of the models ( p > 0.05 ) . this may reflect that genotypes at these variants explain only a small proportion ( < 1% ) of variance in maternal glucose levels . however , a total of 16 genetic variants are now known to explain 34% of the variation in fasting glucose in europeans ( 10 ) . it will therefore be important to repeat these analyses with all known variants . to conclude , variants at the gck and tcf7l2 loci , which predispose to higher fasting glucose and type 2 diabetes in the general population , are associated with 1 ) higher glucose levels from ogtts in pregnant women who do not have overt diabetes and 2 ) gestational diabetes mellitus under the new consensus definition ( 28 ) . further well - powered studies will be important to assess fully the contribution of known genetic variants to maternal glycemia in pregnancy , pregnancy outcomes , and neonatal phenotypes .

objectivecommon genetic variants in gck and tcf7l2 are associated with higher fasting glucose and type 2 diabetes in nonpregnant populations . however , their associations with glucose levels from oral glucose tolerance tests ( ogtts ) in pregnancy have not been assessed in a large sample . we hypothesized that these variants are associated with quantitative measures of glycemia in pregnancy.research design and methodswe analyzed the associations between variants rs1799884 ( gck ) and rs7903146 ( tcf7l2 ) and ogtt outcomes at 2432 weeks ' gestation in 3,811 mothers of european ( u.k . and australia ) and 1,706 mothers of asian ( thailand ) ancestry from the hapo cohort . we also tested associations with offspring birth anthropometrics.resultsthe maternal gck variant was associated with higher fasting glucose in europeans ( p = 0.001 ) and thais ( p < 0.0001 ) , 1-h glucose in europeans ( p = 0.001 ) , and 2-h glucose in thais ( p = 0.005 ) . it was also associated with higher european offspring birth weight , fat mass , and skinfold thicknesses ( p < 0.05 ) . the tcf7l2 variant was associated with all three maternal glucose outcomes ( p = 0.03 , p < 0.0001 , and p < 0.0001 for fasting and 1-h and 2-h glucose , respectively ) in the europeans but not in the thais ( p > 0.05 ) . in both populations , both variants were associated with higher odds of gestational diabetes mellitus according to the new international association of diabetes and pregnancy study groups recommendations ( p = 0.0010.08).conclusionsmaternal gck and tcf7l2 variants are associated with glucose levels known to carry an increased risk of adverse pregnancy outcome in women without overt diabetes . further studies will be important to determine the variance in maternal glucose explained by all known genetic variants .

RESEARCH DESIGN AND METHODS Maternal phenotypes and exclusion criteria. Neonatal phenotypes. Genotyping. Statistical analyses: associations between maternal genotype and maternal glycemia. Associations between maternal genotype and neonatal anthropometric traits. Associations between fetal genotype and birth weight. Power calculations. Comparing the discriminatory impact of maternal genotype with measured glucose on birth weight and neonatal adiposity. RESULTS Associations between maternal genotype and maternal glycemia. Associations between maternal genotype and neonatal anthropometric traits. Associations between fetal genotype and birth weight. Using ROC curves to evaluate the impact of maternal genotype versus maternal glucose on birth weight and neonatal adiposity. DISCUSSION Supplementary Material

we selected the rs1799884 ( gck ) and rs7903146 ( tcf7l2 ) single nucleotide polymorphisms ( snps ) for association analysis . as well as being robustly associated with higher fasting glucose and type 2 diabetes in europeans ( 4,10,19 ) , these snps , or perfectly correlated ( r = 1 ) proxies , show similar associations in east asian and south asian populations ( 3337 ) . however , we also selected tcf7l2 rs290487 and rs11196218 for analysis in our thai samples because they have shown independent evidence of association with type 2 diabetes in east asians ( r < 0.1 with rs7903146 and r = 0.01 with each other in the hapmap han chinese from beijing and japanese from tokyo reference samples ) ( 35,3840 ) and are more common than rs7903146 in these samples , giving greater power to detect associations . we also sought to investigate the association of each snp with maternal glucose levels that carry a substantially higher risk of adverse pregnancy outcome . the high glucose threshold corresponds to an odds ratio ( or ) of 1.75 , relative to the mean glucose level , averaged across three outcomes in the hapo study : offspring birth weight > 90th percentile , cord c - peptide > 90th percentile , offspring percent body fat > 90th percentile , and is the iadpsg - recommended threshold for gestational diabetes mellitus ( 28 ) . using all 5,515 study subjects with both gck and tcf7l2 genotype available ( and adjusting for field center ) , we then analyzed the association between the odds of high glucose and the combined number of t - alleles at both loci ( 0 , 1 , 2 , and 3 or 4 ) , with the same covariates as before . to address whether fetal genotype alters the impact of maternal glucose levels on offspring birth weight , fat mass , or skinfold thickness , we additionally tested for evidence of interaction between fetal genotype and maternal glucose using a likelihood ratio test . to address whether maternal gck and tcf7l2 genotypes improve prediction of birth weight or neonatal adiposity in the presence of different combinations of other known variables , we used logistic regression to model the odds of birth weight , skinfold sum , and percent body fat > 90th percentile against various explanatory variables , including both maternal gck and tcf7l2 genotypes ( included as indicator variables ) . we selected the rs1799884 ( gck ) and rs7903146 ( tcf7l2 ) single nucleotide polymorphisms ( snps ) for association analysis . as well as being robustly associated with higher fasting glucose and type 2 diabetes in europeans ( 4,10,19 ) , these snps , or perfectly correlated ( r = 1 ) proxies , show similar associations in east asian and south asian populations ( 3337 ) . however , we also selected tcf7l2 rs290487 and rs11196218 for analysis in our thai samples because they have shown independent evidence of association with type 2 diabetes in east asians ( r < 0.1 with rs7903146 and r = 0.01 with each other in the hapmap han chinese from beijing and japanese from tokyo reference samples ) ( 35,3840 ) and are more common than rs7903146 in these samples , giving greater power to detect associations . we also sought to investigate the association of each snp with maternal glucose levels that carry a substantially higher risk of adverse pregnancy outcome . the high glucose threshold corresponds to an odds ratio ( or ) of 1.75 , relative to the mean glucose level , averaged across three outcomes in the hapo study : offspring birth weight > 90th percentile , cord c - peptide > 90th percentile , offspring percent body fat > 90th percentile , and is the iadpsg - recommended threshold for gestational diabetes mellitus ( 28 ) . using all 5,515 study subjects with both gck and tcf7l2 genotype available ( and adjusting for field center ) , we then analyzed the association between the odds of high glucose and the combined number of t - alleles at both loci ( 0 , 1 , 2 , and 3 or 4 ) , with the same covariates as before . to address whether fetal genotype alters the impact of maternal glucose levels on offspring birth weight , fat mass , or skinfold thickness , we additionally tested for evidence of interaction between fetal genotype and maternal glucose using a likelihood ratio test . to address whether maternal gck and tcf7l2 genotypes improve prediction of birth weight or neonatal adiposity in the presence of different combinations of other known variables , we used logistic regression to model the odds of birth weight , skinfold sum , and percent body fat > 90th percentile against various explanatory variables , including both maternal gck and tcf7l2 genotypes ( included as indicator variables ) . the associations between continuous measures of maternal glucose , as determined during an ogtt , and the gck and tcf7l2 variants are presented in table 2 and table 3 , respectively . we observed associations between the gck rs1799884 variant and fasting glucose in both the european ( 0.03 mmol / l higher per t - allele [ 95% ci 0.010.05 ] ; p = 0.001 ) and thai samples ( 0.08 mmol / l higher per t - allele [ 0.040.12 ] ; p < 0.0001 ) . we also observed an association with 1-h glucose in the europeans ( p = 0.001 ) but not in the thais ( p = 0.24 ) . there was evidence of association with 2-h glucose in the thai sample ( 0.21 mmol / l higher per t - allele [ 95% ci 0.060.35 ] ; p = 0.005 ) , but not in the europeans ( 0.02 mmol / l per t - allele [ 0.050.09 ] ; p = 0.56 ) . there was evidence of heterogeneity between the european and thai samples for the fasting ( p = 0.03 ) and 2-h ( p = 0.02 ) glucose measures but not the 1-h measure ( p = 0.70 ) . in the european samples , we observed associations between the tcf7l2 rs7903146 variant and both 1-h ( 0.16 mmol / l higher per t - allele [ 95% ci 0.080.24 ] ; p < 0.0001 ) and 2-h ( 0.13 mmol / l higher per t - allele [ 0.070.19 ] ; p < 0.0001 ) glucose levels . we observed no evidence for association at p < 0.05 between the tcf7l2 variant and the maternal glucose measures in the thai samples , reflecting either no association in this population or reduced power due to the lower minor allele frequency . there were also no associations between any of the maternal glucose outcomes and the tcf7l2 rs290487 or rs11196218 snps in the thai sample ( p > 0.05 ; data not shown ) . consistent with the results for continuous glucose measures , we observed associations between both the gck and tcf7l2 variant and the odds of high glucose , defined by the iadpsg ( 28 ) as having one or more of the following : fasting plasma glucose 5.1 26% of women with the gck rs1799884 tt genotype had high glucose , compared with 15% with the cc genotype , and the per - allele or was 1.29 ( 95% ci 1.091.50 ) . association between gck or tcf7l2 genotype and high * maternal glucose levels * defined as fasting glucose 5.1 mmol / l or 1-h glucose 10.0 mmol / l or 2-h glucose 8.5 ors and p values are from logistic regression of high glucose status ( 1 or 0 ) against genotype ( coded 0 , 1 , or 2 t - alleles ) , with field center ( european ancestry only ) , age , bmi , and mean arterial pressure as covariates . inclusion of the first two ancestry principal components as covariates did not change the associations between the gck and tcf7l2 variant and maternal glucose outcomes ( data not shown ) . a sensitivity analysis including only participants whose caregivers meta - analysis of the associations between the maternal gck and tcf7l2 variants and offspring birth weight , using both thai and european study samples , showed that those in the hapo study were consistent in size and direction with previously published studies of europeans ( heterogeneity p values > 0.05 ; i < 20% ) ( fig . the overall p values for association with birth weight were p < 0.0001 for maternal gck rs1799884 ( n = 12,643 ) and p < 0.001 for maternal tcf7l2 rs7903146 ( n = 13,406 ) . meta - analysis of the association between offspring birth weight and maternal gck rs1799884 genotype ( overall p < 0.0001 ) ( a ) or maternal tcf7l2 rs7903146 genotype ( overall p < 0.001 ) ( b ) in the hapo study and previously published studies ( 4,20,22 ) . we analyzed the associations between the maternal gck and tcf7l2 variants and various neonatal traits ( supplementary tables 5 and 6 ) . however , in the europeans , the gck rs1799884 effect size estimates for all traits were in the direction expected , given the association with maternal glucose , and the unadjusted p values for association with birth weight , fat mass , percent body fat , and skinfold thickness were all < 0.05 . we observed no evidence of interaction between fetal genotype and maternal glucose levels on offspring birth weight , fat mass , or skinfold thickness ( p > 0.05 ; data not shown ) . we next addressed 1 ) whether maternal gck and tcf7l2 genotypes improve prediction of birth weight or neonatal adiposity when maternal fasting glucose is not known and 2 ) whether knowledge of maternal genotypes improves prediction of birth weight or neonatal adiposity when maternal fasting glucose is known but maternal 1- and 2-h glucose values are not known . when maternal fasting glucose was added to a model containing various covariates , the observed area under the roc curves for birth weight , skinfold sum , and percent body fat > 90th percentile increased ( p < 0.05 ) . in contrast , the addition of maternal genotype at gck and tcf7l2 did not result in increased discriminatory ability ( p > 0.05 ) . the associations between continuous measures of maternal glucose , as determined during an ogtt , and the gck and tcf7l2 variants are presented in table 2 and table 3 , respectively . we observed associations between the gck rs1799884 variant and fasting glucose in both the european ( 0.03 mmol / l higher per t - allele [ 95% ci 0.010.05 ] ; p = 0.001 ) and thai samples ( 0.08 mmol / l higher per t - allele [ 0.040.12 ] ; p < 0.0001 ) . we also observed an association with 1-h glucose in the europeans ( p = 0.001 ) but not in the thais ( p = 0.24 ) . there was evidence of association with 2-h glucose in the thai sample ( 0.21 mmol / l higher per t - allele [ 95% ci 0.060.35 ] ; p = 0.005 ) , but not in the europeans ( 0.02 mmol / l per t - allele [ 0.050.09 ] ; p = 0.56 ) . there was evidence of heterogeneity between the european and thai samples for the fasting ( p = 0.03 ) and 2-h ( p = 0.02 ) glucose measures but not the 1-h measure ( p = 0.70 ) . in the european samples , we observed associations between the tcf7l2 rs7903146 variant and both 1-h ( 0.16 mmol / l higher per t - allele [ 95% ci 0.080.24 ] ; p < 0.0001 ) and 2-h ( 0.13 mmol / l higher per t - allele [ 0.070.19 ] ; p < 0.0001 ) glucose levels . we observed no evidence for association at p < 0.05 between the tcf7l2 variant and the maternal glucose measures in the thai samples , reflecting either no association in this population or reduced power due to the lower minor allele frequency . there were also no associations between any of the maternal glucose outcomes and the tcf7l2 rs290487 or rs11196218 snps in the thai sample ( p > 0.05 ; data not shown ) . consistent with the results for continuous glucose measures , we observed associations between both the gck and tcf7l2 variant and the odds of high glucose , defined by the iadpsg ( 28 ) as having one or more of the following : fasting plasma glucose 5.1 26% of women with the gck rs1799884 tt genotype had high glucose , compared with 15% with the cc genotype , and the per - allele or was 1.29 ( 95% ci 1.091.50 ) . association between gck or tcf7l2 genotype and high * maternal glucose levels * defined as fasting glucose 5.1 mmol / l or 1-h glucose 10.0 mmol / l or 2-h glucose 8.5 ors and p values are from logistic regression of high glucose status ( 1 or 0 ) against genotype ( coded 0 , 1 , or 2 t - alleles ) , with field center ( european ancestry only ) , age , bmi , and mean arterial pressure as covariates . inclusion of the first two ancestry principal components as covariates did not change the associations between the gck and tcf7l2 variant and maternal glucose outcomes ( data not shown ) . a sensitivity analysis including only participants whose caregivers meta - analysis of the associations between the maternal gck and tcf7l2 variants and offspring birth weight , using both thai and european study samples , showed that those in the hapo study were consistent in size and direction with previously published studies of europeans ( heterogeneity p values > 0.05 ; i < 20% ) ( fig . the overall p values for association with birth weight were p < 0.0001 for maternal gck rs1799884 ( n = 12,643 ) and p < 0.001 for maternal tcf7l2 rs7903146 ( n = 13,406 ) . meta - analysis of the association between offspring birth weight and maternal gck rs1799884 genotype ( overall p < 0.0001 ) ( a ) or maternal tcf7l2 rs7903146 genotype ( overall p < 0.001 ) ( b ) in the hapo study and previously published studies ( 4,20,22 ) . we analyzed the associations between the maternal gck and tcf7l2 variants and various neonatal traits ( supplementary tables 5 and 6 ) . however , in the europeans , the gck rs1799884 effect size estimates for all traits were in the direction expected , given the association with maternal glucose , and the unadjusted p values for association with birth weight , fat mass , percent body fat , and skinfold thickness were all < 0.05 . we observed no evidence of interaction between fetal genotype and maternal glucose levels on offspring birth weight , fat mass , or skinfold thickness ( p > 0.05 ; data not shown ) . we next addressed 1 ) whether maternal gck and tcf7l2 genotypes improve prediction of birth weight or neonatal adiposity when maternal fasting glucose is not known and 2 ) whether knowledge of maternal genotypes improves prediction of birth weight or neonatal adiposity when maternal fasting glucose is known but maternal 1- and 2-h glucose values are not known . when maternal fasting glucose was added to a model containing various covariates , the observed area under the roc curves for birth weight , skinfold sum , and percent body fat > 90th percentile increased ( p < 0.05 ) . in contrast , the addition of maternal genotype at gck and tcf7l2 did not result in increased discriminatory ability ( p > 0.05 ) . in our study of 5,517 women of european and thai ancestry , we have shown for the first time associations between variants at the gck and tcf7l2 loci and continuous ogtt measures of maternal glucose in pregnant women without overt diabetes . an additional new finding in this study is association of these variants with gestational diabetes mellitus , as defined by the new iadpsg consensus recommendation ( 28 ) , itself associated with higher risk of adverse birth outcomes . the association in the thai sample was similar to associations previously observed in nonpregnant east asian subjects ( 37 ) , while the estimated fasting glucose difference per allele in the europeans was smaller than previously observed ( p = 0.02 ) ( 7 ) . however , the rate of -cell glucose metabolism and , hence , insulin secretion is controlled by glucokinase across the full range of glucose levels ( 46,47 ) , and mutations of the glucokinase gene result in a raising of glucose values throughout the glucose tolerance test compared with non mutation - carrying family members ( 48 ) . in keeping with this , gck rs1799884 was associated with higher 1-h glucose levels in the european population in our study and higher 2-h glucose levels in the thais . the tcf7l2 rs7903146 variant showed associations at p < 0.05 with all measures of maternal glucose , but these were largest for 1- and 2-h glucose in keeping with the known association of this locus with glucose - stimulated insulin secretion and incretin signaling in the islet ( 13 ) . in keeping with these , we have shown associations with the new consensus definition of gestational diabetes mellitus ( 28 ) in both the european and thai samples . we observed strong evidence for a trend to higher odds of high glucose with increasing numbers of t - alleles at gck and tcf7l2 ( p < 0.00001 ) but found no evidence for statistical interaction between these loci ( p > 0.2 ) . it is important that we now assess the impact of all confirmed fasting glucose and type 2 diabetes susceptibility loci on maternal glycemia because we are likely to identify extreme groups within the population who are at greatly increased genetic risk of high glucose levels in pregnancy . we hypothesized that maternal gck and tcf7l2 genotypes might add useful information for predicting neonatal birth weight , skinfold sum , and percent body fat > 90th percentile when maternal glucose is not known . however , a total of 16 genetic variants are now known to explain 34% of the variation in fasting glucose in europeans ( 10 ) . to conclude , variants at the gck and tcf7l2 loci , which predispose to higher fasting glucose and type 2 diabetes in the general population , are associated with 1 ) higher glucose levels from ogtts in pregnant women who do not have overt diabetes and 2 ) gestational diabetes mellitus under the new consensus definition ( 28 ) . further well - powered studies will be important to assess fully the contribution of known genetic variants to maternal glycemia in pregnancy , pregnancy outcomes , and neonatal phenotypes .

as well as being robustly associated with higher fasting glucose and type 2 diabetes in europeans ( 4,10,19 ) , these snps , or perfectly correlated ( r = 1 ) proxies , show similar associations in east asian and south asian populations ( 3337 ) . however , we also selected tcf7l2 rs290487 and rs11196218 for analysis in our thai samples because they have shown independent evidence of association with type 2 diabetes in east asians ( r < 0.1 with rs7903146 and r = 0.01 with each other in the hapmap han chinese from beijing and japanese from tokyo reference samples ) ( 35,3840 ) and are more common than rs7903146 in these samples , giving greater power to detect associations . the frequency of the t - allele of gck rs1799884 ( associated with higher glucose levels ) ranged from 17.1 to 18.7% in the european samples and was 10.3% in the thai sample . we analyzed the association between each of the three primary outcome measures ( fasting plasma glucose , 1-h ogtt glucose , and 2-h ogtt glucose ) and genotype using linear regression under an additive genetic model . the high glucose threshold corresponds to an odds ratio ( or ) of 1.75 , relative to the mean glucose level , averaged across three outcomes in the hapo study : offspring birth weight > 90th percentile , cord c - peptide > 90th percentile , offspring percent body fat > 90th percentile , and is the iadpsg - recommended threshold for gestational diabetes mellitus ( 28 ) . we analyzed the association between each snp and the odds of high glucose using logistic regression ( log - additive genetic model ) , adjusting for field center ( european ancestry samples only ) maternal age , bmi , and mean arterial pressure . using all 5,515 study subjects with both gck and tcf7l2 genotype available ( and adjusting for field center ) , we then analyzed the association between the odds of high glucose and the combined number of t - alleles at both loci ( 0 , 1 , 2 , and 3 or 4 ) , with the same covariates as before . we performed the analysis twice : 1 ) a minimally adjusted model with field center ( european ancestry only ) , sex of the baby , and gestational age at delivery as covariates and 2 ) a fully adjusted model including maternal age at ogtt , maternal bmi at ogtt , maternal bmi at ogtt squared , parity , maternal smoking ( yes / no ) , mean arterial pressure , and maternal height as additional covariates . we analyzed the association between birth weight and fetal genotype using linear regression ( additive model ) , adjusting for field center ( european ancestry only ) , sex , and gestational age and again excluding preterm births and unblinded pregnancies . to address whether fetal genotype alters the impact of maternal glucose levels on offspring birth weight , fat mass , or skinfold thickness , we additionally tested for evidence of interaction between fetal genotype and maternal glucose using a likelihood ratio test . to address whether maternal gck and tcf7l2 genotypes improve prediction of birth weight or neonatal adiposity in the presence of different combinations of other known variables , we used logistic regression to model the odds of birth weight , skinfold sum , and percent body fat > 90th percentile against various explanatory variables , including both maternal gck and tcf7l2 genotypes ( included as indicator variables ) . as well as being robustly associated with higher fasting glucose and type 2 diabetes in europeans ( 4,10,19 ) , these snps , or perfectly correlated ( r = 1 ) proxies , show similar associations in east asian and south asian populations ( 3337 ) . however , we also selected tcf7l2 rs290487 and rs11196218 for analysis in our thai samples because they have shown independent evidence of association with type 2 diabetes in east asians ( r < 0.1 with rs7903146 and r = 0.01 with each other in the hapmap han chinese from beijing and japanese from tokyo reference samples ) ( 35,3840 ) and are more common than rs7903146 in these samples , giving greater power to detect associations . the frequency of the t - allele of gck rs1799884 ( associated with higher glucose levels ) ranged from 17.1 to 18.7% in the european samples and was 10.3% in the thai sample . we analyzed the association between each of the three primary outcome measures ( fasting plasma glucose , 1-h ogtt glucose , and 2-h ogtt glucose ) and genotype using linear regression under an additive genetic model . the high glucose threshold corresponds to an odds ratio ( or ) of 1.75 , relative to the mean glucose level , averaged across three outcomes in the hapo study : offspring birth weight > 90th percentile , cord c - peptide > 90th percentile , offspring percent body fat > 90th percentile , and is the iadpsg - recommended threshold for gestational diabetes mellitus ( 28 ) . we analyzed the association between each snp and the odds of high glucose using logistic regression ( log - additive genetic model ) , adjusting for field center ( european ancestry samples only ) maternal age , bmi , and mean arterial pressure . using all 5,515 study subjects with both gck and tcf7l2 genotype available ( and adjusting for field center ) , we then analyzed the association between the odds of high glucose and the combined number of t - alleles at both loci ( 0 , 1 , 2 , and 3 or 4 ) , with the same covariates as before . we performed the analysis twice : 1 ) a minimally adjusted model with field center ( european ancestry only ) , sex of the baby , and gestational age at delivery as covariates and 2 ) a fully adjusted model including maternal age at ogtt , maternal bmi at ogtt , maternal bmi at ogtt squared , parity , maternal smoking ( yes / no ) , mean arterial pressure , and maternal height as additional covariates . we analyzed the association between birth weight and fetal genotype using linear regression ( additive model ) , adjusting for field center ( european ancestry only ) , sex , and gestational age and again excluding preterm births and unblinded pregnancies . to address whether fetal genotype alters the impact of maternal glucose levels on offspring birth weight , fat mass , or skinfold thickness , we additionally tested for evidence of interaction between fetal genotype and maternal glucose using a likelihood ratio test . to address whether maternal gck and tcf7l2 genotypes improve prediction of birth weight or neonatal adiposity in the presence of different combinations of other known variables , we used logistic regression to model the odds of birth weight , skinfold sum , and percent body fat > 90th percentile against various explanatory variables , including both maternal gck and tcf7l2 genotypes ( included as indicator variables ) . we observed associations between the gck rs1799884 variant and fasting glucose in both the european ( 0.03 mmol / l higher per t - allele [ 95% ci 0.010.05 ] ; p = 0.001 ) and thai samples ( 0.08 mmol / l higher per t - allele [ 0.040.12 ] ; p < 0.0001 ) . there was evidence of association with 2-h glucose in the thai sample ( 0.21 mmol / l higher per t - allele [ 95% ci 0.060.35 ] ; p = 0.005 ) , but not in the europeans ( 0.02 mmol / l per t - allele [ 0.050.09 ] ; p = 0.56 ) . there was evidence of heterogeneity between the european and thai samples for the fasting ( p = 0.03 ) and 2-h ( p = 0.02 ) glucose measures but not the 1-h measure ( p = 0.70 ) . in the european samples , we observed associations between the tcf7l2 rs7903146 variant and both 1-h ( 0.16 mmol / l higher per t - allele [ 95% ci 0.080.24 ] ; p < 0.0001 ) and 2-h ( 0.13 mmol / l higher per t - allele [ 0.070.19 ] ; p < 0.0001 ) glucose levels . we observed no evidence for association at p < 0.05 between the tcf7l2 variant and the maternal glucose measures in the thai samples , reflecting either no association in this population or reduced power due to the lower minor allele frequency . there were also no associations between any of the maternal glucose outcomes and the tcf7l2 rs290487 or rs11196218 snps in the thai sample ( p > 0.05 ; data not shown ) . consistent with the results for continuous glucose measures , we observed associations between both the gck and tcf7l2 variant and the odds of high glucose , defined by the iadpsg ( 28 ) as having one or more of the following : fasting plasma glucose 5.1 26% of women with the gck rs1799884 tt genotype had high glucose , compared with 15% with the cc genotype , and the per - allele or was 1.29 ( 95% ci 1.091.50 ) . analysis of the complete dataset , combining information from both gck and tcf7l2 , showed strong evidence for a trend to increased odds of high glucose with an increasing number of t - alleles ( or 1.25 [ 95% ci 1.141.37 ] ; p < 0.00001 ) ( supplementary fig . association between gck or tcf7l2 genotype and high * maternal glucose levels * defined as fasting glucose 5.1 mmol / l or 1-h glucose 10.0 mmol / l or 2-h glucose 8.5 ors and p values are from logistic regression of high glucose status ( 1 or 0 ) against genotype ( coded 0 , 1 , or 2 t - alleles ) , with field center ( european ancestry only ) , age , bmi , and mean arterial pressure as covariates . a sensitivity analysis including only participants whose caregivers meta - analysis of the associations between the maternal gck and tcf7l2 variants and offspring birth weight , using both thai and european study samples , showed that those in the hapo study were consistent in size and direction with previously published studies of europeans ( heterogeneity p values > 0.05 ; i < 20% ) ( fig . the overall p values for association with birth weight were p < 0.0001 for maternal gck rs1799884 ( n = 12,643 ) and p < 0.001 for maternal tcf7l2 rs7903146 ( n = 13,406 ) . meta - analysis of the association between offspring birth weight and maternal gck rs1799884 genotype ( overall p < 0.0001 ) ( a ) or maternal tcf7l2 rs7903146 genotype ( overall p < 0.001 ) ( b ) in the hapo study and previously published studies ( 4,20,22 ) . however , in the europeans , the gck rs1799884 effect size estimates for all traits were in the direction expected , given the association with maternal glucose , and the unadjusted p values for association with birth weight , fat mass , percent body fat , and skinfold thickness were all < 0.05 . we next addressed 1 ) whether maternal gck and tcf7l2 genotypes improve prediction of birth weight or neonatal adiposity when maternal fasting glucose is not known and 2 ) whether knowledge of maternal genotypes improves prediction of birth weight or neonatal adiposity when maternal fasting glucose is known but maternal 1- and 2-h glucose values are not known . we observed associations between the gck rs1799884 variant and fasting glucose in both the european ( 0.03 mmol / l higher per t - allele [ 95% ci 0.010.05 ] ; p = 0.001 ) and thai samples ( 0.08 mmol / l higher per t - allele [ 0.040.12 ] ; p < 0.0001 ) . there was evidence of association with 2-h glucose in the thai sample ( 0.21 mmol / l higher per t - allele [ 95% ci 0.060.35 ] ; p = 0.005 ) , but not in the europeans ( 0.02 mmol / l per t - allele [ 0.050.09 ] ; p = 0.56 ) . there was evidence of heterogeneity between the european and thai samples for the fasting ( p = 0.03 ) and 2-h ( p = 0.02 ) glucose measures but not the 1-h measure ( p = 0.70 ) . in the european samples , we observed associations between the tcf7l2 rs7903146 variant and both 1-h ( 0.16 mmol / l higher per t - allele [ 95% ci 0.080.24 ] ; p < 0.0001 ) and 2-h ( 0.13 mmol / l higher per t - allele [ 0.070.19 ] ; p < 0.0001 ) glucose levels . we observed no evidence for association at p < 0.05 between the tcf7l2 variant and the maternal glucose measures in the thai samples , reflecting either no association in this population or reduced power due to the lower minor allele frequency . there were also no associations between any of the maternal glucose outcomes and the tcf7l2 rs290487 or rs11196218 snps in the thai sample ( p > 0.05 ; data not shown ) . consistent with the results for continuous glucose measures , we observed associations between both the gck and tcf7l2 variant and the odds of high glucose , defined by the iadpsg ( 28 ) as having one or more of the following : fasting plasma glucose 5.1 26% of women with the gck rs1799884 tt genotype had high glucose , compared with 15% with the cc genotype , and the per - allele or was 1.29 ( 95% ci 1.091.50 ) . analysis of the complete dataset , combining information from both gck and tcf7l2 , showed strong evidence for a trend to increased odds of high glucose with an increasing number of t - alleles ( or 1.25 [ 95% ci 1.141.37 ] ; p < 0.00001 ) ( supplementary fig . association between gck or tcf7l2 genotype and high * maternal glucose levels * defined as fasting glucose 5.1 mmol / l or 1-h glucose 10.0 mmol / l or 2-h glucose 8.5 ors and p values are from logistic regression of high glucose status ( 1 or 0 ) against genotype ( coded 0 , 1 , or 2 t - alleles ) , with field center ( european ancestry only ) , age , bmi , and mean arterial pressure as covariates . a sensitivity analysis including only participants whose caregivers meta - analysis of the associations between the maternal gck and tcf7l2 variants and offspring birth weight , using both thai and european study samples , showed that those in the hapo study were consistent in size and direction with previously published studies of europeans ( heterogeneity p values > 0.05 ; i < 20% ) ( fig . the overall p values for association with birth weight were p < 0.0001 for maternal gck rs1799884 ( n = 12,643 ) and p < 0.001 for maternal tcf7l2 rs7903146 ( n = 13,406 ) . meta - analysis of the association between offspring birth weight and maternal gck rs1799884 genotype ( overall p < 0.0001 ) ( a ) or maternal tcf7l2 rs7903146 genotype ( overall p < 0.001 ) ( b ) in the hapo study and previously published studies ( 4,20,22 ) . however , in the europeans , the gck rs1799884 effect size estimates for all traits were in the direction expected , given the association with maternal glucose , and the unadjusted p values for association with birth weight , fat mass , percent body fat , and skinfold thickness were all < 0.05 . we next addressed 1 ) whether maternal gck and tcf7l2 genotypes improve prediction of birth weight or neonatal adiposity when maternal fasting glucose is not known and 2 ) whether knowledge of maternal genotypes improves prediction of birth weight or neonatal adiposity when maternal fasting glucose is known but maternal 1- and 2-h glucose values are not known . in our study of 5,517 women of european and thai ancestry , we have shown for the first time associations between variants at the gck and tcf7l2 loci and continuous ogtt measures of maternal glucose in pregnant women without overt diabetes . the association in the thai sample was similar to associations previously observed in nonpregnant east asian subjects ( 37 ) , while the estimated fasting glucose difference per allele in the europeans was smaller than previously observed ( p = 0.02 ) ( 7 ) . however , the rate of -cell glucose metabolism and , hence , insulin secretion is controlled by glucokinase across the full range of glucose levels ( 46,47 ) , and mutations of the glucokinase gene result in a raising of glucose values throughout the glucose tolerance test compared with non mutation - carrying family members ( 48 ) . the tcf7l2 rs7903146 variant showed associations at p < 0.05 with all measures of maternal glucose , but these were largest for 1- and 2-h glucose in keeping with the known association of this locus with glucose - stimulated insulin secretion and incretin signaling in the islet ( 13 ) . we observed strong evidence for a trend to higher odds of high glucose with increasing numbers of t - alleles at gck and tcf7l2 ( p < 0.00001 ) but found no evidence for statistical interaction between these loci ( p > 0.2 ) . it is important that we now assess the impact of all confirmed fasting glucose and type 2 diabetes susceptibility loci on maternal glycemia because we are likely to identify extreme groups within the population who are at greatly increased genetic risk of high glucose levels in pregnancy . due to the adverse impact of glucose levels per se on offspring phenotype , genetic variants that are only associated with steady - state glucose regulation may be associated with potentially harmful outcomes in pregnancy even if they are not associated with disease risk in nonpregnant adults . our finding that the gck variant showed adiposity associations consistent with the continuous relationship between glucose and neonatal adiposity ( 31 ) is a novel observation that extends the previously demonstrated association of this variant with birth weight . consistent with previous studies ( 4,20,22 ) , we observed no association between birth weight and fetal genotype , suggesting that , unlike rare fetal mutations in gck ( 50 ) , the common variants at gck and tcf7l2 do not influence fetal growth directly . to conclude , variants at the gck and tcf7l2 loci , which predispose to higher fasting glucose and type 2 diabetes in the general population , are associated with 1 ) higher glucose levels from ogtts in pregnant women who do not have overt diabetes and 2 ) gestational diabetes mellitus under the new consensus definition ( 28 ) .

cr(vi ) is a human respiratory carcinogen with documented inhalation exposures in numerous occupational groups . its presence at large toxic waste sites and in many drinking water supplies has also raised concerns about potential adverse health effects of environmental cr(vi ) . chromate , the main form of cr(vi ) in physiological solutions , readily enters cells where it is reduced via direct ( nonenzymatic ) reactions with ascorbate ( asc ) and small cellular thiols glutathione ( gsh ) and cysteine . the final product of chromate reduction is redox - inert cr(iii ) that displays stable binding with proteins and other macromolecules , leading to its long - term intracellular retention . the reduction process also generates cr(v ) and cr(iv ) intermediates , a relative yield of which differs for asc- and thiol - driven reactions . reduction of cr(vi ) by asc involves the initial transfer of two electrons , producing cr(iv ) and no cr(v ) . only under the nonphysiological conditions of insufficient asc for completion of cr(vi ) reduction , there was a detectable formation of cr(v ) resulting from secondary reactions of cr(iv ) . gsh and cys are one - electron reducers of cr(vi ) , yielding cr(v ) as the first intermediate . asc is a dramatically faster reducer of cr(vi ) than thiols in vitro , and it is responsible for the overwhelming majority of chromate metabolism in the lung , kidney , and liver . in contrast to its low millimolar concentrations in cells in vivo , asc concentrations in cultured cells are in low micromolar range , reflecting the absence of vitamin c in synthetic growth medium formulations and the addition of only 1015% serum , which typically lost a majority of vitamin c during processing and storage . restoration of physiological levels of asc in cultured human cells has been found to alter dna damage and cytotoxic responses induced by cr(vi ) . the genotoxicity of cr(vi ) has been linked to the formation of cr - dna adducts and dna oxidation damage by reactive oxygen species ( ros ) and cr(v ) intermediates . ros and cr(v ) are transient products , which makes them difficult to detect and estimate , particularly for environmentally relevant doses of cr(vi ) . widely employed tools for monitoring ros formation in cultured cells are oxidant - sensitive fluorescent dyes . the use of specific probes can allow estimation of the general oxidative stress or the presence of specific ros . the two most popular dyes for the determination of the overall oxidative stress in cells are dihydrorhodamine 123 ( dhr123 ) and dichlorofluorescein ( dcf ) , which have also been used for the assessment of cellular ros after cr(vi ) treatments . however , responses of these probes do not always reflect the presence of ros in cells . for example , dcf fluorescence can be elevated by mitochondria - leaked cytochrome c. the application of dcf and dhr123 for the detection of ros produced by cr(vi ) is also potentially problematic in view of their susceptibility to oxidation by synthetic cr(v ) complexes . it has been proposed that both cr(v ) and cr(iv ) can oxidize dcf and dhr123 . thus , in light of the frequent use of both probes in cr(vi ) toxicology , it is important to identify a type of oxidant that reacts with dhr123 and dcf during cr(vi ) metabolism with biological reducers . two related questions that also need to be addressed are ( 1 ) which cr intermediates can cause oxidation of dhr123 and dcf and ( 2 ) what redox - sensitive probes are unreactive with cr products and can be used for monitoring ros in cr(vi ) reactions . in this work , we examined the responses of seven redox - sensitive dyes during cr(vi ) metabolism . we found no significant ros formation in cr(vi ) reactions with its main reducers and determined that cr(v ) species were responsible for the oxidation of dcf and dhr123 . dihydroethidium , aminophenylfluorescein , and three cellrox dyes were resistant to oxidation by cr(v , iv ) , making them suitable for monitoring ros in cr(vi)/oxidant mixtures . l - ascorbic acid ( 99.9% pure ) , dehydro - l-(+)-ascorbic acid dimer ( dha ) , potassium chromate ( k2cro4 , 99% pure ) , l - buthionine sulfoximine ( bso , 97% pure ) , l - glutathione ( > 98% pure ) , l - cysteine ( > 98% pure ) , nitric acid ( > 99.999% pure ) , 4-morpholinepropanesulfonic acid ( mops ) , and salts were from sigma - aldrich ( st . louis , mo , usa ) . chelex-100 and bio - gel p-30 columns were purchased from bio - rad ( hercules , ca ) . all dyes including dihydrorhodamine 123 ( dhr123 ) , 2,7-dichlorodihydrofluorescein diacetate ( dcf - diacetate ) , dihydroethidium ( dhe ) , aminophenylfluorescein ( apf ) , cellrox deep red , cellrox orange , cellrox green , and 1,2-diamino-4,5-dimethoxybenzene dihydrochloride were from molecular probes ( eugene , or , usa ) . human lung epithelial cells were propagated in rpmi-1640 medium supplemented with 10% ( v / v ) fetal bovine serum and penicillin / streptomycin . hf / sv human fibroblasts were grown in 90% ( v / v ) dmem , 10% ( v / v ) fetal bovine serum , and antibiotics . both cell lines were maintained at 37 c in a humidified atmosphere containing 95% ( v / v ) air and 5% ( v / v ) co2 . h460 cells were depleted of gsh by 24 h preincubation with 0.2 mm bso . h460 cells were incubated for 90 min with 1 mm dha in krebs - hepes buffer [ 30 mm hepes ( ph 7.5 ) , 130 mm nacl , 4 mm kh2po4 , 1 mm mgso4 , and 1 mm cacl2 ] supplemented with 0.5 mm d - glucose . stock solutions of 10 mm dha were freshly prepared in the same buffer and kept on ice . the volume of h460 cells was determined from forward scattering measurements by flow cytometry ( facscalibur , bd biosciences ) . the amounts of cellular gsh and cys were determined by hplc as described earlier . h460 cells were resuspended in cold 40 mm methanesulfonic acid and lysed by two cycles of freezing ( 80 c ) and thawing ( 37 c ) . after centrifugation at 12000 g for 10 min at 4 c , cell extracts were reacted with the thiol - specific dye monobromobimane . the fluorescent gsh and cys monobromobimane conjugates were separated and quantified by hplc . a decrease in chromate absorbance at 372 nm was used to monitor the rates and the extent of cr(vi ) reduction . equal volumes of prewarmed solutions of chromate and reducers were mixed in uv - transparent 96-well plates , and a372 readings were taken every 15 or 20 s. plates were kept at 37 c inside a microplate reader ( the spectramax m5 microplate reader ) for the duration of the reactions . stock solutions of asc , gsh cys , and potassium chromate ( or cr(v)-gsh complex ) were freshly made in deionized water and kept on ice . dcf was activated before usage by reacting 5 mm dcf - diacetate with 10 mm naoh at room temperature for 30 min . the first , made up in 100 mm mops ( ph 7.0 ) and 200 mm nacl , contained 2 the concentration of a reducer and 20 m of a fluorescent dye . the samples were prepared in a black - walled , clear - bottomed costar 96-well plate . the reaction was initiated in the dark by mixing 100 l of the reducer - dye mix with 100 l of chromate in each well . the plates were incubated at 37 c in the dark inside the spectramax m5 plate reader . excitation and emission wavelengths were as follows : 490/530 nm for dcf , 500/535 nm for dhr123 , 395/580 nm for dhe , 490/515 nm for apf , 640/665 nm for cellrox deep red , 545/565 nm for cellrox orange , and 485/530 nm for cellrox green . data in each figure panel were obtained in parallel to avoid batch and autoxidative aging effects in stock solutions of dyes . h460 cells were seeded into black 96-well optical bottom cell culture plates ( 30000 cells / well ) one day before treatments . control , gsh - depleted and dha - treated cells were preloaded with 10 m dcf - diacetate or dhr123 in rpmi-1640 medium for 30 min . after a rinse with warm pbs , cells were incubated with the indicated concentrations of cr(vi ) for 1 h in serum - free rpmi-1640 medium . cellular monolayers were rinsed once with warm pbs and then covered with a modified dpbs solution ( sigma - aldrich , d4031 ) followed by measurements of fluorescence ( dcf ex / em , 490/530 nm ; dhr123 ex / em , 500/535 nm ) . determination of total cellular cr by graphite furnace atomic absorption spectroscopy ( gf - aas ) was based on a previously described procedure . h460 cells were seeded into 6-well plates at a density equivalent to seeding conditions for cellular fluorescence measurements . after removal of cr - containing media , monolayers were rinsed twice with warm pbs followed by collection of cells by trypsinization in the presence of edta ( gibco 15400 - 054 trypsin - edta solution ) . after two washes with cold pbs ( 5 min at 800 g and 4 c ) , cells were extracted with hot 5% ( v / v ) nitric acid followed by centrifugation at 10,000 g for 10 min at 4 c . cr - containing supernatants were diluted to 2% ( v / v ) nitric acid prior to gf - aas analyses ( aanalyst600 atomic absorption spectrometer , perkin - elmer ) . metal - extracted cellular pellets were washed twice with cold 5% ( v / v ) nitric acid ( 10,000 g for 5 min at 4 c ) and then dissolved in 0.5 m naoh at 37 c for 30 min . cytotoxic effects of cr(vi ) treatments were assessed by the celltiter - glo luminescent assay ( promega , madison , wi , usa ) . h460 cells were seeded into 96-well plates ( 30000 cells / well ) and allowed to attach overnight prior to the addition of cr(vi ) . the formation of mutagenic dna damage during the reduction of cr(vi ) with cys was examined using the psp189 vector containing supf as a target gene . reaction mixtures contained 25 mm mops ( ph 7.0 ) , 2 g of psp189 dna , 2 mm cys , and 0 or 100 m chromate in a total volume of 50 l . after 60 min of incubation at 37 c , dna was purified by passage through two bio - gel p-30 columns . the psp189 plasmids were transfected into hf / sv cells , and their progeny were purified 48 h later using a plasmid isolation kit from qiagen . cell - recovered plasmids were electroporated into the e. coli mbl50 , and the total number of transformants was scored on the agar plates containing 30 g / ml ampicillin and 0.5 g / ml chloramphenicol . the supf mutants were detected on plates containing 2 mg / ml l - arabinose and both antibiotics . mutation frequency was calculated by dividing the number of colonies on ampicillin / arabinose - containing plates to the number of ampicillin - only resistant colonies . in our initial studies , we examined the fluorescence of six oxidant - sensitive dyes during the reduction of cr(vi ) with its three main biological reducers ( asc , gsh , and cys ) under in vitro conditions with physiological ionic strength , temperature , and ph . consistent with its principal role in cr(vi ) metabolism in vivo , asc exhibited a dramatically faster metabolism of cr(vi ) in comparison to that of gsh and cys ( figure 1a ) . reduction of cr(vi ) by 2 mm asc was essentially complete after approximately 5 min , whereas even 60-min long incubations with the same concentrations of cys or gsh contained unreduced cr(vi ) ( 7% for cys and 58% for gsh ) . in subsequent experiments with redox probes , fluorescence values were normalized for the amount of cr(vi ) reduced in 60 min reactions . the use of prolonged incubations that are necessary for the completion of cr(vi ) reduction by 2 mm gsh were avoided due to the accumulation of autoxidation products . cr(vi)-reducer reactions were first examined for the presence of oxidizing species using a general oxidant indicator dcf . we found major differences among cr(vi ) reduction reactions in their ability to generate oxidants that were detectable by dcf fluorescence ( figure 1b ) . cys - mediated metabolism of cr(vi ) produced very high amounts of oxidants , whereas the yield of dcf - targeting reactants in gsh reactions was on average 4.5-times lower . samples containing asc showed background dcf fluorescence even at the highest cr(vi ) concentrations . cr(vi ) metabolism by cys and gsh resulted in extensive oxidation of another general redox probe , dhr123 ( figure 1c ) . next , we studied responses of dihydroethidium ( dhe ) , a dye that is commonly used for the detection of superoxide . cr(vi ) reactions with cys displayed only very modest increases in dhe fluorescence ( maximally 2.5-fold over background ) , whereas gsh samples had even lower dhe responses ( figure 1d ) . the use of three other general redox indicators cellrox green , cellrox orange , and cellrox red showed no significant changes in fluorescence above control values for any of the cr(vi)-reducer reactions ( figure 1e ) . oxidation of redox - sensitive dyes in reactions of cr(vi ) with its biological reducers . fluorescence measurements were taken after 60 min incubations and adjusted for the extent of cr(vi ) reduction . ( a ) kinetics of 100 m cr(vi ) reduction at 37 c in the presence of 2 mm asc , cys , or gsh . ( b ) dcf fluorescence and ( c ) dhr123 fluorescence in cr(vi)-reducer reactions . n = 3 . where not visible , error bars were smaller than symbols . ( e ) fluorescence of cellrox green , cellrox orange , and cellrox red probes after incubation in cr(vi)-2 mm reducer reactions for 60 min . error bars are not shown for clarity . although both dcf and dhr123 are known as general oxidative stress probes in cells , their ros responsiveness in buffer solutions lacking peroxidases is limited to hydroxyl radicals . ethanol is a potent scavenger of hydroxyl radicals , but its addition at 100-fold molar excess over dcf had no effect on fluorescence of this dye in cr(vi)-cys and cr(vi)-gsh reactions ( figure 2a , b ) . we next employed a highly reactive species sensor aminophenylfluorescein ( apf ) to test the presence of oh radicals in cr(vi ) reactions . none of our three reduction systems showed increases in apf fluorescence ( figure 2c ) , which taken together with the absence of ethanol effects on dcf fluorescence argues against the formation of hydroxyl radicals during cr(vi ) reduction . ( a ) no effect of ethanol on dcf fluorescence in cr(vi)-cys and ( b ) cr(vi)-gsh reactions ( etoh , 1 mm ethanol ) . thus , testing for the formation of mutagenic dna damage can help determine the presence of genetically important ros . we investigated the production of mutagenic lesions using the psp189 shuttle - vector system . in this approach , the plasmid dna was included in cr(vi ) reduction reactions , purified , and then transfected into human cells for replication and fixation of dna damage as mutations . replicated psp189 molecules were isolated from cells and then electroporated into e. coli for scoring of mutations at the vector - encoded supf gene . in addition to a possible presence of ros or other dna - oxidizing species , reductive metabolism of cr(vi ) is known to produce mutagenic cr - dna adducts . to reveal the presence of a potential adduct - independent mutagenic damage , we compared psp189 mutagenesis in standard reactions and in samples containing edta , which chelates cr(iii ) and completely abrogates the production of cr - dna adducts . we found that reduction of 100 m cr(vi ) with cys resulted in robust mutagenic responses in psp189 vectors , whereas no mutagenicity was observed when reactions contained cr(iii)-sequestering edta ( figure 3a ) . control experiments showed that edta did not significantly change reduction kinetics and did not suppress the production of dcf - oxidizing species ( figure 3b , c ) . overall , the nonresponsiveness of four oxidant - sensitive probes ( three cellrox dyes and apf ) , inability of ethanol to inhibit dcf oxidation , and the absence of cr adduct - independent mutations all indicate that cr(vi)-thiol reactions lacked a significant production of ros . thus , extensive fluorescence of dcf and dhr123 was caused by non - ros oxidants . cr(iii ) chelation by edta blocks the formation of mutagenic dna damage by cr(vi ) . ( a ) mutagenic responses in psp189 plasmids incubated in cr(vi ) reactions with 2 mm cys in the absence or presence of 2 mm edta . ( b ) kinetics of cr(vi ) reduction by 2 mm cys with and without 2 mm edta . ( c ) dcf fluorescence in cr(vi)-cys reactions lacking or containing 2 mm edta . n = 3 . where not visible , error bars were smaller than symbols . cys and gsh - mediated reductions of cr(vi ) proceed through the initial one - electron transfer generating cr(v ) as the first intermediate . in contrast , reduction of cr(vi ) by several - fold molar excess of asc involves a two - electron transfer producing cr(iv ) . thus , large increases in dcf and dhr123 fluorescence in 2 mm thiols - based reactions but no responses with 2 mm asc ( figure 1a , b ) suggest that cr(v ) was probably responsible for the oxidation of both probes . to test whether the formation of cr(v ) in asc reactions would also cause dcf and/or dhr123 fluorescence , we examined reactions with low ratios of asc to cr(vi ) . esr studies have found a significant formation of cr(v ) under conditions of 1:1 and 1:2 molar ratios of asc to cr(vi ) . limited amounts of asc in these reactions permitted the production of cr(v ) via comproportionation and disproportionation of the initially formed cr(iv ) . in agreement with the requirement of 1.5 mol of asc for the reduction of 1 mol of cr(vi ) , we found incomplete reduction of 0.1 and 0.2 mm cr(vi ) by 0.1 mm asc ( figure 4a ) . the amount of reduced cr(vi ) was 70% for 0.1 mm cr(vi ) and 39% for 0.2 mm cr(vi ) . although 0.1 mm asc was theoretically sufficient to complete the reduction of 0.05 mm cr(vi ) , approximately 11% of cr(vi ) remained at the end of 60 min incubations due to a progressive depletion of reduced asc over the course of the reduction and the resulting decrease in the reaction rates . unlike reductions with > 10 molar excess of asc ( 2 mm samples ) that again gave no changes in dcf and dhr123 fluorescence , reactions containing 0.1 mm asc and 0.1 or 0.2 mm cr(vi ) produced oxidants reacting with both dyes ( figure 4b , c ) the observed pattern of fluorescence responses under conditions of asc insufficiency for the completion of cr(vi ) reduction parallels the appearance of cr(v ) in esr studies . thus , our positive findings in reactions with low asc concentrations offer further support that cr(v ) is a principal cause of dcf and dhr123 oxidation during cr(vi ) metabolism by its biological reducers . fluorescence was recorded after 60 min and adjusted for the amount of reduced cr(vi ) . ( b ) dhr123 fluorescence and ( c ) dcf fluorescence in cr(vi ) reactions with 0.1 and 2 mm asc . both cys and gsh reduce cr(vi ) via one - electron transfer generating cr(v ) . similar amounts of oxidized dhr123 in cys and gsh reactions ( figure 1c ) are consistent with the same production of cr(v ) . however , dcf fluorescence in gsh samples was much lower in comparison to that of cys samples , suggesting a potential presence of different cr(v ) species . the main cr(v ) product in gsh reactions is a well - characterized cr(v)-gsh complex , whereas no cr(v)-thiol complex has been detected in reactions with cys . to better understand the responses of both redox probes in gsh reactions , we prepared the cr(v)-gsh complex and tested its reactivity toward dcf and dhr123 . we found that dcf was completely resistant to oxidation by cr(v)-gsh , whereas dhr123 showed a strongly increased fluorescence ( figure 5a ) . next , we examined dcf and dhr123 responses in mixtures of cys and gsh . the presence of two reducers resulted in a slightly larger than additive increase ( 1.14-fold synergism ) in the initial rates of cr(vi ) reduction ( figure 5b ) , suggesting largely independently proceeding reactions for each thiol . however , the inclusion of cys eliminated a slow reduction component that was always present in our gsh - containing reactions and observed earlier in different buffer systems . reduction - adjusted dcf fluorescence in gsh + cys reactions was 3.7-times higher than that in gsh - alone samples and only about 20% lower than that in cys - alone samples ( figure 5c ) . dhr123 fluorescence was equally strong for gsh , cys , and gsh + cys reactions ( figure 5d ) . low dcf fluorescence in gsh - cr(vi ) reactions can be explained by the inability of the main cr(v ) product , cr(v)-gsh complex , to oxidize this dye . the production of cr(v)-gsh probably also accounted for a moderately lower dcf fluorescence in samples containing gsh + cys versus cys alone . a significant oxidation of dcf in gsh reactions despite the complete nonreactivity of cr(v)-gsh suggests the presence of another , less abundant cr(v ) product with a reactivity similar to that of cr(v ) generated in cys reactions . dcf and dhr123 fluorescence in reactions with cr(v)-gsh and gsh / cys mixtures . ( a ) differential susceptibility of dcf and dhr123 to oxidation by cr(v)-gsh complex . background - subtracted fluorescence values recorded after 30 min of incubation are shown ( means sd , n = 3 ) . fluorescence was not adjusted for the extent of cr(v ) to cr(iii ) conversion . ( b ) kinetics of cr(vi ) reduction by 2 mm gsh , 2 mm cys , and a mixture containing both thiols . ( c ) dcf fluorescence and ( d ) dhr123 fluorescence in cr(vi ) reactions with 2 mm thiols alone and together . fluorescence in panels c and d was adjusted for the amount of cr(vi ) reduction at the end of 60 min incubations . ( e ) time - course of dhr123 oxidation and cr(vi ) reduction in reactions containing 2 mm cys and 100 m cr(vi ) . ( f ) time - course of dhr123 oxidation and cr(vi ) reduction in reactions containing 2 mm gsh and 100 m cr(vi ) . for both panels e and f , cr(vi ) absorbance was recorded every 15 s , and dhr123 fluorescence was measured every 1 min . esr studies have found that the kinetics of the cr(v ) signal followed that of the cr(vi ) reduction in cys - driven reactions , whereas the initial cr(v ) accumulation was delayed relative to the loss of chromate absorbance in gsh reactions . consistent with a greater stability of cr(v ) complexed with gsh , the cr(v ) signal remained steady for the duration of the reduction of chromate by gsh but quickly disappeared in cys reactions . since dhr123 appeared to be sensitive to the presence of various of cr(v ) forms ( acting as a sensor of total crv ) , we tested its responsiveness in time - course studies of cr(vi ) reduction by both thiols . we found that the kinetics of dhr123 oxidation and cr(vi ) reduction were essentially identical in cys reactions ( figure 5e ) , indicating a rapid formation of cr(v ) and its fast reactivity with dhr123 . in gsh reactions , oxidation of dhr123 initially lagged the disappearance of cr(vi ) and then showed a steady increase ( figure 5f ) . the observed linear buildup in dhr123 fluorescence after the initial lag is consistent with cr(v ) reaching a steady level at approximately 5 min in gsh reactions monitored by esr . however , the lag time in dhr123 fluorescence appeared longer than that for the cr(v)-esr signal , which probably reflects a slow reactivity of cr(v)-gsh with the probe . lack of dcf oxidation by cr(v)-gsh supports the weak oxidation power of this product . to better understand the potential applications of dcf and dhr123 in the monitoring of cr(vi ) metabolism in human cells , we first examined the fluorescence of these dyes in mixtures of all three biological reducers at their physiological concentrations . asc and gsh are typically present in mammalian cells in low millimolar concentrations , whereas the cellular content of cys is several times lower than that of gsh . therefore , we chose to test cr(vi ) reactions with 2 mm asc , 2 mm gsh , and 0.2 mm cys . as expected based on the preceding results , gsh / cys mixtures caused the oxidation of both dhr123 and dcf ( figure 6a , b ) . similar to the gsh - alone reactions ( figure 1 ) , the overall fluorescence responses for dcf were much lower in comparison to those of dhr123 , which reflected a dominant role of gsh in cr(vi ) reduction when it was present at 10-fold molar excess over cys ( figure 6c ) . the addition of asc to gsh - cys mixtures completely abrogated the oxidation of both probes , which can be attributed to the overwhelming role of asc in cr(vi ) metabolism due to its dramatically faster reduction rates ( figure 1a ) and the absence of the cr(v ) intermediate under the employed toxicologically relevant ratio of asc to cr(vi ) . in the final series of in vitro experiments , we also examined the impact of asc on the oxidation of dhe in thiol - containing cr(vi ) reactions . cys- and to a lesser degree , gsh - containing reactions showed modest responses with this redox indicator ( figure 1d ) . as for dcf and dhr123 , we found that the presence of asc blocked dhe oxidation during cr(vi ) reduction by thiols ( figure 6d ) , suggesting that cr(v ) was also likely responsible for the fluorescence of this probe in cys / gsh - containing reactions . responses of dhr123 , dcf , and dhe in cr(vi ) reactions with asc - thiols mixtures . fluorescence was recorded after 30 min incubations and adjusted for the amount of cr(vi ) reduction . shown are the means sd , n = 3 . ( a ) dhr123 fluorescence and ( b ) dcf fluorescence in reactions containing 2 mm gsh and 0.2 mm cys ( gsh / cys label ) or both thiols plus 2 mm asc ( gsh / cys + asc label ) . ( c ) kinetics of cr(vi ) reduction by 0.2 mm cys , 2 mm gsh , and their mixture . ( d ) dhe fluorescence in cr(vi ) reactions with 2 mm cys ( cys label ) , 2 mm cys and 2 mm asc ( cys + asc label ) , or 2 mm gsh , cys , and asc ( gsh / cys + asc label ) . on the basis of the equally robust responsiveness of dhr123 to the formation of cr(v ) in reactions with cys and gsh , we decided to test this dye for the monitoring of cr(vi ) metabolism in h460 human lung epithelial cells . to assess the role of gsh , cells were treated with the glutathione synthase inhibitor bso for 24 h , which lowered cellular gsh concentrations by > 100-fold , from 2.9 0.2 mm to 26 11 m . time - course measurements showed a moderate increase in dhr123 fluorescence up to approximately 20 min after a 1-h long treatment of control h460 cells with 20 m cr(vi ) ( figure 7a ) . this dose of cr(vi ) corresponded to ic50 for cell viability at 24 h postexposure . in gsh - depleted cells , the amount of oxidized dhr123 was lower at the early time points but continued to rise up to 60 min after cr(vi ) removal , indicating slower reduction rates of cr(vi ) in the absence of the predominant cellular thiol . we determined that control and gsh - depleted cells contained only 2 m asc , which excludes its significant contribution to cr(vi ) reduction . when cellular concentrations of asc were raised to 0.7 mm by preincubation with 1 mm dehydroascorbic acid , we found no changes in either dhr123 or dcf fluorescence at any time point after 30 m cr(vi ) treatment ( figure 7b ) . no significant changes in the fluorescence of both dyes were also detected in asc - restored cells treated with three other doses of cr(vi ) ( 10 , 20 , and 40 m , data not shown ) . control and asc - supplemented cells showed essentially the same accumulation of cr(vi ) ( figure 7c ) , indicating that a different spectrum of cr metabolites was responsible for the lack of oxidized dhr123 in the presence of asc . the unchanged fluorescence of both redox probes in asc - normalized cells is in agreement with the observed absence of dhr123- or dcf - oxidizing species during in vitro reduction of cr(vi ) with an excess of asc . in human a549 cells , preloading with 80 m asc stimulated dcf and dhr123 fluorescence by cr(vi ) , which is consistent with the formation of cr(v ) and oxidation of both dyes in our low - asc reactions ( 100 m asc ) . cells were preloaded with 10 m dye for 30 min prior to a 1-h long treatment with 20 m cr(vi ) . data are the means sd ( n = 3 ) . ( a ) background - subtracted dhr123 fluorescence at different times after cr removal . bso cells were treated with 0.2 mm bso for 24 h to deplete gsh . ( b ) background - subtracted dcf and dhr123 fluorescence in asc - restored cells treated with 30 m cr(vi ) . we found that among seven tested oxidant - sensitive dyes , only dcf and dhr123 showed strongly increased fluorescence ( up to 100-fold over background ) in reactions containing cr(vi ) and its biological reducers gsh and cys . fluorescence of the superoxide probe dhe was also elevated in these reactions , although the overall magnitude was very modest , with a maximal 2.5-fold increase for the highest cr(vi ) doses reduced with cys . several experimental observations strongly indicate that redox probes responded to oxidants other than ros . these include the lack of fluorescence increases for three other general ros indicators and the absence of oxidant - mediated mutagenic damage in psp189 plasmids . in our in vitro conditions that lacked peroxidases , only highly reactive oh radicals but not other ros can cause dcf and dhr123 oxidation . the nonresponsiveness of apf which primarily reacts with oh radicals and the absence of any effect by the radical scavenger ethanol further argue against oh radicals as the cause of dcf and dhr123 fluorescence in cr(vi)-thiol reactions . extensive oxidation of dcf and dhr123 by synthetic cr(v ) complexes has also been found to be ros - independent . the absence of ros points to cr(v ) and/or cr(iv ) intermediates as potential oxidants of redox - sensitive probes . we found no dcf or dhr123 ( or dhe ) oxidation in cr(vi ) reactions with a more than 10-fold excess of the two - electron reducer ascorbate , which lacked a detectable formation of cr(v ) . these findings strongly indicated that cr(iv ) did not react with the redox probes . under the conditions permitting cr(v ) formation due to insufficient amounts of asc for cr(vi ) reduction , we detected the oxidation of both dcf and dhr123 . gsh and cys are one - electron reducers of cr(vi ) , producing abundant amounts of cr(v ) . in agreement with cr(v ) being the principal oxidant , both cys and gsh reactions with cr(vi ) caused large increases in dcf and dhr123 fluorescence . cys- and gsh - based reactions showed similarly high responses with dhr123 , which is consistent with the expected comparable formation of total cr(v ) by both reducers . in the case of the dcf probe , its oxidation in gsh samples was about 4-times lower relative to cys , pointing to a different reactivity of the main cr(v ) species produced by these two thiols . our experiments with a preformed cr(v)-gsh complex showed that it was capable of oxidizing dhr123 but had no reactivity toward dcf . thus , elevated dcf fluorescence in gsh - cr(vi ) reactions indicated the presence of a cr(v ) product other than cr(v)-gsh . a commonly invoked reduction mechanism by gsh involves the initial formation of the cr(vi)-thiolate complex followed by binding of a second gsh molecule leading to one - electron transfer and the production of the cr(v)-gsh complex . a similar reduction process has also been described for cys . however , whereas a relatively stable cr(v)-gsh complex has been readily isolated from in vitro reactions and detected in cr(vi)-treated human a549 cells , efforts to identify the cr(v)-cys complex have proven unsuccessful . thus , probably because of instability of cys binding , it appears that all cr(v ) in cys reactions is reducer - unbound ( free ) cr(v ) . studies on the mechanism of cr(vi ) reduction by gsh have been performed with the supraphysiological concentrations of this reducer , which would favor coordination and reduction of the cr(vi)-thiolate complex by a second gsh molecule . at physiological concentrations , the rate of ternary complex formation is expected to be greatly diminished , which allows a fraction of the initially formed cr(vi)-gsh thiolates to undergo reduction yielding free cr(v ) and the thyil radical . taken together , the presented mechanistic considerations of cr(vi ) reduction and our findings on the average 4.1-fold difference between dcf and dhr112 responses indicate that cr(vi ) metabolism by a physiological 2 mm gsh concentration generated approximately 75% cr(v ) in the form of cr(v)-gsh , which is reactive with dhr123 but not dcf , and 25% free cr(v ) , which is reactive with both dhr123 and dcf . lower yields and instability of free cr(v ) complicate its detection by esr spectroscopy at biologically relevant conditions , although esr studies of some cr(vi)-gsh reactions have previously detected a cr(v ) product with a different g - value than that of cr(v)-gsh . only a single cr(v ) form has been observed in cr(vi ) reactions with cys . similar to our results with cr(v)-gsh , the cr(v)-mannitol complex was able to oxidize dhr123 but not dcf , pointing to a general trend for the nonreactivity of dcf with cr(v ) stabilized by biological ligands . cr(v ) species are weak oxidants , and the variable responses of the tested probes in cr(vi ) reactions can be related to a different susceptibility of target groups to oxidation reactions that are required for the formation of fluorescent products . dcf and dhr123 are converted into their fluorescent forms via two sequential one - electron oxidation reactions . both dyes are initially oxidized via the abstraction of the hydrogen atom at the 9-position , whose location at the central carbon of a triphenylmethane makes it vulnerable to weakly oxidizing species such as cr(v ) . a conversion of apf into fluorescein requires o - dearylation , which is initiated by the abstraction of one electron from the nitrogen atom , and only strongly oxidizing species such as the oh radical have a sufficient reactivity for this reaction . a similar argument about the requirement for a higher oxidizing power than that provided by cr(v ) to cause oxidation of an aromatic amine moiety can be made to explain a very weak responsiveness of dhe in cr(vi ) reactions . cellrox dyes are proprietary probes with unknown chemical structures , which makes it difficult to consider chemical reasons for their insensitivity to the detection of cr(v ) . our findings extend the earlier observations with synthetic cr complexes that intermediate cr forms , which we showed here to be cr(v ) in biological systems , can oxidize dcf and dhr123 . thus , these two popular dyes are inappropriate for ros detection in cr(vi)-treated cells . the concern about the non - ros origins of dcf / dhr123 oxidants is particularly important for standard cell cultures in which asc deficiency leads to one - electron reduction of cr(vi ) by thiols . although cr(vi ) treatments of human cells with restored asc levels did not induce a detectable production of oxidants in this work or biologically significant dna oxidation , coexposure with other redox - active toxicants or phagocytosis of chromate - containing particles can result in elevated ros through chemical or biological mechanisms , respectively . the complete nonresponsiveness of three cellrox dyes and apf and minimal responses of dhe to cr intermediates make these probes suitable for ros monitoring in cr(vi)-exposed cells . a summary of the reactivity of different dyes and their applicability to the detection of specific oxidants in cr(vi ) reactions is shown in figure 8 .

cr(vi ) genotoxicity is caused by products of its reductive metabolism inside the cells . reactive oxygen species ( ros ) and cr(v , iv ) intermediates are potential sources of oxidative damage by cr(vi ) . here , we investigated seven fluorescent probes for the detection of ros and non - ros oxidants in cr(vi ) reactions with its main reducers . we found that cr(v)-skipping metabolism of cr(vi ) by ascorbate in vitro gave no responses with all tested dyes , indicating nonreactivity of cr(iv ) and absence of ros . cr(vi ) reduction with glutathione ( gsh ) or cys strongly enhanced the fluorescence of dichlorofluorescein ( dcf ) and dihydrorhodamine 123 ( dhr123 ) but produced minimal fluorescence with dihydroethidium and no increases with aminophenylfluorescein and cellrox green , orange , and red . several tests showed that cr(vi)-thiol reactions lacked ros and that cr(v ) caused oxidation of dcf and dhr123 . dcf reacted only with free cr(v ) , whereas dhr123 detected both the free cr(v ) and cr(v)-gsh complex . we estimated that cr(vi)-gsh reactions generated approximately 75% cr(v)-gsh and 25% free cr(v ) , whereas cys reactions appeared to produce only free cr(v ) . dhr123 measurements in h460 cells showed that reduction of cr(vi ) was complete within 20 min postexposure , but it lasted at least 1 h without gsh . cells with restored ascorbate levels exhibited no dcf or dhr123 oxidation by cr(vi ) . overall , our results demonstrated that cr(vi ) metabolism with its biological reducers lacked ros and that dhr123 and dcf responses were indicators of total and free cr(v ) , respectively . cellrox dyes , dihydroethidium and aminophenylfluorescein , are insensitive to cr(v , iv ) and can be used for monitoring ros during coexposure to cr(vi ) and oxidants .

Introduction Experimental Procedures Results Discussion

chromate , the main form of cr(vi ) in physiological solutions , readily enters cells where it is reduced via direct ( nonenzymatic ) reactions with ascorbate ( asc ) and small cellular thiols glutathione ( gsh ) and cysteine . reduction of cr(vi ) by asc involves the initial transfer of two electrons , producing cr(iv ) and no cr(v ) . only under the nonphysiological conditions of insufficient asc for completion of cr(vi ) reduction , there was a detectable formation of cr(v ) resulting from secondary reactions of cr(iv ) . gsh and cys are one - electron reducers of cr(vi ) , yielding cr(v ) as the first intermediate . asc is a dramatically faster reducer of cr(vi ) than thiols in vitro , and it is responsible for the overwhelming majority of chromate metabolism in the lung , kidney , and liver . the genotoxicity of cr(vi ) has been linked to the formation of cr - dna adducts and dna oxidation damage by reactive oxygen species ( ros ) and cr(v ) intermediates . ros and cr(v ) are transient products , which makes them difficult to detect and estimate , particularly for environmentally relevant doses of cr(vi ) . the two most popular dyes for the determination of the overall oxidative stress in cells are dihydrorhodamine 123 ( dhr123 ) and dichlorofluorescein ( dcf ) , which have also been used for the assessment of cellular ros after cr(vi ) treatments . for example , dcf fluorescence can be elevated by mitochondria - leaked cytochrome c. the application of dcf and dhr123 for the detection of ros produced by cr(vi ) is also potentially problematic in view of their susceptibility to oxidation by synthetic cr(v ) complexes . it has been proposed that both cr(v ) and cr(iv ) can oxidize dcf and dhr123 . thus , in light of the frequent use of both probes in cr(vi ) toxicology , it is important to identify a type of oxidant that reacts with dhr123 and dcf during cr(vi ) metabolism with biological reducers . two related questions that also need to be addressed are ( 1 ) which cr intermediates can cause oxidation of dhr123 and dcf and ( 2 ) what redox - sensitive probes are unreactive with cr products and can be used for monitoring ros in cr(vi ) reactions . we found no significant ros formation in cr(vi ) reactions with its main reducers and determined that cr(v ) species were responsible for the oxidation of dcf and dhr123 . dihydroethidium , aminophenylfluorescein , and three cellrox dyes were resistant to oxidation by cr(v , iv ) , making them suitable for monitoring ros in cr(vi)/oxidant mixtures . all dyes including dihydrorhodamine 123 ( dhr123 ) , 2,7-dichlorodihydrofluorescein diacetate ( dcf - diacetate ) , dihydroethidium ( dhe ) , aminophenylfluorescein ( apf ) , cellrox deep red , cellrox orange , cellrox green , and 1,2-diamino-4,5-dimethoxybenzene dihydrochloride were from molecular probes ( eugene , or , usa ) . h460 cells were seeded into 96-well plates ( 30000 cells / well ) and allowed to attach overnight prior to the addition of cr(vi ) . in our initial studies , we examined the fluorescence of six oxidant - sensitive dyes during the reduction of cr(vi ) with its three main biological reducers ( asc , gsh , and cys ) under in vitro conditions with physiological ionic strength , temperature , and ph . consistent with its principal role in cr(vi ) metabolism in vivo , asc exhibited a dramatically faster metabolism of cr(vi ) in comparison to that of gsh and cys ( figure 1a ) . reduction of cr(vi ) by 2 mm asc was essentially complete after approximately 5 min , whereas even 60-min long incubations with the same concentrations of cys or gsh contained unreduced cr(vi ) ( 7% for cys and 58% for gsh ) . cys - mediated metabolism of cr(vi ) produced very high amounts of oxidants , whereas the yield of dcf - targeting reactants in gsh reactions was on average 4.5-times lower . next , we studied responses of dihydroethidium ( dhe ) , a dye that is commonly used for the detection of superoxide . cr(vi ) reactions with cys displayed only very modest increases in dhe fluorescence ( maximally 2.5-fold over background ) , whereas gsh samples had even lower dhe responses ( figure 1d ) . the use of three other general redox indicators cellrox green , cellrox orange , and cellrox red showed no significant changes in fluorescence above control values for any of the cr(vi)-reducer reactions ( figure 1e ) . oxidation of redox - sensitive dyes in reactions of cr(vi ) with its biological reducers . ( e ) fluorescence of cellrox green , cellrox orange , and cellrox red probes after incubation in cr(vi)-2 mm reducer reactions for 60 min . in addition to a possible presence of ros or other dna - oxidizing species , reductive metabolism of cr(vi ) is known to produce mutagenic cr - dna adducts . we found that reduction of 100 m cr(vi ) with cys resulted in robust mutagenic responses in psp189 vectors , whereas no mutagenicity was observed when reactions contained cr(iii)-sequestering edta ( figure 3a ) . overall , the nonresponsiveness of four oxidant - sensitive probes ( three cellrox dyes and apf ) , inability of ethanol to inhibit dcf oxidation , and the absence of cr adduct - independent mutations all indicate that cr(vi)-thiol reactions lacked a significant production of ros . thus , extensive fluorescence of dcf and dhr123 was caused by non - ros oxidants . in contrast , reduction of cr(vi ) by several - fold molar excess of asc involves a two - electron transfer producing cr(iv ) . thus , large increases in dcf and dhr123 fluorescence in 2 mm thiols - based reactions but no responses with 2 mm asc ( figure 1a , b ) suggest that cr(v ) was probably responsible for the oxidation of both probes . to test whether the formation of cr(v ) in asc reactions would also cause dcf and/or dhr123 fluorescence , we examined reactions with low ratios of asc to cr(vi ) . in agreement with the requirement of 1.5 mol of asc for the reduction of 1 mol of cr(vi ) , we found incomplete reduction of 0.1 and 0.2 mm cr(vi ) by 0.1 mm asc ( figure 4a ) . although 0.1 mm asc was theoretically sufficient to complete the reduction of 0.05 mm cr(vi ) , approximately 11% of cr(vi ) remained at the end of 60 min incubations due to a progressive depletion of reduced asc over the course of the reduction and the resulting decrease in the reaction rates . unlike reductions with > 10 molar excess of asc ( 2 mm samples ) that again gave no changes in dcf and dhr123 fluorescence , reactions containing 0.1 mm asc and 0.1 or 0.2 mm cr(vi ) produced oxidants reacting with both dyes ( figure 4b , c ) the observed pattern of fluorescence responses under conditions of asc insufficiency for the completion of cr(vi ) reduction parallels the appearance of cr(v ) in esr studies . thus , our positive findings in reactions with low asc concentrations offer further support that cr(v ) is a principal cause of dcf and dhr123 oxidation during cr(vi ) metabolism by its biological reducers . ( b ) dhr123 fluorescence and ( c ) dcf fluorescence in cr(vi ) reactions with 0.1 and 2 mm asc . the main cr(v ) product in gsh reactions is a well - characterized cr(v)-gsh complex , whereas no cr(v)-thiol complex has been detected in reactions with cys . we found that dcf was completely resistant to oxidation by cr(v)-gsh , whereas dhr123 showed a strongly increased fluorescence ( figure 5a ) . low dcf fluorescence in gsh - cr(vi ) reactions can be explained by the inability of the main cr(v ) product , cr(v)-gsh complex , to oxidize this dye . a significant oxidation of dcf in gsh reactions despite the complete nonreactivity of cr(v)-gsh suggests the presence of another , less abundant cr(v ) product with a reactivity similar to that of cr(v ) generated in cys reactions . dcf and dhr123 fluorescence in reactions with cr(v)-gsh and gsh / cys mixtures . ( a ) differential susceptibility of dcf and dhr123 to oxidation by cr(v)-gsh complex . ( b ) kinetics of cr(vi ) reduction by 2 mm gsh , 2 mm cys , and a mixture containing both thiols . ( c ) dcf fluorescence and ( d ) dhr123 fluorescence in cr(vi ) reactions with 2 mm thiols alone and together . fluorescence in panels c and d was adjusted for the amount of cr(vi ) reduction at the end of 60 min incubations . esr studies have found that the kinetics of the cr(v ) signal followed that of the cr(vi ) reduction in cys - driven reactions , whereas the initial cr(v ) accumulation was delayed relative to the loss of chromate absorbance in gsh reactions . since dhr123 appeared to be sensitive to the presence of various of cr(v ) forms ( acting as a sensor of total crv ) , we tested its responsiveness in time - course studies of cr(vi ) reduction by both thiols . we found that the kinetics of dhr123 oxidation and cr(vi ) reduction were essentially identical in cys reactions ( figure 5e ) , indicating a rapid formation of cr(v ) and its fast reactivity with dhr123 . to better understand the potential applications of dcf and dhr123 in the monitoring of cr(vi ) metabolism in human cells , we first examined the fluorescence of these dyes in mixtures of all three biological reducers at their physiological concentrations . therefore , we chose to test cr(vi ) reactions with 2 mm asc , 2 mm gsh , and 0.2 mm cys . the addition of asc to gsh - cys mixtures completely abrogated the oxidation of both probes , which can be attributed to the overwhelming role of asc in cr(vi ) metabolism due to its dramatically faster reduction rates ( figure 1a ) and the absence of the cr(v ) intermediate under the employed toxicologically relevant ratio of asc to cr(vi ) . in the final series of in vitro experiments , we also examined the impact of asc on the oxidation of dhe in thiol - containing cr(vi ) reactions . as for dcf and dhr123 , we found that the presence of asc blocked dhe oxidation during cr(vi ) reduction by thiols ( figure 6d ) , suggesting that cr(v ) was also likely responsible for the fluorescence of this probe in cys / gsh - containing reactions . responses of dhr123 , dcf , and dhe in cr(vi ) reactions with asc - thiols mixtures . ( d ) dhe fluorescence in cr(vi ) reactions with 2 mm cys ( cys label ) , 2 mm cys and 2 mm asc ( cys + asc label ) , or 2 mm gsh , cys , and asc ( gsh / cys + asc label ) . on the basis of the equally robust responsiveness of dhr123 to the formation of cr(v ) in reactions with cys and gsh , we decided to test this dye for the monitoring of cr(vi ) metabolism in h460 human lung epithelial cells . time - course measurements showed a moderate increase in dhr123 fluorescence up to approximately 20 min after a 1-h long treatment of control h460 cells with 20 m cr(vi ) ( figure 7a ) . in gsh - depleted cells , the amount of oxidized dhr123 was lower at the early time points but continued to rise up to 60 min after cr(vi ) removal , indicating slower reduction rates of cr(vi ) in the absence of the predominant cellular thiol . no significant changes in the fluorescence of both dyes were also detected in asc - restored cells treated with three other doses of cr(vi ) ( 10 , 20 , and 40 m , data not shown ) . control and asc - supplemented cells showed essentially the same accumulation of cr(vi ) ( figure 7c ) , indicating that a different spectrum of cr metabolites was responsible for the lack of oxidized dhr123 in the presence of asc . the unchanged fluorescence of both redox probes in asc - normalized cells is in agreement with the observed absence of dhr123- or dcf - oxidizing species during in vitro reduction of cr(vi ) with an excess of asc . in human a549 cells , preloading with 80 m asc stimulated dcf and dhr123 fluorescence by cr(vi ) , which is consistent with the formation of cr(v ) and oxidation of both dyes in our low - asc reactions ( 100 m asc ) . we found that among seven tested oxidant - sensitive dyes , only dcf and dhr123 showed strongly increased fluorescence ( up to 100-fold over background ) in reactions containing cr(vi ) and its biological reducers gsh and cys . the nonresponsiveness of apf which primarily reacts with oh radicals and the absence of any effect by the radical scavenger ethanol further argue against oh radicals as the cause of dcf and dhr123 fluorescence in cr(vi)-thiol reactions . extensive oxidation of dcf and dhr123 by synthetic cr(v ) complexes has also been found to be ros - independent . the absence of ros points to cr(v ) and/or cr(iv ) intermediates as potential oxidants of redox - sensitive probes . we found no dcf or dhr123 ( or dhe ) oxidation in cr(vi ) reactions with a more than 10-fold excess of the two - electron reducer ascorbate , which lacked a detectable formation of cr(v ) . under the conditions permitting cr(v ) formation due to insufficient amounts of asc for cr(vi ) reduction , we detected the oxidation of both dcf and dhr123 . gsh and cys are one - electron reducers of cr(vi ) , producing abundant amounts of cr(v ) . in agreement with cr(v ) being the principal oxidant , both cys and gsh reactions with cr(vi ) caused large increases in dcf and dhr123 fluorescence . cys- and gsh - based reactions showed similarly high responses with dhr123 , which is consistent with the expected comparable formation of total cr(v ) by both reducers . taken together , the presented mechanistic considerations of cr(vi ) reduction and our findings on the average 4.1-fold difference between dcf and dhr112 responses indicate that cr(vi ) metabolism by a physiological 2 mm gsh concentration generated approximately 75% cr(v ) in the form of cr(v)-gsh , which is reactive with dhr123 but not dcf , and 25% free cr(v ) , which is reactive with both dhr123 and dcf . only a single cr(v ) form has been observed in cr(vi ) reactions with cys . similar to our results with cr(v)-gsh , the cr(v)-mannitol complex was able to oxidize dhr123 but not dcf , pointing to a general trend for the nonreactivity of dcf with cr(v ) stabilized by biological ligands . cr(v ) species are weak oxidants , and the variable responses of the tested probes in cr(vi ) reactions can be related to a different susceptibility of target groups to oxidation reactions that are required for the formation of fluorescent products . a similar argument about the requirement for a higher oxidizing power than that provided by cr(v ) to cause oxidation of an aromatic amine moiety can be made to explain a very weak responsiveness of dhe in cr(vi ) reactions . cellrox dyes are proprietary probes with unknown chemical structures , which makes it difficult to consider chemical reasons for their insensitivity to the detection of cr(v ) . the concern about the non - ros origins of dcf / dhr123 oxidants is particularly important for standard cell cultures in which asc deficiency leads to one - electron reduction of cr(vi ) by thiols . although cr(vi ) treatments of human cells with restored asc levels did not induce a detectable production of oxidants in this work or biologically significant dna oxidation , coexposure with other redox - active toxicants or phagocytosis of chromate - containing particles can result in elevated ros through chemical or biological mechanisms , respectively . a summary of the reactivity of different dyes and their applicability to the detection of specific oxidants in cr(vi ) reactions is shown in figure 8 .

chromate , the main form of cr(vi ) in physiological solutions , readily enters cells where it is reduced via direct ( nonenzymatic ) reactions with ascorbate ( asc ) and small cellular thiols glutathione ( gsh ) and cysteine . only under the nonphysiological conditions of insufficient asc for completion of cr(vi ) reduction , there was a detectable formation of cr(v ) resulting from secondary reactions of cr(iv ) . asc is a dramatically faster reducer of cr(vi ) than thiols in vitro , and it is responsible for the overwhelming majority of chromate metabolism in the lung , kidney , and liver . in contrast to its low millimolar concentrations in cells in vivo , asc concentrations in cultured cells are in low micromolar range , reflecting the absence of vitamin c in synthetic growth medium formulations and the addition of only 1015% serum , which typically lost a majority of vitamin c during processing and storage . the genotoxicity of cr(vi ) has been linked to the formation of cr - dna adducts and dna oxidation damage by reactive oxygen species ( ros ) and cr(v ) intermediates . the two most popular dyes for the determination of the overall oxidative stress in cells are dihydrorhodamine 123 ( dhr123 ) and dichlorofluorescein ( dcf ) , which have also been used for the assessment of cellular ros after cr(vi ) treatments . for example , dcf fluorescence can be elevated by mitochondria - leaked cytochrome c. the application of dcf and dhr123 for the detection of ros produced by cr(vi ) is also potentially problematic in view of their susceptibility to oxidation by synthetic cr(v ) complexes . thus , in light of the frequent use of both probes in cr(vi ) toxicology , it is important to identify a type of oxidant that reacts with dhr123 and dcf during cr(vi ) metabolism with biological reducers . two related questions that also need to be addressed are ( 1 ) which cr intermediates can cause oxidation of dhr123 and dcf and ( 2 ) what redox - sensitive probes are unreactive with cr products and can be used for monitoring ros in cr(vi ) reactions . we found no significant ros formation in cr(vi ) reactions with its main reducers and determined that cr(v ) species were responsible for the oxidation of dcf and dhr123 . l - ascorbic acid ( 99.9% pure ) , dehydro - l-(+)-ascorbic acid dimer ( dha ) , potassium chromate ( k2cro4 , 99% pure ) , l - buthionine sulfoximine ( bso , 97% pure ) , l - glutathione ( > 98% pure ) , l - cysteine ( > 98% pure ) , nitric acid ( > 99.999% pure ) , 4-morpholinepropanesulfonic acid ( mops ) , and salts were from sigma - aldrich ( st . equal volumes of prewarmed solutions of chromate and reducers were mixed in uv - transparent 96-well plates , and a372 readings were taken every 15 or 20 s. plates were kept at 37 c inside a microplate reader ( the spectramax m5 microplate reader ) for the duration of the reactions . in our initial studies , we examined the fluorescence of six oxidant - sensitive dyes during the reduction of cr(vi ) with its three main biological reducers ( asc , gsh , and cys ) under in vitro conditions with physiological ionic strength , temperature , and ph . consistent with its principal role in cr(vi ) metabolism in vivo , asc exhibited a dramatically faster metabolism of cr(vi ) in comparison to that of gsh and cys ( figure 1a ) . the use of three other general redox indicators cellrox green , cellrox orange , and cellrox red showed no significant changes in fluorescence above control values for any of the cr(vi)-reducer reactions ( figure 1e ) . none of our three reduction systems showed increases in apf fluorescence ( figure 2c ) , which taken together with the absence of ethanol effects on dcf fluorescence argues against the formation of hydroxyl radicals during cr(vi ) reduction . to reveal the presence of a potential adduct - independent mutagenic damage , we compared psp189 mutagenesis in standard reactions and in samples containing edta , which chelates cr(iii ) and completely abrogates the production of cr - dna adducts . we found that reduction of 100 m cr(vi ) with cys resulted in robust mutagenic responses in psp189 vectors , whereas no mutagenicity was observed when reactions contained cr(iii)-sequestering edta ( figure 3a ) . overall , the nonresponsiveness of four oxidant - sensitive probes ( three cellrox dyes and apf ) , inability of ethanol to inhibit dcf oxidation , and the absence of cr adduct - independent mutations all indicate that cr(vi)-thiol reactions lacked a significant production of ros . thus , large increases in dcf and dhr123 fluorescence in 2 mm thiols - based reactions but no responses with 2 mm asc ( figure 1a , b ) suggest that cr(v ) was probably responsible for the oxidation of both probes . to test whether the formation of cr(v ) in asc reactions would also cause dcf and/or dhr123 fluorescence , we examined reactions with low ratios of asc to cr(vi ) . in agreement with the requirement of 1.5 mol of asc for the reduction of 1 mol of cr(vi ) , we found incomplete reduction of 0.1 and 0.2 mm cr(vi ) by 0.1 mm asc ( figure 4a ) . although 0.1 mm asc was theoretically sufficient to complete the reduction of 0.05 mm cr(vi ) , approximately 11% of cr(vi ) remained at the end of 60 min incubations due to a progressive depletion of reduced asc over the course of the reduction and the resulting decrease in the reaction rates . unlike reductions with > 10 molar excess of asc ( 2 mm samples ) that again gave no changes in dcf and dhr123 fluorescence , reactions containing 0.1 mm asc and 0.1 or 0.2 mm cr(vi ) produced oxidants reacting with both dyes ( figure 4b , c ) the observed pattern of fluorescence responses under conditions of asc insufficiency for the completion of cr(vi ) reduction parallels the appearance of cr(v ) in esr studies . thus , our positive findings in reactions with low asc concentrations offer further support that cr(v ) is a principal cause of dcf and dhr123 oxidation during cr(vi ) metabolism by its biological reducers . to better understand the responses of both redox probes in gsh reactions , we prepared the cr(v)-gsh complex and tested its reactivity toward dcf and dhr123 . the presence of two reducers resulted in a slightly larger than additive increase ( 1.14-fold synergism ) in the initial rates of cr(vi ) reduction ( figure 5b ) , suggesting largely independently proceeding reactions for each thiol . reduction - adjusted dcf fluorescence in gsh + cys reactions was 3.7-times higher than that in gsh - alone samples and only about 20% lower than that in cys - alone samples ( figure 5c ) . low dcf fluorescence in gsh - cr(vi ) reactions can be explained by the inability of the main cr(v ) product , cr(v)-gsh complex , to oxidize this dye . a significant oxidation of dcf in gsh reactions despite the complete nonreactivity of cr(v)-gsh suggests the presence of another , less abundant cr(v ) product with a reactivity similar to that of cr(v ) generated in cys reactions . esr studies have found that the kinetics of the cr(v ) signal followed that of the cr(vi ) reduction in cys - driven reactions , whereas the initial cr(v ) accumulation was delayed relative to the loss of chromate absorbance in gsh reactions . consistent with a greater stability of cr(v ) complexed with gsh , the cr(v ) signal remained steady for the duration of the reduction of chromate by gsh but quickly disappeared in cys reactions . since dhr123 appeared to be sensitive to the presence of various of cr(v ) forms ( acting as a sensor of total crv ) , we tested its responsiveness in time - course studies of cr(vi ) reduction by both thiols . we found that the kinetics of dhr123 oxidation and cr(vi ) reduction were essentially identical in cys reactions ( figure 5e ) , indicating a rapid formation of cr(v ) and its fast reactivity with dhr123 . to better understand the potential applications of dcf and dhr123 in the monitoring of cr(vi ) metabolism in human cells , we first examined the fluorescence of these dyes in mixtures of all three biological reducers at their physiological concentrations . similar to the gsh - alone reactions ( figure 1 ) , the overall fluorescence responses for dcf were much lower in comparison to those of dhr123 , which reflected a dominant role of gsh in cr(vi ) reduction when it was present at 10-fold molar excess over cys ( figure 6c ) . the addition of asc to gsh - cys mixtures completely abrogated the oxidation of both probes , which can be attributed to the overwhelming role of asc in cr(vi ) metabolism due to its dramatically faster reduction rates ( figure 1a ) and the absence of the cr(v ) intermediate under the employed toxicologically relevant ratio of asc to cr(vi ) . in the final series of in vitro experiments , we also examined the impact of asc on the oxidation of dhe in thiol - containing cr(vi ) reactions . as for dcf and dhr123 , we found that the presence of asc blocked dhe oxidation during cr(vi ) reduction by thiols ( figure 6d ) , suggesting that cr(v ) was also likely responsible for the fluorescence of this probe in cys / gsh - containing reactions . ( d ) dhe fluorescence in cr(vi ) reactions with 2 mm cys ( cys label ) , 2 mm cys and 2 mm asc ( cys + asc label ) , or 2 mm gsh , cys , and asc ( gsh / cys + asc label ) . on the basis of the equally robust responsiveness of dhr123 to the formation of cr(v ) in reactions with cys and gsh , we decided to test this dye for the monitoring of cr(vi ) metabolism in h460 human lung epithelial cells . to assess the role of gsh , cells were treated with the glutathione synthase inhibitor bso for 24 h , which lowered cellular gsh concentrations by > 100-fold , from 2.9 0.2 mm to 26 11 m . in gsh - depleted cells , the amount of oxidized dhr123 was lower at the early time points but continued to rise up to 60 min after cr(vi ) removal , indicating slower reduction rates of cr(vi ) in the absence of the predominant cellular thiol . when cellular concentrations of asc were raised to 0.7 mm by preincubation with 1 mm dehydroascorbic acid , we found no changes in either dhr123 or dcf fluorescence at any time point after 30 m cr(vi ) treatment ( figure 7b ) . no significant changes in the fluorescence of both dyes were also detected in asc - restored cells treated with three other doses of cr(vi ) ( 10 , 20 , and 40 m , data not shown ) . control and asc - supplemented cells showed essentially the same accumulation of cr(vi ) ( figure 7c ) , indicating that a different spectrum of cr metabolites was responsible for the lack of oxidized dhr123 in the presence of asc . the unchanged fluorescence of both redox probes in asc - normalized cells is in agreement with the observed absence of dhr123- or dcf - oxidizing species during in vitro reduction of cr(vi ) with an excess of asc . in human a549 cells , preloading with 80 m asc stimulated dcf and dhr123 fluorescence by cr(vi ) , which is consistent with the formation of cr(v ) and oxidation of both dyes in our low - asc reactions ( 100 m asc ) . we found that among seven tested oxidant - sensitive dyes , only dcf and dhr123 showed strongly increased fluorescence ( up to 100-fold over background ) in reactions containing cr(vi ) and its biological reducers gsh and cys . fluorescence of the superoxide probe dhe was also elevated in these reactions , although the overall magnitude was very modest , with a maximal 2.5-fold increase for the highest cr(vi ) doses reduced with cys . the nonresponsiveness of apf which primarily reacts with oh radicals and the absence of any effect by the radical scavenger ethanol further argue against oh radicals as the cause of dcf and dhr123 fluorescence in cr(vi)-thiol reactions . we found no dcf or dhr123 ( or dhe ) oxidation in cr(vi ) reactions with a more than 10-fold excess of the two - electron reducer ascorbate , which lacked a detectable formation of cr(v ) . in the case of the dcf probe , its oxidation in gsh samples was about 4-times lower relative to cys , pointing to a different reactivity of the main cr(v ) species produced by these two thiols . a commonly invoked reduction mechanism by gsh involves the initial formation of the cr(vi)-thiolate complex followed by binding of a second gsh molecule leading to one - electron transfer and the production of the cr(v)-gsh complex . however , whereas a relatively stable cr(v)-gsh complex has been readily isolated from in vitro reactions and detected in cr(vi)-treated human a549 cells , efforts to identify the cr(v)-cys complex have proven unsuccessful . studies on the mechanism of cr(vi ) reduction by gsh have been performed with the supraphysiological concentrations of this reducer , which would favor coordination and reduction of the cr(vi)-thiolate complex by a second gsh molecule . at physiological concentrations , the rate of ternary complex formation is expected to be greatly diminished , which allows a fraction of the initially formed cr(vi)-gsh thiolates to undergo reduction yielding free cr(v ) and the thyil radical . taken together , the presented mechanistic considerations of cr(vi ) reduction and our findings on the average 4.1-fold difference between dcf and dhr112 responses indicate that cr(vi ) metabolism by a physiological 2 mm gsh concentration generated approximately 75% cr(v ) in the form of cr(v)-gsh , which is reactive with dhr123 but not dcf , and 25% free cr(v ) , which is reactive with both dhr123 and dcf . lower yields and instability of free cr(v ) complicate its detection by esr spectroscopy at biologically relevant conditions , although esr studies of some cr(vi)-gsh reactions have previously detected a cr(v ) product with a different g - value than that of cr(v)-gsh . similar to our results with cr(v)-gsh , the cr(v)-mannitol complex was able to oxidize dhr123 but not dcf , pointing to a general trend for the nonreactivity of dcf with cr(v ) stabilized by biological ligands . cr(v ) species are weak oxidants , and the variable responses of the tested probes in cr(vi ) reactions can be related to a different susceptibility of target groups to oxidation reactions that are required for the formation of fluorescent products . both dyes are initially oxidized via the abstraction of the hydrogen atom at the 9-position , whose location at the central carbon of a triphenylmethane makes it vulnerable to weakly oxidizing species such as cr(v ) . a conversion of apf into fluorescein requires o - dearylation , which is initiated by the abstraction of one electron from the nitrogen atom , and only strongly oxidizing species such as the oh radical have a sufficient reactivity for this reaction . a similar argument about the requirement for a higher oxidizing power than that provided by cr(v ) to cause oxidation of an aromatic amine moiety can be made to explain a very weak responsiveness of dhe in cr(vi ) reactions . our findings extend the earlier observations with synthetic cr complexes that intermediate cr forms , which we showed here to be cr(v ) in biological systems , can oxidize dcf and dhr123 . the concern about the non - ros origins of dcf / dhr123 oxidants is particularly important for standard cell cultures in which asc deficiency leads to one - electron reduction of cr(vi ) by thiols . although cr(vi ) treatments of human cells with restored asc levels did not induce a detectable production of oxidants in this work or biologically significant dna oxidation , coexposure with other redox - active toxicants or phagocytosis of chromate - containing particles can result in elevated ros through chemical or biological mechanisms , respectively . a summary of the reactivity of different dyes and their applicability to the detection of specific oxidants in cr(vi ) reactions is shown in figure 8 .

female nod / ltj and nod.scid mice were obtained from the jackson laboratory ( bar harbor , me ) and maintained under pathogen - free conditions . nod mice were screened for hyperglycemia at 12 weeks of age and diagnosed with diabetes when two consecutive glucose levels were > 200 mg / dl ( 2 ) . glucose levels were measured in whole blood from the tail vein using a glucometer elite xl ( bayer , elkhart , in ) . prediabetic nod mice were treated with intradermal phloridzin ( phz ) injections at 0.4 mg / kg per day for 14 days ( 15 ) . glycosuria induced by phz was detected for as long as 9 h after the injection . diabetic nod mice were treated with 10 g / day intraperitoneally ( i.p . ) of the mab 145 - 2c11 ( anti - mouse cd3 ) for 5 days beginning at the onset of hyperglycemia , as previously described ( 2 ) . reversal of diabetes was defined when random glucose levels were < 200 mg / dl for 2 weeks after diagnosis and persisted for > 10 days . glucose was injected i.p . at a dose of 4 g / kg per day . anti - cd3treated mice with reversed hyperglycemia for at least 10 days received daily injections of glucose or vehicle for 14 days . on day 14 , mice were subjected to an intraperitoneal glucose tolerance test ( ipgtt ) , and sera and pancreata were collected for additional analysis . in a separate set of experiments , nondiabetic 12- to 14-week - old nod.scid mice and 8- to 10-week - old nod mice were subjected to 14 days of either glucose or vehicle treatment . whole - blood glucose levels were measured from the tail vein taken before and 15 , 30 , 60 , and , in some experiments , 120 min after the injection . after fixation , pancreatic tissue was placed in a sucrose gradient and snap frozen in liquid nitrogen . noncontiguous 14-m pancreatic sections were stained with antibodies to insulin ( invitrogen , carlsbad , ca ) , 4,6-diamidine-2-phenylindole dihydrochloride ( dapi ) , cd31 ( bd biosciences , san jose , ca ) , and either ki67 or vegf ( abcam , cambridge , ma ) . the bound antibodies were detected by immunofluorescent secondary antibodies ( jackson immunoresearch , west grove , pa ) . for whole - tissue sections , 5 images were captured using mosaix software ( carl zeiss , thornwood , ny ) . numbers of single - color and dual - color labeled cells were counted using functions in imagej ( colocalization , watershed , and analyze particles ) . the total pancreatic section area and insulin cells were measure using imagej ( 16 ) . insulin concentration was measured on a continuous scale using automated quantitative analysis ( aqua ) , as has been previously described ( 17 ) . in brief , a series of high - resolution monochromatic images were captured using an automated microscope platform ( pm-2000tm ; historx , new haven , ct ) . fluorescent chromagens ( dapi , cy3-insulin ) were used to demarcate insulin cells within each whole - section image . dapi staining of cell nuclei was used to generate the nuclear compartment . subtracting the generated nuclear compartment from the masked cell area created a nonnuclear compartment , which included both the cytoplasm and the membrane . to generate an aqua score , each pixel was recorded on a scale of 0 ( black ) to 255 ( white ) , and pixel intensity was defined using the following equation : ( target pixel intensity ) ( compartment pixel intensity/255 ) . using the target and compartment pixel intensities , the following equation was generated : ( sum of all target pixel intensities)/(sum of all compartment pixel intensities/255 ) = the aqua score . all scores are normalized for image - exposure time and bit depth , allowing direct comparison of different histospot / whole - section scores . islets were isolated from nod or nod.scid mice after digestion with collagenase ( liberase ; roche , indianapolis , in ) ( 18 ) . islets were handpicked twice with an inverted microscope . to examine the effects of increasing glucose concentrations on vegf expression , islets were cultured in 10% fetal calf serum glucose - free rpmi ( mediatech , manassas , va ) supplemented with indicated glucose concentrations for 24 h. in some experiments , preconditioned islet culture media was collected and added to cultures of freshly isolated nod.scid islets . after incubation , islets were harvested and total rna was extracted using an rnaeasy kit according to the manufacturer s protocol ( qiagen , valencia , ca ) . purified islets from either nod or nod.scid donors were incubated with 5 or 10 mmol / l glucose for 7 days and then dispersed using trypsin digestion . we used bs-1 rather than cd31 for identification of endothelial cells because of cd31 sensitivity to trypsin digestion . examination of vegf-120 , vegf-164 , and vegf-188 levels was quantitated by real - time pcr using sybr green ( qiagen , valencia , ca ) on the iq-5 multicolor real - time pcr system ( bio - rad , hercules , ca ) . pcr primer pairs were as follows : gapdh : forward ctgcaccaccaactgcttag , reverse gatggcatggactgtggtcat ; and vegf : exon 3 common forward primer atcttcaagccgtcctgtgtgc , 120 reverse ttggcttgtcacatttttctgg , 164 reverse caaggctcacagtgattttctgg , and 188 reverse atcttcaagccgtcctgtgtgc . prediabetic nod mice were treated with intradermal phloridzin ( phz ) injections at 0.4 mg / kg per day for 14 days ( 15 ) . glycosuria induced by phz was detected for as long as 9 h after the injection . diabetic nod mice were treated with 10 g / day intraperitoneally ( i.p . ) of the mab 145 - 2c11 ( anti - mouse cd3 ) for 5 days beginning at the onset of hyperglycemia , as previously described ( 2 ) . reversal of diabetes was defined when random glucose levels were < 200 mg / dl for 2 weeks after diagnosis and persisted for > 10 days . glucose was injected i.p . at a dose of 4 g / kg per day . anti - cd3treated mice with reversed hyperglycemia for at least 10 days received daily injections of glucose or vehicle for 14 days . on day 14 , mice were subjected to an intraperitoneal glucose tolerance test ( ipgtt ) , and sera and pancreata were collected for additional analysis . in a separate set of experiments , nondiabetic 12- to 14-week - old nod.scid mice and 8- to 10-week - old nod mice were subjected to 14 days of either glucose or vehicle treatment . mice undergoing an ipgtt were fasted overnight . they received a 2 g / kg i.p . whole - blood glucose levels were measured from the tail vein taken before and 15 , 30 , 60 , and , in some experiments , 120 min after the injection . noncontiguous 14-m pancreatic sections were stained with antibodies to insulin ( invitrogen , carlsbad , ca ) , 4,6-diamidine-2-phenylindole dihydrochloride ( dapi ) , cd31 ( bd biosciences , san jose , ca ) , and either ki67 or vegf ( abcam , cambridge , ma ) . the bound antibodies were detected by immunofluorescent secondary antibodies ( jackson immunoresearch , west grove , pa ) . for whole - tissue sections , 5 images were captured using mosaix software ( carl zeiss , thornwood , ny ) . numbers of single - color and dual - color labeled cells were counted using functions in imagej ( colocalization , watershed , and analyze particles ) . the total pancreatic section area and insulin cells were measure using imagej ( 16 ) . insulin concentration was measured on a continuous scale using automated quantitative analysis ( aqua ) , as has been previously described ( 17 ) . in brief , a series of high - resolution monochromatic images were captured using an automated microscope platform ( pm-2000tm ; historx , new haven , ct ) . fluorescent chromagens ( dapi , cy3-insulin ) were used to demarcate insulin cells within each whole - section image . dapi staining of cell nuclei was used to generate the nuclear compartment . subtracting the generated nuclear compartment from the masked cell area created a nonnuclear compartment , which included both the cytoplasm and the membrane . to generate an aqua score , each pixel was recorded on a scale of 0 ( black ) to 255 ( white ) , and pixel intensity was defined using the following equation : ( target pixel intensity ) ( compartment pixel intensity/255 ) . using the target and compartment pixel intensities , the following equation was generated : ( sum of all target pixel intensities)/(sum of all compartment pixel intensities/255 ) = the aqua score . all scores are normalized for image - exposure time and bit depth , allowing direct comparison of different histospot / whole - section scores . islets were isolated from nod or nod.scid mice after digestion with collagenase ( liberase ; roche , indianapolis , in ) ( 18 ) . islets were handpicked twice with an inverted microscope . to examine the effects of increasing glucose concentrations on vegf expression , islets were cultured in 10% fetal calf serum glucose - free rpmi ( mediatech , manassas , va ) supplemented with indicated glucose concentrations for 24 h. in some experiments , preconditioned islet culture media was collected and added to cultures of freshly isolated nod.scid islets . after incubation , islets were harvested and total rna was extracted using an rnaeasy kit according to the manufacturer s protocol ( qiagen , valencia , ca ) . purified islets from either nod or nod.scid donors were incubated with 5 or 10 mmol / l glucose for 7 days and then dispersed using trypsin digestion . we used bs-1 rather than cd31 for identification of endothelial cells because of cd31 sensitivity to trypsin digestion . examination of vegf-120 , vegf-164 , and vegf-188 levels was quantitated by real - time pcr using sybr green ( qiagen , valencia , ca ) on the iq-5 multicolor real - time pcr system ( bio - rad , hercules , ca ) . pcr primer pairs were as follows : gapdh : forward ctgcaccaccaactgcttag , reverse gatggcatggactgtggtcat ; and vegf : exon 3 common forward primer atcttcaagccgtcctgtgtgc , 120 reverse ttggcttgtcacatttttctgg , 164 reverse caaggctcacagtgattttctgg , and 188 reverse atcttcaagccgtcctgtgtgc . examination of prediabetic mice showed impaired glucose tolerance in 13-week - old nod mice when compared with 9-week - old nod mice ( fig . 1a ; area under the curve at 9 weeks = 8,702 vs. at 13 weeks = 11,195 ; p < 0.05 ) . previous studies have described a decline in islet vascular area in nod mice during the progression of diabetes ( 9 ) , but the relationship between these changes and the impairment of glucose tolerance in nod mice has not been directly evaluated . we therefore studied the islet vascular area in prediabetic mice and after recovery from hyperglycemia in diabetic nod mice that were treated with anti - cd3 mabs . reduced cd31 area was evident as early as 9 weeks of age when compared with nonautoimmune nod.scid animals ( fig . although there was not a direct relationship between changes in glucose tolerance and islet vasculature during prediabetes , there was a sharp decline in cd31 area in the islets at the time of diabetes onset ( fig . anti - cd3treated diabetic nod mice fail to normalize their ipgtts and endothelial cell area in the islet . a : ipgtts of prediabetic mice at the ages of 9 , 11 , and 13 weeks ( n = 5 each group ) and after anti - cd3 treatment ( n = 12 ) . b : quantitative histomorphic analysis of cd31 in pancreatic sections ( n = 3 and four mice of islets from prediabetic , diabetic , and anti - cd3treated nod mice and n = 3 and four mice at each time point ) ( mean se ) . the cd31 area in the islets was compared with the same in nonautoimmune nod.scid mice by anova ( top line , p < 0.0001 ) and by the dunnett multiple comparison test ( * p < 0.05 ; * * * p < 0.001 ) and with mice with new - onset diabetes ( bottom line , anova p < 0.0001 and * * p < 0.01 ; * * * p < 0.001 ) . c : immunofluoresence staining samples of representative islets from 13-week - old nonautoimmune nod.scid mice , prediabetic nod mice , and nod mice with new - onset diabetes . blue , dapi ; red , insulin ; green , cd31 . ( a high - quality digital representation of this figure is available in the online issue . ) next , we treated newly diabetic nod mice with anti - cd3 mabs and found that the nonfasting glucose levels returned to nondiabetic levels of < 200 mg / dl for a period > 2 consecutive weeks ( 154.9 8.2 mg / dl ) . however , when challenged with an ipgtt , these mice showed glucose intolerance when compared with 9-week - old nod mice ( fig . 1a ; area under the curve at 9 weeks = 8,702 vs. anti - cd3treated = 16,578 ; p < 0.0001 ) . in addition , the endothelial cell density was not normalized after anti - cd3 mab therapy and remained significantly reduced when compared with nod.scid or 9-week - old nod mice ( fig . these results show a loss of endothelial cells in the islet during prediabetes that is concomitant with deterioration of glucose tolerance , both of which are not fully corrected following anti - cd3 treatment . to determine the relationship between changes in glucose and the cd31 cell density , we treated prediabetic nod mice with normal ( 8 weeks old ) or impaired ( 12 weeks old ) results from the ipgtt with daily injections of phz for a period of 14 days . first , we tested phz effects on glucose load in normoglycemic balb / c during the ipgtt . phz injection increased clearance of blood glucose , as indicated by a more rapid return to baseline glucose levels ( supplementary fig . daily phz injections of 12-week - old nod mice reduced endothelial cell density when compared with vehicle - treated mice ( fig . interestingly , phz also decreased the rates of -cell proliferation ( table 1 and supplementary fig . 3 ) , which is consistent with the previously described effects of glucose on -cell proliferation in prediabetic mice ( 13 ) . in contrast , phz treatment of 8-week - old nod mice , with normal ipgtt results , did not reduce endothelial cell density ( fig . histomorphic analysis of pancreata ( n 3 per group ) cd31 area in 8-week - old ( a ) and 12-week - old ( b ) prediabetic nod mice after daily injections of phz for a period of 14 days ( * p < 0.04 ) . c : endothelial cell area in diabetic and anti - cd3treated nod mice receiving either vehicle or glucose injections for 14 days . d : as in c but with immunodeficient nod.scid mice . * p < 0.05 ; * * p < 0.001 . proliferation levels in islets of prediabetic and anti - cd3treated mice data are means se . we also tested whether an increase in glucose in mice with impaired glucose tolerance would have the opposite effect . recovered anti - cd3treated nod mice showing a minimum of 10 consecutive days of basal euglycemia received daily injections of glucose ( 4 mg / kg i.p . ) for 14 days . glucose injections significantly increased endothelial cell density to a density similar in prediabetic nod mice when compared with vehicle - treated controls ( fig . in contrast , similar treatment of nod.scid mice with daily glucose injections failed to increase endothelial cell density in islets ( fig . these data suggest that fluctuation in daily glucose may modulate endothelial cell density under the conditions of islet inflammation . next , we tested the physiological consequences of increased endothelial cell density in glucose - treated anti - cd3treated nod mice . interestingly , despite the presence of impaired glucose tolerance before treatment , glucose - treated anti - cd3 mice showed improved ipgtt results when compared with vehicle - treated mice ( fig . 3a and b ; p < 0.05 ) . fasting insulin levels in glucose - treated mice were higher than in placebo - treated mice ( vehicle = 0.61 ng / ml vs. glucose = 0.90 ng / ml ; p < 0.06 ) ; however , no increase in -cell mass was detected ( fig . 3c ) . therefore , to examine the effect of glucose on insulin content in individual -cells , we used the aqua score to measure the content of insulin . the aqua score has been validated for protein quantization in histological section of malignant tissues in which good reproducibility and linearity with protein content of tissues has been validated ( 1823 ) . aqua score analysis revealed increased levels of insulin staining per -cell in glucose - treated mice ( fig . glucose treatment of anti - cd3treated mice did not increase -cell proliferation , albeit proliferation was at relatively high levels in both vehicle- and glucose - treated mice ( table 1).these results suggest that increased islet endothelial cell density may promote -cell function by increasing insulin content rather than -cell proliferation after treatment with anti - cd3 antibodies . daily glucose injections improve glucose tolerance and increase insulin content in anti - cd3treated nod mice . a : representative ipgtts of anti - cd3treated mice with normal basal glucose levels receiving daily injections of either vehicle ( left panel ) or glucose ( right panel ) intraperitoneally ( , pretreatment ; , posttreatment ) . b : summary of changes in ipgtt results of anti - cd3treated diabetic mice with normal basal glucose levels after a 14-day challenge of daily injections of either glucose or vehicle . * p < 0.04 ( n = 12 per group ) . stained pancreatic section of vehicle- and glucose - treated mice for insulin cells . d : aqua scores of insulin levels per -cell as described in research design and methods . * p < 0.03 . ins , insulin . to understand the basis for increased endothelial cell density after glucose treatment , we measured vegf production in the islets of glucose - treated anti - cd3treated mice by histomorphic analysis . preliminary immunofluorescence staining revealed an increase in vegf levels in prediabetic nod islets compared with nod.scid islets ( supplementary fig . , daily glucose injections resulted in increased vegf expression in the islets when compared with vehicle - treated mice ( fig . 4a ; p < 0.02 ) , suggesting that vegf production by resident or infiltrating islet cells can promote islets in response to glucose . to directly examine the effects of glucose on endothelial cell numbers , we incubated purified islets from either nod or nod.scid donors at either 5 mmol / l or 10 mmol / l glucose for 7 days followed by fluorescence - activated cell sorter analysis of bs-1 endothelial cells . nod islets incubated at 10 mmol / l glucose showed better maintenance of endothelial cells when compared with islets incubated at 5 mmol , high glucose levels failed to maintain endothelial cell levels in islets derived from nod / scid mice when compared with islets incubated at 5 mmol 4b ) . quantitative pcr analysis of purified endothelial cells from nod islets cultured at 10 mmol / l glucose showed a 4.2-fold increase in the expression of the antiapoptotic bcl - xl gene ( cycle difference from gapdh : nod = 1.87 0.73 vs. nod.scid = 0.44 0.07 ; p = 0.10 ) , whereas ki67 gene expression remained unchanged ( data not shown ) . high glucose levels stimulate vegf expression under the conditions of islet inflammation . a : histomorphic analysis of islets from the pancreatic section of vehicle- or glucose - treated anti - cd3treated nod mice ( p < 0.02 ) . b : purified islets from 8- to 10-week - old nod or nod.scid mice were incubated in either 5 or 10 mmol / l glucose for 7 days . after incubation , islets were dispersed and endothelial cell frequency was analyzed using fluorescence - activated cell sorter of bs-1 cells as described in research design and methods . left panel : a representative histogram depicting a sample of two nod or nod.scid islet cultures stained for bs-1 . right panel : summary of the percentage of bs-1 cells after culture in 5 or 10 mmol / l glucose . c : purified islets from 8- to 10-week - old nod ( left ) or nod.scid ( right ) mice as in b were incubated with increasing concentrations of glucose . quantitative rt - pcr was done for vegf-120 and vegf-164 isoforms in the islets ( * p < 0.05 ) . n 3 per group . because vegf - a is the main regulator of endothelial cell maintenance , we tested whether increased glucose had a differential effect on vegf production by islets from nod and nod.scid mice ex vivo . purified islets from 8- to 10-week - old mice were incubated with increasing concentrations of glucose for 24 h followed by mrna extraction . rt - pcr analysis of vegf-120 , vegf-164 , and vegf-188 mrna isoforms revealed a fivefold increase in vegf-120 and vegf-164 production in the islets of nod mice , whereas vegf-188 was undetectable ( fig . this increase was evident at glucose concentrations of 5 , 10 , 20 , and 40 mmol / l ( fig . in contrast , hyperglycemia did not induce vegf production in cultured islets form nod.scid mice ( fig . these findings were consistent with the lack of an effect of glucose on vascular area in nod.scid mice treated with glucose but left open the possibility that during inflammation , islets could acquire the ability to produce vegf after direct immune injury or in response to soluble factors produced by the infiltrating cells . to test this , we cultured nod.scid islets with supernatants collected after a 72-h incubation of nod islets in 10 mmol / l glucose and measured vegf production by real - time pcr . there was a significant increase in vegf-120 and vegf-164 after incubation with islets from nod but not control nod.scid mice ( fig . this increase was more pronounced in the vegf-164 isoform , suggesting that the vegf-120 isoform may derive from a non - islet resident cell ( fig . these data suggest that inflammation can promote the production of vegf - a in the islet by a yet - unidentified soluble factor . examination of prediabetic mice showed impaired glucose tolerance in 13-week - old nod mice when compared with 9-week - old nod mice ( fig . 1a ; area under the curve at 9 weeks = 8,702 vs. at 13 weeks = 11,195 ; p < 0.05 ) . previous studies have described a decline in islet vascular area in nod mice during the progression of diabetes ( 9 ) , but the relationship between these changes and the impairment of glucose tolerance in nod mice has not been directly evaluated . we therefore studied the islet vascular area in prediabetic mice and after recovery from hyperglycemia in diabetic nod mice that were treated with anti - cd3 mabs . reduced cd31 area was evident as early as 9 weeks of age when compared with nonautoimmune nod.scid animals ( fig . although there was not a direct relationship between changes in glucose tolerance and islet vasculature during prediabetes , there was a sharp decline in cd31 area in the islets at the time of diabetes onset ( fig . anti - cd3treated diabetic nod mice fail to normalize their ipgtts and endothelial cell area in the islet . a : ipgtts of prediabetic mice at the ages of 9 , 11 , and 13 weeks ( n = 5 each group ) and after anti - cd3 treatment ( n = 12 ) . b : quantitative histomorphic analysis of cd31 in pancreatic sections ( n = 3 and four mice of islets from prediabetic , diabetic , and anti - cd3treated nod mice and n = 3 and four mice at each time point ) ( mean se ) . the cd31 area in the islets was compared with the same in nonautoimmune nod.scid mice by anova ( top line , p < 0.0001 ) and by the dunnett multiple comparison test ( * p < 0.05 ; * * * p < 0.001 ) and with mice with new - onset diabetes ( bottom line , anova p < 0.0001 and * * p < 0.01 ; * * * p < 0.001 ) . c : immunofluoresence staining samples of representative islets from 13-week - old nonautoimmune nod.scid mice , prediabetic nod mice , and nod mice with new - onset diabetes . blue , dapi ; red , insulin ; green , cd31 . ( a high - quality digital representation of this figure is available in the online issue . ) next , we treated newly diabetic nod mice with anti - cd3 mabs and found that the nonfasting glucose levels returned to nondiabetic levels of < 200 mg / dl for a period > 2 consecutive weeks ( 154.9 8.2 mg / dl ) . however , when challenged with an ipgtt , these mice showed glucose intolerance when compared with 9-week - old nod mice ( fig . 1a ; area under the curve at 9 weeks = 8,702 vs. anti - cd3treated = 16,578 ; p < 0.0001 ) . in addition , the endothelial cell density was not normalized after anti - cd3 mab therapy and remained significantly reduced when compared with nod.scid or 9-week - old nod mice ( fig . these results show a loss of endothelial cells in the islet during prediabetes that is concomitant with deterioration of glucose tolerance , both of which are not fully corrected following anti - cd3 treatment . to determine the relationship between changes in glucose and the cd31 cell density , we treated prediabetic nod mice with normal ( 8 weeks old ) or impaired ( 12 weeks old ) results from the ipgtt with daily injections of phz for a period of 14 days . first , we tested phz effects on glucose load in normoglycemic balb / c during the ipgtt . phz injection increased clearance of blood glucose , as indicated by a more rapid return to baseline glucose levels ( supplementary fig . daily phz injections of 12-week - old nod mice reduced endothelial cell density when compared with vehicle - treated mice ( fig . interestingly , phz also decreased the rates of -cell proliferation ( table 1 and supplementary fig . 3 ) , which is consistent with the previously described effects of glucose on -cell proliferation in prediabetic mice ( 13 ) . in contrast , phz treatment of 8-week - old nod mice , with normal ipgtt results , did not reduce endothelial cell density ( fig . histomorphic analysis of pancreata ( n 3 per group ) cd31 area in 8-week - old ( a ) and 12-week - old ( b ) prediabetic nod mice after daily injections of phz for a period of 14 days ( * p < 0.04 ) . c : endothelial cell area in diabetic and anti - cd3treated nod mice receiving either vehicle or glucose injections for 14 days . d : as in c but with immunodeficient nod.scid mice . * p < 0.05 ; * * p < 0.001 . proliferation levels in islets of prediabetic and anti - cd3treated mice data are means se . we also tested whether an increase in glucose in mice with impaired glucose tolerance would have the opposite effect . recovered anti - cd3treated nod mice showing a minimum of 10 consecutive days of basal euglycemia received daily injections of glucose ( 4 mg / kg i.p . ) for 14 days . glucose injections significantly increased endothelial cell density to a density similar in prediabetic nod mice when compared with vehicle - treated controls ( fig . in contrast , similar treatment of nod.scid mice with daily glucose injections failed to increase endothelial cell density in islets ( fig . these data suggest that fluctuation in daily glucose may modulate endothelial cell density under the conditions of islet inflammation . next , we tested the physiological consequences of increased endothelial cell density in glucose - treated anti - cd3treated nod mice . interestingly , despite the presence of impaired glucose tolerance before treatment , glucose - treated anti - cd3 mice showed improved ipgtt results when compared with vehicle - treated mice ( fig . 3a and b ; p < 0.05 ) . fasting insulin levels in glucose - treated mice were higher than in placebo - treated mice ( vehicle = 0.61 ng / ml vs. glucose = 0.90 ng / ml ; p < 0.06 ) ; however , no increase in -cell mass was detected ( fig . 3c ) . therefore , to examine the effect of glucose on insulin content in individual -cells , we used the aqua score to measure the content of insulin . the aqua score has been validated for protein quantization in histological section of malignant tissues in which good reproducibility and linearity with protein content of tissues has been validated ( 1823 ) . aqua score analysis revealed increased levels of insulin staining per -cell in glucose - treated mice ( fig . glucose treatment of anti - cd3treated mice did not increase -cell proliferation , albeit proliferation was at relatively high levels in both vehicle- and glucose - treated mice ( table 1).these results suggest that increased islet endothelial cell density may promote -cell function by increasing insulin content rather than -cell proliferation after treatment with anti - cd3 antibodies . daily glucose injections improve glucose tolerance and increase insulin content in anti - cd3treated nod mice . a : representative ipgtts of anti - cd3treated mice with normal basal glucose levels receiving daily injections of either vehicle ( left panel ) or glucose ( right panel ) intraperitoneally ( , pretreatment ; , posttreatment ) . b : summary of changes in ipgtt results of anti - cd3treated diabetic mice with normal basal glucose levels after a 14-day challenge of daily injections of either glucose or vehicle . * p < 0.04 ( n = 12 per group ) . stained pancreatic section of vehicle- and glucose - treated mice for insulin cells . d : aqua scores of insulin levels per -cell as described in research design and methods . * p < 0.03 . ins , insulin . to understand the basis for increased endothelial cell density after glucose treatment , we measured vegf production in the islets of glucose - treated anti - cd3treated mice by histomorphic analysis . preliminary immunofluorescence staining revealed an increase in vegf levels in prediabetic nod islets compared with nod.scid islets ( supplementary fig . , daily glucose injections resulted in increased vegf expression in the islets when compared with vehicle - treated mice ( fig . 4a ; p < 0.02 ) , suggesting that vegf production by resident or infiltrating islet cells can promote islets in response to glucose . to directly examine the effects of glucose on endothelial cell numbers , we incubated purified islets from either nod or nod.scid donors at either 5 mmol / l or 10 mmol / l glucose for 7 days followed by fluorescence - activated cell sorter analysis of bs-1 endothelial cells . nod islets incubated at 10 mmol / l glucose showed better maintenance of endothelial cells when compared with islets incubated at 5 mmol in contrast , high glucose levels failed to maintain endothelial cell levels in islets derived from nod / scid mice when compared with islets incubated at 5 mmol 4b ) . quantitative pcr analysis of purified endothelial cells from nod islets cultured at 10 mmol / l glucose showed a 4.2-fold increase in the expression of the antiapoptotic bcl - xl gene ( cycle difference from gapdh : nod = 1.87 0.73 vs. nod.scid = 0.44 0.07 ; p = 0.10 ) , whereas ki67 gene expression remained unchanged ( data not shown ) . high glucose levels stimulate vegf expression under the conditions of islet inflammation . a : histomorphic analysis of islets from the pancreatic section of vehicle- or glucose - treated anti - cd3treated nod mice ( p < 0.02 ) . b : purified islets from 8- to 10-week - old nod or nod.scid mice were incubated in either 5 or 10 mmol / l glucose for 7 days . after incubation , islets were dispersed and endothelial cell frequency was analyzed using fluorescence - activated cell sorter of bs-1 cells as described in research design and methods . left panel : a representative histogram depicting a sample of two nod or nod.scid islet cultures stained for bs-1 . right panel : summary of the percentage of bs-1 cells after culture in 5 or 10 mmol / l glucose . c : purified islets from 8- to 10-week - old nod ( left ) or nod.scid ( right ) mice as in b were incubated with increasing concentrations of glucose . d : supernatants from nod or nod.scid islets incubated at 10 mmol / l glucose for 72 h were added to freshly purified nod.scid islets . quantitative rt - pcr was done for vegf-120 and vegf-164 isoforms in the islets ( * p < 0.05 ) . n 3 per group . because vegf - a is the main regulator of endothelial cell maintenance , we tested whether increased glucose had a differential effect on vegf production by islets from nod and nod.scid mice ex vivo . purified islets from 8- to 10-week - old mice were incubated with increasing concentrations of glucose for 24 h followed by mrna extraction . rt - pcr analysis of vegf-120 , vegf-164 , and vegf-188 mrna isoforms revealed a fivefold increase in vegf-120 and vegf-164 production in the islets of nod mice , whereas vegf-188 was undetectable ( fig . this increase was evident at glucose concentrations of 5 , 10 , 20 , and 40 mmol / l ( fig . , hyperglycemia did not induce vegf production in cultured islets form nod.scid mice ( fig . these findings were consistent with the lack of an effect of glucose on vascular area in nod.scid mice treated with glucose but left open the possibility that during inflammation , islets could acquire the ability to produce vegf after direct immune injury or in response to soluble factors produced by the infiltrating cells . to test this , we cultured nod.scid islets with supernatants collected after a 72-h incubation of nod islets in 10 mmol / l glucose and measured vegf production by real - time pcr . there was a significant increase in vegf-120 and vegf-164 after incubation with islets from nod but not control nod.scid mice ( fig . this increase was more pronounced in the vegf-164 isoform , suggesting that the vegf-120 isoform may derive from a non - islet resident cell ( fig . these data suggest that inflammation can promote the production of vegf - a in the islet by a yet - unidentified soluble factor . the improvement in insulin secretion that is seen after immune therapy in nod mice and most likely during the honeymoon of type 1 diabetes reflects the functional recovery of existing -cells , possibly with growth of new cells , but the factor(s ) that controls these responses is not well understood ( 3 ) . in this study , we have confirmed previous findings that in addition to -cells , the islet vasculature density is reduced prior to the presentation of diabetes and that a significant decrease in -cell mass and islet endothelial cells after presentation of diabetes in nod mice further promote glucose intolerance . moreover , treatment with the anti - inflammatory anti - cd3 mabs , which reverse frank hyperglycemia , fails to correct the impaired ipgtt results , which are associated with reduced cd31 cells . in addition , we have found that glucose is a regulator of islet vascular density both in the prediabetic period and after treatment of diabetic mice with anti - cd3 mabs , where glucose treatment augments cd31 density within the islets . this increase in islet vasculature occurs in conjunction with an increase in vegf production , resulting in enhanced insulin content and islet vasculature , but not -cell proliferation , in islets that have recovered or been spared destruction by the immune attack . the increase in endothelial cell density may be a result of decreased rates of cell death because the levels of the antiapoptotic gene bcl - xl were increased in inflamed endothelial cells under the condition of hyperglycemia . susceptibility to the effects of glucose is induced by soluble factor(s ) secreted by the inflamed islets in response to hyperglycemia . this soluble factor produced by inflamed islets controls vegf production by resident islet cells . the ongoing immunological assault on insulin - producing -cells in the islet during the progression of diabetes not only leads to the amplification of the immune response to islet antigens but also results in a gradual deterioration of glucose tolerance in prediabetic nod mice . this increase in metabolic demand imposes a new challenge for remaining -cells to compensate for increased glucose levels , as seen in situations of type 2 diabetes and during pregnancy ( 12 ) . our finding showing the ability of treatment with the glycosuric agent phz to reduce islet vasculature suggests that glucose levels may control the islet vasculature in the prediabetic state by increasing it with hyperglycemia and conversely by contracting it with reduced glucose load . the control of islet capillaries by glucose would be predicted because their unique structure , which consists of a basement membrane and fenestrations and is important for the control of glucose flow to the endocrine cells ( 25 ) . islet endothelial cells are highly sensitive to changes in glucose levels because increased glucose levels in gk rats can increase islet capillary blood pressure ( 26 ) . ( 8) have recently shown that islet blood flow is regulated by glucose in normal mice without insulitis . in their studies ( 8) , blood flow to the islet was dramatically increased after the hyperglycemic clamp . however , the effects of inflammation on imparting sensitivity of the islet vasculature to glucose had not been previously identified . in anti - cd3 mab treated mice , which show improved basal glucose levels but exhibit impaired glucose tolerance , islet vasculature improves modestly following anti - cd3 treatment . this recovery of endothelial cell density is enhanced by daily glucose injections , leading to improved glucose tolerance and increased insulin content per -cell . based on our previous studies ( 27 ) , it is likely that the improved islet vasculature increases the insulin content of recovered degranulated -cells , which accounts for the majority of -cell mass recovery after treatment with anti - cd3 ( 2 ) . inflammation has been suggested as a stimulant of -cell replication because -cell proliferation is increased in prediabetic nod mice and is highest at the time of the greatest -cell destruction ( 2,28 ) . in addition , the microvasculture is also dependent on inflammation and can increase by a variety of cytokines . these permissive roles of inflammation on -cell proliferation and islet vasculature are supported by our findings showing no increase in -cell replication and islet vasculature after glucose treatment of immunodeficient nod.scid mice . our new findings suggest that inflammation also affects the islet vasculature and that soluble products from inflamed islets render islet cells themselves responsive to glucose because nod.scid islets cultured with supernatants from nod islets acquired responsiveness of vegf production to glucose . one candidate cytokine capable of stimulating vegf production and vessel remodeling is tumor necrosis factor ( tnf)- ( 29 ) . the addition of tnf or its closely related family member , lymphotoxin , can lead to increased vegf production and stimulation of angiogenesis ( 30 ) . the ability of the islet endothelium to respond to increasing vegf levels may relate to increased expression of vegf receptors on endothelial cells during inflammation . the addition of tnf to endothelial cells in culture can indeed result in increased vegfr2 expression , leading to altered endothelial cell morphology and angiogenesis ( 31 ) , which , when combined with increased glucose levels , can lead to increased vasculature . the effects of the inflammatory response on the extracellular matrix ( ecm ) also must be considered . normal endothelial cell interactions are largely mediated via integrins , suggesting that inflammation may alter these conditions . recently , the role of matrix metalloproteinase-9 was documented in mediating the release of vegf , thus increasing the effective concentration of vegf-165 at the site of inflammation ( 32 ) . the breakdown of the ecm may further potentiate angiogenesis and endothelial cell survival under the conditions of inflammation ( 33 ) . based on our finding of different proportions of vegf isoforms produced in response to glucose by nod islets and nod.scid islets cultured with nod culture supernatants , we speculate that both the islet and immune cells are the sources of vegf in inflamed islets stimulated with glucose , and the possibility of ecm disturbance and increased accessibility of vegf to activated endothelial cells require additional investigation . our findings with phz are consistent with a recent report by pechhold et al . ( 13 ) in which islet transplantation or insulin treatment reduced -cell proliferation in nod mice . however , we were unable to detect an increase in -cell replication when we treated previously diabetic mice with glucose or even in prediabetic mice that were treated with glucose ( data not shown ) . one factor that may account for these discrepancies may be the way in which glucose was administered . in addition , the rates of -cell replication were already high in our prediabetic and postdiabetic mice treated with glucose , and , thus , it may not have been possible to increase the rates of replication further . our data suggest a novel role for glucose in the maintenance of islet vasculature during the progression of and recovery from diabetes and the effects of inflammation on imparting sensitivity to these effects . the factor(s ) that render the islet vasculature susceptible to glucose may identify a means of increasing -cell function and/or mass in settings where -cell mass is compromised .

objective-cell and islet endothelial cell destruction occurs during the progression of type 1 diabetes , but , paradoxically , -cell proliferation is increased during this period . altered glucose tolerance may affect -cell mass and its association with endothelial cells . our objective was to study the effects of glucose and inflammation on islet vascularity and on function , mass , and insulin in immunologically tolerant anti - cd3 monoclonal antibody ( mab)-treated and prediabetic nod mice.research design and methodsthe effects of phloridzin or glucose injections on -cells and endothelial cells were tested in prediabetic and previously diabetic nod mice treated with anti - cd3 mabs . glucose tolerance , immunofluorescence staining , and examination of islet cultures ex vivo were evaluated.resultsislet endothelial cell density decreased in nod mice and failed to recover after anti - cd3 mab treatment despite baseline euglycemia . glucose treatment of anti - cd3 mab treated mice showed increased islet vascular density and increased insulin content , which was associated with improved glucose tolerance . the increase in the vascular area was dependent on islet inflammation . increased islet endothelial cell density was associated with increased production of vascular endothelial growth factor ( vegf ) by islets from nod mice . this response was recapitulated ex vivo by the transfer of supernatants from nod islets cultured in high - glucose levels.conclusionsour results demonstrate a novel role for glucose and inflammation in the control of islet vasculature and insulin content of -cells in prediabetic and anti - cd3treated nod mice . vegf production by the islets is affected by glucose levels and is imparted by soluble factors released by inflamed islets .

RESEARCH DESIGN AND METHODS Treatment with phloridzin. Treatment with anti-CD3 mAbs. Treatment with glucose. IPGTT. Immunofluorescence. Image capture and automated quantitative analysis. Islet isolation. Studies of glucose effect on endothelial cell number. Real-time PCR. RESULTS Islet vasculature density is reduced in prediabetic NOD mice and fails to recover after anti-CD3 treatment. Variations in glucose load affect endothelial cell density in prediabetic and anti-CD3treated NOD mice. Increased endothelial cell density after a glucose load is associated with improved glucose tolerance and insulin content in anti-CD3treated NOD mice. DISCUSSION

previous studies have described a decline in islet vascular area in nod mice during the progression of diabetes ( 9 ) , but the relationship between these changes and the impairment of glucose tolerance in nod mice has not been directly evaluated . we therefore studied the islet vascular area in prediabetic mice and after recovery from hyperglycemia in diabetic nod mice that were treated with anti - cd3 mabs . although there was not a direct relationship between changes in glucose tolerance and islet vasculature during prediabetes , there was a sharp decline in cd31 area in the islets at the time of diabetes onset ( fig . anti - cd3treated diabetic nod mice fail to normalize their ipgtts and endothelial cell area in the islet . a : ipgtts of prediabetic mice at the ages of 9 , 11 , and 13 weeks ( n = 5 each group ) and after anti - cd3 treatment ( n = 12 ) . b : quantitative histomorphic analysis of cd31 in pancreatic sections ( n = 3 and four mice of islets from prediabetic , diabetic , and anti - cd3treated nod mice and n = 3 and four mice at each time point ) ( mean se ) . c : immunofluoresence staining samples of representative islets from 13-week - old nonautoimmune nod.scid mice , prediabetic nod mice , and nod mice with new - onset diabetes . next , we treated newly diabetic nod mice with anti - cd3 mabs and found that the nonfasting glucose levels returned to nondiabetic levels of < 200 mg / dl for a period > 2 consecutive weeks ( 154.9 8.2 mg / dl ) . in addition , the endothelial cell density was not normalized after anti - cd3 mab therapy and remained significantly reduced when compared with nod.scid or 9-week - old nod mice ( fig . these results show a loss of endothelial cells in the islet during prediabetes that is concomitant with deterioration of glucose tolerance , both of which are not fully corrected following anti - cd3 treatment . to determine the relationship between changes in glucose and the cd31 cell density , we treated prediabetic nod mice with normal ( 8 weeks old ) or impaired ( 12 weeks old ) results from the ipgtt with daily injections of phz for a period of 14 days . daily phz injections of 12-week - old nod mice reduced endothelial cell density when compared with vehicle - treated mice ( fig . 3 ) , which is consistent with the previously described effects of glucose on -cell proliferation in prediabetic mice ( 13 ) . in contrast , phz treatment of 8-week - old nod mice , with normal ipgtt results , did not reduce endothelial cell density ( fig . c : endothelial cell area in diabetic and anti - cd3treated nod mice receiving either vehicle or glucose injections for 14 days . proliferation levels in islets of prediabetic and anti - cd3treated mice data are means se . recovered anti - cd3treated nod mice showing a minimum of 10 consecutive days of basal euglycemia received daily injections of glucose ( 4 mg / kg i.p . ) glucose injections significantly increased endothelial cell density to a density similar in prediabetic nod mice when compared with vehicle - treated controls ( fig . in contrast , similar treatment of nod.scid mice with daily glucose injections failed to increase endothelial cell density in islets ( fig . these data suggest that fluctuation in daily glucose may modulate endothelial cell density under the conditions of islet inflammation . next , we tested the physiological consequences of increased endothelial cell density in glucose - treated anti - cd3treated nod mice . interestingly , despite the presence of impaired glucose tolerance before treatment , glucose - treated anti - cd3 mice showed improved ipgtt results when compared with vehicle - treated mice ( fig . glucose treatment of anti - cd3treated mice did not increase -cell proliferation , albeit proliferation was at relatively high levels in both vehicle- and glucose - treated mice ( table 1).these results suggest that increased islet endothelial cell density may promote -cell function by increasing insulin content rather than -cell proliferation after treatment with anti - cd3 antibodies . daily glucose injections improve glucose tolerance and increase insulin content in anti - cd3treated nod mice . a : representative ipgtts of anti - cd3treated mice with normal basal glucose levels receiving daily injections of either vehicle ( left panel ) or glucose ( right panel ) intraperitoneally ( , pretreatment ; , posttreatment ) . b : summary of changes in ipgtt results of anti - cd3treated diabetic mice with normal basal glucose levels after a 14-day challenge of daily injections of either glucose or vehicle . to understand the basis for increased endothelial cell density after glucose treatment , we measured vegf production in the islets of glucose - treated anti - cd3treated mice by histomorphic analysis . preliminary immunofluorescence staining revealed an increase in vegf levels in prediabetic nod islets compared with nod.scid islets ( supplementary fig . , daily glucose injections resulted in increased vegf expression in the islets when compared with vehicle - treated mice ( fig . to directly examine the effects of glucose on endothelial cell numbers , we incubated purified islets from either nod or nod.scid donors at either 5 mmol / l or 10 mmol / l glucose for 7 days followed by fluorescence - activated cell sorter analysis of bs-1 endothelial cells . nod islets incubated at 10 mmol / l glucose showed better maintenance of endothelial cells when compared with islets incubated at 5 mmol , high glucose levels failed to maintain endothelial cell levels in islets derived from nod / scid mice when compared with islets incubated at 5 mmol 4b ) . quantitative pcr analysis of purified endothelial cells from nod islets cultured at 10 mmol / l glucose showed a 4.2-fold increase in the expression of the antiapoptotic bcl - xl gene ( cycle difference from gapdh : nod = 1.87 0.73 vs. nod.scid = 0.44 0.07 ; p = 0.10 ) , whereas ki67 gene expression remained unchanged ( data not shown ) . a : histomorphic analysis of islets from the pancreatic section of vehicle- or glucose - treated anti - cd3treated nod mice ( p < 0.02 ) . after incubation , islets were dispersed and endothelial cell frequency was analyzed using fluorescence - activated cell sorter of bs-1 cells as described in research design and methods . because vegf - a is the main regulator of endothelial cell maintenance , we tested whether increased glucose had a differential effect on vegf production by islets from nod and nod.scid mice ex vivo . rt - pcr analysis of vegf-120 , vegf-164 , and vegf-188 mrna isoforms revealed a fivefold increase in vegf-120 and vegf-164 production in the islets of nod mice , whereas vegf-188 was undetectable ( fig . these findings were consistent with the lack of an effect of glucose on vascular area in nod.scid mice treated with glucose but left open the possibility that during inflammation , islets could acquire the ability to produce vegf after direct immune injury or in response to soluble factors produced by the infiltrating cells . to test this , we cultured nod.scid islets with supernatants collected after a 72-h incubation of nod islets in 10 mmol / l glucose and measured vegf production by real - time pcr . examination of prediabetic mice showed impaired glucose tolerance in 13-week - old nod mice when compared with 9-week - old nod mice ( fig . previous studies have described a decline in islet vascular area in nod mice during the progression of diabetes ( 9 ) , but the relationship between these changes and the impairment of glucose tolerance in nod mice has not been directly evaluated . we therefore studied the islet vascular area in prediabetic mice and after recovery from hyperglycemia in diabetic nod mice that were treated with anti - cd3 mabs . although there was not a direct relationship between changes in glucose tolerance and islet vasculature during prediabetes , there was a sharp decline in cd31 area in the islets at the time of diabetes onset ( fig . anti - cd3treated diabetic nod mice fail to normalize their ipgtts and endothelial cell area in the islet . b : quantitative histomorphic analysis of cd31 in pancreatic sections ( n = 3 and four mice of islets from prediabetic , diabetic , and anti - cd3treated nod mice and n = 3 and four mice at each time point ) ( mean se ) . next , we treated newly diabetic nod mice with anti - cd3 mabs and found that the nonfasting glucose levels returned to nondiabetic levels of < 200 mg / dl for a period > 2 consecutive weeks ( 154.9 8.2 mg / dl ) . in addition , the endothelial cell density was not normalized after anti - cd3 mab therapy and remained significantly reduced when compared with nod.scid or 9-week - old nod mice ( fig . these results show a loss of endothelial cells in the islet during prediabetes that is concomitant with deterioration of glucose tolerance , both of which are not fully corrected following anti - cd3 treatment . to determine the relationship between changes in glucose and the cd31 cell density , we treated prediabetic nod mice with normal ( 8 weeks old ) or impaired ( 12 weeks old ) results from the ipgtt with daily injections of phz for a period of 14 days . daily phz injections of 12-week - old nod mice reduced endothelial cell density when compared with vehicle - treated mice ( fig . 3 ) , which is consistent with the previously described effects of glucose on -cell proliferation in prediabetic mice ( 13 ) . in contrast , phz treatment of 8-week - old nod mice , with normal ipgtt results , did not reduce endothelial cell density ( fig . c : endothelial cell area in diabetic and anti - cd3treated nod mice receiving either vehicle or glucose injections for 14 days . proliferation levels in islets of prediabetic and anti - cd3treated mice data are means se . recovered anti - cd3treated nod mice showing a minimum of 10 consecutive days of basal euglycemia received daily injections of glucose ( 4 mg / kg i.p . ) glucose injections significantly increased endothelial cell density to a density similar in prediabetic nod mice when compared with vehicle - treated controls ( fig . in contrast , similar treatment of nod.scid mice with daily glucose injections failed to increase endothelial cell density in islets ( fig . these data suggest that fluctuation in daily glucose may modulate endothelial cell density under the conditions of islet inflammation . next , we tested the physiological consequences of increased endothelial cell density in glucose - treated anti - cd3treated nod mice . interestingly , despite the presence of impaired glucose tolerance before treatment , glucose - treated anti - cd3 mice showed improved ipgtt results when compared with vehicle - treated mice ( fig . glucose treatment of anti - cd3treated mice did not increase -cell proliferation , albeit proliferation was at relatively high levels in both vehicle- and glucose - treated mice ( table 1).these results suggest that increased islet endothelial cell density may promote -cell function by increasing insulin content rather than -cell proliferation after treatment with anti - cd3 antibodies . daily glucose injections improve glucose tolerance and increase insulin content in anti - cd3treated nod mice . a : representative ipgtts of anti - cd3treated mice with normal basal glucose levels receiving daily injections of either vehicle ( left panel ) or glucose ( right panel ) intraperitoneally ( , pretreatment ; , posttreatment ) . to understand the basis for increased endothelial cell density after glucose treatment , we measured vegf production in the islets of glucose - treated anti - cd3treated mice by histomorphic analysis . preliminary immunofluorescence staining revealed an increase in vegf levels in prediabetic nod islets compared with nod.scid islets ( supplementary fig . , daily glucose injections resulted in increased vegf expression in the islets when compared with vehicle - treated mice ( fig . to directly examine the effects of glucose on endothelial cell numbers , we incubated purified islets from either nod or nod.scid donors at either 5 mmol / l or 10 mmol / l glucose for 7 days followed by fluorescence - activated cell sorter analysis of bs-1 endothelial cells . nod islets incubated at 10 mmol / l glucose showed better maintenance of endothelial cells when compared with islets incubated at 5 mmol in contrast , high glucose levels failed to maintain endothelial cell levels in islets derived from nod / scid mice when compared with islets incubated at 5 mmol 4b ) . quantitative pcr analysis of purified endothelial cells from nod islets cultured at 10 mmol / l glucose showed a 4.2-fold increase in the expression of the antiapoptotic bcl - xl gene ( cycle difference from gapdh : nod = 1.87 0.73 vs. nod.scid = 0.44 0.07 ; p = 0.10 ) , whereas ki67 gene expression remained unchanged ( data not shown ) . a : histomorphic analysis of islets from the pancreatic section of vehicle- or glucose - treated anti - cd3treated nod mice ( p < 0.02 ) . after incubation , islets were dispersed and endothelial cell frequency was analyzed using fluorescence - activated cell sorter of bs-1 cells as described in research design and methods . because vegf - a is the main regulator of endothelial cell maintenance , we tested whether increased glucose had a differential effect on vegf production by islets from nod and nod.scid mice ex vivo . rt - pcr analysis of vegf-120 , vegf-164 , and vegf-188 mrna isoforms revealed a fivefold increase in vegf-120 and vegf-164 production in the islets of nod mice , whereas vegf-188 was undetectable ( fig . these findings were consistent with the lack of an effect of glucose on vascular area in nod.scid mice treated with glucose but left open the possibility that during inflammation , islets could acquire the ability to produce vegf after direct immune injury or in response to soluble factors produced by the infiltrating cells . to test this , we cultured nod.scid islets with supernatants collected after a 72-h incubation of nod islets in 10 mmol / l glucose and measured vegf production by real - time pcr . the improvement in insulin secretion that is seen after immune therapy in nod mice and most likely during the honeymoon of type 1 diabetes reflects the functional recovery of existing -cells , possibly with growth of new cells , but the factor(s ) that controls these responses is not well understood ( 3 ) . in this study , we have confirmed previous findings that in addition to -cells , the islet vasculature density is reduced prior to the presentation of diabetes and that a significant decrease in -cell mass and islet endothelial cells after presentation of diabetes in nod mice further promote glucose intolerance . moreover , treatment with the anti - inflammatory anti - cd3 mabs , which reverse frank hyperglycemia , fails to correct the impaired ipgtt results , which are associated with reduced cd31 cells . in addition , we have found that glucose is a regulator of islet vascular density both in the prediabetic period and after treatment of diabetic mice with anti - cd3 mabs , where glucose treatment augments cd31 density within the islets . this increase in islet vasculature occurs in conjunction with an increase in vegf production , resulting in enhanced insulin content and islet vasculature , but not -cell proliferation , in islets that have recovered or been spared destruction by the immune attack . the increase in endothelial cell density may be a result of decreased rates of cell death because the levels of the antiapoptotic gene bcl - xl were increased in inflamed endothelial cells under the condition of hyperglycemia . susceptibility to the effects of glucose is induced by soluble factor(s ) secreted by the inflamed islets in response to hyperglycemia . this soluble factor produced by inflamed islets controls vegf production by resident islet cells . the ongoing immunological assault on insulin - producing -cells in the islet during the progression of diabetes not only leads to the amplification of the immune response to islet antigens but also results in a gradual deterioration of glucose tolerance in prediabetic nod mice . the control of islet capillaries by glucose would be predicted because their unique structure , which consists of a basement membrane and fenestrations and is important for the control of glucose flow to the endocrine cells ( 25 ) . however , the effects of inflammation on imparting sensitivity of the islet vasculature to glucose had not been previously identified . in anti - cd3 mab treated mice , which show improved basal glucose levels but exhibit impaired glucose tolerance , islet vasculature improves modestly following anti - cd3 treatment . this recovery of endothelial cell density is enhanced by daily glucose injections , leading to improved glucose tolerance and increased insulin content per -cell . based on our previous studies ( 27 ) , it is likely that the improved islet vasculature increases the insulin content of recovered degranulated -cells , which accounts for the majority of -cell mass recovery after treatment with anti - cd3 ( 2 ) . inflammation has been suggested as a stimulant of -cell replication because -cell proliferation is increased in prediabetic nod mice and is highest at the time of the greatest -cell destruction ( 2,28 ) . these permissive roles of inflammation on -cell proliferation and islet vasculature are supported by our findings showing no increase in -cell replication and islet vasculature after glucose treatment of immunodeficient nod.scid mice . our new findings suggest that inflammation also affects the islet vasculature and that soluble products from inflamed islets render islet cells themselves responsive to glucose because nod.scid islets cultured with supernatants from nod islets acquired responsiveness of vegf production to glucose . the addition of tnf to endothelial cells in culture can indeed result in increased vegfr2 expression , leading to altered endothelial cell morphology and angiogenesis ( 31 ) , which , when combined with increased glucose levels , can lead to increased vasculature . based on our finding of different proportions of vegf isoforms produced in response to glucose by nod islets and nod.scid islets cultured with nod culture supernatants , we speculate that both the islet and immune cells are the sources of vegf in inflamed islets stimulated with glucose , and the possibility of ecm disturbance and increased accessibility of vegf to activated endothelial cells require additional investigation . however , we were unable to detect an increase in -cell replication when we treated previously diabetic mice with glucose or even in prediabetic mice that were treated with glucose ( data not shown ) . in addition , the rates of -cell replication were already high in our prediabetic and postdiabetic mice treated with glucose , and , thus , it may not have been possible to increase the rates of replication further . our data suggest a novel role for glucose in the maintenance of islet vasculature during the progression of and recovery from diabetes and the effects of inflammation on imparting sensitivity to these effects .

of the mab 145 - 2c11 ( anti - mouse cd3 ) for 5 days beginning at the onset of hyperglycemia , as previously described ( 2 ) . subtracting the generated nuclear compartment from the masked cell area created a nonnuclear compartment , which included both the cytoplasm and the membrane . to examine the effects of increasing glucose concentrations on vegf expression , islets were cultured in 10% fetal calf serum glucose - free rpmi ( mediatech , manassas , va ) supplemented with indicated glucose concentrations for 24 h. in some experiments , preconditioned islet culture media was collected and added to cultures of freshly isolated nod.scid islets . of the mab 145 - 2c11 ( anti - mouse cd3 ) for 5 days beginning at the onset of hyperglycemia , as previously described ( 2 ) . subtracting the generated nuclear compartment from the masked cell area created a nonnuclear compartment , which included both the cytoplasm and the membrane . to examine the effects of increasing glucose concentrations on vegf expression , islets were cultured in 10% fetal calf serum glucose - free rpmi ( mediatech , manassas , va ) supplemented with indicated glucose concentrations for 24 h. in some experiments , preconditioned islet culture media was collected and added to cultures of freshly isolated nod.scid islets . examination of prediabetic mice showed impaired glucose tolerance in 13-week - old nod mice when compared with 9-week - old nod mice ( fig . previous studies have described a decline in islet vascular area in nod mice during the progression of diabetes ( 9 ) , but the relationship between these changes and the impairment of glucose tolerance in nod mice has not been directly evaluated . we therefore studied the islet vascular area in prediabetic mice and after recovery from hyperglycemia in diabetic nod mice that were treated with anti - cd3 mabs . although there was not a direct relationship between changes in glucose tolerance and islet vasculature during prediabetes , there was a sharp decline in cd31 area in the islets at the time of diabetes onset ( fig . a : ipgtts of prediabetic mice at the ages of 9 , 11 , and 13 weeks ( n = 5 each group ) and after anti - cd3 treatment ( n = 12 ) . b : quantitative histomorphic analysis of cd31 in pancreatic sections ( n = 3 and four mice of islets from prediabetic , diabetic , and anti - cd3treated nod mice and n = 3 and four mice at each time point ) ( mean se ) . the cd31 area in the islets was compared with the same in nonautoimmune nod.scid mice by anova ( top line , p < 0.0001 ) and by the dunnett multiple comparison test ( * p < 0.05 ; * * * p < 0.001 ) and with mice with new - onset diabetes ( bottom line , anova p < 0.0001 and * * p < 0.01 ; * * * p < 0.001 ) . c : immunofluoresence staining samples of representative islets from 13-week - old nonautoimmune nod.scid mice , prediabetic nod mice , and nod mice with new - onset diabetes . next , we treated newly diabetic nod mice with anti - cd3 mabs and found that the nonfasting glucose levels returned to nondiabetic levels of < 200 mg / dl for a period > 2 consecutive weeks ( 154.9 8.2 mg / dl ) . in addition , the endothelial cell density was not normalized after anti - cd3 mab therapy and remained significantly reduced when compared with nod.scid or 9-week - old nod mice ( fig . these results show a loss of endothelial cells in the islet during prediabetes that is concomitant with deterioration of glucose tolerance , both of which are not fully corrected following anti - cd3 treatment . to determine the relationship between changes in glucose and the cd31 cell density , we treated prediabetic nod mice with normal ( 8 weeks old ) or impaired ( 12 weeks old ) results from the ipgtt with daily injections of phz for a period of 14 days . first , we tested phz effects on glucose load in normoglycemic balb / c during the ipgtt . 3 ) , which is consistent with the previously described effects of glucose on -cell proliferation in prediabetic mice ( 13 ) . histomorphic analysis of pancreata ( n 3 per group ) cd31 area in 8-week - old ( a ) and 12-week - old ( b ) prediabetic nod mice after daily injections of phz for a period of 14 days ( * p < 0.04 ) . glucose injections significantly increased endothelial cell density to a density similar in prediabetic nod mice when compared with vehicle - treated controls ( fig . in contrast , similar treatment of nod.scid mice with daily glucose injections failed to increase endothelial cell density in islets ( fig . next , we tested the physiological consequences of increased endothelial cell density in glucose - treated anti - cd3treated nod mice . interestingly , despite the presence of impaired glucose tolerance before treatment , glucose - treated anti - cd3 mice showed improved ipgtt results when compared with vehicle - treated mice ( fig . fasting insulin levels in glucose - treated mice were higher than in placebo - treated mice ( vehicle = 0.61 ng / ml vs. glucose = 0.90 ng / ml ; p < 0.06 ) ; however , no increase in -cell mass was detected ( fig . therefore , to examine the effect of glucose on insulin content in individual -cells , we used the aqua score to measure the content of insulin . glucose treatment of anti - cd3treated mice did not increase -cell proliferation , albeit proliferation was at relatively high levels in both vehicle- and glucose - treated mice ( table 1).these results suggest that increased islet endothelial cell density may promote -cell function by increasing insulin content rather than -cell proliferation after treatment with anti - cd3 antibodies . b : summary of changes in ipgtt results of anti - cd3treated diabetic mice with normal basal glucose levels after a 14-day challenge of daily injections of either glucose or vehicle . to understand the basis for increased endothelial cell density after glucose treatment , we measured vegf production in the islets of glucose - treated anti - cd3treated mice by histomorphic analysis . to directly examine the effects of glucose on endothelial cell numbers , we incubated purified islets from either nod or nod.scid donors at either 5 mmol / l or 10 mmol / l glucose for 7 days followed by fluorescence - activated cell sorter analysis of bs-1 endothelial cells . nod islets incubated at 10 mmol / l glucose showed better maintenance of endothelial cells when compared with islets incubated at 5 mmol , high glucose levels failed to maintain endothelial cell levels in islets derived from nod / scid mice when compared with islets incubated at 5 mmol 4b ) . quantitative pcr analysis of purified endothelial cells from nod islets cultured at 10 mmol / l glucose showed a 4.2-fold increase in the expression of the antiapoptotic bcl - xl gene ( cycle difference from gapdh : nod = 1.87 0.73 vs. nod.scid = 0.44 0.07 ; p = 0.10 ) , whereas ki67 gene expression remained unchanged ( data not shown ) . a : histomorphic analysis of islets from the pancreatic section of vehicle- or glucose - treated anti - cd3treated nod mice ( p < 0.02 ) . because vegf - a is the main regulator of endothelial cell maintenance , we tested whether increased glucose had a differential effect on vegf production by islets from nod and nod.scid mice ex vivo . rt - pcr analysis of vegf-120 , vegf-164 , and vegf-188 mrna isoforms revealed a fivefold increase in vegf-120 and vegf-164 production in the islets of nod mice , whereas vegf-188 was undetectable ( fig . these findings were consistent with the lack of an effect of glucose on vascular area in nod.scid mice treated with glucose but left open the possibility that during inflammation , islets could acquire the ability to produce vegf after direct immune injury or in response to soluble factors produced by the infiltrating cells . these data suggest that inflammation can promote the production of vegf - a in the islet by a yet - unidentified soluble factor . previous studies have described a decline in islet vascular area in nod mice during the progression of diabetes ( 9 ) , but the relationship between these changes and the impairment of glucose tolerance in nod mice has not been directly evaluated . we therefore studied the islet vascular area in prediabetic mice and after recovery from hyperglycemia in diabetic nod mice that were treated with anti - cd3 mabs . although there was not a direct relationship between changes in glucose tolerance and islet vasculature during prediabetes , there was a sharp decline in cd31 area in the islets at the time of diabetes onset ( fig . a : ipgtts of prediabetic mice at the ages of 9 , 11 , and 13 weeks ( n = 5 each group ) and after anti - cd3 treatment ( n = 12 ) . b : quantitative histomorphic analysis of cd31 in pancreatic sections ( n = 3 and four mice of islets from prediabetic , diabetic , and anti - cd3treated nod mice and n = 3 and four mice at each time point ) ( mean se ) . the cd31 area in the islets was compared with the same in nonautoimmune nod.scid mice by anova ( top line , p < 0.0001 ) and by the dunnett multiple comparison test ( * p < 0.05 ; * * * p < 0.001 ) and with mice with new - onset diabetes ( bottom line , anova p < 0.0001 and * * p < 0.01 ; * * * p < 0.001 ) . c : immunofluoresence staining samples of representative islets from 13-week - old nonautoimmune nod.scid mice , prediabetic nod mice , and nod mice with new - onset diabetes . next , we treated newly diabetic nod mice with anti - cd3 mabs and found that the nonfasting glucose levels returned to nondiabetic levels of < 200 mg / dl for a period > 2 consecutive weeks ( 154.9 8.2 mg / dl ) . in addition , the endothelial cell density was not normalized after anti - cd3 mab therapy and remained significantly reduced when compared with nod.scid or 9-week - old nod mice ( fig . these results show a loss of endothelial cells in the islet during prediabetes that is concomitant with deterioration of glucose tolerance , both of which are not fully corrected following anti - cd3 treatment . to determine the relationship between changes in glucose and the cd31 cell density , we treated prediabetic nod mice with normal ( 8 weeks old ) or impaired ( 12 weeks old ) results from the ipgtt with daily injections of phz for a period of 14 days . histomorphic analysis of pancreata ( n 3 per group ) cd31 area in 8-week - old ( a ) and 12-week - old ( b ) prediabetic nod mice after daily injections of phz for a period of 14 days ( * p < 0.04 ) . glucose injections significantly increased endothelial cell density to a density similar in prediabetic nod mice when compared with vehicle - treated controls ( fig . next , we tested the physiological consequences of increased endothelial cell density in glucose - treated anti - cd3treated nod mice . interestingly , despite the presence of impaired glucose tolerance before treatment , glucose - treated anti - cd3 mice showed improved ipgtt results when compared with vehicle - treated mice ( fig . fasting insulin levels in glucose - treated mice were higher than in placebo - treated mice ( vehicle = 0.61 ng / ml vs. glucose = 0.90 ng / ml ; p < 0.06 ) ; however , no increase in -cell mass was detected ( fig . therefore , to examine the effect of glucose on insulin content in individual -cells , we used the aqua score to measure the content of insulin . glucose treatment of anti - cd3treated mice did not increase -cell proliferation , albeit proliferation was at relatively high levels in both vehicle- and glucose - treated mice ( table 1).these results suggest that increased islet endothelial cell density may promote -cell function by increasing insulin content rather than -cell proliferation after treatment with anti - cd3 antibodies . b : summary of changes in ipgtt results of anti - cd3treated diabetic mice with normal basal glucose levels after a 14-day challenge of daily injections of either glucose or vehicle . to understand the basis for increased endothelial cell density after glucose treatment , we measured vegf production in the islets of glucose - treated anti - cd3treated mice by histomorphic analysis . to directly examine the effects of glucose on endothelial cell numbers , we incubated purified islets from either nod or nod.scid donors at either 5 mmol / l or 10 mmol / l glucose for 7 days followed by fluorescence - activated cell sorter analysis of bs-1 endothelial cells . nod islets incubated at 10 mmol / l glucose showed better maintenance of endothelial cells when compared with islets incubated at 5 mmol in contrast , high glucose levels failed to maintain endothelial cell levels in islets derived from nod / scid mice when compared with islets incubated at 5 mmol 4b ) . quantitative pcr analysis of purified endothelial cells from nod islets cultured at 10 mmol / l glucose showed a 4.2-fold increase in the expression of the antiapoptotic bcl - xl gene ( cycle difference from gapdh : nod = 1.87 0.73 vs. nod.scid = 0.44 0.07 ; p = 0.10 ) , whereas ki67 gene expression remained unchanged ( data not shown ) . a : histomorphic analysis of islets from the pancreatic section of vehicle- or glucose - treated anti - cd3treated nod mice ( p < 0.02 ) . because vegf - a is the main regulator of endothelial cell maintenance , we tested whether increased glucose had a differential effect on vegf production by islets from nod and nod.scid mice ex vivo . rt - pcr analysis of vegf-120 , vegf-164 , and vegf-188 mrna isoforms revealed a fivefold increase in vegf-120 and vegf-164 production in the islets of nod mice , whereas vegf-188 was undetectable ( fig . these findings were consistent with the lack of an effect of glucose on vascular area in nod.scid mice treated with glucose but left open the possibility that during inflammation , islets could acquire the ability to produce vegf after direct immune injury or in response to soluble factors produced by the infiltrating cells . the improvement in insulin secretion that is seen after immune therapy in nod mice and most likely during the honeymoon of type 1 diabetes reflects the functional recovery of existing -cells , possibly with growth of new cells , but the factor(s ) that controls these responses is not well understood ( 3 ) . in this study , we have confirmed previous findings that in addition to -cells , the islet vasculature density is reduced prior to the presentation of diabetes and that a significant decrease in -cell mass and islet endothelial cells after presentation of diabetes in nod mice further promote glucose intolerance . in addition , we have found that glucose is a regulator of islet vascular density both in the prediabetic period and after treatment of diabetic mice with anti - cd3 mabs , where glucose treatment augments cd31 density within the islets . this increase in islet vasculature occurs in conjunction with an increase in vegf production , resulting in enhanced insulin content and islet vasculature , but not -cell proliferation , in islets that have recovered or been spared destruction by the immune attack . the increase in endothelial cell density may be a result of decreased rates of cell death because the levels of the antiapoptotic gene bcl - xl were increased in inflamed endothelial cells under the condition of hyperglycemia . the ongoing immunological assault on insulin - producing -cells in the islet during the progression of diabetes not only leads to the amplification of the immune response to islet antigens but also results in a gradual deterioration of glucose tolerance in prediabetic nod mice . this increase in metabolic demand imposes a new challenge for remaining -cells to compensate for increased glucose levels , as seen in situations of type 2 diabetes and during pregnancy ( 12 ) . our finding showing the ability of treatment with the glycosuric agent phz to reduce islet vasculature suggests that glucose levels may control the islet vasculature in the prediabetic state by increasing it with hyperglycemia and conversely by contracting it with reduced glucose load . the control of islet capillaries by glucose would be predicted because their unique structure , which consists of a basement membrane and fenestrations and is important for the control of glucose flow to the endocrine cells ( 25 ) . based on our previous studies ( 27 ) , it is likely that the improved islet vasculature increases the insulin content of recovered degranulated -cells , which accounts for the majority of -cell mass recovery after treatment with anti - cd3 ( 2 ) . inflammation has been suggested as a stimulant of -cell replication because -cell proliferation is increased in prediabetic nod mice and is highest at the time of the greatest -cell destruction ( 2,28 ) . our new findings suggest that inflammation also affects the islet vasculature and that soluble products from inflamed islets render islet cells themselves responsive to glucose because nod.scid islets cultured with supernatants from nod islets acquired responsiveness of vegf production to glucose . the addition of tnf to endothelial cells in culture can indeed result in increased vegfr2 expression , leading to altered endothelial cell morphology and angiogenesis ( 31 ) , which , when combined with increased glucose levels , can lead to increased vasculature . recently , the role of matrix metalloproteinase-9 was documented in mediating the release of vegf , thus increasing the effective concentration of vegf-165 at the site of inflammation ( 32 ) . based on our finding of different proportions of vegf isoforms produced in response to glucose by nod islets and nod.scid islets cultured with nod culture supernatants , we speculate that both the islet and immune cells are the sources of vegf in inflamed islets stimulated with glucose , and the possibility of ecm disturbance and increased accessibility of vegf to activated endothelial cells require additional investigation . however , we were unable to detect an increase in -cell replication when we treated previously diabetic mice with glucose or even in prediabetic mice that were treated with glucose ( data not shown ) . in addition , the rates of -cell replication were already high in our prediabetic and postdiabetic mice treated with glucose , and , thus , it may not have been possible to increase the rates of replication further . our data suggest a novel role for glucose in the maintenance of islet vasculature during the progression of and recovery from diabetes and the effects of inflammation on imparting sensitivity to these effects .

polycystic kidney diseases ( pkds ) are a large family of disorders characterized by the formation and growth of renal cysts often leading to end - stage renal disease ( esrd ) . the most common inherited pkds can be transmitted as autosomal dominant or autosomal recessive traits . autosomal dominant polycystic kidney disease ( adpkd ) occurs with an incidence of 1 : 1000 and is characterized by the development of fluid - filled cysts in the kidneys , liver , pancreas and other organs , accounting for up to 10%ofesrdcases . adpkd is a systemic disorder with cardiovascular manifestations including cardiac valve abnormalities , cardiac hypertrophy and intracranial aneurisms . complications include hypertension , abdominal pain , hematuria and urinary tract infections [ 2 , 3 ] . the disease manifests in adulthood , with 50% of patients developing renal failure by age 60 [ 4 , 5 ] . autosomal recessive polycystic kidney disease ( arpkd ) is a less frequent childhood disease with an incidence of 1 : 20 000 . arpkd is characterized by cystic kidneys and congenital hepatic fibrosis with a high level of mortality in affected newborns . . a smaller number of patients display later onset of disease with portal hypertension or cholangitis . there are several other less common inherited recessive cystic diseases including nephronophthisis ( nphp ) , bardet - biedl syndrome ( bbs ) , joubert syndrome ( jbts ) and meckel - gruber syndrome ( mks ) that will not be discussed here and are described in other excellent reports [ 710].this review will mainly focus on recent advances in understanding the molecular pathogenesis of pkds and development of novel therapeutic options . approximately 85% of all adpkd cases are caused by mutations in the pkd1 gene , encoding polycystin-1 , which maps to human chromosome 16p13.3 [ 11 14 ] . the remaining 15%can be attributed to the pkd2 gene , encoding polycystin-2 , which maps to 4q21 [ 15 , 16 ] . clinical presentations of the pkd1 and pkd2 mutations are very similar , although the disease phenotype in the latter is significantly milder . typically , individuals with pkd2 display later mean age at diagnosis , hypertension and esrd [ 17 , 18 ] . heterogeneity is also seen within families in age of onset , rate of cystic disease progression and extrarenal manifestations , suggesting the role of genetic and environmental factors . the pkd1 genomic region of 53 kb is organized into 46 exons encoding a 14 kb mrna . the pkd1 genomic region contains a number of unusual structural features , including high gc content and multiple simple repeats . such lengthy polypyrimidine elements may interfere with replication , transcription or rna processing . because of the complex structure of the pkd1 gene , comprehensive mutation screening is difficult . most mutations are predicted to produce truncated protein and are unique to a single family , although missense mutations have also been identified . while genotype / phenotype correlations in thepkd1gene suggest that mutations in the 5 portion of the gene are associated with a more severe phenotype , no obvious correlations have been found in the pkd2 gene . the combination of the unique unstable structural elements in the pkd1 gene with intrafamilial heterogeneity and the focal nature of cyst formation has led to the two - hit hypothesis for cyst formation , which suggests that cysts form in cells with an inherited mutation in one allele and a somatic mutation occurring in the other allele . this hypothesis received experimental support through identification of somatic mutations in a subset of kidney cysts [ 23 , 24 ] . a similar mechanism was also found to play a role in pkd2 renal and hepatic cystogenesis . cystogenesis in humans and mice occurs in trans - heterozygotes with pkd1 and pkd2 mutations [ 26 , 27 ] . it is possible that reduced expression of the normal pkd1 allele below a critical level due to genetic or environmental factors may lead to cyst formation in the kidneys of adpkd patients . on the other hand , over - expression of polycystin-1 in transgenic animals these data suggest that abnormal levels of polycystin-1 expression can trigger pathogenic mechanisms leading to cyst formation . genetic data have shown that mutations in a single gene , pkhd1 , located on chromosome 6p21 ( encoding fibrocystin / polyductin ) cause arpkd [ 31 , 32 ] . the pkhd1 gene spans a region of 500 kb , consists of 67 exons and encodes a large 16 kb mrna . genotype / phenotype correlations show that individuals with two truncating mutations die in the perinatal period , while one or two missense changes are associated with milder disease . this finding was further supported by a study of neonatal survivors in which no two truncating mutations were identified , suggesting that missense mutations are required for survival of newborns . the majority of pkhd1 mutations are unique to a single family , with no obvious hot spots identified . polycystin-1 , the product of the pkd1 gene , is a large transmembrane protein with an estimated molecular weight of 500 kd ( fig . the large extracellular n - terminal region contains several specific motifs including leucine - rich repeats ( lrrs ) , c - type lectin domain , ldl - a region , multiple ig - like domains ( or pkd domains ) , rej domain and gps domain . it has 11 transmembrane domains , with a plat domain located in the first cytoplasmic loop and a small cytoplasmic tail with a g - protein - binding motif and coiled - coil region ( fig . the 16 ig - like domains are segmented such that the first ig - like domain is localized between the lrrs and the c - type lectin domain , while the remaining 15 iglike domains are clustered together between ldl - a and rej domains . this ig - like domain cluster forms strong homophilic interactions that are important for cell - cell adhesion [ 36 , 37 ] . polycystin is likely a multifunctional protein with important roles in cell - cell / matrix adhesion and ciliary functions [ 10 , 38 ] . polycystin-1 undergoes partial cleavage at the gps domain such that n - terminal and c - terminal polypeptides remain non - covalently linked [ 39 , 40 ] . it is subsequently cleaved at the second site , which releases its c - terminal tail . this signaling function of polycystin-1 is regulated by polycystin-2 and may possibly be initiated by mechanical stimuli . figure 1the structure of polycystin-1 , polycystin-2 and fibrocystin ( lrr , leucine - rich repeats ; wsc , cell wall integrity and stress response component 1 ; pkd ( ig - like ) , ig - like domains ; ldl , low density lipoprotein domain ; rej , receptor for egg jelly ; gps , proteolytic g protein - coupled receptor proteolytic site ; plat , lipoxygenase domain ; ef , ef hand domain ; tig , immunoglobulin - like domains ; tmem2 , homology with tmem2 protein ) . the structure of polycystin-1 , polycystin-2 and fibrocystin ( lrr , leucine - rich repeats ; wsc , cell wall integrity and stress response component 1 ; pkd ( ig - like ) , ig - like domains ; ldl , low density lipoprotein domain ; rej , receptor for egg jelly ; gps , proteolytic g protein - coupled receptor proteolytic site ; plat , lipoxygenase domain ; ef , ef hand domain ; tig , immunoglobulin - like domains ; tmem2 , homology with tmem2 protein ) . polycystin-2 is a protein of 110 kd with six transmembrane domains and cytoplasmic n- and c - terminal domains ( fig . 1 ) . polycystin-2 ( or trpp2 ) is thought to be a new member of the transient receptor potential ( trp ) family of ion channels . it was shown to be a cation channel with some selectivity for ca and functions in multiple subcellular locations including plasma membrane [ 43 , 44 ] , endoplasmic reticulum and the primary cilia . polycystin-1 and -2 can function together as a complex as well as independently in a variety of subcellular compartments . direct interaction between the cytoplasmic tails of the polycystins has been shown using yeast two - hybrid assay [ 47 , 48 ] . fibrocystin is a large receptorlike membrane - associated protein of 450 kd with a single transmembrane domain , large extracellular n - terminal region and small cytoplasmic c - terminal tail . its extracellular portion consists of several tig domains ( immunoglobulin - like ) and a short cytoplasmic c - terminus with putative phosphorylation sites ( fig . 1 ) [ 49 , 50 ] . based on similarities with other tig - containing proteins such as the hepatocyte growth factor receptor and the plexins , fibrocystin was suggested to function as a receptor or a ligand , since secreted forms may be generated from alternatively spliced transcripts . fibrocystin is expressed in cortical and medullary collecting ducts of the kidney as well as biliary and pancreatic ducts in a pattern consistent with the histologic features seen in arpkd . similar to polycystins , fibrocystin is expressed in multiple subcellular locations including the basolateral membrane , cytoplasm and cilia . it has been shown that fibrocystin can undergo notch - like processing with release of the ectodomain from primary cilia . an independent study also showed cleavage of the ectodomain of fibrocystin as well as generation of a cytoplasmic fragment that translocates to the nucleus . such proteolytic cleavage can be elicited by stimulation of intracellular ca release or protein kinase c activation . fibrocystin may form a complex with polycystin-2 to regulate calcium responses in kidney epithelia , but its exact role in normal and cystic epithelia is not known . the formation and growth of cysts in pkd is accompanied by increased proliferation and apoptosis of cyst - lining epithelia , loss of epithelial polarity and de - differentiation , dysregulation of cell / matrix interactions and transformation of the absorptive epithelial phenotype to a secretory phenotype [ 54 56 ] . epidermal growth factor ( egf ) , transforming growth factor ( tgf ) alpha and egf receptor ( egfr ) play important roles in promoting cystic epithelial proliferation . in human pkds and several animal models , apoptosis is also essential for cystogenesis : deletion of the anti - apoptotic bcl-2 and ap-2 genes and overexpression of the pro - apoptotic gene c - myc in mice results in renal cyst formation . cystic fluid is derived from glomerular filtrate in the early stages of adpkd , when cysts are still attached to the parent tubule . cysts separate from the tubule of origin when they reach 200 m in diameter and continue to expand through a transepithelial chloride secretion mechanism mediated by camp . chloride enters cells via the basolateral na - k - cl cotransporter and accumulates in the cytoplasm . a chloride channel in the apical membrane , cftr , mediates movement of chloride into the cystic lumen . chloride secretion drives sodium into the cystic cavity through paracellular mechanisms ; this causes movement of water through aguaporins . in contrast to adpkd , cysts in arpkd do not separate from affected collecting ducts . therefore , proliferation but not transepithelial secretion is a major component causing cystic kidney volume enlargement in arpkd . impaired cell - cell / matrix adhesion . the overlapping expression and localization patterns of polycystin-1 and both proteins are found in basolateral membranes and the primary cilium , where they may act together to regulate cellular adhesion and ca signaling . on the other hand , polycystin-2 is mainly expressed in endoplasmic reticulum , where it functions as a ca release channel . in addition , polycystin-1 is highly expressed during development , with significant down - regulation of its expression in adult tissues . in contrast , expression of polycystin-2 seems to persist into adult life . experimental evidence from several groups has established an important role for polycystins in epithelial cell morphogenesis , including differentiation and maturation in vivo [ 63 , 64 ] . studies using in vitro models of tubulogenesis and cystogenesis based on mdck cells demonstrated that expression of polycystin-1 at cell - cell junctions at controlled levels is critical for proper tubular differentiation . it has been shown that polycystin-1 is directly involved in intercellular adhesion via formation of strong homophilic interactions of its pkd ( ig - like ) domains as shown in fig . a direct role for ig - like domains in cell - cell adhesion was demonstrated by specific perturbation of intercellular adhesion using antibodies against ig - like domains in cell cultures [ 36 , 37 ] . polycystin-1 was localized to the cell - cell adhesion complexes with adherens junctions and desmosomal junctions in epithelial cells of different origin [ 65 67 ] . because alterations in polycystin-1-mediated adhesion may cause the abnormal epithelial cell phenotype observed in adpkd cells , including dedifferentiation and loss of epithelial polarity , several studies examined cell - cell adhesion junctions in primary cells derived from adpkd kidneys [ 37 , 68 , 69 ] . as shown in fig . 2 , abnormal adherens and desmosomal junctions were found in adpkd : intracellular junctions were devoid of desmosomal cadherins and associated proteins , which were sequestered to the cytoplasmic pools , and adherens junctions appeared disrupted , accompanied by a great reduction of ecadherin expression and partial compensatory expression of n - cadherin . ( a ) polycystin-1 ( pc-1 ) mediates cell - cell adhesion through homophilic interactions of its ig - like domains . the components of djs are shown : desmoplakin ( dp ) , desmogleins ( dsg ) , desmocollins ( dsc ) and plakoglobin ( pg ) . ajs consist of e - cadherin ( e - cad ) as well as , and -catenins ( catenins ) . the aj complex provides a linkage to the actin cytoskeleton , and dj link together intermediate filaments ( if ) of epithelial cells . in adpkd epithelial cells , dysfunctional pc-1 ( crossed red lines ) and desmosomal proteins are lost from cell - cell contacts and remain in intracellular vesicles . in addition , e - cadherin expression is reduced , resulting in compensatory expression of n - cadherin ( n - cad ) . thus , ajs and djs are disrupted in adpkd cells , while tight junctions ( tj ) remain intact . ( b ) expression of pc-1 in cell - matrix focal adhesion contacts . in normal epithelial cells , pc-1 is found in a complex with talin ( tal ) , paxillin ( pax ) , vinculin ( vinc ) , focal adhesion kinase ( fak ) , c - src ( src ) , p130-cas ( cas ) , nephrocystin ( nph1 ) and tensin ( ten ) . ( a ) polycystin-1 ( pc-1 ) mediates cell - cell adhesion through homophilic interactions of its ig - like domains . the components of djs are shown : desmoplakin ( dp ) , desmogleins ( dsg ) , desmocollins ( dsc ) and plakoglobin ( pg ) . ajs consist of e - cadherin ( e - cad ) as well as , and -catenins ( catenins ) . the aj complex provides a linkage to the actin cytoskeleton , and dj link together intermediate filaments ( if ) of epithelial cells . in adpkd epithelial cells , dysfunctional pc-1 ( crossed red lines ) and desmosomal proteins are lost from cell - cell contacts and remain in intracellular vesicles . in addition , e - cadherin expression is reduced , resulting in compensatory expression of n - cadherin ( n - cad ) . thus , ajs and djs are disrupted in adpkd cells , while tight junctions ( tj ) remain intact . ( b ) expression of pc-1 in cell - matrix focal adhesion contacts . in normal epithelial cells , pc-1 is found in a complex with talin ( tal ) , paxillin ( pax ) , vinculin ( vinc ) , focal adhesion kinase ( fak ) , c - src ( src ) , p130-cas ( cas ) , nephrocystin ( nph1 ) and tensin ( ten ) . interestingly , co - immunoprecipitation studies in adpkd cells using an anti - polycystin-1 antibody showed that e - cadherin was lost from the complex , while beta - catenin remained associated with polycystin-1 . moreover , beta - catenin was still expressed at the plasma membrane despite the loss of e - cadherin , suggesting the presence of an alternative cadherin . n - cadherin , but not k - cadherin , was overexpressed in adpkd cells and formed a complex with beta - catenin . in normal kidney , e - cadherin is expressed in distal segments of the nephron , while ncadherin is expressed in proximal tubules . in the adpkd kidney , n - cadherin is markedly increased in cysts of distal origin . however , the expression of n - cadherin in place of e - cadherin in adpkd cells was not sufficient to maintain epithelial cell - cell adhesion [ 37 , 69 ] . 2 ) [ 54 , 71 ] . abnormalities in the basement - membrane composition and expression of matrix metalloproteases and their inhibitors were identified in pkd kidneys . interestingly , the inactivation of tensin and insertional mutation in laminin alpha five result in cystogenesis [ 72 , 73 ] . it has been shown that polycystin-1 interacts with focal adhesion complex molecules including 12 integrin , vinculin , paxillin , p130-cas , talin and focal adhesion kinase ( fak ) . in adpkd cells , the focal adhesion complex polycystins , fibrocystin and numerous other proteins or cystoproteins such as nephrocystin-1 , -3 , -4 , -5 , inversin , alms1 , ofd1 , bbs1 - 8 , cystin , polaris and nek8 , which cause pkd in humans and animals , were recently discovered in a distinct subcellular compartment , the primary cilium [ 10 , 74 , 75 ] . cilia are microtubule - containing organelles that project from the surface of most eukaryotic cells . the primary ( non - motile ) cilia are known to transduce sensory stimuli such as concentrations of growth factors , osmolarity and fluid flow . they primary cilia are formed in fully differentiated cells during interphase and grow out of the basal bodies ( fig . the ciliary basal body is formed by the centrosomes , more specifically by the mother centriole that moves to the membrane where the axoneme , the structural core , is formed . construction and maintenance of the axoneme requires a bidirectional intraflagellar transport system ( ift ) to deliver structural components from the cell body to the tip of the cilium . it was recently discovered that defects in ciliary structure or function underlie multiple human diseases with diverse phenotypes , including retinal degeneration , neural tube defects , obesity , polydactyly and pkd . mutations in the lov-1 and pkd-2 genes of c. elegans , which are closely related to human polycystins , were associated with mechanosensation defects of ciliated sensory neurons . direct evidence linking defects in ciliogenesis and pkd came from the study of renal tissue - specific inactivation of kif3a , a subunit of kinesin - ii essential for cilia formation : inactivation of kif3a in renal tubular epithelial cells resulted in development of pkd . it was demonstrated that the primary cilia in the cystic kidney of mice with a mutation in the tg737 gene ( homologous to the ift gene of chlamydomonas , ift88 ) are shorter than normal . on the other hand , the primary cilia of cystic epithelial cells in jck mice were found to be significantly longer than the cilia in wild - type mice ; this study suggested that ciliary protein nek8 , which is mutated in the jck mice , may play a role in control of ciliary length . cilia and mechanosensation in pkd . a number of studies support a role for the primary cilium as a mechanosensor in kidney tubular epithelia . reported that the primary cilia of renal epithelial mdck cells can serve as a flow sensor . bending the authors concluded that the primary cilium in mdck cells is mechanically sensitive and responds to flow by increasing intracellular calcium . a subsequent study demonstrated that polycystins can serve as flow - sensitive ciliary mechanosensors in kidney epithelia . more specifically , ciliary polycystin-1 and polycystin-2 function together with ryanodine receptors to mediate mechanotransduction into the intracellular ca signaling response ( fig . the influx of ca across the plasma membrane constitutes the initial response to mechanical stimulation , and downstream signaling is mediated through intracellular ca release . it is possible that polycystin-1 functions as a sensor of ciliary bending , while polycystin-2 transduces themechanical signal into a calcium response . changes in intracellular ca concentration are known to regulate multiple cellular functions including gene expression , cell cycle , differentiation and apoptosis . cells with mutated polycystin-1 fail to respond to the fluid flow : it was shown that in adpkd - derived cells , the ciliary mechanosensation of fluid - flow shear stress by poly - cystins is absent . figure 3ciliary functions of cystoproteins : mechanosensation and cell cycle control . polycystin-1 ( pc-1 ) , polycystin-2 ( pc-2 ) and other cystoproteins ( not shown ) are expressed in primary cilia , basal bodies or centrosomes . the primary cilium forms in fully differentiated cells in the g0 phase of the cell cycle . a cilium protrudes from the basal body ( centrosome ) formed by two centrioles , a mother centriole ( blue cylinder ) and a daughter centriole ( orange cylinder ) . as cells enter the cell cycle , the cilium is resorbed , and the centrosome can act as a mitotic spindle pole organizer . flow - induced bending of cilia in renal epithelial cells triggers ca influx mediated by pc-2 in association with pc-1.the role of ciliary proteins in cell cycle control is supported by several findings : ( 1 ) in the normal state , pc-2 sequesters id2 [ proproliferative helix - loop - helix ( hlh ) protein ] in the cytoplasm , preventing it from entering the nucleus . in pkd , when pc-1 or pc-2 are inactivated , id2 translocates to the nucleus and interacts with e - protein , blocking its ability to induce growth - suppressive genes and resulting in increased activity of cdk2 and activation of cell proliferation ; ( 2 ) pc-1 can activate the jak / stat signaling pathway , resulting in the induction of p21 and cell cycle arrest in g0 and g1 ; ( 3 ) the intraflagellar transport component ift88/polaris is capable of controlling g1/s transition of the cell cycle . ift88/polaris remains associated with the centrosome throughout the cell cycle , where it forms a complex with che-1 , thus modulating its binding to rb protein . polycystin-1 ( pc-1 ) , polycystin-2 ( pc-2 ) and other cystoproteins ( not shown ) are expressed in primary cilia , basal bodies or centrosomes . the primary cilium forms in fully differentiated cells in the g0 phase of the cell cycle . a cilium protrudes from the basal body ( centrosome ) formed by two centrioles , a mother centriole ( blue cylinder ) and a daughter centriole ( orange cylinder ) . as cells enter the cell cycle , the cilium is resorbed , and the centrosome can act as a mitotic spindle pole organizer . flow - induced bending of cilia in renal epithelial cells triggers ca influx mediated by pc-2 in association with pc-1.the role of ciliary proteins in cell cycle control is supported by several findings : ( 1 ) in the normal state , pc-2 sequesters id2 [ proproliferative helix - loop - helix ( hlh ) protein ] in the cytoplasm , preventing it from entering the nucleus . in pkd , when pc-1 or pc-2 are inactivated , id2 translocates to the nucleus and interacts with e - protein , blocking its ability to induce growth - suppressive genes and resulting in increased activity of cdk2 and activation of cell proliferation ; ( 2 ) pc-1 can activate the jak / stat signaling pathway , resulting in the induction of p21 and cell cycle arrest in g0 and g1 ; ( 3 ) the intraflagellar transport component ift88/polaris is capable of controlling g1/s transition of the cell cycle . ift88/polaris remains associated with the centrosome throughout the cell cycle , where it forms a complex with che-1 , thus modulating its binding to rb protein . the basal bodies / centrosomes of the cilia act as organizers of the mitotic spindle poles during cell division , directly connecting ciliogenesis with cell cycle regulation , and cilia are resorbed when cells enter the cell cycle ( fig . a number of cystoproteins causing pkd in humans and animals are expressed , at least partially , in cilia or the basal body of the cilia . polycystins and other cystoproteins may play an important role in connecting the mechanosensory function of the cilia to the centrosome and thus influence cell cycle control . disruption of cystoproteins associated with cilia or basal bodies could , therefore , lead to dysregulation of the cell cycle and proliferation , resulting in cystic disease . several lines of evidence support this hypothesis : overexpression of exogenous polycystin-1 in cultured cells resulted in growth arrest in g0/g1 phase of the cell cycle ( fig . polycystin-1 activates the jak - stat pathway , thereby up - regulating p21waf1 and inducing cell cycle arrest in g0/g1 . other cystoproteins localized to cilia have also been recently shown to directly regulate the cell cycle . for example , the ift88/polaris protein , a component of the ift , was shown to be tightly associated with the centrosome during cell cycle transitions . similar to polycystin-1 , overexpression of polaris resulted in cell cycle arrest at the g0/g1 stage ( fig . polycystin-2 has been shown to participate in cell cycle regulation as well : it can associate with the helix - loop - helix protein id2 and block its translocation to the nucleus , preventing proliferation . the translocation of id2 in cells with mutated polycystins is associated with downregulation of p21 expression , leading to an increase in cdk2 activity and cell cycle progression ( fig . 3 ) . thus , evidence of a direct link between cystoproteins , ciliary dysfunction and cell cycle dysregulation continues to accumulate . as we discussed previously , pkds in humans and animals can be caused by different cystogenes and can be distinguished by the patterns of inheritance and clinical manifestation . on the other hand , cyst formation in these disorders common cellular abnormalities of cystic epithelia include increased proliferation , apoptosis , loss of epithelial polarity accompanied by missorting of proteins and intracystic fluid secretion . ciliary dysfunction may also constitute one of the common features for cystic diseases , since most known cystoproteins are associated with cilia / centrosome . for example , polycystin-2 was found to directly interact with fibrocystin to regulate calcium responses in the kidneys . genetic interaction between murine genes encoding polycystin-1 and fibrocystin was shown , suggesting that polycystin-1 and fibrocystin function in the same molecular pathway to maintain tubular integrity . in addition , genetic interaction was also identified between cystogenes encoding polycystin-1 and nek8 ( mutated in the jck mouse model of pkd ) ( natoli et al . , unpublished observations ) . therefore , elucidation of common pathogenic mechanisms involved in pkds is an important step toward developing new therapies for effective treatment of cystic diseases . studies using in vitro - cultured cells from adpkd cysts and animal models of pkd have revealed an important role for camp - activated pathways . camp was shown to block proliferation of normal kidney epithelial cells through inhibition of the ras / raf / mek / erk pathway . in sharp contrast , camp was able to induce proliferation of adpkd - derived cystic epithelial cells through activation of the b - raf / mek / erk pathway . this switch from conventional camp - induced inhibition of growth to the camp - induced stimulation of growth in cystic epithelia was mediated by decreased intracellular calcium levels . mutated polycystins disrupted ca signaling , which led to abnormal camp - stimulated proliferation of cystic cells . cystic kidneys were shown to accumulate high levels of camp inmultiple models of pkd , making it plausible that activated camp pathways lead to both the increased proliferation and fluid secretion seen in cystic epithelia [ 86 , 88 , 89 ] . wnt signaling in pkd . activated wnt signaling was initially implicated in pkd through studies demonstrating that overexpression of the cytoplasmic tail of polycystin-1 stabilizes endogenous beta - catenin and stimulates tcf - dependent gene transcription in vitro . microinjection of the polycystin-1 cytoplasmic tail induced dorsalization in zebrafish , suggesting that polycystin-1 is involved in modulating wnt signaling during renal development . other studies supported this finding by connecting dysregulation of the beta - catenin pathway with pkd . overexpression of beta - catenin in transgenic animals or inactivation of apc resulted in the development of cystic kidneys [ 91 , 92 ] . canonical beta - catenin - dependent wnt signaling is required for kidney development , while constitutive beta - catenin signaling in maturing tubules leads to formation of cysts . flow - induced signaling switches the wnt pathway to a non - canonical beta - catenin - independent pathway through increased expression of inversin , a protein mutated in nephronophthisis type ii . this , in turn , results in reduced levels of the cytoplasmic dishevelled protein that activates destruction of the beta - catenin complex . the pkd1 gene is located in close proximity to the tsc2 gene , which encodes the tuberin protein responsible for the tuberous sclerosis complex ( tsc ) , in a tail - to - tail orientation . a functional link between polycystin-1 and tuberin was established by a study showing that tuberin is required for membrane trafficking of polycystin-1 . polycystin-1 appeared to form a complex with tuberin and regulate the activity of mtor , an important regulator of protein synthesis and cellular differentiation . activation of mtor was also found in animal models of recessive pkd such as the bpk and orpk - rescue , suggesting that mutations in polycystin-1 are not critical form mtor activation . it is possible that signaling through other membrane receptors could lead to stimulation of mtor pathway , which is likely to be a late stage of the cystogenesis cascade . greater understanding of the molecular mechanisms of cyst formation and growth has led to the discovery of novel potential therapeutic targets . animal models of pkd have been critical in supporting studies of disease pathogenesis and in testing potential therapies . the availability of well - characterized and disease - relevant models is instrumental for successful development of viable therapies . several preclinical pkd models have been described , some with spontaneous mutations and others generated through chemical / genetic manipulations . no existing model is ideal for preclinical testing in pkd ; however , each represents a facet of human pathobiology ( table 1 ) . in general , an ideal pkd model should carry a mutation in a gene orthologous to the human disease - carrying gene.the development of the disease in such orthologous models should reproduce human pkd relative to the severity , segmental origin of kidney cysts , extrarenal manifestations and known cellular and molecular abnormalities including increased proliferation , apoptosis , camp , protein missorting as well as activation of known signaling pathways such as wnt , b - raf / mek / erk and mtor . a dozen different mouse models with targeted disruption of the pkd1 gene have been created , including animals with conditional gene inactivation ( reviewed in ) . mice with heterozygous pkd1 and pkd2 mutations develop a very mild and heterogeneous kidney cystic disease late in life and therefore are not suitable for therapeutic testing . homozygous animals develop renal and pancreatic cysts at embryonic day 15.5 and die during embryogenesis . mice with conditional inactivation of the pkd1 gene develop kidney and liver cysts , but disease is too mild to be informative in preclinical testing . an interesting mouse model with a mutation in the pkd2 gene ( pkd2 ) carrying an unstable allele combined with a pkd2-null allele although these animals develop kidney and liver cystic disease that mimics human adpkd , the model has not been widely used for therapeutic testing due to a very high degree of phenotypic heterogeneity in combination with a slow course of progression with no measurable loss of renal function as late as 46 months of age . the pkd2 model should be most useful as a secondary confirmatory model rather than the primary model for proof of concept therapeutic testing . several spontaneous mouse models of pkd , such as cpk , bpk and orpk , resemble human recessive disease with respect to cyst localization and the rapid rate of disease progression ( table 1 ) . cystic disease in the pcy mouse and han : sprd rat is slowly progressive with cyst formation similar to adpkd . the jck mouse model is characterized by the development of cysts in multiple nephron segments and , despite the autosomal recessive mode of inheritance , resembles human adpkd phenotypically and can be used for relatively rapid therapeutic testing . since no single model fully recapitulates all aspects of human pkd pathogenesis , it is important to test a potential therapy in more than one pkd model to determine its utility for clinical testing . table 1rodent models of polycystic kidney diseases used in therapeutic intervention studies.modelgeneproteinprogressionrenal pathologykidney phenotypemousecpkcys1cystinrapidpt , cdarpkdbpkbicc1bicaudal crapidpt , cdarpkdorpktgn737polarisrapidpt , cdarpkdpcynphp3nephrocystin-3slownephron cdadpkdjcknek8nek8moderatelh , dt , cdadpkdpkd1pkd1polycystin-1el arpkdpkd2pkd2polycystin-2slowpt , lh , dt , cdadpkdrathan : sprd - cypkdr1samcystinslowptadpkdpckpkhd1fibrocystinslowcdarpkdpt , proximal tubule ; dt , distal tubule ; cd , collecting duct ; lh , loop of henle ; el , embryonic lethal . pt , proximal tubule ; dt , distal tubule ; cd , collecting duct ; lh , loop of henle ; el , embryonic lethal . an important role for epidermal growth factor ( egf ) and its receptor ( egfr ) in contributing to cystic epithelial proliferation has been demonstrated . in human pkd and multiple animal models , egfr is overexpressed and mislocalized to the apical membrane in cystic epithelial cells . transforming growth factor alpha ( tgf alpha ) , a member of egf family , also signals through egfr . the role of tgf alpha in cystogenesis was addressed by renal expression of a tgf alpha transgene in the mouse . preclinical studies showed that inhibition of egfr tyrosine kinase activity significantly reduced cystogenesis in mouse models of pkd , including bpk and orpk , as well as in the han : sprd rat model [ 99 , 100 ] , all models characterized by overexpression and mislocalization of egfr to the apical membrane . however , no therapeutic effect was detected in the pck rat ; interestingly , although the pck rat is an orthologous model of arpkd , egfr was not found to be mislocated to the apical membrane of cystic cells , which may explain the lack of therapeutic efficacy in this model . growth of cysts in 3-d collagen cultures of epithelial cells in vitro proceeds with increased apoptosis . moreover , deletion of the anti - apoptotic bcl-2 and ap-2 beta genes as well as overexpression of pro - apoptotic c - myc in mice leads to renal cyst formation [ 103 , 104 ] . to test the effect of inhibition of apoptosis in pkd this study showed inhibition of pkd progression and attenuation of the loss of renal function . the critical role of the camp - activated b - raf / mek / erk pathway was confirmed by the successful preclinical testing of a mek inhibitor : oral administration the map / erk kinase inhibitor pd184352 to pcy mice significantly decreased kidney weight , serum creatinine levels and water intake and significantly increased urine osmolality . thus , inhibitors of the b - raf / mek / erk pathway may prove to be promising agents for pkd therapy . studies were conducted to specifically target the cystogenic camp and vasopressin pathways using vasopressin v2 receptor ( vpv2r ) inhibitors , and efficacy was shown in four different pkd animal models . treatment with tolvaptan ( human vpv2r antagonist , otsuka pharmaceutical ) resulted in significant lowering of camp levels in pck rat kidneys and inhibition of renal cystogenesis and fibrosis . tolvaptan has entered human clinical trials in adpkd , and phase ii results have shown that it is well tolerated , with thirst and polyuria as side effects [ 88 , 108 ] . it is important to note that effective reduction of plasma vasopressin levels through increased water intake decreased vpv2r expression and slowed pkd progression in pck rats . as somatostatin is capable of inhibiting camp - stimulated fluid secretion , it constitutes an alternative approach to the targeting of abnormal camp signaling in cysts . in a randomized placebo - controlled human adpkd trial , the benefit of a 6-month treatment with long - acting somatostatin ( octreotide - lar , 40 mg intramuscularly every 28 days ) was assessed . treatment of the bpk and orpk - rescue mouse models with the mtor inhibitor rapamycin showed effective inhibition of pkd . similar results were previously observed in a separate study with rapamycin performed in han : sprd rats . treatment of human adpkd transplant recipient patients with rapamycin resulted in a significant reduction in polycystic kidney volumes . several clinical trials are currently underway to test the efficacy of rapamycin and everolimus in adpkd patients . an important role of ciliary dysfunction in pkd pathology is highlighted by findings of the disrupted connection between cilia and the cell cycle . this dysregulation between cilia and cell cycle progression seems to occur early in cystogenesis and represents an attractive therapeutic target . the cdk inhibitor roscovitine was tested in cystic assay in vitro and in the preclinical pkd mouse models jck and cpk . roscovitine ( seliciclib , cyc202 ) is a potent and remarkably selective inhibitor of cdk2/cyclin e , cdk7/cyclin h , cdk9/cyclin t1 and cdk5/p35-p25 [ 112 , 113 ] . robust inhibition of pkd was shown in the jck mouse model of slowly progressive disease as well as in the cpk mouse model of aggressive pkd . pulse treatment resulted in a persistent long - lasting effect such that continuous daily administration of the drug was not required to achieve efficacy . roscovitine was equally effective against cysts formed in different segments of the nephron , a desirable feature for a potential drug against adpkd , where cysts are formed in multiple parts of the nephron . the effect of roscovitine at the molecular level was mediated through cell cycle blockade , transcriptional inhibition and apoptotic arrest . thus , the apparent therapeutic benefit of cdk inhibition for pkd may be due to integrative effects on several key aspects of disease pathology . seliciclib , an orally bioavailable compound , is currently in clinical trials as a cancer drug candidate . known adverse events include transient elevations in serum creatinine , transient hypokalemia and reversible elevations in liver enzymes . the deregulation of ca signaling in cystic cells is believed to be mediated by polycystin-2 in complex with polycystin-1 . the first example of a therapeutic strategy to promote an increase in cytosolic ca in pkd has been demonstrated by treatment with triptolide , an active diterpene used in traditional chinese medicine . triptolide treatment of pkd1-null homozygous embryos via maternal administration of the drug resulted in arrest of cellular proliferation and inhibition of cyst formation through restoration of ca signaling . the majority if not all of the drugs successfully tested in animal models so far were not specifically designed to treat pkd but rather were originally developed for other indications . some of these drugs may require further optimization to achieve efficacy / toxicity profiles that are more appropriate for the pkd patient population . because multiple optimized analogues would need to be tested , direct assessment of their efficacy in animal models is not feasible . the availability of high - throughput in vitro screening assays that are disease - relevant , robust , rapid and reproducible is crucial . in addition , such assays can be directly used for discovery of pkd - specific compounds that antagonize cyst development . it has been shown that mdck cysts grown in a 3-d collagen matrix in vitro adequately reflect several aspects of cystogenesis in vivo and can be successfully used for drug testing ( fig . 4 ) [ 65 , 116 , 117 ] ; the cystic assay can be used directly for high - throughput screening of a compound library or as a drug optimization assay . recently it was shown that an embryonic cystic kidney organ culture assay can be a very valuable testing platform for rapid assessment of efficacy as well as preliminary testing for organ toxicity before entering into time- and material - consuming trials in animals ( and t. natoli , unpublished results ) ( fig . 4 ) . with the availability of such a screening cascade and successful candidates already emerging from preclinical testing , the field is poised to suggest viable therapeutic options for the treatment of pkd . high - throughput drug screening ( hts ) of a small compound library can be performed using an in vitro assay of cystogenesis . identified hits ( active drugs ) can then be rapidly assayed for preliminary efficacy and organ toxicity using a readily available cystic kidney organ culture assay that utilizes pkd1 animals . effective compounds can be selected for in vivo efficacy testing in animal models of pkd . further optimization of compounds using medicinal chemistry and sar ( structure - activity relationship ) can proceed through the same screening platforms . the discovery process can also be initiated through proof of concept in vivo testing ( drug candidate validation ) , followed by the drug optimization cycle . high - throughput drug screening ( hts ) of a small compound library can be performed using an in vitro assay of cystogenesis . identified hits ( active drugs ) can then be rapidly assayed for preliminary efficacy and organ toxicity using a readily available cystic kidney organ culture assay that utilizes pkd1 animals . effective compounds can be selected for in vivo efficacy testing in animal models of pkd . further optimization of compounds using medicinal chemistry and sar ( structure - activity relationship ) can proceed through the same screening platforms . the discovery process can also be initiated through proof of concept in vivo testing ( drug candidate validation ) , followed by the drug optimization cycle . several potential therapies , including tolvaptan , sirolimus , everolimus and octreotide ( as discussed above ) , are being tested in ongoing adpkd clinical trials . in addition , a large clinical trial ( halt - pkd ) has been implemented to address the benefit of inhibition of the renin - angiotensin system ; this trial will test whether using ace inhibitors and angiotensin receptor blockers in combination is beneficial in comparison with ace inhibitors alone . the main challenge in adpkd clinical trials because disease develops slowly over several decades , renal function begins to decline late in the course of the disease when significant and likely irreversible damage to the kidney occurs . more beneficial early intervention trials require availability of reliable endpoints predictive of the subsequent renal function decline . results of the crisp ( consortium for radiologic imaging studies of pkd ) study have demonstrated that measurement of kidney volume by magnetic resonance imaging ( mri ) can be a reliable indicator of clinical outcome . recent advances in our fundamental understanding of the molecular pathogenesis of pkd have allowed selection of disease - relevant targets for therapeutic testing . several successful studies in animal models that are currently being translated into human clinical trials strengthen the need to further explore disease - specific therapeutic targets . a rapidly growing number of well - characterized in vitro and in vivo models of cystic disease open opportunities for systematic drug discovery efforts . the availability of magnetic resonance imaging techniques to accurately measure pkd progression and , subsequently , the effect of therapeutic agents in a short period of time greatly enhances options for clinical trial design . it is likely that future treatment for pkd will require a combination therapy integrating several key pathways of cystogenesis .

abstract.polycystic kidney diseases ( pkds ) represent a large group of progressive renal disorders characterized by the development of renal cysts leading to end - stage renal disease . enormous strides have been made in understanding the pathogenesis of pkds and the development of new therapies . studies of autosomal dominant and recessive polycystic kidney diseases converge on molecular mechanisms of cystogenesis , including ciliary abnormalities and intracellular calcium dysregulation , ultimately leading to increased proliferation , apoptosis and dedifferentiation . here we review the pathobiology of pkd , highlighting recent progress in elucidating common molecular pathways of cystogenesis . we discuss available models and challenges for therapeutic discovery as well as summarize the results from preclinical experimental treatments targeting key disease - specific pathways .

Introduction Molecular genetics of PKDs PKD proteins Cellular and molecular pathogenesis of PKDs Other signaling pathways affected in PKD Therapeutic approaches for the treatment of PKD: preclinical and clinical testing Conclusions

polycystic kidney diseases ( pkds ) are a large family of disorders characterized by the formation and growth of renal cysts often leading to end - stage renal disease ( esrd ) . the most common inherited pkds can be transmitted as autosomal dominant or autosomal recessive traits . autosomal dominant polycystic kidney disease ( adpkd ) occurs with an incidence of 1 : 1000 and is characterized by the development of fluid - filled cysts in the kidneys , liver , pancreas and other organs , accounting for up to 10%ofesrdcases . autosomal recessive polycystic kidney disease ( arpkd ) is a less frequent childhood disease with an incidence of 1 : 20 000 . arpkd is characterized by cystic kidneys and congenital hepatic fibrosis with a high level of mortality in affected newborns . there are several other less common inherited recessive cystic diseases including nephronophthisis ( nphp ) , bardet - biedl syndrome ( bbs ) , joubert syndrome ( jbts ) and meckel - gruber syndrome ( mks ) that will not be discussed here and are described in other excellent reports [ 710].this review will mainly focus on recent advances in understanding the molecular pathogenesis of pkds and development of novel therapeutic options . heterogeneity is also seen within families in age of onset , rate of cystic disease progression and extrarenal manifestations , suggesting the role of genetic and environmental factors . the pkd1 genomic region contains a number of unusual structural features , including high gc content and multiple simple repeats . while genotype / phenotype correlations in thepkd1gene suggest that mutations in the 5 portion of the gene are associated with a more severe phenotype , no obvious correlations have been found in the pkd2 gene . the combination of the unique unstable structural elements in the pkd1 gene with intrafamilial heterogeneity and the focal nature of cyst formation has led to the two - hit hypothesis for cyst formation , which suggests that cysts form in cells with an inherited mutation in one allele and a somatic mutation occurring in the other allele . on the other hand , over - expression of polycystin-1 in transgenic animals these data suggest that abnormal levels of polycystin-1 expression can trigger pathogenic mechanisms leading to cyst formation . the pkhd1 gene spans a region of 500 kb , consists of 67 exons and encodes a large 16 kb mrna . polycystin-1 , the product of the pkd1 gene , is a large transmembrane protein with an estimated molecular weight of 500 kd ( fig . the large extracellular n - terminal region contains several specific motifs including leucine - rich repeats ( lrrs ) , c - type lectin domain , ldl - a region , multiple ig - like domains ( or pkd domains ) , rej domain and gps domain . the 16 ig - like domains are segmented such that the first ig - like domain is localized between the lrrs and the c - type lectin domain , while the remaining 15 iglike domains are clustered together between ldl - a and rej domains . it was shown to be a cation channel with some selectivity for ca and functions in multiple subcellular locations including plasma membrane [ 43 , 44 ] , endoplasmic reticulum and the primary cilia . polycystin-1 and -2 can function together as a complex as well as independently in a variety of subcellular compartments . fibrocystin is a large receptorlike membrane - associated protein of 450 kd with a single transmembrane domain , large extracellular n - terminal region and small cytoplasmic c - terminal tail . 1 ) [ 49 , 50 ] . based on similarities with other tig - containing proteins such as the hepatocyte growth factor receptor and the plexins , fibrocystin was suggested to function as a receptor or a ligand , since secreted forms may be generated from alternatively spliced transcripts . fibrocystin is expressed in cortical and medullary collecting ducts of the kidney as well as biliary and pancreatic ducts in a pattern consistent with the histologic features seen in arpkd . an independent study also showed cleavage of the ectodomain of fibrocystin as well as generation of a cytoplasmic fragment that translocates to the nucleus . the formation and growth of cysts in pkd is accompanied by increased proliferation and apoptosis of cyst - lining epithelia , loss of epithelial polarity and de - differentiation , dysregulation of cell / matrix interactions and transformation of the absorptive epithelial phenotype to a secretory phenotype [ 54 56 ] . in human pkds and several animal models , apoptosis is also essential for cystogenesis : deletion of the anti - apoptotic bcl-2 and ap-2 genes and overexpression of the pro - apoptotic gene c - myc in mice results in renal cyst formation . therefore , proliferation but not transepithelial secretion is a major component causing cystic kidney volume enlargement in arpkd . the overlapping expression and localization patterns of polycystin-1 and both proteins are found in basolateral membranes and the primary cilium , where they may act together to regulate cellular adhesion and ca signaling . experimental evidence from several groups has established an important role for polycystins in epithelial cell morphogenesis , including differentiation and maturation in vivo [ 63 , 64 ] . because alterations in polycystin-1-mediated adhesion may cause the abnormal epithelial cell phenotype observed in adpkd cells , including dedifferentiation and loss of epithelial polarity , several studies examined cell - cell adhesion junctions in primary cells derived from adpkd kidneys [ 37 , 68 , 69 ] . ajs consist of e - cadherin ( e - cad ) as well as , and -catenins ( catenins ) . ajs consist of e - cadherin ( e - cad ) as well as , and -catenins ( catenins ) . the ciliary basal body is formed by the centrosomes , more specifically by the mother centriole that moves to the membrane where the axoneme , the structural core , is formed . it was recently discovered that defects in ciliary structure or function underlie multiple human diseases with diverse phenotypes , including retinal degeneration , neural tube defects , obesity , polydactyly and pkd . direct evidence linking defects in ciliogenesis and pkd came from the study of renal tissue - specific inactivation of kif3a , a subunit of kinesin - ii essential for cilia formation : inactivation of kif3a in renal tubular epithelial cells resulted in development of pkd . a number of studies support a role for the primary cilium as a mechanosensor in kidney tubular epithelia . reported that the primary cilia of renal epithelial mdck cells can serve as a flow sensor . bending the authors concluded that the primary cilium in mdck cells is mechanically sensitive and responds to flow by increasing intracellular calcium . as cells enter the cell cycle , the cilium is resorbed , and the centrosome can act as a mitotic spindle pole organizer . flow - induced bending of cilia in renal epithelial cells triggers ca influx mediated by pc-2 in association with pc-1.the role of ciliary proteins in cell cycle control is supported by several findings : ( 1 ) in the normal state , pc-2 sequesters id2 [ proproliferative helix - loop - helix ( hlh ) protein ] in the cytoplasm , preventing it from entering the nucleus . in pkd , when pc-1 or pc-2 are inactivated , id2 translocates to the nucleus and interacts with e - protein , blocking its ability to induce growth - suppressive genes and resulting in increased activity of cdk2 and activation of cell proliferation ; ( 2 ) pc-1 can activate the jak / stat signaling pathway , resulting in the induction of p21 and cell cycle arrest in g0 and g1 ; ( 3 ) the intraflagellar transport component ift88/polaris is capable of controlling g1/s transition of the cell cycle . as cells enter the cell cycle , the cilium is resorbed , and the centrosome can act as a mitotic spindle pole organizer . in pkd , when pc-1 or pc-2 are inactivated , id2 translocates to the nucleus and interacts with e - protein , blocking its ability to induce growth - suppressive genes and resulting in increased activity of cdk2 and activation of cell proliferation ; ( 2 ) pc-1 can activate the jak / stat signaling pathway , resulting in the induction of p21 and cell cycle arrest in g0 and g1 ; ( 3 ) the intraflagellar transport component ift88/polaris is capable of controlling g1/s transition of the cell cycle . disruption of cystoproteins associated with cilia or basal bodies could , therefore , lead to dysregulation of the cell cycle and proliferation , resulting in cystic disease . polycystin-2 has been shown to participate in cell cycle regulation as well : it can associate with the helix - loop - helix protein id2 and block its translocation to the nucleus , preventing proliferation . the translocation of id2 in cells with mutated polycystins is associated with downregulation of p21 expression , leading to an increase in cdk2 activity and cell cycle progression ( fig . as we discussed previously , pkds in humans and animals can be caused by different cystogenes and can be distinguished by the patterns of inheritance and clinical manifestation . on the other hand , cyst formation in these disorders common cellular abnormalities of cystic epithelia include increased proliferation , apoptosis , loss of epithelial polarity accompanied by missorting of proteins and intracystic fluid secretion . in addition , genetic interaction was also identified between cystogenes encoding polycystin-1 and nek8 ( mutated in the jck mouse model of pkd ) ( natoli et al . therefore , elucidation of common pathogenic mechanisms involved in pkds is an important step toward developing new therapies for effective treatment of cystic diseases . studies using in vitro - cultured cells from adpkd cysts and animal models of pkd have revealed an important role for camp - activated pathways . this switch from conventional camp - induced inhibition of growth to the camp - induced stimulation of growth in cystic epithelia was mediated by decreased intracellular calcium levels . cystic kidneys were shown to accumulate high levels of camp inmultiple models of pkd , making it plausible that activated camp pathways lead to both the increased proliferation and fluid secretion seen in cystic epithelia [ 86 , 88 , 89 ] . overexpression of beta - catenin in transgenic animals or inactivation of apc resulted in the development of cystic kidneys [ 91 , 92 ] . greater understanding of the molecular mechanisms of cyst formation and growth has led to the discovery of novel potential therapeutic targets . animal models of pkd have been critical in supporting studies of disease pathogenesis and in testing potential therapies . the availability of well - characterized and disease - relevant models is instrumental for successful development of viable therapies . several preclinical pkd models have been described , some with spontaneous mutations and others generated through chemical / genetic manipulations . in general , an ideal pkd model should carry a mutation in a gene orthologous to the human disease - carrying gene.the development of the disease in such orthologous models should reproduce human pkd relative to the severity , segmental origin of kidney cysts , extrarenal manifestations and known cellular and molecular abnormalities including increased proliferation , apoptosis , camp , protein missorting as well as activation of known signaling pathways such as wnt , b - raf / mek / erk and mtor . a dozen different mouse models with targeted disruption of the pkd1 gene have been created , including animals with conditional gene inactivation ( reviewed in ) . mice with heterozygous pkd1 and pkd2 mutations develop a very mild and heterogeneous kidney cystic disease late in life and therefore are not suitable for therapeutic testing . an interesting mouse model with a mutation in the pkd2 gene ( pkd2 ) carrying an unstable allele combined with a pkd2-null allele although these animals develop kidney and liver cystic disease that mimics human adpkd , the model has not been widely used for therapeutic testing due to a very high degree of phenotypic heterogeneity in combination with a slow course of progression with no measurable loss of renal function as late as 46 months of age . several spontaneous mouse models of pkd , such as cpk , bpk and orpk , resemble human recessive disease with respect to cyst localization and the rapid rate of disease progression ( table 1 ) . the jck mouse model is characterized by the development of cysts in multiple nephron segments and , despite the autosomal recessive mode of inheritance , resembles human adpkd phenotypically and can be used for relatively rapid therapeutic testing . table 1rodent models of polycystic kidney diseases used in therapeutic intervention studies.modelgeneproteinprogressionrenal pathologykidney phenotypemousecpkcys1cystinrapidpt , cdarpkdbpkbicc1bicaudal crapidpt , cdarpkdorpktgn737polarisrapidpt , cdarpkdpcynphp3nephrocystin-3slownephron cdadpkdjcknek8nek8moderatelh , dt , cdadpkdpkd1pkd1polycystin-1el arpkdpkd2pkd2polycystin-2slowpt , lh , dt , cdadpkdrathan : sprd - cypkdr1samcystinslowptadpkdpckpkhd1fibrocystinslowcdarpkdpt , proximal tubule ; dt , distal tubule ; cd , collecting duct ; lh , loop of henle ; el , embryonic lethal . in human pkd and multiple animal models , egfr is overexpressed and mislocalized to the apical membrane in cystic epithelial cells . preclinical studies showed that inhibition of egfr tyrosine kinase activity significantly reduced cystogenesis in mouse models of pkd , including bpk and orpk , as well as in the han : sprd rat model [ 99 , 100 ] , all models characterized by overexpression and mislocalization of egfr to the apical membrane . moreover , deletion of the anti - apoptotic bcl-2 and ap-2 beta genes as well as overexpression of pro - apoptotic c - myc in mice leads to renal cyst formation [ 103 , 104 ] . to test the effect of inhibition of apoptosis in pkd this study showed inhibition of pkd progression and attenuation of the loss of renal function . the critical role of the camp - activated b - raf / mek / erk pathway was confirmed by the successful preclinical testing of a mek inhibitor : oral administration the map / erk kinase inhibitor pd184352 to pcy mice significantly decreased kidney weight , serum creatinine levels and water intake and significantly increased urine osmolality . treatment with tolvaptan ( human vpv2r antagonist , otsuka pharmaceutical ) resulted in significant lowering of camp levels in pck rat kidneys and inhibition of renal cystogenesis and fibrosis . in a randomized placebo - controlled human adpkd trial , the benefit of a 6-month treatment with long - acting somatostatin ( octreotide - lar , 40 mg intramuscularly every 28 days ) was assessed . treatment of the bpk and orpk - rescue mouse models with the mtor inhibitor rapamycin showed effective inhibition of pkd . treatment of human adpkd transplant recipient patients with rapamycin resulted in a significant reduction in polycystic kidney volumes . an important role of ciliary dysfunction in pkd pathology is highlighted by findings of the disrupted connection between cilia and the cell cycle . robust inhibition of pkd was shown in the jck mouse model of slowly progressive disease as well as in the cpk mouse model of aggressive pkd . the availability of high - throughput in vitro screening assays that are disease - relevant , robust , rapid and reproducible is crucial . in addition , such assays can be directly used for discovery of pkd - specific compounds that antagonize cyst development . it has been shown that mdck cysts grown in a 3-d collagen matrix in vitro adequately reflect several aspects of cystogenesis in vivo and can be successfully used for drug testing ( fig . recently it was shown that an embryonic cystic kidney organ culture assay can be a very valuable testing platform for rapid assessment of efficacy as well as preliminary testing for organ toxicity before entering into time- and material - consuming trials in animals ( and t. natoli , unpublished results ) ( fig . with the availability of such a screening cascade and successful candidates already emerging from preclinical testing , the field is poised to suggest viable therapeutic options for the treatment of pkd . high - throughput drug screening ( hts ) of a small compound library can be performed using an in vitro assay of cystogenesis . effective compounds can be selected for in vivo efficacy testing in animal models of pkd . the discovery process can also be initiated through proof of concept in vivo testing ( drug candidate validation ) , followed by the drug optimization cycle . high - throughput drug screening ( hts ) of a small compound library can be performed using an in vitro assay of cystogenesis . effective compounds can be selected for in vivo efficacy testing in animal models of pkd . the discovery process can also be initiated through proof of concept in vivo testing ( drug candidate validation ) , followed by the drug optimization cycle . several potential therapies , including tolvaptan , sirolimus , everolimus and octreotide ( as discussed above ) , are being tested in ongoing adpkd clinical trials . in addition , a large clinical trial ( halt - pkd ) has been implemented to address the benefit of inhibition of the renin - angiotensin system ; this trial will test whether using ace inhibitors and angiotensin receptor blockers in combination is beneficial in comparison with ace inhibitors alone . results of the crisp ( consortium for radiologic imaging studies of pkd ) study have demonstrated that measurement of kidney volume by magnetic resonance imaging ( mri ) can be a reliable indicator of clinical outcome . recent advances in our fundamental understanding of the molecular pathogenesis of pkd have allowed selection of disease - relevant targets for therapeutic testing . several successful studies in animal models that are currently being translated into human clinical trials strengthen the need to further explore disease - specific therapeutic targets . it is likely that future treatment for pkd will require a combination therapy integrating several key pathways of cystogenesis .

autosomal dominant polycystic kidney disease ( adpkd ) occurs with an incidence of 1 : 1000 and is characterized by the development of fluid - filled cysts in the kidneys , liver , pancreas and other organs , accounting for up to 10%ofesrdcases . there are several other less common inherited recessive cystic diseases including nephronophthisis ( nphp ) , bardet - biedl syndrome ( bbs ) , joubert syndrome ( jbts ) and meckel - gruber syndrome ( mks ) that will not be discussed here and are described in other excellent reports [ 710].this review will mainly focus on recent advances in understanding the molecular pathogenesis of pkds and development of novel therapeutic options . approximately 85% of all adpkd cases are caused by mutations in the pkd1 gene , encoding polycystin-1 , which maps to human chromosome 16p13.3 [ 11 14 ] . while genotype / phenotype correlations in thepkd1gene suggest that mutations in the 5 portion of the gene are associated with a more severe phenotype , no obvious correlations have been found in the pkd2 gene . the combination of the unique unstable structural elements in the pkd1 gene with intrafamilial heterogeneity and the focal nature of cyst formation has led to the two - hit hypothesis for cyst formation , which suggests that cysts form in cells with an inherited mutation in one allele and a somatic mutation occurring in the other allele . on the other hand , over - expression of polycystin-1 in transgenic animals these data suggest that abnormal levels of polycystin-1 expression can trigger pathogenic mechanisms leading to cyst formation . genetic data have shown that mutations in a single gene , pkhd1 , located on chromosome 6p21 ( encoding fibrocystin / polyductin ) cause arpkd [ 31 , 32 ] . the large extracellular n - terminal region contains several specific motifs including leucine - rich repeats ( lrrs ) , c - type lectin domain , ldl - a region , multiple ig - like domains ( or pkd domains ) , rej domain and gps domain . it has 11 transmembrane domains , with a plat domain located in the first cytoplasmic loop and a small cytoplasmic tail with a g - protein - binding motif and coiled - coil region ( fig . the 16 ig - like domains are segmented such that the first ig - like domain is localized between the lrrs and the c - type lectin domain , while the remaining 15 iglike domains are clustered together between ldl - a and rej domains . figure 1the structure of polycystin-1 , polycystin-2 and fibrocystin ( lrr , leucine - rich repeats ; wsc , cell wall integrity and stress response component 1 ; pkd ( ig - like ) , ig - like domains ; ldl , low density lipoprotein domain ; rej , receptor for egg jelly ; gps , proteolytic g protein - coupled receptor proteolytic site ; plat , lipoxygenase domain ; ef , ef hand domain ; tig , immunoglobulin - like domains ; tmem2 , homology with tmem2 protein ) . the structure of polycystin-1 , polycystin-2 and fibrocystin ( lrr , leucine - rich repeats ; wsc , cell wall integrity and stress response component 1 ; pkd ( ig - like ) , ig - like domains ; ldl , low density lipoprotein domain ; rej , receptor for egg jelly ; gps , proteolytic g protein - coupled receptor proteolytic site ; plat , lipoxygenase domain ; ef , ef hand domain ; tig , immunoglobulin - like domains ; tmem2 , homology with tmem2 protein ) . based on similarities with other tig - containing proteins such as the hepatocyte growth factor receptor and the plexins , fibrocystin was suggested to function as a receptor or a ligand , since secreted forms may be generated from alternatively spliced transcripts . fibrocystin is expressed in cortical and medullary collecting ducts of the kidney as well as biliary and pancreatic ducts in a pattern consistent with the histologic features seen in arpkd . the formation and growth of cysts in pkd is accompanied by increased proliferation and apoptosis of cyst - lining epithelia , loss of epithelial polarity and de - differentiation , dysregulation of cell / matrix interactions and transformation of the absorptive epithelial phenotype to a secretory phenotype [ 54 56 ] . in human pkds and several animal models , apoptosis is also essential for cystogenesis : deletion of the anti - apoptotic bcl-2 and ap-2 genes and overexpression of the pro - apoptotic gene c - myc in mice results in renal cyst formation . the overlapping expression and localization patterns of polycystin-1 and both proteins are found in basolateral membranes and the primary cilium , where they may act together to regulate cellular adhesion and ca signaling . studies using in vitro models of tubulogenesis and cystogenesis based on mdck cells demonstrated that expression of polycystin-1 at cell - cell junctions at controlled levels is critical for proper tubular differentiation . a direct role for ig - like domains in cell - cell adhesion was demonstrated by specific perturbation of intercellular adhesion using antibodies against ig - like domains in cell cultures [ 36 , 37 ] . polycystin-1 was localized to the cell - cell adhesion complexes with adherens junctions and desmosomal junctions in epithelial cells of different origin [ 65 67 ] . because alterations in polycystin-1-mediated adhesion may cause the abnormal epithelial cell phenotype observed in adpkd cells , including dedifferentiation and loss of epithelial polarity , several studies examined cell - cell adhesion junctions in primary cells derived from adpkd kidneys [ 37 , 68 , 69 ] . 2 , abnormal adherens and desmosomal junctions were found in adpkd : intracellular junctions were devoid of desmosomal cadherins and associated proteins , which were sequestered to the cytoplasmic pools , and adherens junctions appeared disrupted , accompanied by a great reduction of ecadherin expression and partial compensatory expression of n - cadherin . in normal epithelial cells , pc-1 is found in a complex with talin ( tal ) , paxillin ( pax ) , vinculin ( vinc ) , focal adhesion kinase ( fak ) , c - src ( src ) , p130-cas ( cas ) , nephrocystin ( nph1 ) and tensin ( ten ) . in normal epithelial cells , pc-1 is found in a complex with talin ( tal ) , paxillin ( pax ) , vinculin ( vinc ) , focal adhesion kinase ( fak ) , c - src ( src ) , p130-cas ( cas ) , nephrocystin ( nph1 ) and tensin ( ten ) . interestingly , co - immunoprecipitation studies in adpkd cells using an anti - polycystin-1 antibody showed that e - cadherin was lost from the complex , while beta - catenin remained associated with polycystin-1 . moreover , beta - catenin was still expressed at the plasma membrane despite the loss of e - cadherin , suggesting the presence of an alternative cadherin . however , the expression of n - cadherin in place of e - cadherin in adpkd cells was not sufficient to maintain epithelial cell - cell adhesion [ 37 , 69 ] . in adpkd cells , the focal adhesion complex polycystins , fibrocystin and numerous other proteins or cystoproteins such as nephrocystin-1 , -3 , -4 , -5 , inversin , alms1 , ofd1 , bbs1 - 8 , cystin , polaris and nek8 , which cause pkd in humans and animals , were recently discovered in a distinct subcellular compartment , the primary cilium [ 10 , 74 , 75 ] . mutations in the lov-1 and pkd-2 genes of c. elegans , which are closely related to human polycystins , were associated with mechanosensation defects of ciliated sensory neurons . direct evidence linking defects in ciliogenesis and pkd came from the study of renal tissue - specific inactivation of kif3a , a subunit of kinesin - ii essential for cilia formation : inactivation of kif3a in renal tubular epithelial cells resulted in development of pkd . it was demonstrated that the primary cilia in the cystic kidney of mice with a mutation in the tg737 gene ( homologous to the ift gene of chlamydomonas , ift88 ) are shorter than normal . on the other hand , the primary cilia of cystic epithelial cells in jck mice were found to be significantly longer than the cilia in wild - type mice ; this study suggested that ciliary protein nek8 , which is mutated in the jck mice , may play a role in control of ciliary length . cells with mutated polycystin-1 fail to respond to the fluid flow : it was shown that in adpkd - derived cells , the ciliary mechanosensation of fluid - flow shear stress by poly - cystins is absent . flow - induced bending of cilia in renal epithelial cells triggers ca influx mediated by pc-2 in association with pc-1.the role of ciliary proteins in cell cycle control is supported by several findings : ( 1 ) in the normal state , pc-2 sequesters id2 [ proproliferative helix - loop - helix ( hlh ) protein ] in the cytoplasm , preventing it from entering the nucleus . in pkd , when pc-1 or pc-2 are inactivated , id2 translocates to the nucleus and interacts with e - protein , blocking its ability to induce growth - suppressive genes and resulting in increased activity of cdk2 and activation of cell proliferation ; ( 2 ) pc-1 can activate the jak / stat signaling pathway , resulting in the induction of p21 and cell cycle arrest in g0 and g1 ; ( 3 ) the intraflagellar transport component ift88/polaris is capable of controlling g1/s transition of the cell cycle . flow - induced bending of cilia in renal epithelial cells triggers ca influx mediated by pc-2 in association with pc-1.the role of ciliary proteins in cell cycle control is supported by several findings : ( 1 ) in the normal state , pc-2 sequesters id2 [ proproliferative helix - loop - helix ( hlh ) protein ] in the cytoplasm , preventing it from entering the nucleus . in pkd , when pc-1 or pc-2 are inactivated , id2 translocates to the nucleus and interacts with e - protein , blocking its ability to induce growth - suppressive genes and resulting in increased activity of cdk2 and activation of cell proliferation ; ( 2 ) pc-1 can activate the jak / stat signaling pathway , resulting in the induction of p21 and cell cycle arrest in g0 and g1 ; ( 3 ) the intraflagellar transport component ift88/polaris is capable of controlling g1/s transition of the cell cycle . the basal bodies / centrosomes of the cilia act as organizers of the mitotic spindle poles during cell division , directly connecting ciliogenesis with cell cycle regulation , and cilia are resorbed when cells enter the cell cycle ( fig . several lines of evidence support this hypothesis : overexpression of exogenous polycystin-1 in cultured cells resulted in growth arrest in g0/g1 phase of the cell cycle ( fig . on the other hand , cyst formation in these disorders common cellular abnormalities of cystic epithelia include increased proliferation , apoptosis , loss of epithelial polarity accompanied by missorting of proteins and intracystic fluid secretion . this switch from conventional camp - induced inhibition of growth to the camp - induced stimulation of growth in cystic epithelia was mediated by decreased intracellular calcium levels . cystic kidneys were shown to accumulate high levels of camp inmultiple models of pkd , making it plausible that activated camp pathways lead to both the increased proliferation and fluid secretion seen in cystic epithelia [ 86 , 88 , 89 ] . activated wnt signaling was initially implicated in pkd through studies demonstrating that overexpression of the cytoplasmic tail of polycystin-1 stabilizes endogenous beta - catenin and stimulates tcf - dependent gene transcription in vitro . flow - induced signaling switches the wnt pathway to a non - canonical beta - catenin - independent pathway through increased expression of inversin , a protein mutated in nephronophthisis type ii . the pkd1 gene is located in close proximity to the tsc2 gene , which encodes the tuberin protein responsible for the tuberous sclerosis complex ( tsc ) , in a tail - to - tail orientation . activation of mtor was also found in animal models of recessive pkd such as the bpk and orpk - rescue , suggesting that mutations in polycystin-1 are not critical form mtor activation . in general , an ideal pkd model should carry a mutation in a gene orthologous to the human disease - carrying gene.the development of the disease in such orthologous models should reproduce human pkd relative to the severity , segmental origin of kidney cysts , extrarenal manifestations and known cellular and molecular abnormalities including increased proliferation , apoptosis , camp , protein missorting as well as activation of known signaling pathways such as wnt , b - raf / mek / erk and mtor . an interesting mouse model with a mutation in the pkd2 gene ( pkd2 ) carrying an unstable allele combined with a pkd2-null allele although these animals develop kidney and liver cystic disease that mimics human adpkd , the model has not been widely used for therapeutic testing due to a very high degree of phenotypic heterogeneity in combination with a slow course of progression with no measurable loss of renal function as late as 46 months of age . several spontaneous mouse models of pkd , such as cpk , bpk and orpk , resemble human recessive disease with respect to cyst localization and the rapid rate of disease progression ( table 1 ) . the jck mouse model is characterized by the development of cysts in multiple nephron segments and , despite the autosomal recessive mode of inheritance , resembles human adpkd phenotypically and can be used for relatively rapid therapeutic testing . since no single model fully recapitulates all aspects of human pkd pathogenesis , it is important to test a potential therapy in more than one pkd model to determine its utility for clinical testing . table 1rodent models of polycystic kidney diseases used in therapeutic intervention studies.modelgeneproteinprogressionrenal pathologykidney phenotypemousecpkcys1cystinrapidpt , cdarpkdbpkbicc1bicaudal crapidpt , cdarpkdorpktgn737polarisrapidpt , cdarpkdpcynphp3nephrocystin-3slownephron cdadpkdjcknek8nek8moderatelh , dt , cdadpkdpkd1pkd1polycystin-1el arpkdpkd2pkd2polycystin-2slowpt , lh , dt , cdadpkdrathan : sprd - cypkdr1samcystinslowptadpkdpckpkhd1fibrocystinslowcdarpkdpt , proximal tubule ; dt , distal tubule ; cd , collecting duct ; lh , loop of henle ; el , embryonic lethal . preclinical studies showed that inhibition of egfr tyrosine kinase activity significantly reduced cystogenesis in mouse models of pkd , including bpk and orpk , as well as in the han : sprd rat model [ 99 , 100 ] , all models characterized by overexpression and mislocalization of egfr to the apical membrane . however , no therapeutic effect was detected in the pck rat ; interestingly , although the pck rat is an orthologous model of arpkd , egfr was not found to be mislocated to the apical membrane of cystic cells , which may explain the lack of therapeutic efficacy in this model . to test the effect of inhibition of apoptosis in pkd this study showed inhibition of pkd progression and attenuation of the loss of renal function . the critical role of the camp - activated b - raf / mek / erk pathway was confirmed by the successful preclinical testing of a mek inhibitor : oral administration the map / erk kinase inhibitor pd184352 to pcy mice significantly decreased kidney weight , serum creatinine levels and water intake and significantly increased urine osmolality . thus , inhibitors of the b - raf / mek / erk pathway may prove to be promising agents for pkd therapy . in a randomized placebo - controlled human adpkd trial , the benefit of a 6-month treatment with long - acting somatostatin ( octreotide - lar , 40 mg intramuscularly every 28 days ) was assessed . roscovitine ( seliciclib , cyc202 ) is a potent and remarkably selective inhibitor of cdk2/cyclin e , cdk7/cyclin h , cdk9/cyclin t1 and cdk5/p35-p25 [ 112 , 113 ] . roscovitine was equally effective against cysts formed in different segments of the nephron , a desirable feature for a potential drug against adpkd , where cysts are formed in multiple parts of the nephron . recently it was shown that an embryonic cystic kidney organ culture assay can be a very valuable testing platform for rapid assessment of efficacy as well as preliminary testing for organ toxicity before entering into time- and material - consuming trials in animals ( and t. natoli , unpublished results ) ( fig . in addition , a large clinical trial ( halt - pkd ) has been implemented to address the benefit of inhibition of the renin - angiotensin system ; this trial will test whether using ace inhibitors and angiotensin receptor blockers in combination is beneficial in comparison with ace inhibitors alone . results of the crisp ( consortium for radiologic imaging studies of pkd ) study have demonstrated that measurement of kidney volume by magnetic resonance imaging ( mri ) can be a reliable indicator of clinical outcome .

a central question in neuroscience is the mechanism by which memories can be preserved for years . long - term memories are at least in part encoded as specific patterns , or engrams , of strengthened synapses [ 1 , 2 ] , and long - term synaptic potentiation ( ltp ) persists for months in vivo . how can specific groups of synapses remain strong for months or years despite turnover of macromolecules and fluctuations in the size and shape of synaptic structures ? numerous mathematical models have been developed that describe maintenance of long - term memory ( ltm ) as dependent on bistability of synaptic weights , mediated by positive feedback loops of biochemical reactions , typically thought of as operative in dendritic spines . proposed feedback mechanisms have relied on self - sustaining autophosphorylation of cam kinase ii [ 4 , 5 ] , persistent phosphorylation of ampa receptors by protein kinase a , enhanced translation of protein kinase m , or self - sustaining clustering of a translation activator , cytoplasmic polyadenylation element binding protein . with these models , ltp switches a synapse from a state of low basal weight to a high weight state and turns on the positive feedback loop . the loop then operates autonomously to keep the synapse in the high weight state indefinitely . however , despite extensive investigation , empirical evidence of a bistable distribution of two distinct synaptic weight states has not , in fact , been obtained . although some studies [ 9 , 10 ] have suggested two distinct strength states for synapses , as measured by the amplitude of excitatory postsynaptic currents before and after a stimulus protocol , these studies have only examined the early phase of ltp ( < 1 h ) , which does not depend on protein synthesis or other processes necessary for long - term memory storage . therefore , those data do not address the dynamics of long - term memory storage . in addition , a demonstration of synaptic bistability would require not only finding two distinct synaptic strength states but also finding that a set of different protocols for ltp induction ( e.g. , different patterns of stimuli , or localized application of pharmacological agents ) commonly switched synaptic weights between the same two stable states . in addition , modeling suggests that stochastic fluctuations of macromolecule numbers within a small volume such as a spine head are likely to destabilize steady states of biochemical positive feedback loops , causing randomly timed state switches (; see for an opposing view ) . finally , in hippocampal neuron cultures , continuous and extensive fluctuations of postsynaptic density ( psd ) morphology are observed and are driven in part by synaptic activity . such dynamics would seem difficult to reconcile with only two , or a few , stable weight states . empirical distributions of the weights of excitatory synapses onto cortical or hippocampal pyramidal neurons appear unimodal ( a single peak ) rather than bimodal and are commonly heavy - tailed ( skewed towards high weights ) [ 1318 ] . some histograms are based on relatively small numbers of measurements , so that some bimodality might be present but hidden in variability among bins . however , the weight distribution of song et al . is based on measurements of several hundred excitatory postsynaptic potential ( epsp ) amplitudes and appears to particularly disfavor the bimodal hypothesis . a large number of measurements are fit well by a log - normal distribution ( i.e. , a normal distribution with the logarithm of the volume on the x - axis ) . in addition , a histogram of the volume of dendritic spines , based on a large number of individual measurements ( ~10,000 ) in mouse auditory cortex , is clearly unimodal , heavy - tailed , and approximately log - normal . spine volume is approximately proportional to the postsynaptic density size [ 2022 ] and to the number of postsynaptic ampa receptors as well as to the amplitude of the epsc measured following localized glutamate uncaging . thus it is plausible , and we assume that increases / decreases in spine volume can serve as a proxy for ltp / ltd . if synaptic weights and correlated spine volumes are not in fact bistable , how can patterns of strong synapses be maintained for very long times ? two observations support a mechanism based on metastability of small clusters of large dendritic spines , corresponding to groups of strong synaptic contacts . the first observation is that although spine volumes fluctuate , some large spines are extremely stable , existing for months ( in sensory cortex or in motor cortex ) . the second is that induction of late , protein synthesis - dependent ltp ( l - ltp ) at a spine facilitates l - ltp expression at nearby spines , on the same dendritic branch , that coincidentally receive stimuli too weak to support l - ltp if given alone . this observation supports the clustered plasticity hypothesis in which clusters of spines on a single dendritic branch , rather than single spines , may serve as primary functional units for storage of ltm . for example ( 1 ) in motor cortex , learning induces coordinated formation of small spine clusters on a given dendritic branch ( 2 ) morphologically , spines are grouped into small clusters on pyramidal dendrites and ( 3 ) in rat hippocampal slice cultures , spontaneous coactivation of dendritic spines is frequent and is clustered , occurring more often for spines within 8 m of each other . here an initial , relatively phenomenological model is developed describing synaptic weight changes due to competing processes of ltp ( corresponding to spine growth ) and long - term synaptic depression ( ltd ) ( corresponding to spine shrinkage ) . assuming volume changes are a proxy for weight changes , weight changes are simulated for discrete intervals of 1 day , over times of months or years . the discrete intervals were chosen to simulate the dynamics observed in experiments where volumes are imaged at intervals of ~1 day [ 19 , 32 ] . in the model each day the magnitude of ltp is governed by a gaussian random variable , as is that of ltd ( methods ) . these magnitudes are also approximately proportional to the preexisting weight . as suggested by recent data ( [ 24 , 25 , 32 ] but see ) , a volatility factor was introduced so that the weights of larger synapses fluctuate less . this factor proved necessary for large synapses to remain stable for months ( see discussion ) . model parameter sensitivity was lessened when synapses were modeled as coupled into small clusters ( ~10 spines , modeled as on the same dendritic branch and able to interact ) . in accordance with data [ 32 , 33 ] , the model also incorporates disappearance or silencing of small synapses and compensatory regeneration of new synapses . when the dynamics of 1,000 small clusters were simulated , the weight distribution of the entire ensemble of individual synapses converged to a steady - state , log - normal form . individual clusters remained stable for many years , with the average number of active synapses maintained in a range consistent with empirical data . the magnitude of daily changes in synaptic weight approximated a normal distribution except for an extra peak at w = 0 , which constitutes a model prediction . if a subset of synapses was reinitialized to have large weights , this subset maintained large average weights for ~2 yrs , corresponding to persistence of a pattern of strong synapses that might serve as the engram for a memory . some memories however persist for even longer times . in support of the clustered plasticity hypothesis , our simulations suggest that such memories might be encoded as a pattern of specific , stable clusters of active synapses . in simulations , each cluster consists of ncl synapses , corresponding to ncl individual spines . at a given time , most of these synapses are active , with synaptic weight w ranging from ~0.2 to 10 . the remaining synapses are silent , with a very low , basal weight of 0.05 . weight evolution is simulated using discrete , large time steps t , considered to correspond to 24 h. at each time step , all weights are synchronously updated . the size and direction of a weight update at a given synapse are assumed uncorrelated with that at a neighboring synapse and with the preceding update at the given synapse . , two independent processes change synaptic weights during each time step . an ltp process increases w and an ltd process decreases w. strong synapses consume more resources for maintenance , corresponding to locally available mrnas / proteins . the model thus assumes that the more strong synapses present in a cluster , the fewer resources are available to support synaptic growth ( ltp ) . thus , the model assumes that the amplitude of ltp is also proportional to a volatility factor that decreases as w increases ( 1)-(2 ) as is that of ltd ( 3 ) . ltp and ltd add to give the net change in w per time step ( 4 ) . for each time step , the ltp and ltd amplitudes are proportional , respectively , to gaussian random variables r1 and r2 . these variables have respective means a1 and a2 , and sd1 and sd2 are the standard deviations . a1 and a2 are substantially larger , by a fixed factor of 4 , than are sd1 and sd2 . thus r1 and r2 are very rarely negative , but if either becomes negative it is reset to zero . the average ltp amplitude a1 is a decreasing function of the number of strong synapses in a given cluster , denoted as nst . with ncl the total number of synapses in a cluster , the average ltp amplitude a1 decreases linearly with nst , from a maximum amplitude x2 ( for nst = 0 ) to a minimum x1 ( for nst = ncl ) . for the simulation of figure 2(b ) with this amplitude decrease removed , a1 and thus sd1 are fixed parameters . for comparison , simulations were also carried out in which r1 and r2 were drawn from exponential distributions . with exponential distributions r1 and r2 are always nonnegative , with probability densities that peak at 0 and decay exponentially for increasing positive values . ltp and ltd are also proportional to a volatility factor vow , decreasing with w:(1)vow = vhivhivloww+wmed.from ( 1 ) , vow decreases from the parameter vhi ( for w = 0 ) to vlo ( for w wmed ) . when w = wmed , vow is midway between vhi and vlo . ltp and ltd amplitudes are also multiplied by the preexisting value of w. to keep w bounded the ltp amplitude is also multiplied by a decreasing function of w that has two parameters , khi and whi . combining factors ltd amplitude is(3)altd = wr2vow.at each time step , for each active synapse,(4)wnew = wold+altpaltd.if w falls below a threshold twk , the synapse is reset to be silent . for each silent synapse at each time step , the probability for regeneration pact increases with the number of strong synapses in its cluster , to a maximal value pbas:(5)pact = pbasnstncl.in addition , regeneration only occurs if an adjacent synapse is strong . in a cluster , synapse 1 can only switch to active if synapse 2 is strong , and synapse 5 can only switch if synapse 4 and/or 6 is strong . a switch resets w to wreset , above twk . for all simulations , to ensure initial weight , distributions were at steady state and distributions and other quantities were computed only after 50,000 simulated days . it is necessary that simulated time steps plausibly correspond to the common empirical interval of 1 day between spine imaging sessions . therefore , it was necessary to scale ltp and ltd amplitudes so that for a time step the simulated per cent change in w , averaged over all synapses , agreed with an average daily empirical change in w. empirically yasumatsu et al . presented a piecewise - linear relationship between spine volume v and its change v ( figure 7(b ) of ) under control conditions ( normal synaptic activity ) . this model , which they denote c-1 , was also able to predict an empirical steady - state spine volume distribution ( figure 8(e ) of ) . is(6)v=0.16v+0.01 for v0.25 m3 ( most spines),v=0.12v0.06 for 0.25 m3<v0.5 m3,v=0 for v>0.5 m3.from this relationship , combined with the plotted volumes in figures 1(b ) and 1(c ) of , it can be inferred that the average percent daily change in v lies within the range of ~1420% . one can not infer a more precise value from these data . for comparison , in the model 's steady - state weight distribution of figure 2(a ) , the percent change in w during a time step , averaged over all 10,000 synapses , is 16.5% . assuming v is a proxy for w , this qualitative agreement between simulated and empirical average weight changes suggests that the fixed time step in figures 25 , which is otherwise arbitrary , can be taken to correspond to approximately 1 day of weight dynamics . let xi denote the total set of n synaptic weights at a reference time , with i the indexing variable from 1 to n. for 1,000 10-synapse clusters , n = 10 , 000 . as t increases from the reference time , yi will evolve and r will decline from 1 . let x- , y- denote the means of xi , yi . the standard equation was used:(7)r=i=1nxixyiyi=1nxix2i=1nyiy2.standard parameter values , used in all simulations unless stated otherwise , are as follows:(8)ncl=10 , wreset=0.4 , twk=0.08,tst=0.8 , vhi=4.0 , vlo=0.2,wmed=0.4 , x1=0.144 , x2=0.18,a2=0.16 , khi=0.05 , whi=20.0 , pbas=0.1.in supplementary material ( available online at http://dx.doi.org/10.1155/2015/185410 ) , a java program is given that executes simulations from figures 24 . figure 2(a ) illustrates the approximately log - normal distribution of synaptic weights obtained at steady state . here , 1,000 clusters of synapses were simulated , with ncl = 10 . the black trace is the resulting histogram of synaptic weight w ; the red trace is a log - normal distribution fitted to the histogram . the range of w spans approximately 5 natural log units ( i.e. , a multiplicative factor of ~150 ) . this range is similar to data describing the range of dendritic spine volumes [ 19 , 32 ] . in this steady state , the relative synaptic weight change per time step , averaged over all synapses , is 16.5% . this agreement is necessary for the model time step to represent the empirical interval of 1 day between imaging sessions . to prevent this distribution from being overly sensitive to the average daily ltp amplitude , the model assumes this amplitude decreases with the number of strong synapses in the cluster to which the synapse belongs . starting from figure 2(a ) , when the mean ltp amplitude was decreased by 5% , the mean of w decreased by only 16% . in contrast , figure 2(b ) illustrates that in a model variant with mean and standard deviation of the ltp amplitude fixed , the weight histogram was shifted to the right of the log - normal distribution from figure 2(a ) and has a shape clearly distorted from log - normal with a much steeper cutoff at high w. to attempt to improve the histogram , the mean ltp amplitude was decreased by 2% . this small change resulted in a large shift of the histogram to the left ( blue trace ) , and 56% of the synapses became silent ( not included in the histogram ) . a similar distribution , with extreme sensitivity to mean ltp amplitude , resulted from a model variant in which synaptic clustering was deleted by fixing the mean and standard deviation of the ltp amplitude and also fixing the synapse regeneration probability pact ( 5 ) . the distribution is strongly bimodal , with almost 50% of synapses silenced at the low basal weight , unable to regenerate , and the remainder in a narrow distribution centered at very high weights . one other model variant was also simulated , in which the gaussian random variables r1 and r2 that govern the amplitudes of individual ltp and ltd increments were replaced by random variables drawn from exponential distributions ( methods ) . however , these simulations failed to produce synaptic weight distributions that resembled experimental data . if the decay rate constants for the two exponential distributions differed by more than twofold , almost all synaptic weights either ran to infinity ( if the mean ltp amplitude was greater ) or converged to a low basal synaptic weight imposed as a floor in the model ( if the mean ltd amplitude was greater ) . if the rate constants were similar a positive synaptic weight distribution could be obtained , but the shape of this distribution was itself exponential . all following results are therefore based on the first model variant described above , that of figure 2(a ) , with synaptic regeneration and an ltp amplitude that decreases with the number of strong synapses in a cluster . equations and parameter values are in methods . typical time courses for clusters with regeneration are illustrated in figures 3(a ) and 3(b ) . strong synapses are much more stable on average , in agreement with data demonstrating that large dendritic spines are more persistent [ 32 , 34 , 35 ] . strong synapses often maintain high values of w for a year or more , whereas weak synapses show much larger relative ( percent ) fluctuations in w. weak synapses often drop to a very low basal weight . silent synapses can regenerate , evident as vertical jumps in time courses near the bottom of figures 3(a)-3(b ) ( e.g. , arrowheads below x - axes ) . figure 3(c ) illustrates a typical time course for the number of strong synapses nst in a cluster ( with weights greater than a threshold tst = 0.8 ) . clusters are very stable in that , for ncl = 10 synapses per cluster , nst almost always remains between 4 and 7 for years . simulations with ncl = 15 or 20 also had very stable clusters . however , for a smaller ncl = 5 , the model no longer simulated a log - normal synaptic weight distribution at steady state . recorded synaptic dynamics for many days in cortical neuron cultures . rather than spine volume , they examined variations in the size of psd-95:gfp puncta , that is , localized concentrations , with a size corresponding to spines , of the postsynaptic density protein psd-95 fused to a gfp fluorophore . their dynamics are qualitatively consistent with those illustrated in figures 2(a ) and 3 in that ( 1 ) new synapses were continually formed , ( 2 ) synapses whose size was reduced beneath some threshold , analogous to the model threshold twk , were eliminated , and ( 3 ) large synapses tended to shrink and small synapses to grow . in accordance with ( 3 ) , stability of a simulated steady state , unimodal distribution such as that of figure 2(a ) requires that individual synapses with weights above the peak of the distribution decrease their weight on average and vice versa for those with low weights . empirically matsuzaki et al . found that smaller spines are more likely to undergo stable enlargement in response to an ltp induction protocol . figure 4(a ) illustrates a simulated histogram of the amplitudes of the daily changes in synaptic weight , w , at steady state . the majority of the histogram is fitted well by a normal distribution with the clear exception of the narrow peak close to w = 0 . a scatter plot of w versus w revealed that over almost the entire range of w , the amplitude of w varied from near zero to a peak value of ~0.30.5 . the plot is qualitatively linear , which is not surprising because , in the model , the average daily ltd and ltp amplitudes contain terms proportional to w ( methods , ( 2 ) , ( 3 ) ) . however , the plot further illustrates that the average change in w varies much less than does w itself . as w increases from 0.05 to 2.0 , the absolute value of w increases only from ~0.05 to 0.12 . thus , the relative change in w ( i.e. , w / w ) decreases substantially as w increases . this prediction of the model appears in accordance with the data from yasumatsu et al . but not with the data of loewenstein et al . for which this relative change appears nearly constant ( see discussion ) . however , what if the weight distribution is altered by an imposed large perturbation that sets high weights for a specified subset of synapses ? such a perturbation might correspond to formation of a specific , long - term memory engram . will the subset of strong synaptic weights remain elevated for months , corresponding to long - lasting storage of a memory ? starting from the distribution of figure 2(a ) , for all of the 1000 10-synapse clusters , synapses 15 were reset to a high weight of 5.0 at t = 200 days . this weight is well above the steady - state mean w. the other 5 synapses were reset to a low weight ( 0.5 ) . after the reset , the weights fluctuate but 400 days later , all but one of the strong synapses have maintained w above the steady - state average . figure 5(b ) illustrates the time course of resetting and decay for all 5,000 synapses that were reset to w = 5.0 . their average weight decays slowly such that 700 days after reset this average is still about twice the steady - state average of w. the lower red trace , one standard deviation below the average weight , is also still above the steady - state average . if the steady - state distribution of w is evolved without any perturbation , the time scale for decorrelation of synaptic weights from their specific values at a given time is , perhaps surprisingly , quite long , similar to the time scale for the decay of perturbed synaptic weights . starting from the distribution of figure 2(a ) , the pearson correlation coefficient between the weights of the 10,000 synapses decays with a time constant of approximately 500 days ( figure 5(c ) ) , similar to the dynamics of figure 5(b ) . with simulations of isolated , uncoupled synapses ( figure 2(b ) ) , weight distributions were extremely sensitive to model parameters . this model variant was therefore not a plausible description of synaptic dynamics , because biophysical parameters are expected to vary somewhat between spines , dendritic branches , and individual neurons . this sensitivity was eliminated when synapses were modeled as coupled into clusters ( ~10 synapses ) . in the model have reported similar spine clusters on primate cortical pyramidal neurons . on a scale of ~410 m along dendritic branches , numerous clusters of 515 spines were identified algorithmically . this distance scale and cluster number appears to correspond to the observations of takahashi et al . that spontaneous coactivation of dendritic spines is clustered , occurring more often for spines within 8 m of each other . we do note recent analysis by a different group failed to support clustering of this type and scale . with coupling into clusters of ~10 synapses , when the dynamics of 1,000 clusters were simulated , the weight distribution of individual synapses converged to a stable steady - state , log - normal form ( figure 2(a ) ) . data illustrates that spines compete for ltp expression ; that is , local resources ( possibly amounts of key proteins ) are limited such that the amount of ltp at a given spine decreases if ltp is simultaneously induced at multiple spines close together on the same dendritic branch . our mechanism of coupling synapses into clusters is similar in that it corresponds to an additional form of resource competition . however , in the model , competition is generated by ongoing maintenance of multiple synapses rather than only by simultaneous ltp of multiple synapses . thus , the mean magnitude of ltp at a given synapse during a simulated day was assumed to be a decreasing function of the number of other large synapses in the same cluster ( methods ) . ongoing maintenance of large spines is assumed to consume resources ( proteins , rna ) that would otherwise be available for strengthening of neighboring spines , so that their mean ltp magnitude decreased . the competition described by govindarajan et al . occurs over a distance scale of 1020 m , comparable to , although slightly larger than , the scale of 510 m posited by yadav et al . for clusters of ~510 spines . however , they did determine this rate for another measure of synaptic cross talk , the ability of a weakly stimulated spine to capture plasticity - related factors synthesized in response to strong stimulation of a nearby spine ( synaptic tagging and capture , resulting in ltp at the weakly stimulated spine ) . these data suggest that , for clusters of ~10 spines as simulated here , distance between spines might not limit the efficacy of resource competition . however , these data do not address whether competition could occur over a longer distance scale , for example , 50 m . to assess this question groups of spines spaced along a dendritic branch would need to be concurrently stimulated , and ltp quantified . to simulate a distribution spanning a broad range of synaptic weights , as is observed empirically , it was critical to model synaptic regeneration . to obtain distributions similar to that of figure 2(a ) , synapses that fell to a low basal weight needed to have , on successive days , a probability of weight reset to a higher , active value . this method of simulating regeneration was chosen to maintain equal average numbers of synaptic loss and regeneration events within any given cluster . empirically , the number of active spines in a cluster is relatively low ( ~37 ) . thus for clusters with low numbers of active spines to serve as functional memory storage units , as suggested by the clustered plasticity hypothesis , the average cluster size would need to be stable to avoid disappearance of clusters . an important question in neuroscience is how the observed regeneration of spines avoids disrupting stored memories or forming spurious memories . it is plausible that new , or regenerated , spines are generically not strong ( as in our model ) and therefore can not participate in memory storage unless they are potentiated by stimuli that induce long - term memory . however , further empirical investigation of this question , in conjunction with modeling , is needed . the model of loewenstein et al . simulates daily changes in the volume of dendritic spines and obtains a steady - state log - normal distribution of spine volumes very similar to the empirical distribution found by these authors . these important results have clarified the necessity of reconciling unimodal weight distributions with stable storage of long - term memory . their model consists of an ornstein - uhlenbeck stochastic process in which the logarithm of the volume of any given spine is directly incremented each day . the model presented here may constitute a further advance , in that it represents more biophysical elements , such as synapse loss / regeneration and synapse clustering . the ltp and ltd processes in the model ( 1 ) act directly on the synaptic weight rather than on its logarithm and ( 2 ) can be thought of as due to a large number of individual biochemical events occurring during a simulated day and corresponding to insertion and removal of individual molecular complexes or slots . point ( 2 ) connects the current model with the more detailed molecular model of lisman and raghavachari , in which ltp and ltd correspond , respectively , to insertion and removal of slot complexes , each consisting of a small number of ampa receptors together with associated scaffolding proteins and , possibly , kinases or other signaling proteins . additional elements of our model that may be consistent with that of lisman and raghavachari are as follows . if over a day , the time intervals between individual insertions of slot complexes as well as the intervals between removals of complexes follow poisson distributions , and if there are on average more than ~20 insertions and removals / day , then by the central limit theorem the sums of these poisson processes will approximate gaussian random variables . these gaussian variables would correspond to the model 's gaussian random variables r1 and r2 , to which the daily ltp and ltd amplitudes are proportional ( 2 ) , ( 3 ) . similarly , if the individual time intervals were gaussian random variables , their sum would be gaussian . it also appears plausible that average numbers of slot insertions and removals are proportional to the preexisting size of a spine , corresponding to the model assumption that mean ltp and ltd amplitudes are approximately proportional to synaptic weight . recent data are consistent with these ideas and also suggest such slot complexes include presynaptic components and release sites . current data has not directly examined whether there is spatial clustering of presynaptic boutons at the distance scale of ~5 m that corresponds to putative postsynaptic spine clusters . however , at this distance scale , such clusters would correspond to a single axon and spines in a cluster would thus be coactivated by a common input , consistent with the hypothesis that such clusters constitute functional units in the formation and storage of specific memories . in the model , the average relative ( percent ) change in w over a day decreases substantially as w increases ( figure 4(b ) ) . this result is essential for the model to represent stable long - term memory storage , because selective stability of strong synapses is only found if the relative change in w decreases in this manner . are these dynamics supported by current data ? relevant data describes the relationship of spine volume changes v to v. these data are , however , contradictory . illustrate a substantially larger relative variation in v ( figure 4(c ) of ) , such that v and v appear approximately proportional . however , yasumatsu et al . show a different relationship , for which smaller spines have an average v similar to large spines ( figure 1(c ) of ) . it is plausible that the difference in synaptic dynamics in these studies was generated , at least in part , by substantial differences in experimental conditions . imaged ca1 pyramidal neurons in cultured rat hippocampal slices at postnatal days 1723 , whereas loewenstein et al . did craniotomies to allow in vivo imaging of dendrites in auditory cortex of mice approximately 6 months old . in addition , filopodia ( protrusions without spine heads or with very small heads ) were eliminated from the former study , but not the latter . clearly further empirical investigation is needed to clarify these critical aspects of synaptic dynamics . at steady state the magnitudes of the daily changes in weight were distributed approximately normally except for an extra peak centered at w = 0 ( figure 4(a ) ) . however , data does indicate that a minor percentage of spines are stable for months or years [ 24 , 25 ] . these data suggest that a subpopulation of spines with very small daily weight changes might exist , but not yet be resolved due to empirical sensitivity limits . the current model does not represent the biochemical processes that might underlie long - term stability of such a subpopulation . however , recent studies support a relevant hypothesis that ongoing spontaneous neuronal activity is critical for long - term maintenance of synaptic strength . models have suggested that such activity can preferentially maintain synapses that are already strong possibly by preferentially reactivating stored patterns of strengthened synapses . empirically , a temporary induced knockdown of nmda receptor function can irreversibly eliminate remote memories , plausibly by preventing spontaneous activity from potentiating and thereby maintaining synapses . ongoing ltp increments that are necessary to maintain strong synapses , whether single or clustered ( as in the current model ) , may correspond to frequent spontaneous or environmentally induced activity , which may induce repeated cycles of nmda receptor - dependent ltp . simulations ( figures 5(a)-5(b ) ) illustrate that the model can store a specific memory trace , for ~1 yr , corresponding to persistence of a pattern of strong synapses . however , in humans , some memories persist for many years . in accordance with the hypothesis of govindarajan et al . , such memories might be encoded at the cluster level rather than the single - synapse level , as a set of specific , stable clusters of active synapses . in simulations , they maintained a range of strong synapse numbers ( ~47 ) similar to the range suggested by data demonstrating clustering on neocortical pyramidal dendrites . because each cluster was stable indefinitely , if a pattern of such clusters was established , that pattern would persist . in simulations , stable clusters were found when the total number of synapses in a cluster was 10 ( figure 3(c ) ) , or 1520 . however , for five synapses per cluster , anomalous dynamics resulted . a bimodal synaptic weight distribution , not resembling empirical data , we do not take this result to be a prediction of a minimum empirical cluster size . instead , we believe that the model should be improved in future work to avoid or reduce this dramatic change in dynamics . the model makes additional predictions . when comparing spines of similar sizes in different clusters , the average volume change between imaging sessions is predicted to be less positive ( or more negative ) if other spines in a cluster are large . in addition , because synaptic weights and spine volumes are not considered bistable , different induction protocols for late , protein - synthesis dependent ltp are predicted to induce clearly different amplitudes of synaptic weight increase or of average spine volume increase . without bistability , repeated applications of stimulus protocols should , at least in some cases , further enhance l - ltp .

memories are stored , at least partly , as patterns of strong synapses . given molecular turnover , how can synapses maintain strong for the years that memories can persist ? some models postulate that biochemical bistability maintains strong synapses . however , bistability should give a bimodal distribution of synaptic strength or weight , whereas current data show unimodal distributions for weights and for a correlated variable , dendritic spine volume . thus it is important for models to simulate both unimodal distributions and long - term memory persistence . here a model is developed that connects ongoing , competing processes of synaptic growth and weakening to stochastic processes of receptor insertion and removal in dendritic spines . the model simulates long - term ( > 1 yr ) persistence of groups of strong synapses . a unimodal weight distribution results . for stability of this distribution it proved essential to incorporate resource competition between synapses organized into small clusters . with competition , these clusters are stable for years . these simulations concur with recent data to support the clustered plasticity hypothesis which suggests clusters , rather than single synaptic contacts , may be a fundamental unit for storage of long - term memory . the model makes empirical predictions and may provide a framework to investigate mechanisms maintaining the balance between synaptic plasticity and stability of memory .

1. Introduction 2. Methods 3. Results 4. Discussion

a central question in neuroscience is the mechanism by which memories can be preserved for years . long - term memories are at least in part encoded as specific patterns , or engrams , of strengthened synapses [ 1 , 2 ] , and long - term synaptic potentiation ( ltp ) persists for months in vivo . how can specific groups of synapses remain strong for months or years despite turnover of macromolecules and fluctuations in the size and shape of synaptic structures ? numerous mathematical models have been developed that describe maintenance of long - term memory ( ltm ) as dependent on bistability of synaptic weights , mediated by positive feedback loops of biochemical reactions , typically thought of as operative in dendritic spines . however , despite extensive investigation , empirical evidence of a bistable distribution of two distinct synaptic weight states has not , in fact , been obtained . although some studies [ 9 , 10 ] have suggested two distinct strength states for synapses , as measured by the amplitude of excitatory postsynaptic currents before and after a stimulus protocol , these studies have only examined the early phase of ltp ( < 1 h ) , which does not depend on protein synthesis or other processes necessary for long - term memory storage . therefore , those data do not address the dynamics of long - term memory storage . in addition , a demonstration of synaptic bistability would require not only finding two distinct synaptic strength states but also finding that a set of different protocols for ltp induction ( e.g. , different patterns of stimuli , or localized application of pharmacological agents ) commonly switched synaptic weights between the same two stable states . however , the weight distribution of song et al . in addition , a histogram of the volume of dendritic spines , based on a large number of individual measurements ( ~10,000 ) in mouse auditory cortex , is clearly unimodal , heavy - tailed , and approximately log - normal . spine volume is approximately proportional to the postsynaptic density size [ 2022 ] and to the number of postsynaptic ampa receptors as well as to the amplitude of the epsc measured following localized glutamate uncaging . thus it is plausible , and we assume that increases / decreases in spine volume can serve as a proxy for ltp / ltd . if synaptic weights and correlated spine volumes are not in fact bistable , how can patterns of strong synapses be maintained for very long times ? two observations support a mechanism based on metastability of small clusters of large dendritic spines , corresponding to groups of strong synaptic contacts . the second is that induction of late , protein synthesis - dependent ltp ( l - ltp ) at a spine facilitates l - ltp expression at nearby spines , on the same dendritic branch , that coincidentally receive stimuli too weak to support l - ltp if given alone . this observation supports the clustered plasticity hypothesis in which clusters of spines on a single dendritic branch , rather than single spines , may serve as primary functional units for storage of ltm . for example ( 1 ) in motor cortex , learning induces coordinated formation of small spine clusters on a given dendritic branch ( 2 ) morphologically , spines are grouped into small clusters on pyramidal dendrites and ( 3 ) in rat hippocampal slice cultures , spontaneous coactivation of dendritic spines is frequent and is clustered , occurring more often for spines within 8 m of each other . here an initial , relatively phenomenological model is developed describing synaptic weight changes due to competing processes of ltp ( corresponding to spine growth ) and long - term synaptic depression ( ltd ) ( corresponding to spine shrinkage ) . the discrete intervals were chosen to simulate the dynamics observed in experiments where volumes are imaged at intervals of ~1 day [ 19 , 32 ] . in the model each day the magnitude of ltp is governed by a gaussian random variable , as is that of ltd ( methods ) . as suggested by recent data ( [ 24 , 25 , 32 ] but see ) , a volatility factor was introduced so that the weights of larger synapses fluctuate less . this factor proved necessary for large synapses to remain stable for months ( see discussion ) . model parameter sensitivity was lessened when synapses were modeled as coupled into small clusters ( ~10 spines , modeled as on the same dendritic branch and able to interact ) . in accordance with data [ 32 , 33 ] , the model also incorporates disappearance or silencing of small synapses and compensatory regeneration of new synapses . when the dynamics of 1,000 small clusters were simulated , the weight distribution of the entire ensemble of individual synapses converged to a steady - state , log - normal form . individual clusters remained stable for many years , with the average number of active synapses maintained in a range consistent with empirical data . if a subset of synapses was reinitialized to have large weights , this subset maintained large average weights for ~2 yrs , corresponding to persistence of a pattern of strong synapses that might serve as the engram for a memory . in support of the clustered plasticity hypothesis , our simulations suggest that such memories might be encoded as a pattern of specific , stable clusters of active synapses . an ltp process increases w and an ltd process decreases w. strong synapses consume more resources for maintenance , corresponding to locally available mrnas / proteins . the model thus assumes that the more strong synapses present in a cluster , the fewer resources are available to support synaptic growth ( ltp ) . thus r1 and r2 are very rarely negative , but if either becomes negative it is reset to zero . the average ltp amplitude a1 is a decreasing function of the number of strong synapses in a given cluster , denoted as nst . for the simulation of figure 2(b ) with this amplitude decrease removed , a1 and thus sd1 are fixed parameters . for each silent synapse at each time step , the probability for regeneration pact increases with the number of strong synapses in its cluster , to a maximal value pbas:(5)pact = pbasnstncl.in addition , regeneration only occurs if an adjacent synapse is strong . for all simulations , to ensure initial weight , distributions were at steady state and distributions and other quantities were computed only after 50,000 simulated days . therefore , it was necessary to scale ltp and ltd amplitudes so that for a time step the simulated per cent change in w , averaged over all synapses , agreed with an average daily empirical change in w. empirically yasumatsu et al . presented a piecewise - linear relationship between spine volume v and its change v ( figure 7(b ) of ) under control conditions ( normal synaptic activity ) . this model , which they denote c-1 , was also able to predict an empirical steady - state spine volume distribution ( figure 8(e ) of ) . for comparison , in the model 's steady - state weight distribution of figure 2(a ) , the percent change in w during a time step , averaged over all 10,000 synapses , is 16.5% . let xi denote the total set of n synaptic weights at a reference time , with i the indexing variable from 1 to n. for 1,000 10-synapse clusters , n = 10 , 000 . figure 2(a ) illustrates the approximately log - normal distribution of synaptic weights obtained at steady state . the black trace is the resulting histogram of synaptic weight w ; the red trace is a log - normal distribution fitted to the histogram . this range is similar to data describing the range of dendritic spine volumes [ 19 , 32 ] . this agreement is necessary for the model time step to represent the empirical interval of 1 day between imaging sessions . to prevent this distribution from being overly sensitive to the average daily ltp amplitude , the model assumes this amplitude decreases with the number of strong synapses in the cluster to which the synapse belongs . a similar distribution , with extreme sensitivity to mean ltp amplitude , resulted from a model variant in which synaptic clustering was deleted by fixing the mean and standard deviation of the ltp amplitude and also fixing the synapse regeneration probability pact ( 5 ) . the distribution is strongly bimodal , with almost 50% of synapses silenced at the low basal weight , unable to regenerate , and the remainder in a narrow distribution centered at very high weights . however , these simulations failed to produce synaptic weight distributions that resembled experimental data . if the decay rate constants for the two exponential distributions differed by more than twofold , almost all synaptic weights either ran to infinity ( if the mean ltp amplitude was greater ) or converged to a low basal synaptic weight imposed as a floor in the model ( if the mean ltd amplitude was greater ) . if the rate constants were similar a positive synaptic weight distribution could be obtained , but the shape of this distribution was itself exponential . all following results are therefore based on the first model variant described above , that of figure 2(a ) , with synaptic regeneration and an ltp amplitude that decreases with the number of strong synapses in a cluster . strong synapses are much more stable on average , in agreement with data demonstrating that large dendritic spines are more persistent [ 32 , 34 , 35 ] . strong synapses often maintain high values of w for a year or more , whereas weak synapses show much larger relative ( percent ) fluctuations in w. weak synapses often drop to a very low basal weight . figure 3(c ) illustrates a typical time course for the number of strong synapses nst in a cluster ( with weights greater than a threshold tst = 0.8 ) . clusters are very stable in that , for ncl = 10 synapses per cluster , nst almost always remains between 4 and 7 for years . however , for a smaller ncl = 5 , the model no longer simulated a log - normal synaptic weight distribution at steady state . rather than spine volume , they examined variations in the size of psd-95:gfp puncta , that is , localized concentrations , with a size corresponding to spines , of the postsynaptic density protein psd-95 fused to a gfp fluorophore . in accordance with ( 3 ) , stability of a simulated steady state , unimodal distribution such as that of figure 2(a ) requires that individual synapses with weights above the peak of the distribution decrease their weight on average and vice versa for those with low weights . figure 4(a ) illustrates a simulated histogram of the amplitudes of the daily changes in synaptic weight , w , at steady state . the plot is qualitatively linear , which is not surprising because , in the model , the average daily ltd and ltp amplitudes contain terms proportional to w ( methods , ( 2 ) , ( 3 ) ) . however , what if the weight distribution is altered by an imposed large perturbation that sets high weights for a specified subset of synapses ? such a perturbation might correspond to formation of a specific , long - term memory engram . will the subset of strong synaptic weights remain elevated for months , corresponding to long - lasting storage of a memory ? starting from the distribution of figure 2(a ) , for all of the 1000 10-synapse clusters , synapses 15 were reset to a high weight of 5.0 at t = 200 days . after the reset , the weights fluctuate but 400 days later , all but one of the strong synapses have maintained w above the steady - state average . their average weight decays slowly such that 700 days after reset this average is still about twice the steady - state average of w. the lower red trace , one standard deviation below the average weight , is also still above the steady - state average . if the steady - state distribution of w is evolved without any perturbation , the time scale for decorrelation of synaptic weights from their specific values at a given time is , perhaps surprisingly , quite long , similar to the time scale for the decay of perturbed synaptic weights . starting from the distribution of figure 2(a ) , the pearson correlation coefficient between the weights of the 10,000 synapses decays with a time constant of approximately 500 days ( figure 5(c ) ) , similar to the dynamics of figure 5(b ) . this model variant was therefore not a plausible description of synaptic dynamics , because biophysical parameters are expected to vary somewhat between spines , dendritic branches , and individual neurons . that spontaneous coactivation of dendritic spines is clustered , occurring more often for spines within 8 m of each other . we do note recent analysis by a different group failed to support clustering of this type and scale . with coupling into clusters of ~10 synapses , when the dynamics of 1,000 clusters were simulated , the weight distribution of individual synapses converged to a stable steady - state , log - normal form ( figure 2(a ) ) . our mechanism of coupling synapses into clusters is similar in that it corresponds to an additional form of resource competition . however , in the model , competition is generated by ongoing maintenance of multiple synapses rather than only by simultaneous ltp of multiple synapses . thus , the mean magnitude of ltp at a given synapse during a simulated day was assumed to be a decreasing function of the number of other large synapses in the same cluster ( methods ) . however , they did determine this rate for another measure of synaptic cross talk , the ability of a weakly stimulated spine to capture plasticity - related factors synthesized in response to strong stimulation of a nearby spine ( synaptic tagging and capture , resulting in ltp at the weakly stimulated spine ) . however , these data do not address whether competition could occur over a longer distance scale , for example , 50 m . to assess this question groups of spines spaced along a dendritic branch would need to be concurrently stimulated , and ltp quantified . to simulate a distribution spanning a broad range of synaptic weights , as is observed empirically , it was critical to model synaptic regeneration . this method of simulating regeneration was chosen to maintain equal average numbers of synaptic loss and regeneration events within any given cluster . thus for clusters with low numbers of active spines to serve as functional memory storage units , as suggested by the clustered plasticity hypothesis , the average cluster size would need to be stable to avoid disappearance of clusters . it is plausible that new , or regenerated , spines are generically not strong ( as in our model ) and therefore can not participate in memory storage unless they are potentiated by stimuli that induce long - term memory . however , further empirical investigation of this question , in conjunction with modeling , is needed . the model of loewenstein et al . simulates daily changes in the volume of dendritic spines and obtains a steady - state log - normal distribution of spine volumes very similar to the empirical distribution found by these authors . these important results have clarified the necessity of reconciling unimodal weight distributions with stable storage of long - term memory . the model presented here may constitute a further advance , in that it represents more biophysical elements , such as synapse loss / regeneration and synapse clustering . the ltp and ltd processes in the model ( 1 ) act directly on the synaptic weight rather than on its logarithm and ( 2 ) can be thought of as due to a large number of individual biochemical events occurring during a simulated day and corresponding to insertion and removal of individual molecular complexes or slots . point ( 2 ) connects the current model with the more detailed molecular model of lisman and raghavachari , in which ltp and ltd correspond , respectively , to insertion and removal of slot complexes , each consisting of a small number of ampa receptors together with associated scaffolding proteins and , possibly , kinases or other signaling proteins . additional elements of our model that may be consistent with that of lisman and raghavachari are as follows . these gaussian variables would correspond to the model 's gaussian random variables r1 and r2 , to which the daily ltp and ltd amplitudes are proportional ( 2 ) , ( 3 ) . it also appears plausible that average numbers of slot insertions and removals are proportional to the preexisting size of a spine , corresponding to the model assumption that mean ltp and ltd amplitudes are approximately proportional to synaptic weight . recent data are consistent with these ideas and also suggest such slot complexes include presynaptic components and release sites . current data has not directly examined whether there is spatial clustering of presynaptic boutons at the distance scale of ~5 m that corresponds to putative postsynaptic spine clusters . however , at this distance scale , such clusters would correspond to a single axon and spines in a cluster would thus be coactivated by a common input , consistent with the hypothesis that such clusters constitute functional units in the formation and storage of specific memories . in the model , the average relative ( percent ) change in w over a day decreases substantially as w increases ( figure 4(b ) ) . this result is essential for the model to represent stable long - term memory storage , because selective stability of strong synapses is only found if the relative change in w decreases in this manner . relevant data describes the relationship of spine volume changes v to v. these data are , however , contradictory . it is plausible that the difference in synaptic dynamics in these studies was generated , at least in part , by substantial differences in experimental conditions . imaged ca1 pyramidal neurons in cultured rat hippocampal slices at postnatal days 1723 , whereas loewenstein et al . however , data does indicate that a minor percentage of spines are stable for months or years [ 24 , 25 ] . the current model does not represent the biochemical processes that might underlie long - term stability of such a subpopulation . however , recent studies support a relevant hypothesis that ongoing spontaneous neuronal activity is critical for long - term maintenance of synaptic strength . models have suggested that such activity can preferentially maintain synapses that are already strong possibly by preferentially reactivating stored patterns of strengthened synapses . ongoing ltp increments that are necessary to maintain strong synapses , whether single or clustered ( as in the current model ) , may correspond to frequent spontaneous or environmentally induced activity , which may induce repeated cycles of nmda receptor - dependent ltp . simulations ( figures 5(a)-5(b ) ) illustrate that the model can store a specific memory trace , for ~1 yr , corresponding to persistence of a pattern of strong synapses . however , in humans , some memories persist for many years . , such memories might be encoded at the cluster level rather than the single - synapse level , as a set of specific , stable clusters of active synapses . in simulations , they maintained a range of strong synapse numbers ( ~47 ) similar to the range suggested by data demonstrating clustering on neocortical pyramidal dendrites . a bimodal synaptic weight distribution , not resembling empirical data , we do not take this result to be a prediction of a minimum empirical cluster size . instead , we believe that the model should be improved in future work to avoid or reduce this dramatic change in dynamics . the model makes additional predictions . when comparing spines of similar sizes in different clusters , the average volume change between imaging sessions is predicted to be less positive ( or more negative ) if other spines in a cluster are large . in addition , because synaptic weights and spine volumes are not considered bistable , different induction protocols for late , protein - synthesis dependent ltp are predicted to induce clearly different amplitudes of synaptic weight increase or of average spine volume increase . without bistability , repeated applications of stimulus protocols should , at least in some cases , further enhance l - ltp .

long - term memories are at least in part encoded as specific patterns , or engrams , of strengthened synapses [ 1 , 2 ] , and long - term synaptic potentiation ( ltp ) persists for months in vivo . how can specific groups of synapses remain strong for months or years despite turnover of macromolecules and fluctuations in the size and shape of synaptic structures ? numerous mathematical models have been developed that describe maintenance of long - term memory ( ltm ) as dependent on bistability of synaptic weights , mediated by positive feedback loops of biochemical reactions , typically thought of as operative in dendritic spines . proposed feedback mechanisms have relied on self - sustaining autophosphorylation of cam kinase ii [ 4 , 5 ] , persistent phosphorylation of ampa receptors by protein kinase a , enhanced translation of protein kinase m , or self - sustaining clustering of a translation activator , cytoplasmic polyadenylation element binding protein . with these models , ltp switches a synapse from a state of low basal weight to a high weight state and turns on the positive feedback loop . however , despite extensive investigation , empirical evidence of a bistable distribution of two distinct synaptic weight states has not , in fact , been obtained . although some studies [ 9 , 10 ] have suggested two distinct strength states for synapses , as measured by the amplitude of excitatory postsynaptic currents before and after a stimulus protocol , these studies have only examined the early phase of ltp ( < 1 h ) , which does not depend on protein synthesis or other processes necessary for long - term memory storage . therefore , those data do not address the dynamics of long - term memory storage . in addition , a demonstration of synaptic bistability would require not only finding two distinct synaptic strength states but also finding that a set of different protocols for ltp induction ( e.g. , different patterns of stimuli , or localized application of pharmacological agents ) commonly switched synaptic weights between the same two stable states . in addition , modeling suggests that stochastic fluctuations of macromolecule numbers within a small volume such as a spine head are likely to destabilize steady states of biochemical positive feedback loops , causing randomly timed state switches (; see for an opposing view ) . finally , in hippocampal neuron cultures , continuous and extensive fluctuations of postsynaptic density ( psd ) morphology are observed and are driven in part by synaptic activity . empirical distributions of the weights of excitatory synapses onto cortical or hippocampal pyramidal neurons appear unimodal ( a single peak ) rather than bimodal and are commonly heavy - tailed ( skewed towards high weights ) [ 1318 ] . in addition , a histogram of the volume of dendritic spines , based on a large number of individual measurements ( ~10,000 ) in mouse auditory cortex , is clearly unimodal , heavy - tailed , and approximately log - normal . spine volume is approximately proportional to the postsynaptic density size [ 2022 ] and to the number of postsynaptic ampa receptors as well as to the amplitude of the epsc measured following localized glutamate uncaging . thus it is plausible , and we assume that increases / decreases in spine volume can serve as a proxy for ltp / ltd . if synaptic weights and correlated spine volumes are not in fact bistable , how can patterns of strong synapses be maintained for very long times ? two observations support a mechanism based on metastability of small clusters of large dendritic spines , corresponding to groups of strong synaptic contacts . the first observation is that although spine volumes fluctuate , some large spines are extremely stable , existing for months ( in sensory cortex or in motor cortex ) . the second is that induction of late , protein synthesis - dependent ltp ( l - ltp ) at a spine facilitates l - ltp expression at nearby spines , on the same dendritic branch , that coincidentally receive stimuli too weak to support l - ltp if given alone . this observation supports the clustered plasticity hypothesis in which clusters of spines on a single dendritic branch , rather than single spines , may serve as primary functional units for storage of ltm . for example ( 1 ) in motor cortex , learning induces coordinated formation of small spine clusters on a given dendritic branch ( 2 ) morphologically , spines are grouped into small clusters on pyramidal dendrites and ( 3 ) in rat hippocampal slice cultures , spontaneous coactivation of dendritic spines is frequent and is clustered , occurring more often for spines within 8 m of each other . here an initial , relatively phenomenological model is developed describing synaptic weight changes due to competing processes of ltp ( corresponding to spine growth ) and long - term synaptic depression ( ltd ) ( corresponding to spine shrinkage ) . assuming volume changes are a proxy for weight changes , weight changes are simulated for discrete intervals of 1 day , over times of months or years . in the model each day the magnitude of ltp is governed by a gaussian random variable , as is that of ltd ( methods ) . when the dynamics of 1,000 small clusters were simulated , the weight distribution of the entire ensemble of individual synapses converged to a steady - state , log - normal form . individual clusters remained stable for many years , with the average number of active synapses maintained in a range consistent with empirical data . if a subset of synapses was reinitialized to have large weights , this subset maintained large average weights for ~2 yrs , corresponding to persistence of a pattern of strong synapses that might serve as the engram for a memory . in support of the clustered plasticity hypothesis , our simulations suggest that such memories might be encoded as a pattern of specific , stable clusters of active synapses . the model thus assumes that the more strong synapses present in a cluster , the fewer resources are available to support synaptic growth ( ltp ) . thus , the model assumes that the amplitude of ltp is also proportional to a volatility factor that decreases as w increases ( 1)-(2 ) as is that of ltd ( 3 ) . with ncl the total number of synapses in a cluster , the average ltp amplitude a1 decreases linearly with nst , from a maximum amplitude x2 ( for nst = 0 ) to a minimum x1 ( for nst = ncl ) . ltp and ltd are also proportional to a volatility factor vow , decreasing with w:(1)vow = vhivhivloww+wmed.from ( 1 ) , vow decreases from the parameter vhi ( for w = 0 ) to vlo ( for w wmed ) . combining factors ltd amplitude is(3)altd = wr2vow.at each time step , for each active synapse,(4)wnew = wold+altpaltd.if w falls below a threshold twk , the synapse is reset to be silent . for each silent synapse at each time step , the probability for regeneration pact increases with the number of strong synapses in its cluster , to a maximal value pbas:(5)pact = pbasnstncl.in addition , regeneration only occurs if an adjacent synapse is strong . in a cluster , synapse 1 can only switch to active if synapse 2 is strong , and synapse 5 can only switch if synapse 4 and/or 6 is strong . therefore , it was necessary to scale ltp and ltd amplitudes so that for a time step the simulated per cent change in w , averaged over all synapses , agreed with an average daily empirical change in w. empirically yasumatsu et al . is(6)v=0.16v+0.01 for v0.25 m3 ( most spines),v=0.12v0.06 for 0.25 m3<v0.5 m3,v=0 for v>0.5 m3.from this relationship , combined with the plotted volumes in figures 1(b ) and 1(c ) of , it can be inferred that the average percent daily change in v lies within the range of ~1420% . for comparison , in the model 's steady - state weight distribution of figure 2(a ) , the percent change in w during a time step , averaged over all 10,000 synapses , is 16.5% . assuming v is a proxy for w , this qualitative agreement between simulated and empirical average weight changes suggests that the fixed time step in figures 25 , which is otherwise arbitrary , can be taken to correspond to approximately 1 day of weight dynamics . let xi denote the total set of n synaptic weights at a reference time , with i the indexing variable from 1 to n. for 1,000 10-synapse clusters , n = 10 , 000 . the standard equation was used:(7)r=i=1nxixyiyi=1nxix2i=1nyiy2.standard parameter values , used in all simulations unless stated otherwise , are as follows:(8)ncl=10 , wreset=0.4 , twk=0.08,tst=0.8 , vhi=4.0 , vlo=0.2,wmed=0.4 , x1=0.144 , x2=0.18,a2=0.16 , khi=0.05 , whi=20.0 , pbas=0.1.in supplementary material ( available online at http://dx.doi.org/10.1155/2015/185410 ) , a java program is given that executes simulations from figures 24 . to prevent this distribution from being overly sensitive to the average daily ltp amplitude , the model assumes this amplitude decreases with the number of strong synapses in the cluster to which the synapse belongs . in contrast , figure 2(b ) illustrates that in a model variant with mean and standard deviation of the ltp amplitude fixed , the weight histogram was shifted to the right of the log - normal distribution from figure 2(a ) and has a shape clearly distorted from log - normal with a much steeper cutoff at high w. to attempt to improve the histogram , the mean ltp amplitude was decreased by 2% . this small change resulted in a large shift of the histogram to the left ( blue trace ) , and 56% of the synapses became silent ( not included in the histogram ) . a similar distribution , with extreme sensitivity to mean ltp amplitude , resulted from a model variant in which synaptic clustering was deleted by fixing the mean and standard deviation of the ltp amplitude and also fixing the synapse regeneration probability pact ( 5 ) . the distribution is strongly bimodal , with almost 50% of synapses silenced at the low basal weight , unable to regenerate , and the remainder in a narrow distribution centered at very high weights . if the decay rate constants for the two exponential distributions differed by more than twofold , almost all synaptic weights either ran to infinity ( if the mean ltp amplitude was greater ) or converged to a low basal synaptic weight imposed as a floor in the model ( if the mean ltd amplitude was greater ) . all following results are therefore based on the first model variant described above , that of figure 2(a ) , with synaptic regeneration and an ltp amplitude that decreases with the number of strong synapses in a cluster . figure 3(c ) illustrates a typical time course for the number of strong synapses nst in a cluster ( with weights greater than a threshold tst = 0.8 ) . however , for a smaller ncl = 5 , the model no longer simulated a log - normal synaptic weight distribution at steady state . rather than spine volume , they examined variations in the size of psd-95:gfp puncta , that is , localized concentrations , with a size corresponding to spines , of the postsynaptic density protein psd-95 fused to a gfp fluorophore . their dynamics are qualitatively consistent with those illustrated in figures 2(a ) and 3 in that ( 1 ) new synapses were continually formed , ( 2 ) synapses whose size was reduced beneath some threshold , analogous to the model threshold twk , were eliminated , and ( 3 ) large synapses tended to shrink and small synapses to grow . in accordance with ( 3 ) , stability of a simulated steady state , unimodal distribution such as that of figure 2(a ) requires that individual synapses with weights above the peak of the distribution decrease their weight on average and vice versa for those with low weights . figure 4(a ) illustrates a simulated histogram of the amplitudes of the daily changes in synaptic weight , w , at steady state . the majority of the histogram is fitted well by a normal distribution with the clear exception of the narrow peak close to w = 0 . the plot is qualitatively linear , which is not surprising because , in the model , the average daily ltd and ltp amplitudes contain terms proportional to w ( methods , ( 2 ) , ( 3 ) ) . will the subset of strong synaptic weights remain elevated for months , corresponding to long - lasting storage of a memory ? starting from the distribution of figure 2(a ) , for all of the 1000 10-synapse clusters , synapses 15 were reset to a high weight of 5.0 at t = 200 days . this weight is well above the steady - state mean w. the other 5 synapses were reset to a low weight ( 0.5 ) . after the reset , the weights fluctuate but 400 days later , all but one of the strong synapses have maintained w above the steady - state average . their average weight decays slowly such that 700 days after reset this average is still about twice the steady - state average of w. the lower red trace , one standard deviation below the average weight , is also still above the steady - state average . if the steady - state distribution of w is evolved without any perturbation , the time scale for decorrelation of synaptic weights from their specific values at a given time is , perhaps surprisingly , quite long , similar to the time scale for the decay of perturbed synaptic weights . starting from the distribution of figure 2(a ) , the pearson correlation coefficient between the weights of the 10,000 synapses decays with a time constant of approximately 500 days ( figure 5(c ) ) , similar to the dynamics of figure 5(b ) . this model variant was therefore not a plausible description of synaptic dynamics , because biophysical parameters are expected to vary somewhat between spines , dendritic branches , and individual neurons . with coupling into clusters of ~10 synapses , when the dynamics of 1,000 clusters were simulated , the weight distribution of individual synapses converged to a stable steady - state , log - normal form ( figure 2(a ) ) . data illustrates that spines compete for ltp expression ; that is , local resources ( possibly amounts of key proteins ) are limited such that the amount of ltp at a given spine decreases if ltp is simultaneously induced at multiple spines close together on the same dendritic branch . however , in the model , competition is generated by ongoing maintenance of multiple synapses rather than only by simultaneous ltp of multiple synapses . thus , the mean magnitude of ltp at a given synapse during a simulated day was assumed to be a decreasing function of the number of other large synapses in the same cluster ( methods ) . however , they did determine this rate for another measure of synaptic cross talk , the ability of a weakly stimulated spine to capture plasticity - related factors synthesized in response to strong stimulation of a nearby spine ( synaptic tagging and capture , resulting in ltp at the weakly stimulated spine ) . these data suggest that , for clusters of ~10 spines as simulated here , distance between spines might not limit the efficacy of resource competition . to obtain distributions similar to that of figure 2(a ) , synapses that fell to a low basal weight needed to have , on successive days , a probability of weight reset to a higher , active value . thus for clusters with low numbers of active spines to serve as functional memory storage units , as suggested by the clustered plasticity hypothesis , the average cluster size would need to be stable to avoid disappearance of clusters . it is plausible that new , or regenerated , spines are generically not strong ( as in our model ) and therefore can not participate in memory storage unless they are potentiated by stimuli that induce long - term memory . simulates daily changes in the volume of dendritic spines and obtains a steady - state log - normal distribution of spine volumes very similar to the empirical distribution found by these authors . the model presented here may constitute a further advance , in that it represents more biophysical elements , such as synapse loss / regeneration and synapse clustering . the ltp and ltd processes in the model ( 1 ) act directly on the synaptic weight rather than on its logarithm and ( 2 ) can be thought of as due to a large number of individual biochemical events occurring during a simulated day and corresponding to insertion and removal of individual molecular complexes or slots . point ( 2 ) connects the current model with the more detailed molecular model of lisman and raghavachari , in which ltp and ltd correspond , respectively , to insertion and removal of slot complexes , each consisting of a small number of ampa receptors together with associated scaffolding proteins and , possibly , kinases or other signaling proteins . if over a day , the time intervals between individual insertions of slot complexes as well as the intervals between removals of complexes follow poisson distributions , and if there are on average more than ~20 insertions and removals / day , then by the central limit theorem the sums of these poisson processes will approximate gaussian random variables . it also appears plausible that average numbers of slot insertions and removals are proportional to the preexisting size of a spine , corresponding to the model assumption that mean ltp and ltd amplitudes are approximately proportional to synaptic weight . however , at this distance scale , such clusters would correspond to a single axon and spines in a cluster would thus be coactivated by a common input , consistent with the hypothesis that such clusters constitute functional units in the formation and storage of specific memories . this result is essential for the model to represent stable long - term memory storage , because selective stability of strong synapses is only found if the relative change in w decreases in this manner . it is plausible that the difference in synaptic dynamics in these studies was generated , at least in part , by substantial differences in experimental conditions . ongoing ltp increments that are necessary to maintain strong synapses , whether single or clustered ( as in the current model ) , may correspond to frequent spontaneous or environmentally induced activity , which may induce repeated cycles of nmda receptor - dependent ltp . , such memories might be encoded at the cluster level rather than the single - synapse level , as a set of specific , stable clusters of active synapses . in simulations , stable clusters were found when the total number of synapses in a cluster was 10 ( figure 3(c ) ) , or 1520 . when comparing spines of similar sizes in different clusters , the average volume change between imaging sessions is predicted to be less positive ( or more negative ) if other spines in a cluster are large . in addition , because synaptic weights and spine volumes are not considered bistable , different induction protocols for late , protein - synthesis dependent ltp are predicted to induce clearly different amplitudes of synaptic weight increase or of average spine volume increase .

drug addiction is a multifactorial disorder caused by the interaction of individual and environmental factors . among the underlying factors that contribute to an enhanced predisposition to drug addiction are the existence of a vulnerable personality or phenotype [ 15 ] , early exposure to drugs of abuse [ 68 ] , and the presence of adverse environmental conditions such as exposure to stress [ 912 ] . in fact , evidence suggests that individual differences in susceptibility to addiction involve integrated neurocircuits underlying stress , reward , and behavioural inhibitory processes . one of the most recognised factors facilitating the transition from voluntary , recreational drug use to dependence and addiction is exposure to drugs of abuse early on in life . adolescence is a critical developmental period characterized by immaturity of inhibitory control brain systems related with planning , evaluation of consequences , decision - making , and control of behaviour , such as the prefrontal cortex ( pfc ) [ 14 , 15 ] . moreover , the adolescent brain exhibits more plasticity and adolescents are more sensitive than adults to the rewarding effects of drugs and less sensitive to their aversive properties , all of which facilitate drug consumption at this age [ 1417 ] . in fact , the characteristic behaviour of adolescents ( impulsivity , emotional liability , increased risk - taking , enhanced novelty - seeking , etc . ) that can favour drug use is due to this lack of prefrontal cortical maturation and hyperactivity of limbic structures involved in the processing of rewarding , emotional , and stressful stimuli , such as the nucleus accumbens ( nacc ) and amygdala [ 14 , 15 ] . consumption of cannabis , the most used illegal drug , usually begins during adolescence , and an increase in the problematic use of this drug among adolescents has been reported in recent years [ 1820 ] . regular heavy use has more negative consequences at this early age than during adulthood , including enhanced vulnerability to develop dependence , suggesting that the adolescent brain is particularly vulnerable to the effects of cannabis exposure [ 2224 ] . furthermore , adolescent cannabis abuse seems to enhance vulnerability to later consumption of other drugs [ 25 , 26 ] . early onset of cannabis consumption has been shown to be a proximal trigger of later cocaine use [ 2729 ] and increases the severity of cocaine withdrawal symptoms and relapse to cocaine dependence . similarly , in animal models , exposure to cannabinoid agonists ( thc and cp 55940 ) during adolescence induces an upregulation of dat in the caudate - putamen , increased self - administration of opioids , cocaine , and nicotine [ 3237 ] , and enhanced locomotor responses to cocaine . in line with this , previous studies in our laboratory have demonstrated that preexposure to the cannabinoid agonist win 55212 - 2 increases the cpp induced by morphine and the acquisition , persistence , and reinstatement of mdma - induced cpp . however , no previous studies have evaluated whether preexposure to cannabinoids during adolescence modifies the subsequent acquisition and reinstatement of the cpp induced by cannabinoids or cocaine . regarding the individual factors that contribute to drug addiction , differences in response to novelty and impulsivity that exist before the first experience of the drug have been related to differences in sensitivity to drug reward and vulnerability to addiction [ 1 , 3 , 4 , 9 ] . in previous studies by our group we have observed that the novelty - seeking trait predicts greater sensitivity to the conditioned rewarding effects of cocaine [ 41 , 42 ] . in particular , the hole - board test is a highly effective paradigm of novelty - seeking and predicts said sensitivity in adolescent male mice , since only high novelty seeker ( hns ) adolescent mice have been shown to acquire the cpp induced by a low dose of cocaine , which is ineffective in inducing cpp in low novelty seeker ( lns ) mice . moreover , we have observed a higher sensitivity of hns to the conditioned rewarding effects of low doses of cocaine and mdma in mice exposed to cocaine , ethanol , or mdma . however , the influence of the novelty - seeking phenotype on sensitivity to the rewarding effects of cannabinoids has not been studied to date . thus , the first aim of the present study was to evaluate the influence of the novelty - seeking phenotype on the sensitivity of adolescent mice to the rewarding effects of low doses of the cb1 agonist win 55212 - 2 in the cpp paradigm . we hypothesised that only hns mice would acquire cpp after conditioning with a low dose of win 55212 - 2 , as occurs with other drugs of abuse . the second aim was to study whether the stimulation or blockade of cb1 receptors during adolescence modifies the conditioned rewarding effects of win 55212 - 2 or cocaine and if such effects are modulated by the novelty - seeking phenotype . we expected preexposure to a cannabinoid agonist during adolescence to increase the cpp induced by low doses of win 55212 - 2 and cocaine and for this effect to be more pronounced in hns mice . a total of 250 male mice of the of1 strain were acquired commercially from charles river ( barcelona , spain ) at 21 days of age . they were housed in groups of four in plastic cages ( 25 25 14.5 cm ) for 5 days before experiments were initiated , under the following conditions : constant temperature ( 21c ) , a reversed light schedule ( white lights on 19.3007.30 h ) , and food and water available ad libitum , except during behavioural tests . animals were handled on each of the 3 days immediately prior to the preconditioning ( pre - c ) phase in order to reduce their stress levels in response to experimental manipulations . procedures involving mice and their care were conducted in conformity with national , regional , and local laws and regulations , which are in compliance with the european directive 2010/63/eu . the hole - board test was carried out in a square box ( 28 28 20.5 cm ) with transparent plexiglas walls and 16 equidistant holes of 3 cm in diameter in the floor . photocells below the surface of the holes detected the number of times mice performed a head dip . frequency of head dips was recorded automatically by the apparatus ( cibertec , sa , spain ) . for place conditioning , we employed twelve identical plexiglas boxes with two equal sized compartments ( length 30.7 cm , width 31.5 cm , and height 34.5 cm ) separated by a grey central area ( length 13.8 cm , width 31.5 cm , and height 34.5 cm ) . the compartments of these boxes had different coloured walls ( black versus white ) and distinct floor textures ( fine grid in the black compartment and wide grid in the white one ) . four infrared light beams in each compartment of the box and six in the central area allowed the position of the animal and its crossings from one compartment to the other to be recorded . the equipment was controlled using three pcs and monpre 2z software ( cibertec , sa , spain ) . ( laboratorio alcaliber sa , madrid , spain ) , win 55212 - 2 ( tocris , biogen cientfica , s.l . , madrid , spain ) , or rimonabant ( sr 141716a , sanofi recherche , montpellier , france ) in a volume of 0.01 ml / g . control groups were injected with the physiological saline used to dissolve cocaine ( nacl 0.9% ) or with tween-80 ( sigma - aldrich , madrid , spain ) , which was used to dissolve win 55212 - 2 and rimonabant ( 0.01% , 0.01 ml of tween dissolved in 100 ml of saline ) . the doses of cocaine we administered were selected on the basis of previous studies showing that 1 mg / kg is a subthreshold dose for inducing cpp in nave mice , while 6 mg / kg is effective in inducing cpp acquisition but not in producing reinstatement after extinction of cpp [ 41 , 46 ] . similarly , the doses of cannabinoid drugs administered were selected on the basis of previous studies on the effects of win 55212 - 2 in the cpp paradigm [ 39 , 47 ] and on the effects of cannabinoid pretreatment on the cpp induced by other drugs of abuse . pretreatment of mice with 0.1 mg / kg of win 55212 - 2 is effective in increasing the cpp induced by mdma , while 1 mg / kg of rimonabant specifically blocks cb1 receptors and does not act as an inverse agonist . at the beginning of the test , mice were placed in the same corner of the box and were allowed to freely explore the apparatus for 10 min . the illumination in the experimental room consisted of four neon tubes fixed to the ceiling ( light intensity of 110 lux at 50 cm above floor level ) . in all experiments , animals were first tested in the hole board on pnd 26 ( prior to any treatment ) and defined as high novelty seekers ( hns ) or low novelty seekers ( lns ) according to whether the number of head dips they performed was higher or lower than the median of the group . we have previously used this median - split analysis to study the effects of novelty - seeking on the behavioural effects of different drugs of abuse [ 4245 ] . in experiment 1 , mice initiated the place conditioning procedure six days after the hole - board test ( on pnd 32 ) . in the other experiments , given that mice received a pretreatment after being classified as hns or lns , the place conditioning procedure began two days after ( on pnd 34 ) . thus , the window of adolescence taken into account for the experiments included from pnd 26 to pnd 45 ( see a timeline of the experiments in figure 1 ) . place conditioning , consisting of three phases , took place during the dark cycle . during the first phase , or preconditioning ( pre - c ) , mice were allowed access to both compartments of the apparatus for 15 min ( 900 s ) per day for 3 days . on day 3 , the time spent by the animal in each compartment during 900 s period was recorded . animals showing strong unconditioned aversion for any compartment ( less than 33% of the session time ) were excluded from the rest of the procedure so that the cpp procedure was unbiased in terms of initial spontaneous preference [ 46 , 47 ] . one compartment was paired with the drug and the other with the vehicle using a counterbalanced design such that half the animals in each group received the treatment in one compartment and the other half in the other compartment . after assigning compartments , no significant differences were observed between the time spent in the drug - paired and vehicle - paired compartments during the preconditioning phase . this is an important step in the experimental procedure that avoids any preference bias before conditioning . in the second phase ( conditioning ) , animals were conditioned with win 55212 - 2 or cocaine , as described in previous studies [ 46 , 47 ] . in brief , in the case of win 55212 - 2 , mice were treated with win 55212 - 2 immediately before confinement for 30 min to the drug - paired compartment ( days 4 , 6 , 8 , and 10 ) and with vehicle before confinement to the vehicle - paired compartment ( days 5 , 7 , 9 , and 11 ) . in the case of cocaine , mice underwent two pairings per day on days 4 , 5 , 6 , and 7 , receiving an injection of physiological saline immediately before being confined for 30 min to the vehicle - paired compartment and receiving an injection of cocaine after an interval of 4 h , immediately before confinement to the drug - paired compartment . during the third phase ( postconditioning , post - c ) , which took place on day 8 ( in the case of cocaine ) or day 12 ( in the case of win 55212 - 2 ) , the guillotine door separating the two compartments was removed and the time spent by the untreated mice in each compartment was recorded during a 900 s observation period . the difference in seconds between the times spent in the drug - paired compartment in the post - c versus pre - c test is a measure of the degree of conditioning induced by the drug . if this difference is positive , then the drug is considered to have induced a preference for the drug - paired compartment . groups showing cpp in post - c underwent an extinction session every three days ( on mondays , wednesday , and friday ) during which they were placed in the apparatus ( without the guillotine doors separating the compartments ) for 15 min until the time spent by each group in the drug - paired compartment was similar to that of pre - c and differed from that of post - c ( student 's t - test ) . after extinction had been confirmed in an additional session , a reinstatement test was performed 15 min after administration of a priming dose ( half of that used during conditioning ) of the respective drug ( 0.0375 mg / kg of win 55212 - 2 , 0.5 or 3 mg / kg of cocaine ) . in study 1 , two experiments were performed in order to study the influence of the novelty - seeking phenotype on the effects of cannabinoid exposure on the acquisition of the cpp induced by win 55212 - 2 . in the first experiment , 60 male mice performed the hole - board test in order to be classified as hns or lns and were randomly assigned a drug treatment ( 0.075 or 0.05 mg / kg of win 55212 - 2 ) . thus , four groups of mice were formed according to novelty - seeking profile and the dose of win 55212 - 2 received during cpp conditioning ( hns+win 0.075 , hns+win 0.05 , lns+win 0.075 , and lns+win 0.05 ) . the cpp procedure began on pnd 32 and conditioning took place from pnd 35 to pnd 42 . after the post - c test , groups showing cpp underwent extinction and reinstatement tests . in the second experiment , 80 male mice performed the hole - board test in order to be classified as hns or lns and were randomly assigned a drug treatment ( vehicle , 0.1 mg / kg of win 55212 - 2 or 1 mg / kg of rimonabant ) . mice received one daily injection of their respective treatment for 5 days ( pnd 2630 ) and , after an interval of 3 days without any treatment , underwent the cpp procedure following conditioning with the same dose of win 55212 - 2 ( 0.05 mg / kg ) . thus , six groups of mice were formed according to novelty - seeking profile and the pretreatment received before conditioning with win 55212 - 2 ( hns - veh - win , hns - win - win , hns+sr - win , lns - veh - win , lns - win - win , and lns - sr - win ) . the place conditioning procedure began on pnd 34 , and conditioning took place from pnd 37 to pnd 44 . study 2 was performed in order to study the influence of the novelty - seeking phenotype on the effects of cannabinoid exposure on the acquisition of the cpp induced by cocaine . eighty male mice performed the hole - board test and were defined as hns or lns and randomly assigned a drug treatment ( vehicle , 0.1 mg / kg of win 55212 - 2 or 1 mg / kg of rimonabant ) . the animals received a daily injection of their respective treatment for 5 days ( pnd 2630 ) and , after an interval of 3 days without any treatment , underwent the cpp procedure having been conditioned with the same dose of cocaine ( 1 mg / kg ) . thus , six groups of mice were formed according to novelty - seeking profile and the pretreatment received before conditioning with cocaine ( hns - veh - coc , hns - win - coc , hns+sr - coc , lns - veh - coc , lns - win - coc , and lns - sr - coc ) . following the post - c test , groups showing cpp underwent extinction and reinstatement tests . with the objective of corroborating the results obtained in the groups receiving pretreatment with win 55212 - 2 and conditioned with 1 mg / kg of cocaine , two additional groups were included in the procedure . thirty mice performed the hole - board test in order to be classified as hns or lns and were then treated with 0.1 mg / kg of win 55212 - 2 for 5 days . after an interval of 3 days without any treatment , the mice underwent the cpp procedure having been conditioned with 6 mg / kg of cocaine ( hns - win - coc6 and lns - win - coc6 ) . in this study all the groups began the cpp procedure on pnd 34 , and conditioning took place from pnd 37 to pnd 40 . differences between the number of dips performed by hns and lns groups were analysed with student 's t - tests . to evaluate the influence of the novelty - seeking trait on the cpp induced by win 55212 - 2 ( study 1 , experiment 1 ) , data of the time spent by the animals in the drug - paired compartment were analysed by means of a mixed anova with two between - subjects variables : novelty - seeking , with two levels ( hns and lns ) , and treatment , with two levels ( win 0.05 and win 0.075 ) , and one within - subjects variable : days , with two levels ( pre - c and post - c ) . to evaluate the effect of pretreatment with cannabinoid drugs on the subsequent cpp induced by win 55212 - 2 or cocaine in hns and lns mice ( study 1 , experiment 2 ; and study 2 ) , data of the time spent in the drug - paired compartment were analysed with a mixed anova with two between - subjects variables : novelty - seeking , with two levels ( hns and lns ) , and pre - treatment , with three levels ( veh , win , and sr ) , and one within - subjects variable : days , with two levels ( pre - c and post - c ) . in the abovementioned experiments , extinction and reinstatement values in the groups showing cpp to evaluate the effect of pretreatment of hns and lns mice with win 55212 - 2 on the subsequent cpp induced by 6 mg / kg of cocaine ( additional groups of study 2 ) , data of the time spent in the drug - paired compartment were analysed with a mixed anova with one between - subjects variable : novelty - seeking , with two levels ( hns and lns ) , and one within - subjects variable : days , with four levels ( pre - c , post - c , extinction , and reinstatement ) . the time required for preference to be extinguished in each animal was analysed by means of the kaplan - meier test , with breslow ( generalized wilcoxon ) comparisons when appropriate . in all the anovas , linear and logistic regression analysis was employed to determine the association between the level of novelty - seeking and the development of preference . the novelty scores for each mouse , identified by group , are represented in figure 2 . although the distribution is not completely bimodal ( some mice had a similar novelty - seeking score in lns and hns groups ) , they are clearly different with respect to the median scores . in all experiments , student 's t - tests showed significant differences between the number of dips performed by hns and lns groups ( ps < 0.01 ) . study 1 explains the influence of the novelty - seeking phenotype on the effects of cannabinoid exposure on acquisition of the cpp induced by win 55212 - 2 . experiment 1 is about the influence of the novelty - seeking phenotype on the sensitivity of mice to the rewarding effects of win 55212 - 2 . the anova of the data obtained with the mice conditioned with 0.05 and 0.075 mg / kg of win 55212 - 2 revealed that the interaction days treatment novelty - seeking [ f(1,45 ) = 4.175 ; p < 0.05 ] was significant . post hoc comparisons showed that only the group of hns mice conditioned with the high dose of win 55212 - 2 spent more time in the drug - paired compartment in post - c than during pre - c ( p < 0.05 ) . this cpp disappeared after two extinction sessions and was not reinstated by priming with 0.0375 mg / kg of win 55212 - 2 . thus , the hns trait would seem to increase the sensitivity of mice to the rewarding effects of win 55212 - 2 ( see figure 3 ) . linear and logistic regression analysis did not show any significant correlation between the novelty - seeking trait and development of the cpp induced by 0.075 mg / kg of win 55212 - 2 . experiment 2 is about the effects of exposure of hns and lns mice to agonist and antagonist cannabinoids on acquisition of the cpp induced by a subthreshold dose of win 55212 - 2 . the anova did not show any significant effect , thus indicating that pretreatment with a cannabinoid agonist or antagonist did not increase the sensitivity of mice to the conditioned rewarding effects of win 55212 - 2 ( see figure 4 ) . study 2 explains the influence of the novelty - seeking phenotype on the effects of agonist and antagonist cannabinoid on acquisition of the cpp induced by cocaine . the anova of the data obtained with the mice conditioned with 1 mg / kg of cocaine revealed a significant effect of the interaction post hoc comparisons showed a significant increase in the time spent by hns and lns mice pretreated with win 55212 - 2 in the drug - paired compartment in post - c with respect to pre - c ( ps < 0.01 ) . after extinction of cpp ( 7 sessions ) , a priming dose of 0.5 mg / kg of cocaine induced reinstatement of cpp only in hns mice ( p < 0.01 ) . thus , pretreatment with win 55212 - 2 increased the rewarding effects of cocaine irrespective of the novelty - seeking profile of the mice , but only hns animals were more sensitive to reinstatement after cocaine priming ( see figure 5 ) . the anova of the data obtained with the mice pretreated with win 55212 - 2 and conditioned with 6 mg / kg of cocaine revealed a significant effect of the variable days [ f(3,72 ) = 17.772 ; p < 0.01 ] , with mice spending more time in the drug - paired compartment in post - c than during pre - c ( p < 0.01 ) and in the reinstatement test than during the previous extinction test ( p < 0.05 ) . post hoc comparisons showed a significant increase in the time spent by hns and lns mice in the drug - paired compartment in post - c with respect to pre - c ( ps < 0.01 ) and revealed a reinstatement of cpp ( ps < 0.05 ) after a priming dose of 3 mg / kg of cocaine ( see figure 6 ) . the kaplan - meier test showed that the time required for extinction was longer in hns than in lns mice ( 14 versus 7 days , x = 3.995 , p < 0.05 ) ( see figure 7 ) . linear and logistic regression analysis did not show any significant correlation between the novelty - seeking trait and development of the cpp induced by 1 or 6 mg / kg of cocaine , in accordance with a previous study carried out in our laboratory . animal models are a vital tool for increasing our understanding of the behavioural traits ( e.g. , novelty - seeking ) and environmental events ( e.g. , early drug exposure ) associated with the individual vulnerability of subjects to repeated drug consumption and how these factors interact to facilitate the development of drug addiction . the results of the present study demonstrate for the first time that adolescent hns mice are more vulnerable to the rewarding effects of cannabinoids . even more importantly , given the high risk of adverse effects associated with cocaine , there was some indication that adolescent mice with this phenotype are more vulnerable to the reinstating effects of a low dose of cocaine if they have been previously exposed to a cannabinoid agonist . the first contribution of this study is the demonstration that the hns phenotype increases the sensitivity of mice to the conditioned rewarding effects of the cannabinoid agonist win 55212 - 2 . we have observed that hns mice acquire cpp after conditioning with 0.075 mg / kg , a dose that is ineffective in lns . although no previous studies have evaluated the influence of the novelty - seeking trait on the rewarding effects of cannabinoids , our results are in accordance with those observed with other drugs of abuse , such as cocaine or mdma , which have demonstrated that hns mice are more sensitive to the conditioned rewarding effects of these drugs [ 4143 ] . it is not clear if the ability of hns mice to develop cpp after administration of the cannabinoid agonist is related with an increase in the reinforcing / rewarding value of this drug for these animals or whether they acquire incentive learning in a more efficient way than lns mice . either way , increased levels of incentive salience attributed to drugs and/or drug - associated cues can enhance the intensity and duration of incentive motivation for drugs of abuse ( higher unconscious wanting and conscious craving ) , thus facilitating the transition to drug addiction , as suggested by robinson and berridge . it has been reported that hns animals have a characteristic striatal da profile ( higher endogenous levels , stronger responses to reward cues , and lower availability of d2/d3/d4 receptors ) , which may contribute to the tendency of these animals to exhibit approach reactions towards novel stimuli and may explain the increased cpp observed in the present study . the second important result of the present study is that even though exposure to the cannabinoid agonist win 55212 - 2 during adolescence did not enhance the acquisition of cpp induced by win 55212 - 2 itself at the doses tested , it did enhance the acquisition of cpp induced by a low dose of cocaine . mice pretreated with win 55212 - 2 exhibited cpp after conditioning with a low dose of cocaine that was ineffective in inducing cpp in animals pretreated with vehicle . moreover , mice pretreated with win 55212 - 2 showed priming - induced reinstatement of the cpp induced by 6 mg / kg of cocaine , an effect that has not been observed in nave mice . although clinical and epidemiologic studies show that cannabis consumption usually precedes the initiation of cocaine use [ 2729 ] , only two studies have evaluated the effect of stimulation of the endocannabinoid system ( ecs ) during adolescence on the subsequent effects of cocaine . in line with the results of the present study , adolescent rats pretreated with cannabinoid agonists showed increased locomotor responses to cocaine challenge and a higher rate of cocaine self - administration . thc preexposure also increases the rewarding effects of nicotine , morphine , and mdma [ 40 , 47 , 51 ] . conversely , other studies with adult animals have shown that cannabinoid agonists reduce cocaine reward [ 52 , 53 ] . usually , genetic ablation or antagonism of cb1 receptors decreases the self - administration [ 5456 ] , cpp [ 5760 ] , and sensitization [ 55 , 61 ] induced by cocaine , although some studies have found no effects [ 55 , 62 , 63 ] . thus , the ecs plays a complex role in the behavioural effects of different drugs of abuse . the present study extends these results by demonstrating that exposure to a cb1 agonist during adolescence increases the conditioned rewarding effects of cocaine , thus suggesting sensitization of the brain reward system . ecs plays an important role in adolescent brain development , and the strong stimulation of this system by cannabinoids might induce long - lasting neurobiological consequences , such as alterations in emotional and cognitive performance , increased risk of developing schizophrenia , and enhanced vulnerability to the use of drugs of abuse . in particular , changes in the da reward system induced by exposure to cannabinoids could underlie the behavioural effects observed . cannabinoid adolescent exposure induces an upregulation of dat in the caudate - putamen , an increase in d1rs content in the nacc shell , and a reduction in the expression of d2rs in ca1 . these changes can contribute to an increase in the reinforcing / rewarding value of the drug , leading to a greater risk of developing compulsive drug seeking . moreover , neuroadaptations in the ecs may be part of the neuroplasticity associated with the development of cocaine addiction . on the other hand , neither exposure to the cannabinoid agonist win 55212 - 2 during adolescence nor pretreatment with rimonabant modified the subsequent effect of win 55212 - 2 in the cpp paradigm , in contrast with that observed with cocaine in this study or in previous studies with morphine or mdma [ 39 , 40 ] . there is not a clear reason for the absence of an increase in vulnerability to the cpp induced by win 55212 - 2 after adolescent preexposure to this drug . it is possible that the particular profile of win 55212 - 2 in the cpp paradigm underlies the results observed . this drug only induces cpp with specific doses , with higher or lower doses being ineffective . given that our main objective was to detect differences in the influence of adolescent cannabinoid exposure between hns and lns , we used a very low dose of win 55212 - 2 ( 0.05 mg / kg ) , as we expected that cannabinoid pretreatment would cause a shift to the right of the dose - response curve . for example , in a previous study we observed that older adolescent mice ( pnd 52 ) developed cpp after conditioning with 0.05 mg / kg of win 55212 - 2 , suggesting that adolescent maturation is a factor that increases sensitivity to this drug . in any case , it is possible that if higher doses were used during conditioning , we would observe a potentiation of the rewarding effects of win 55212 - 2 in mice pretreated with this cannabinoid during adolescence . in fact , the effects of novelty - seeking phenotype ( experiment 1 ) were only apparent when the dose of win 55212 - 2 was higher ( 0.075 mg / kg ) than the dose used in this experiment . this warrants a tentative conclusion concerning the effects of adolescent exposure to win 55212 - 2 on a subsequent win 55212 - 2 cpp . future studies using higher doses of win 55212 - 2 during conditioning or different procedures of preexposure to this drug are necessary to determine whether or not adolescent exposure to cannabinoids alters the effects of win 55212 - 2 in the cpp paradigm . the main result of the present work is that the novelty - seeking phenotype determines the influence of adolescent cannabinoid exposure on the subsequent rewarding effects of cocaine in the cpp paradigm . not all mice are equally vulnerable to the sensitization of the brain reward system induced by stimulation of the cannabinoid system during adolescence . hns mice are particularly affected by pretreatment with win 55212 - 2 , showing a priming - induced reinstatement of the cpp induced by 1 mg / kg of cocaine and an enhanced duration of the cpp induced by 6 mg / kg of this drug ( effects that are not observed in lns mice exposed to win 55212 - 2 ) . our results support the idea that exposure to cannabis during adolescence , though it can increase the rewarding effects of subthreshold doses of cocaine six days after pretreatment , is not enough to promote long - lasting brain changes that increase the likelihood of the development of cocaine addiction ( which can be evaluated by the maintenance of cpp or its reinstatement after extinction ) . genetic and behavioural predispositions for example , a novelty - seeking phenotype may underlie increased adolescent drug experimentation , enhanced reward when the subject is exposed to the drug , and the development of neuroadaptations that lead to later adult addiction . animal models allow us to answer questions that can not be explored in human subjects due to ethical constraints and can be useful for analysing possible neurobiological substrates underlying interactions between environmental and biological factors that contribute to individual vulnerability to drug abuse and addiction . the phenotypic causation gateway hypothesis proposes a sequential progression of drug use in which early initiation of cannabis use is a risk factor for the future consumption of other drugs of abuse , such as cocaine . in support of this hypothesis , we have observed that mice exposed to the cannabinoid agonist during adolescence show an increased acquisition and reinstatement of cocaine cpp . the alternative common liability hypothesis proposes that cannabis and use of other illicit drugs are influenced by correlated genetic and environmental factors . our research has shown that the novelty - seeking trait is associated with an enhanced acquisition of win 55212 - 2 cpp , as we have observed previously with cocaine , and also with an increase in the effects of adolescent cannabinoid exposure on reinstatement and maintenance of cocaine cpp . thus , the results of the present study support the formulation of a vulnerability model that integrates the gateway and common liability hypotheses in order to explain the increased likelihood of transition from regular cannabis use to that of other substances ( such as cocaine or heroin ) . there is a subpopulation of adolescents which engages in extremely risky cannabis and drug use early in life and which seems to run a greater risk of abuse and addiction later in life . the results of the present study suggest that there is not a direct causal mechanism between adolescent drug exposure and the subsequent development of addiction . instead , there are individual brain and behavioural differences that are present prior to the onset of drug use , such as a higher propensity for sensation - seeking , which influence both the tendency to experiment with drugs of abuse early in life and the later development of addiction . these subjects appear to be more vulnerable to the appearance of permanent neurobiological changes following drug exposure that may lead to the transition from voluntary to compulsive drug use . thus , specific preventive programs aimed at these more vulnerable subjects could reduce drug consumption and later addiction .

adolescent exposure to cannabinoids enhances the behavioural effects of cocaine , and high novelty - seeking trait predicts greater sensitivity to the conditioned place preference ( cpp ) induced by this drug . our aim was to evaluate the influence of novelty - seeking on the effects of adolescent cannabinoid exposure . adolescent male mice were classified as high or low novelty seekers ( hns and lns ) in the hole - board test . first , we evaluated the cpp induced by the cannabinoid agonist win 55212 - 2 ( 0.05 and 0.075 mg / kg , i.p . ) in hns and lns mice . then , hns and lns mice were pretreated i.p . with vehicle , win 55212 - 2 ( 0.1 mg / kg ) , or cannabinoid antagonist rimonabant ( 1 mg / kg ) and were subsequently conditioned with win 55212 - 2 ( 0.05 mg / kg , i.p . ) or cocaine ( 1 or 6 mg / kg , i.p . ) . only hns mice conditioned with the 0.075 mg / kg dose acquired cpp with win 55212 - 2 . adolescent exposure to this cannabinoid agonist increased the rewarding effects of 1 mg / kg of cocaine in both hns and lns mice , and in hns mice it also increased the reinstating effect of a low dose of cocaine . our results endorse a role for individual differences such as a higher propensity for sensation - seeking in the development of addiction .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

in line with this , previous studies in our laboratory have demonstrated that preexposure to the cannabinoid agonist win 55212 - 2 increases the cpp induced by morphine and the acquisition , persistence , and reinstatement of mdma - induced cpp . in previous studies by our group we have observed that the novelty - seeking trait predicts greater sensitivity to the conditioned rewarding effects of cocaine [ 41 , 42 ] . in particular , the hole - board test is a highly effective paradigm of novelty - seeking and predicts said sensitivity in adolescent male mice , since only high novelty seeker ( hns ) adolescent mice have been shown to acquire the cpp induced by a low dose of cocaine , which is ineffective in inducing cpp in low novelty seeker ( lns ) mice . moreover , we have observed a higher sensitivity of hns to the conditioned rewarding effects of low doses of cocaine and mdma in mice exposed to cocaine , ethanol , or mdma . however , the influence of the novelty - seeking phenotype on sensitivity to the rewarding effects of cannabinoids has not been studied to date . thus , the first aim of the present study was to evaluate the influence of the novelty - seeking phenotype on the sensitivity of adolescent mice to the rewarding effects of low doses of the cb1 agonist win 55212 - 2 in the cpp paradigm . we hypothesised that only hns mice would acquire cpp after conditioning with a low dose of win 55212 - 2 , as occurs with other drugs of abuse . the second aim was to study whether the stimulation or blockade of cb1 receptors during adolescence modifies the conditioned rewarding effects of win 55212 - 2 or cocaine and if such effects are modulated by the novelty - seeking phenotype . we expected preexposure to a cannabinoid agonist during adolescence to increase the cpp induced by low doses of win 55212 - 2 and cocaine and for this effect to be more pronounced in hns mice . ( laboratorio alcaliber sa , madrid , spain ) , win 55212 - 2 ( tocris , biogen cientfica , s.l . control groups were injected with the physiological saline used to dissolve cocaine ( nacl 0.9% ) or with tween-80 ( sigma - aldrich , madrid , spain ) , which was used to dissolve win 55212 - 2 and rimonabant ( 0.01% , 0.01 ml of tween dissolved in 100 ml of saline ) . the doses of cocaine we administered were selected on the basis of previous studies showing that 1 mg / kg is a subthreshold dose for inducing cpp in nave mice , while 6 mg / kg is effective in inducing cpp acquisition but not in producing reinstatement after extinction of cpp [ 41 , 46 ] . similarly , the doses of cannabinoid drugs administered were selected on the basis of previous studies on the effects of win 55212 - 2 in the cpp paradigm [ 39 , 47 ] and on the effects of cannabinoid pretreatment on the cpp induced by other drugs of abuse . pretreatment of mice with 0.1 mg / kg of win 55212 - 2 is effective in increasing the cpp induced by mdma , while 1 mg / kg of rimonabant specifically blocks cb1 receptors and does not act as an inverse agonist . in all experiments , animals were first tested in the hole board on pnd 26 ( prior to any treatment ) and defined as high novelty seekers ( hns ) or low novelty seekers ( lns ) according to whether the number of head dips they performed was higher or lower than the median of the group . we have previously used this median - split analysis to study the effects of novelty - seeking on the behavioural effects of different drugs of abuse [ 4245 ] . in the second phase ( conditioning ) , animals were conditioned with win 55212 - 2 or cocaine , as described in previous studies [ 46 , 47 ] . in brief , in the case of win 55212 - 2 , mice were treated with win 55212 - 2 immediately before confinement for 30 min to the drug - paired compartment ( days 4 , 6 , 8 , and 10 ) and with vehicle before confinement to the vehicle - paired compartment ( days 5 , 7 , 9 , and 11 ) . during the third phase ( postconditioning , post - c ) , which took place on day 8 ( in the case of cocaine ) or day 12 ( in the case of win 55212 - 2 ) , the guillotine door separating the two compartments was removed and the time spent by the untreated mice in each compartment was recorded during a 900 s observation period . after extinction had been confirmed in an additional session , a reinstatement test was performed 15 min after administration of a priming dose ( half of that used during conditioning ) of the respective drug ( 0.0375 mg / kg of win 55212 - 2 , 0.5 or 3 mg / kg of cocaine ) . in study 1 , two experiments were performed in order to study the influence of the novelty - seeking phenotype on the effects of cannabinoid exposure on the acquisition of the cpp induced by win 55212 - 2 . in the first experiment , 60 male mice performed the hole - board test in order to be classified as hns or lns and were randomly assigned a drug treatment ( 0.075 or 0.05 mg / kg of win 55212 - 2 ) . thus , four groups of mice were formed according to novelty - seeking profile and the dose of win 55212 - 2 received during cpp conditioning ( hns+win 0.075 , hns+win 0.05 , lns+win 0.075 , and lns+win 0.05 ) . in the second experiment , 80 male mice performed the hole - board test in order to be classified as hns or lns and were randomly assigned a drug treatment ( vehicle , 0.1 mg / kg of win 55212 - 2 or 1 mg / kg of rimonabant ) . mice received one daily injection of their respective treatment for 5 days ( pnd 2630 ) and , after an interval of 3 days without any treatment , underwent the cpp procedure following conditioning with the same dose of win 55212 - 2 ( 0.05 mg / kg ) . thus , six groups of mice were formed according to novelty - seeking profile and the pretreatment received before conditioning with win 55212 - 2 ( hns - veh - win , hns - win - win , hns+sr - win , lns - veh - win , lns - win - win , and lns - sr - win ) . study 2 was performed in order to study the influence of the novelty - seeking phenotype on the effects of cannabinoid exposure on the acquisition of the cpp induced by cocaine . eighty male mice performed the hole - board test and were defined as hns or lns and randomly assigned a drug treatment ( vehicle , 0.1 mg / kg of win 55212 - 2 or 1 mg / kg of rimonabant ) . the animals received a daily injection of their respective treatment for 5 days ( pnd 2630 ) and , after an interval of 3 days without any treatment , underwent the cpp procedure having been conditioned with the same dose of cocaine ( 1 mg / kg ) . thus , six groups of mice were formed according to novelty - seeking profile and the pretreatment received before conditioning with cocaine ( hns - veh - coc , hns - win - coc , hns+sr - coc , lns - veh - coc , lns - win - coc , and lns - sr - coc ) . with the objective of corroborating the results obtained in the groups receiving pretreatment with win 55212 - 2 and conditioned with 1 mg / kg of cocaine , two additional groups were included in the procedure . thirty mice performed the hole - board test in order to be classified as hns or lns and were then treated with 0.1 mg / kg of win 55212 - 2 for 5 days . after an interval of 3 days without any treatment , the mice underwent the cpp procedure having been conditioned with 6 mg / kg of cocaine ( hns - win - coc6 and lns - win - coc6 ) . to evaluate the influence of the novelty - seeking trait on the cpp induced by win 55212 - 2 ( study 1 , experiment 1 ) , data of the time spent by the animals in the drug - paired compartment were analysed by means of a mixed anova with two between - subjects variables : novelty - seeking , with two levels ( hns and lns ) , and treatment , with two levels ( win 0.05 and win 0.075 ) , and one within - subjects variable : days , with two levels ( pre - c and post - c ) . to evaluate the effect of pretreatment with cannabinoid drugs on the subsequent cpp induced by win 55212 - 2 or cocaine in hns and lns mice ( study 1 , experiment 2 ; and study 2 ) , data of the time spent in the drug - paired compartment were analysed with a mixed anova with two between - subjects variables : novelty - seeking , with two levels ( hns and lns ) , and pre - treatment , with three levels ( veh , win , and sr ) , and one within - subjects variable : days , with two levels ( pre - c and post - c ) . in the abovementioned experiments , extinction and reinstatement values in the groups showing cpp to evaluate the effect of pretreatment of hns and lns mice with win 55212 - 2 on the subsequent cpp induced by 6 mg / kg of cocaine ( additional groups of study 2 ) , data of the time spent in the drug - paired compartment were analysed with a mixed anova with one between - subjects variable : novelty - seeking , with two levels ( hns and lns ) , and one within - subjects variable : days , with four levels ( pre - c , post - c , extinction , and reinstatement ) . study 1 explains the influence of the novelty - seeking phenotype on the effects of cannabinoid exposure on acquisition of the cpp induced by win 55212 - 2 . experiment 1 is about the influence of the novelty - seeking phenotype on the sensitivity of mice to the rewarding effects of win 55212 - 2 . the anova of the data obtained with the mice conditioned with 0.05 and 0.075 mg / kg of win 55212 - 2 revealed that the interaction days treatment novelty - seeking [ f(1,45 ) = 4.175 ; p < 0.05 ] was significant . post hoc comparisons showed that only the group of hns mice conditioned with the high dose of win 55212 - 2 spent more time in the drug - paired compartment in post - c than during pre - c ( p < 0.05 ) . this cpp disappeared after two extinction sessions and was not reinstated by priming with 0.0375 mg / kg of win 55212 - 2 . thus , the hns trait would seem to increase the sensitivity of mice to the rewarding effects of win 55212 - 2 ( see figure 3 ) . linear and logistic regression analysis did not show any significant correlation between the novelty - seeking trait and development of the cpp induced by 0.075 mg / kg of win 55212 - 2 . experiment 2 is about the effects of exposure of hns and lns mice to agonist and antagonist cannabinoids on acquisition of the cpp induced by a subthreshold dose of win 55212 - 2 . the anova did not show any significant effect , thus indicating that pretreatment with a cannabinoid agonist or antagonist did not increase the sensitivity of mice to the conditioned rewarding effects of win 55212 - 2 ( see figure 4 ) . study 2 explains the influence of the novelty - seeking phenotype on the effects of agonist and antagonist cannabinoid on acquisition of the cpp induced by cocaine . the anova of the data obtained with the mice conditioned with 1 mg / kg of cocaine revealed a significant effect of the interaction post hoc comparisons showed a significant increase in the time spent by hns and lns mice pretreated with win 55212 - 2 in the drug - paired compartment in post - c with respect to pre - c ( ps < 0.01 ) . after extinction of cpp ( 7 sessions ) , a priming dose of 0.5 mg / kg of cocaine induced reinstatement of cpp only in hns mice ( p < 0.01 ) . thus , pretreatment with win 55212 - 2 increased the rewarding effects of cocaine irrespective of the novelty - seeking profile of the mice , but only hns animals were more sensitive to reinstatement after cocaine priming ( see figure 5 ) . the anova of the data obtained with the mice pretreated with win 55212 - 2 and conditioned with 6 mg / kg of cocaine revealed a significant effect of the variable days [ f(3,72 ) = 17.772 ; p < 0.01 ] , with mice spending more time in the drug - paired compartment in post - c than during pre - c ( p < 0.01 ) and in the reinstatement test than during the previous extinction test ( p < 0.05 ) . post hoc comparisons showed a significant increase in the time spent by hns and lns mice in the drug - paired compartment in post - c with respect to pre - c ( ps < 0.01 ) and revealed a reinstatement of cpp ( ps < 0.05 ) after a priming dose of 3 mg / kg of cocaine ( see figure 6 ) . linear and logistic regression analysis did not show any significant correlation between the novelty - seeking trait and development of the cpp induced by 1 or 6 mg / kg of cocaine , in accordance with a previous study carried out in our laboratory . the results of the present study demonstrate for the first time that adolescent hns mice are more vulnerable to the rewarding effects of cannabinoids . even more importantly , given the high risk of adverse effects associated with cocaine , there was some indication that adolescent mice with this phenotype are more vulnerable to the reinstating effects of a low dose of cocaine if they have been previously exposed to a cannabinoid agonist . the first contribution of this study is the demonstration that the hns phenotype increases the sensitivity of mice to the conditioned rewarding effects of the cannabinoid agonist win 55212 - 2 . we have observed that hns mice acquire cpp after conditioning with 0.075 mg / kg , a dose that is ineffective in lns . although no previous studies have evaluated the influence of the novelty - seeking trait on the rewarding effects of cannabinoids , our results are in accordance with those observed with other drugs of abuse , such as cocaine or mdma , which have demonstrated that hns mice are more sensitive to the conditioned rewarding effects of these drugs [ 4143 ] . it is not clear if the ability of hns mice to develop cpp after administration of the cannabinoid agonist is related with an increase in the reinforcing / rewarding value of this drug for these animals or whether they acquire incentive learning in a more efficient way than lns mice . the second important result of the present study is that even though exposure to the cannabinoid agonist win 55212 - 2 during adolescence did not enhance the acquisition of cpp induced by win 55212 - 2 itself at the doses tested , it did enhance the acquisition of cpp induced by a low dose of cocaine . mice pretreated with win 55212 - 2 exhibited cpp after conditioning with a low dose of cocaine that was ineffective in inducing cpp in animals pretreated with vehicle . moreover , mice pretreated with win 55212 - 2 showed priming - induced reinstatement of the cpp induced by 6 mg / kg of cocaine , an effect that has not been observed in nave mice . the present study extends these results by demonstrating that exposure to a cb1 agonist during adolescence increases the conditioned rewarding effects of cocaine , thus suggesting sensitization of the brain reward system . in particular , changes in the da reward system induced by exposure to cannabinoids could underlie the behavioural effects observed . on the other hand , neither exposure to the cannabinoid agonist win 55212 - 2 during adolescence nor pretreatment with rimonabant modified the subsequent effect of win 55212 - 2 in the cpp paradigm , in contrast with that observed with cocaine in this study or in previous studies with morphine or mdma [ 39 , 40 ] . there is not a clear reason for the absence of an increase in vulnerability to the cpp induced by win 55212 - 2 after adolescent preexposure to this drug . given that our main objective was to detect differences in the influence of adolescent cannabinoid exposure between hns and lns , we used a very low dose of win 55212 - 2 ( 0.05 mg / kg ) , as we expected that cannabinoid pretreatment would cause a shift to the right of the dose - response curve . for example , in a previous study we observed that older adolescent mice ( pnd 52 ) developed cpp after conditioning with 0.05 mg / kg of win 55212 - 2 , suggesting that adolescent maturation is a factor that increases sensitivity to this drug . in any case , it is possible that if higher doses were used during conditioning , we would observe a potentiation of the rewarding effects of win 55212 - 2 in mice pretreated with this cannabinoid during adolescence . in fact , the effects of novelty - seeking phenotype ( experiment 1 ) were only apparent when the dose of win 55212 - 2 was higher ( 0.075 mg / kg ) than the dose used in this experiment . this warrants a tentative conclusion concerning the effects of adolescent exposure to win 55212 - 2 on a subsequent win 55212 - 2 cpp . future studies using higher doses of win 55212 - 2 during conditioning or different procedures of preexposure to this drug are necessary to determine whether or not adolescent exposure to cannabinoids alters the effects of win 55212 - 2 in the cpp paradigm . the main result of the present work is that the novelty - seeking phenotype determines the influence of adolescent cannabinoid exposure on the subsequent rewarding effects of cocaine in the cpp paradigm . hns mice are particularly affected by pretreatment with win 55212 - 2 , showing a priming - induced reinstatement of the cpp induced by 1 mg / kg of cocaine and an enhanced duration of the cpp induced by 6 mg / kg of this drug ( effects that are not observed in lns mice exposed to win 55212 - 2 ) . our results support the idea that exposure to cannabis during adolescence , though it can increase the rewarding effects of subthreshold doses of cocaine six days after pretreatment , is not enough to promote long - lasting brain changes that increase the likelihood of the development of cocaine addiction ( which can be evaluated by the maintenance of cpp or its reinstatement after extinction ) . genetic and behavioural predispositions for example , a novelty - seeking phenotype may underlie increased adolescent drug experimentation , enhanced reward when the subject is exposed to the drug , and the development of neuroadaptations that lead to later adult addiction . our research has shown that the novelty - seeking trait is associated with an enhanced acquisition of win 55212 - 2 cpp , as we have observed previously with cocaine , and also with an increase in the effects of adolescent cannabinoid exposure on reinstatement and maintenance of cocaine cpp . instead , there are individual brain and behavioural differences that are present prior to the onset of drug use , such as a higher propensity for sensation - seeking , which influence both the tendency to experiment with drugs of abuse early in life and the later development of addiction .

among the underlying factors that contribute to an enhanced predisposition to drug addiction are the existence of a vulnerable personality or phenotype [ 15 ] , early exposure to drugs of abuse [ 68 ] , and the presence of adverse environmental conditions such as exposure to stress [ 912 ] . adolescence is a critical developmental period characterized by immaturity of inhibitory control brain systems related with planning , evaluation of consequences , decision - making , and control of behaviour , such as the prefrontal cortex ( pfc ) [ 14 , 15 ] . moreover , the adolescent brain exhibits more plasticity and adolescents are more sensitive than adults to the rewarding effects of drugs and less sensitive to their aversive properties , all of which facilitate drug consumption at this age [ 1417 ] . that can favour drug use is due to this lack of prefrontal cortical maturation and hyperactivity of limbic structures involved in the processing of rewarding , emotional , and stressful stimuli , such as the nucleus accumbens ( nacc ) and amygdala [ 14 , 15 ] . consumption of cannabis , the most used illegal drug , usually begins during adolescence , and an increase in the problematic use of this drug among adolescents has been reported in recent years [ 1820 ] . similarly , in animal models , exposure to cannabinoid agonists ( thc and cp 55940 ) during adolescence induces an upregulation of dat in the caudate - putamen , increased self - administration of opioids , cocaine , and nicotine [ 3237 ] , and enhanced locomotor responses to cocaine . in line with this , previous studies in our laboratory have demonstrated that preexposure to the cannabinoid agonist win 55212 - 2 increases the cpp induced by morphine and the acquisition , persistence , and reinstatement of mdma - induced cpp . regarding the individual factors that contribute to drug addiction , differences in response to novelty and impulsivity that exist before the first experience of the drug have been related to differences in sensitivity to drug reward and vulnerability to addiction [ 1 , 3 , 4 , 9 ] . in particular , the hole - board test is a highly effective paradigm of novelty - seeking and predicts said sensitivity in adolescent male mice , since only high novelty seeker ( hns ) adolescent mice have been shown to acquire the cpp induced by a low dose of cocaine , which is ineffective in inducing cpp in low novelty seeker ( lns ) mice . moreover , we have observed a higher sensitivity of hns to the conditioned rewarding effects of low doses of cocaine and mdma in mice exposed to cocaine , ethanol , or mdma . thus , the first aim of the present study was to evaluate the influence of the novelty - seeking phenotype on the sensitivity of adolescent mice to the rewarding effects of low doses of the cb1 agonist win 55212 - 2 in the cpp paradigm . the second aim was to study whether the stimulation or blockade of cb1 receptors during adolescence modifies the conditioned rewarding effects of win 55212 - 2 or cocaine and if such effects are modulated by the novelty - seeking phenotype . the doses of cocaine we administered were selected on the basis of previous studies showing that 1 mg / kg is a subthreshold dose for inducing cpp in nave mice , while 6 mg / kg is effective in inducing cpp acquisition but not in producing reinstatement after extinction of cpp [ 41 , 46 ] . similarly , the doses of cannabinoid drugs administered were selected on the basis of previous studies on the effects of win 55212 - 2 in the cpp paradigm [ 39 , 47 ] and on the effects of cannabinoid pretreatment on the cpp induced by other drugs of abuse . pretreatment of mice with 0.1 mg / kg of win 55212 - 2 is effective in increasing the cpp induced by mdma , while 1 mg / kg of rimonabant specifically blocks cb1 receptors and does not act as an inverse agonist . in all experiments , animals were first tested in the hole board on pnd 26 ( prior to any treatment ) and defined as high novelty seekers ( hns ) or low novelty seekers ( lns ) according to whether the number of head dips they performed was higher or lower than the median of the group . in brief , in the case of win 55212 - 2 , mice were treated with win 55212 - 2 immediately before confinement for 30 min to the drug - paired compartment ( days 4 , 6 , 8 , and 10 ) and with vehicle before confinement to the vehicle - paired compartment ( days 5 , 7 , 9 , and 11 ) . in the case of cocaine , mice underwent two pairings per day on days 4 , 5 , 6 , and 7 , receiving an injection of physiological saline immediately before being confined for 30 min to the vehicle - paired compartment and receiving an injection of cocaine after an interval of 4 h , immediately before confinement to the drug - paired compartment . during the third phase ( postconditioning , post - c ) , which took place on day 8 ( in the case of cocaine ) or day 12 ( in the case of win 55212 - 2 ) , the guillotine door separating the two compartments was removed and the time spent by the untreated mice in each compartment was recorded during a 900 s observation period . the difference in seconds between the times spent in the drug - paired compartment in the post - c versus pre - c test is a measure of the degree of conditioning induced by the drug . groups showing cpp in post - c underwent an extinction session every three days ( on mondays , wednesday , and friday ) during which they were placed in the apparatus ( without the guillotine doors separating the compartments ) for 15 min until the time spent by each group in the drug - paired compartment was similar to that of pre - c and differed from that of post - c ( student 's t - test ) . after extinction had been confirmed in an additional session , a reinstatement test was performed 15 min after administration of a priming dose ( half of that used during conditioning ) of the respective drug ( 0.0375 mg / kg of win 55212 - 2 , 0.5 or 3 mg / kg of cocaine ) . in study 1 , two experiments were performed in order to study the influence of the novelty - seeking phenotype on the effects of cannabinoid exposure on the acquisition of the cpp induced by win 55212 - 2 . in the first experiment , 60 male mice performed the hole - board test in order to be classified as hns or lns and were randomly assigned a drug treatment ( 0.075 or 0.05 mg / kg of win 55212 - 2 ) . thus , four groups of mice were formed according to novelty - seeking profile and the dose of win 55212 - 2 received during cpp conditioning ( hns+win 0.075 , hns+win 0.05 , lns+win 0.075 , and lns+win 0.05 ) . in the second experiment , 80 male mice performed the hole - board test in order to be classified as hns or lns and were randomly assigned a drug treatment ( vehicle , 0.1 mg / kg of win 55212 - 2 or 1 mg / kg of rimonabant ) . mice received one daily injection of their respective treatment for 5 days ( pnd 2630 ) and , after an interval of 3 days without any treatment , underwent the cpp procedure following conditioning with the same dose of win 55212 - 2 ( 0.05 mg / kg ) . thus , six groups of mice were formed according to novelty - seeking profile and the pretreatment received before conditioning with win 55212 - 2 ( hns - veh - win , hns - win - win , hns+sr - win , lns - veh - win , lns - win - win , and lns - sr - win ) . study 2 was performed in order to study the influence of the novelty - seeking phenotype on the effects of cannabinoid exposure on the acquisition of the cpp induced by cocaine . eighty male mice performed the hole - board test and were defined as hns or lns and randomly assigned a drug treatment ( vehicle , 0.1 mg / kg of win 55212 - 2 or 1 mg / kg of rimonabant ) . the animals received a daily injection of their respective treatment for 5 days ( pnd 2630 ) and , after an interval of 3 days without any treatment , underwent the cpp procedure having been conditioned with the same dose of cocaine ( 1 mg / kg ) . thus , six groups of mice were formed according to novelty - seeking profile and the pretreatment received before conditioning with cocaine ( hns - veh - coc , hns - win - coc , hns+sr - coc , lns - veh - coc , lns - win - coc , and lns - sr - coc ) . with the objective of corroborating the results obtained in the groups receiving pretreatment with win 55212 - 2 and conditioned with 1 mg / kg of cocaine , two additional groups were included in the procedure . after an interval of 3 days without any treatment , the mice underwent the cpp procedure having been conditioned with 6 mg / kg of cocaine ( hns - win - coc6 and lns - win - coc6 ) . to evaluate the influence of the novelty - seeking trait on the cpp induced by win 55212 - 2 ( study 1 , experiment 1 ) , data of the time spent by the animals in the drug - paired compartment were analysed by means of a mixed anova with two between - subjects variables : novelty - seeking , with two levels ( hns and lns ) , and treatment , with two levels ( win 0.05 and win 0.075 ) , and one within - subjects variable : days , with two levels ( pre - c and post - c ) . to evaluate the effect of pretreatment with cannabinoid drugs on the subsequent cpp induced by win 55212 - 2 or cocaine in hns and lns mice ( study 1 , experiment 2 ; and study 2 ) , data of the time spent in the drug - paired compartment were analysed with a mixed anova with two between - subjects variables : novelty - seeking , with two levels ( hns and lns ) , and pre - treatment , with three levels ( veh , win , and sr ) , and one within - subjects variable : days , with two levels ( pre - c and post - c ) . in the abovementioned experiments , extinction and reinstatement values in the groups showing cpp to evaluate the effect of pretreatment of hns and lns mice with win 55212 - 2 on the subsequent cpp induced by 6 mg / kg of cocaine ( additional groups of study 2 ) , data of the time spent in the drug - paired compartment were analysed with a mixed anova with one between - subjects variable : novelty - seeking , with two levels ( hns and lns ) , and one within - subjects variable : days , with four levels ( pre - c , post - c , extinction , and reinstatement ) . study 1 explains the influence of the novelty - seeking phenotype on the effects of cannabinoid exposure on acquisition of the cpp induced by win 55212 - 2 . the anova of the data obtained with the mice conditioned with 0.05 and 0.075 mg / kg of win 55212 - 2 revealed that the interaction days treatment novelty - seeking [ f(1,45 ) = 4.175 ; p < 0.05 ] was significant . post hoc comparisons showed that only the group of hns mice conditioned with the high dose of win 55212 - 2 spent more time in the drug - paired compartment in post - c than during pre - c ( p < 0.05 ) . linear and logistic regression analysis did not show any significant correlation between the novelty - seeking trait and development of the cpp induced by 0.075 mg / kg of win 55212 - 2 . experiment 2 is about the effects of exposure of hns and lns mice to agonist and antagonist cannabinoids on acquisition of the cpp induced by a subthreshold dose of win 55212 - 2 . the anova of the data obtained with the mice conditioned with 1 mg / kg of cocaine revealed a significant effect of the interaction post hoc comparisons showed a significant increase in the time spent by hns and lns mice pretreated with win 55212 - 2 in the drug - paired compartment in post - c with respect to pre - c ( ps < 0.01 ) . thus , pretreatment with win 55212 - 2 increased the rewarding effects of cocaine irrespective of the novelty - seeking profile of the mice , but only hns animals were more sensitive to reinstatement after cocaine priming ( see figure 5 ) . the anova of the data obtained with the mice pretreated with win 55212 - 2 and conditioned with 6 mg / kg of cocaine revealed a significant effect of the variable days [ f(3,72 ) = 17.772 ; p < 0.01 ] , with mice spending more time in the drug - paired compartment in post - c than during pre - c ( p < 0.01 ) and in the reinstatement test than during the previous extinction test ( p < 0.05 ) . post hoc comparisons showed a significant increase in the time spent by hns and lns mice in the drug - paired compartment in post - c with respect to pre - c ( ps < 0.01 ) and revealed a reinstatement of cpp ( ps < 0.05 ) after a priming dose of 3 mg / kg of cocaine ( see figure 6 ) . the kaplan - meier test showed that the time required for extinction was longer in hns than in lns mice ( 14 versus 7 days , x = 3.995 , p < 0.05 ) ( see figure 7 ) . linear and logistic regression analysis did not show any significant correlation between the novelty - seeking trait and development of the cpp induced by 1 or 6 mg / kg of cocaine , in accordance with a previous study carried out in our laboratory . even more importantly , given the high risk of adverse effects associated with cocaine , there was some indication that adolescent mice with this phenotype are more vulnerable to the reinstating effects of a low dose of cocaine if they have been previously exposed to a cannabinoid agonist . the first contribution of this study is the demonstration that the hns phenotype increases the sensitivity of mice to the conditioned rewarding effects of the cannabinoid agonist win 55212 - 2 . although no previous studies have evaluated the influence of the novelty - seeking trait on the rewarding effects of cannabinoids , our results are in accordance with those observed with other drugs of abuse , such as cocaine or mdma , which have demonstrated that hns mice are more sensitive to the conditioned rewarding effects of these drugs [ 4143 ] . it is not clear if the ability of hns mice to develop cpp after administration of the cannabinoid agonist is related with an increase in the reinforcing / rewarding value of this drug for these animals or whether they acquire incentive learning in a more efficient way than lns mice . either way , increased levels of incentive salience attributed to drugs and/or drug - associated cues can enhance the intensity and duration of incentive motivation for drugs of abuse ( higher unconscious wanting and conscious craving ) , thus facilitating the transition to drug addiction , as suggested by robinson and berridge . it has been reported that hns animals have a characteristic striatal da profile ( higher endogenous levels , stronger responses to reward cues , and lower availability of d2/d3/d4 receptors ) , which may contribute to the tendency of these animals to exhibit approach reactions towards novel stimuli and may explain the increased cpp observed in the present study . the second important result of the present study is that even though exposure to the cannabinoid agonist win 55212 - 2 during adolescence did not enhance the acquisition of cpp induced by win 55212 - 2 itself at the doses tested , it did enhance the acquisition of cpp induced by a low dose of cocaine . although clinical and epidemiologic studies show that cannabis consumption usually precedes the initiation of cocaine use [ 2729 ] , only two studies have evaluated the effect of stimulation of the endocannabinoid system ( ecs ) during adolescence on the subsequent effects of cocaine . in line with the results of the present study , adolescent rats pretreated with cannabinoid agonists showed increased locomotor responses to cocaine challenge and a higher rate of cocaine self - administration . ecs plays an important role in adolescent brain development , and the strong stimulation of this system by cannabinoids might induce long - lasting neurobiological consequences , such as alterations in emotional and cognitive performance , increased risk of developing schizophrenia , and enhanced vulnerability to the use of drugs of abuse . cannabinoid adolescent exposure induces an upregulation of dat in the caudate - putamen , an increase in d1rs content in the nacc shell , and a reduction in the expression of d2rs in ca1 . on the other hand , neither exposure to the cannabinoid agonist win 55212 - 2 during adolescence nor pretreatment with rimonabant modified the subsequent effect of win 55212 - 2 in the cpp paradigm , in contrast with that observed with cocaine in this study or in previous studies with morphine or mdma [ 39 , 40 ] . given that our main objective was to detect differences in the influence of adolescent cannabinoid exposure between hns and lns , we used a very low dose of win 55212 - 2 ( 0.05 mg / kg ) , as we expected that cannabinoid pretreatment would cause a shift to the right of the dose - response curve . for example , in a previous study we observed that older adolescent mice ( pnd 52 ) developed cpp after conditioning with 0.05 mg / kg of win 55212 - 2 , suggesting that adolescent maturation is a factor that increases sensitivity to this drug . in fact , the effects of novelty - seeking phenotype ( experiment 1 ) were only apparent when the dose of win 55212 - 2 was higher ( 0.075 mg / kg ) than the dose used in this experiment . the main result of the present work is that the novelty - seeking phenotype determines the influence of adolescent cannabinoid exposure on the subsequent rewarding effects of cocaine in the cpp paradigm . hns mice are particularly affected by pretreatment with win 55212 - 2 , showing a priming - induced reinstatement of the cpp induced by 1 mg / kg of cocaine and an enhanced duration of the cpp induced by 6 mg / kg of this drug ( effects that are not observed in lns mice exposed to win 55212 - 2 ) . our results support the idea that exposure to cannabis during adolescence , though it can increase the rewarding effects of subthreshold doses of cocaine six days after pretreatment , is not enough to promote long - lasting brain changes that increase the likelihood of the development of cocaine addiction ( which can be evaluated by the maintenance of cpp or its reinstatement after extinction ) . our research has shown that the novelty - seeking trait is associated with an enhanced acquisition of win 55212 - 2 cpp , as we have observed previously with cocaine , and also with an increase in the effects of adolescent cannabinoid exposure on reinstatement and maintenance of cocaine cpp . thus , the results of the present study support the formulation of a vulnerability model that integrates the gateway and common liability hypotheses in order to explain the increased likelihood of transition from regular cannabis use to that of other substances ( such as cocaine or heroin ) . instead , there are individual brain and behavioural differences that are present prior to the onset of drug use , such as a higher propensity for sensation - seeking , which influence both the tendency to experiment with drugs of abuse early in life and the later development of addiction .

the pol3/cdc2 gene of saccharomyces cerevisiae encodes the catalytic subunit of the dna polymerase . the coding sequence includes a catalytic domain , a nucleotide binding domain , and an exonuclease proofreading site . pol has a primase activity and is involved in initiation of both the leading and lagging strands . both pol and pol can extend the primers formed by pol [ 3 , 4 ] and are proposed to be involved in nucleotide excision repair and base excision repair [ 6 , 7 ] . in addition , the dna polymerase exonuclease is involved in postreplication repair [ 8 , 9 ] . yeast strains lacking the proofreading exonuclease activity of the polymerase have a strong mutator phenotype . the pol3-t mutation is located near the catalytic domain outside the exonuclease domain in a region probably involved in nucleotide binding . the pol3-t mutant allele , initially isolated as tex1 mutant because it increased the rate of excision of a bacterial transposon within the yeast lys2 gene , also enhances intrachromosomal deletion recombination between short repeats of several base pairs separated by long inverted repeats . the molecular analysis of the transposon excision events indicates that dna replication slippage is most likely responsible for these excision events [ 11 , 12 ] . furthermore , the frequency of deletions between distant short repeats within lys2 or the can1 gene is also increased many fold . finally , it has been shown that the same mutator phenotype as observed in the pol3-t mutation exists after repression of the pol3 gene , indicating that the mutator phenotype may be due to low levels of pol3 rather than to faulty effects of the pol3 mutant proteins . rad50 is involved in dna double strand break repair by nonhomologous end joining and homologous recombination such as sister chromatid recombination and double strand break ( dsb ) processing . rad50 together with xrs2 and mre11 is part of the mrx complex which localizes to dsbs [ 18 , 19 ] . whereas wild type cells are much more radiation - sensitive in g1 ( 1.5% survival at 150 gy ) compared to g2 ( 70% survival ) , mre11 mutant cells show about the same survival rate in both phases ( 0.6% versus 1% ) , indicating preferential mrx - mediated repair when sister chromatids are present in g2 . in addition the gene products 46/47 of the bacteriophage t4 , homologs of mre11/rad50 , are required for recombination - induced replication [ 2325 ] . recombination - induced replication also may be involved in dsb repair during g2 by synthesis dependent strand annealing , possibly explaining the mrx - mediated preferential repair in g2 cells . several mutants with elevated spontaneous intrachromosomal recombination frequencies have been isolated in saccharomyces cerevisiae [ 27 , 28 ] . among them , a mutant allele of cdc2/pol3 , which encodes the catalytic subunit of the dna polymerase , increases deletion events . intrachromosomal deletion events between duplicated sequences may occur by several mechanisms such as intrachromatid exchange , single - strand annealing , one - sided invasion , unequal sister chromatid exchange or , sister chromatid conversion [ 13 , 2931 ] . this hyperrecombination phenotype is partially dependent of rad1 and rad52 because the pol3-t mutation still enhances intrachromosomal recombination in the rad1rad52 double mutant . this suggests that the hyperrecombination phenotype may depend on dna genes other than rad52 or rad1 . here , we report a further characterization of the pol3-t mutant . we investigated the effect of the rad50 gene involved in dsb repair , on the pol3-t phenotype by measuring methyl methanesulfonate ( mms ) sensitivity , the spontaneous as well as mms- , uv- , and -ray - induced intrachromosomal recombination and , finally , the effect on homologous integration . complete media ( ypad ) , synthetic complete ( sc ) , and drop - out ( sd ) media were prepared according to standard procedures . magic column ( promega , madison , wi ) was used for preparation of small - scale dna . the names and the genotypes of the strains used are listed in table 1 . because pol3-t confers a temperature sensitive phenotype , strains agy34 , agy38 , agy39 , and agy40 were constructed by introducing the pol3-t mutation into strains yr50 - 1 , rsy12 , yr50 - 12 , and ymg1 , respectively . this was done by transformation of the cells with plasmid p171 ( a gift from michael resnick , national institute of environmental health sciences , research triangle park , nc ) , which contains a 2.2-kb ecorv - hindiii fragment containing the pol3-t allele . temperature - sensitive ura colonies that contained the full - length pol3-t allele and a truncated pol3 allele flanking the ura3 gene were isolated . ura temperature - sensitive strains carrying the pol3-t allele were selected after selection on medium containing 5-foa . single colonies of strain rsy6 and its derivatives mag1 , rad50 , pol3 t , mag1pol3 t and rad50 pol3 t were inoculated into ypad at 25 for 24 hours . thereafter , cells were washed , resuspended in 5 ml of fresh yapd at the concentration of 3 10 cells / ml and exposed to mms for 4 hours at 30. then cells were washed twice , counted , and plated in ypad at the concentration of 200 cells per plate . this substrate consists of two his3 alleles , one with a deletion at the 3 end and the other with a deletion at the 5 end , which share 400 bp of homology . these two alleles are separated by the leu2 marker and by the plasmid dna sequence . an intrachromosomal recombination event between the two his3 alleles leads to his3 reversion and loss of leu2 . to determine the frequency of spontaneous intrachromosomal recombination , single colonies were inoculated into 5 ml of sc - leu , so that recombinants can not grow , and incubated at 25 or 30 for 24 hours . thereafter , cultures were washed twice and counted and appropriate numbers were plated onto sc and sc - his plates to determine the surviving fraction and the frequency of intrachromosomal recombination events , respectively . intrachromosomal recombination was measured following uv , -ray , and mms exposure . for uv exposure thereafter , cells were washed , resuspended in fresh sc - leu for 4 hours at 30c . 10 ml aliquots containing 3 10 cells / ml were irradiated in distilled water using a uv source at the dose rate of 3.5 ergs / m / sec . the same number of cells were exposed to -rays using a co -ray source at 9.1 cgy per second [ 30 , 34 ] . following irradiation , thereafter , cells were washed , resuspended in 5 ml of fresh sc - leu at the concentration of 3 10 cells / ml and exposed to mms for 4 hours at 30. then cells were washed , counted and plated as described . at the highest mms dose only the wild type strain grew for one generation ; at low doses all strains grew an average of 2 - 3 generations . the gene targeting events were determined in the rsy12 strain and its derivatives y50 - 12 , agy38 , and agy39 which carry the complete deletion of the ura3 gene . the ecori - hindiii fragment from plasmid pjz102 carrying the lys2 gene disrupted by ura3 insertion was transformed in all the rsy12 derivative strains as previously described . transformants were selected on sc - ura plates and , then , replicated in sc - lys medium . the frequency of homologous gene replacement was calculated as number of total ura3lys2 colonies 10 transformed cells per g dna . the number of transformed cells per g dna was determined using the episomal plasmid yeplac195 as previously described results were statistically analysed using the student 's t - test . probabilities are shown as * p < .05 , * * p < .01 , * * * p < .001 . the rate of dna damage - induced recombination was extraplotated as follows : for each strain , we measured the number of recombination events induced for a range of increasing dosages in single experiment . from one experiment , we fitted the best - fit line to the data and took the slope of this line as the rate of induction . we used a student 's t test to compare between individually extrapolated rate of induction values between strains ( for the same dna damaging agent ) . the main lesion mms produced in dna is methylation , primarily producing 3-methyladenine ( 3mea ) . 3mea is mainly repaired by base excision repair ( ber ) , but some lesions can be converted to dsbs which are repaired by nonhomologous end joining or homologous recombination [ 3538 ] . it has previously been shown that pol3 mutant cells are sensitive to mms which is taken as evidence for involvement of dna polymerase in the base excision repair pathway . the 3mea dna glycosylase , encoded by the mag1 gene , has been shown to be very important for 3mea removal from dna [ 39 , 40 ] . the pol3-t mutant is sensitive to the alkylating agent mms as reported for other pol3 mutants [ 6 , 14 ] . the deletion of the rad50 gene also confers high sensitivity to mms . in the present study , we determined the epistasis of the mms sensitivity of the pol3-t mutant with the base excision repair mutation mag1 and the double strand break repair gene rad50 . previously , it has been shown that the mag1 and the rad50 mutations show a synergistic interaction with respect to mms sensitivity implying that mag1 and rad50 act in distinct repair pathways . here the pol3-t mutant was more sensitive to mms than wild type ( figure 1 ) , and the double mutant mag1pol3-t was more sensitive to mms than each single mutant indicating that mag1 and pol3 belong to different repair pathways ( figure 1(a ) ) . moreover , the double mutant rad50pol3-t showed the same sensitivity to mms as the rad50 single mutant . this suggests that rad50 and pol3 may belong to the same pathway for repairing mms - induced lesions . we previously have shown that the pol3-t mutation causes a hyperrecombination phenotype in yeast that is partially dependent on rad52 and rad1 . this suggests that replication slippage or a single - strand annealing pathway that is rad52 and rad1 independent could be responsible for the hyperrecombination phenotype . the rad50 gene product is involved in dna replication slippage between distant repeats [ 11 , 42 ] . moreover , the deletion of the rad50 gene in the pol3-t background decreases the frequency of excision of tn5 . to investigate the effect of rad50 on pol3-t - mediated recombination , we constructed the haploid strain agy34 which contains an intrachromosomal recombination substrate ( see materials and methods ) . the pol3-t mutation confers a temperature - sensitive phenotype and growth arrest at 37c ; therefore we measured the effect of the rad50 deletion mutation after growth at 25c and 30c . single colonies of rsy6 , agy30 , yr50 - 1 , and agy34 were inoculated into sc - leu medium for 24 hours at 25c and 30c . during this period rsy6 ( wild type ) and yr50 - 1 ( rad50 ) underwent 4 to 5 cell divisions at both temperatures . agy30 ( pol3-t ) and agy34 ( rad50pol3-t ) underwent 3 cell divisions at 25c and 2 cell divisions at 30c . in the pol3-t strain , intrachromosomal recombination increased 14-fold at 25c and 32-fold at 30c confirming that pol3-t confers a hyperrecombination phenotype ( table 2 , ) . in the rad50 background strain , the pol3-t mutation did not increase intrachromosomal recombination at either 25c or 30c ( table 2 ) demonstrating that the rad50 deletion completely abolished the pol3-t - mediated hyperrecombination phenotype . integration of linear dna fragment by homologous recombination into a chromosomal gene is thought to occur by two independent strand invasion events leading to the replacement of the chromosomal target with the dna fragment [ 43 , 44 ] . the homologous integration is reduced in the rad1 , rad51 , rad52 , and rad57 deletion mutant while it is unaffected in the rad50 . we , therefore , measured the homologous integration in the pol3-t and in the rad50pol3-t mutant . the rsy12 , agy38 , agy39 , and y50 - 12 strains were transformed with the ura3 fragment flanked by lys2 sequence . as an homologous integration event leads to the replacement of chromosomal lys2 gene with the ura3 fragment , the frequency of homologous integration was determined as number of ura3lys2 colonies 10 transformed cells per g dna . in the pol3-t mutant , the frequency of homologous integration increased 11-fold as compared to the wild type ( table 3 ) . in the double mutant rad50pol3-t , the frequency has increased 10.5-fold as compared to the wild type indicating that the elevated level of integration is not rad50 dependent ( table 3 ) . as previously reported , the rad50 mutation did not affect the frequency of homologous integration as compared to the wild type ( table 3 ) . mms , uv , and -rays induced intrachromosomal recombination in the pol3-t mutant ( tables 4 , 5 , and 6 ) . to further characterize the pol3-t phenotype , we looked at whether the rad50 deletion could also suppress mutagen - induced intrachromosomal recombination events in the pol3-t mutant . single colonies of both yr50 - 1 ( rad50 ) and agy34 ( rad50pol3-t ) strains were grown at 25c for 17 hours and then incubated at 30c for 4 hours before mms , uv , and -ray exposure after which survival and intrachromosomal events were scored for a range of doses of each mutagen . the rate of intrachromosomal recombination induction with dose for each experiment was found by linear regression ; comparison of the induction rate between strains was made using student 's t - test . to see if pol3-t strains are defective in dna damage - induced intrachromosomal recombination , we compared the rate of recombination induction among strains . then , we measured the number of recombination events induced for a range of increasing dosages in single experiment . from one experiment , we fitted the best - fit line to the data and took the slope of this line as the rate of induction . each experiment was done in at least triplicate , and thus , for each mutagen and each strain there are at least three separate measures of the rate of induction with dose . survival and intrachromosomal recombination events were measured in wild type and pol3-t strains to 0 , 10 , 100 , 200 , and 500 g / ml mms and in rad50 and rad50pol3-t strains to 0 , 1 , 10 , 50 , 100 , and 200 g / ml . 10 g / ml mms induced a significant increase of intrachromosomal recombination in the wild type but none in each of the mutants ( table 4 ) . yet 100 g / ml mms was the lowest dose used that significantly increased intrachromosomal recombination in each of the strains . the rate of intrachromosomal recombination induction was 19.3 1.9 ( 10 per g / ml mms ) in the wild type and 20.1 1.3 ( 10 per g / ml mms ) in the pol3-t . the rad50 mutation resulted in a significantly lower induction rates of 1.3 0.3 ( 10 per g / ml mms ) ( p < .005 ) and was partially restored to the wild type level in the double mutant rad50pol3-t with 5.0 1.1 ( 10 per g / ml mms ) ( p < .005 when compared to rad50 ) . this suggests that rad50 is required for mms - induced intrachromosomal recombination more so in the wild type than in the pol3-t mutant background . a fluence of 100 j / m induced a significant increase in each of the strains except the rad50 mutant for which a significant increase was not observed below 500 j / m ( table 5 ) . the intrachromosomal recombination induction rate was 42.1 5.5 ( 10 per j / m uv ) in the wild type and 17.3 3.9 ( 10 per j / m uv ) in the pol3-t strain . this rate was significantly lower in both the rad50 and rad50pol3-t strains which exhibited induction rates of 2.1 0.4 and 3.8 1.0 ( 10 per j / m uv ) , respectively ( p this suggests that rad50 had a reducing effect in the pol3 as well as in the pol3-t mutant to uv - induced intrachromosomal recombination events ( table 5 ) . the rad50 and the rad50pol3-t strains are much more sensitive to -rays than the wild type and the pol3-t single mutant strains , respectively , yet little difference in -ray sensitivity was found among the rad50 and rad50pol3-t strains ( table 6 ) . the rad50 phenotypic sensitivity to ionizing radiation is severe ; the dose corresponding to 5%10% survival , 50 gy , is approximately 20x less than an equitoxic dose in the wild type and pol3-t strains . because rad50 and rad50pol3-t strains exhibit extremely low survival to -rays at doses > 50 gy , the rate of intrachromosomal recombination from 0 and 50 gy was compared between each of the four strains , a dose range at which recombination was found to sharply increase with dose . within this dose range , the rate of intrachromosomal recombination in each of the strains was as follows : 79.3 35.5 ( 10 per gy ) in wild type , 101.6 46.9 ( 10 per gy ) in pol3-t 69.5 20.1 ( 10per gy ) in rad50 , and 77.8 22.6 ( 10 per gy ) in the rad50pol3-t double mutant . thus between the range 050 gy , all strains exhibited a similar rate of intrachromosomal recombination . the pol3-t toxicity to ionizing radiation is similar to wild - type , and thus the rate of -ray - induced recombination was compared between these strains between 50 and 1000 gy , a range at which recombination is induced at a lower rate ( table 6 ) . here , the pol3-t strain showed a trend of a lower induction rate than that of wild type : 19.8 4.3 versus 35.6 13.2 ( 10 per gy ) ( p = .06 ) . we found that pol3-t was synergistic with mag1 for mms toxicity but epistatic with rad50 . this suggests that pol3 may participate in the rad50 pathway for repair of mms damage . the mechanism by which the mms - induced lesions are converted to dsbs is not completely understood except that dsb repair - deficient mutants are also sensitive to mms . in theory , it is possible that mms damaged sites are converted into dsbs , and pol3 is involved in their repair even though there is no published evidence for that . however , in our experiments the pol3-t mutant was not more sensitive to ionizing radiation that causes dsbs arguing against pol3 involvement in dsb repair . recently , it has been shown that mms does not induce dsbs in both yeast and mammalian cells . the number of alkylated sites converted to single - strand breaks and dsbs , however , could be too few to be detected by their assay , but enough to require the involvement of the dsb repair pathway . the authors also suggested that the alkylation damage may stall the replication fork leading to the formation of a chicken foot structure which resembles a holliday junction . this may explain the reason why the dsb repair mutants that are also deficient in recombination are sensitive to mms . this would imply some involvement of rad50 in resolution of stalled replication forks , which may involve recombination [ 21 , 22 ] . in fact , the gene products 46/47 , the bacteriophage t4 homologs of mre11/rad50 , are required for recombination - induced replication [ 2325 ] . both pol3 as well as rad50 may be involved in replication on the mms - damaged template explaining their epistasis for mms sensitivity . we also found that the elevated level of intrachromosomal deletion recombination events in the pol3-t mutant is dependent on the rad50 gene . the rad50 protein is part of a complex that plays a major role in processing of dna dsb ends ; therefore dna dsb processing may be necessary for conferring the hyperrecombination phenotype of pol3-t . on the other hand , as discussed above for mms toxicity , rad50 may be involved in recombinational resolution of replication forks stalled by dna damage . in the presence of the pol3-t mutation , when replication is stalled at the restrictive temperature , such recombinational resolution may become important . if the second copy of the his3 repeat is accidentally used as template for such resolution rather than the copy at which replication has stalled , the pol3-t - caused hyperrecombination phenotype may be mediated by rad50 . in a similar way for the involvement of rad50 in the pol3-t - mediated replication slippage at direct repeats within lys2 a possible replication function of rad50 has been proposed , rather than dsbs being involved in the slippage events . we , indeed , found that the elevated frequency of homologous integration conferred by the pol3-t mutation was not affected by rad50 . this may indicate that the slow replication rate of the pol3-t mutant may favor homologous recombination events between the chromosomal dna and an exogenous dna fragment . moreover , rad50 that is primarly involved in dsb processing did not affect this phenotype . we found that the rad50 mutant is almost completely deficient in mms , and uv - induced recombination but no difference was observed in -rays - induced recombination . it has also previously been found that rad50 is involved in mms but not uv - induced recombination between homologs in a diploid . we have previously shown that intrachromosomal recombination between repeats is induced by site specific dsbs in g1 , g2 , and dividing cells . a site - specific single strand break , however , induced recombination only in dividing but not in g1 or g2 arrested cells . in a similar fashion , mms , ems , 4-nqo , and most but not all ionizing radiation - induced dna damage were dependent on dna replication for induction of intrachromosomal recombination this indicates that either replication turns the various dna damages into dsbs , which then induces intrachromosomal recombination by single - strand annealing , or that recombination is induced by resolution of dna - damaged replication forks as mentioned above . the latter explanation would be in agreement with rad50 being involved in dna damage - induced resolution of stalled replication forks since most of the damage - induced recombination was rad50-dependent similar to pol3-t induced recombination . at greater levels of dna damage some dsbs may be formed accounting for the low level of recombination induction in the rad50 mutant . interestingly , no difference was observed in -ray - induced recombination events . in the wild type the rate of -ray - induced recombination was observed to be biphasic with the number of events increasing sharply between 0 and 50 gy , and at doses beyond 50 gy the rate of recombination was substantially lower . because the rad50 mutation is extra sensitive to ionizing radiation , the rate of recombination events was not scored at doses above 50 gy . between 0 and 50 gy neither rad50 nor pol3-t mutations had an effect on the rate of -ray - induced recombination . it is possible that rad50 is involved in a second phase of -ray induced recombination at doses above 50 gy or that the types of damage primarily produced by ionizing radiation are not repaired though the rad50 pathway . either of such would explain why no difference in rate of intrachromosomal recombination was observed between 0 and 50 gy . in summary , furthermore , the epistasis for mms sensitivity and the deficiency in dna damage induced as well as complete block in pol3-t induced intrachromosomal recombination by rad50 is in agreement with involvement of rad50 in repair by recombination resolution of stalled replication forks .

the dna polymerase ( pol3/cdc2 ) allele pol3-t of saccharomyces cerevisiae has previously been shown to be sensitive to methylmethanesulfonate ( mms ) and has been proposed to be involved in base excision repair . our results , however , show that the pol3-t mutation is synergistic for mms sensitivity with mag1 , a known base excision repair gene , but it is epistatic with rad50 , suggesting that pol3 may be involved not only in base excision repair but also in a rad50 dependent function . we further studied the interaction of pol3-t with rad50 by examining their effect on spontaneous , mms- , uv- , and ionizing radiation - induced intrachromosomal recombination . we found that rad50 completely abolishes the elevated spontaneous frequency of intrachromosomal recombination in the pol3-t mutant and significantly decreases uv- and mms - induced recombination in both pol3 and pol3-t strains . interestingly , rad50 had no effect on -ray - induced recombination in both backgrounds between 0 and 50 gy . finally , the deletion of rad50 had no effect on the elevated frequency of homologous integration conferred by the pol3-t mutation . rad50 is possibly involved in resolution of replication forks that are stalled by mutagen - induced external dna damage , or internal dna damage produced by growing the pol3-t mutant at the restrictive temperature .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

the pol3/cdc2 gene of saccharomyces cerevisiae encodes the catalytic subunit of the dna polymerase . the coding sequence includes a catalytic domain , a nucleotide binding domain , and an exonuclease proofreading site . both pol and pol can extend the primers formed by pol [ 3 , 4 ] and are proposed to be involved in nucleotide excision repair and base excision repair [ 6 , 7 ] . in addition , the dna polymerase exonuclease is involved in postreplication repair [ 8 , 9 ] . the pol3-t mutation is located near the catalytic domain outside the exonuclease domain in a region probably involved in nucleotide binding . the pol3-t mutant allele , initially isolated as tex1 mutant because it increased the rate of excision of a bacterial transposon within the yeast lys2 gene , also enhances intrachromosomal deletion recombination between short repeats of several base pairs separated by long inverted repeats . furthermore , the frequency of deletions between distant short repeats within lys2 or the can1 gene is also increased many fold . finally , it has been shown that the same mutator phenotype as observed in the pol3-t mutation exists after repression of the pol3 gene , indicating that the mutator phenotype may be due to low levels of pol3 rather than to faulty effects of the pol3 mutant proteins . rad50 is involved in dna double strand break repair by nonhomologous end joining and homologous recombination such as sister chromatid recombination and double strand break ( dsb ) processing . whereas wild type cells are much more radiation - sensitive in g1 ( 1.5% survival at 150 gy ) compared to g2 ( 70% survival ) , mre11 mutant cells show about the same survival rate in both phases ( 0.6% versus 1% ) , indicating preferential mrx - mediated repair when sister chromatids are present in g2 . recombination - induced replication also may be involved in dsb repair during g2 by synthesis dependent strand annealing , possibly explaining the mrx - mediated preferential repair in g2 cells . several mutants with elevated spontaneous intrachromosomal recombination frequencies have been isolated in saccharomyces cerevisiae [ 27 , 28 ] . among them , a mutant allele of cdc2/pol3 , which encodes the catalytic subunit of the dna polymerase , increases deletion events . this hyperrecombination phenotype is partially dependent of rad1 and rad52 because the pol3-t mutation still enhances intrachromosomal recombination in the rad1rad52 double mutant . here , we report a further characterization of the pol3-t mutant . we investigated the effect of the rad50 gene involved in dsb repair , on the pol3-t phenotype by measuring methyl methanesulfonate ( mms ) sensitivity , the spontaneous as well as mms- , uv- , and -ray - induced intrachromosomal recombination and , finally , the effect on homologous integration . because pol3-t confers a temperature sensitive phenotype , strains agy34 , agy38 , agy39 , and agy40 were constructed by introducing the pol3-t mutation into strains yr50 - 1 , rsy12 , yr50 - 12 , and ymg1 , respectively . single colonies of strain rsy6 and its derivatives mag1 , rad50 , pol3 t , mag1pol3 t and rad50 pol3 t were inoculated into ypad at 25 for 24 hours . thereafter , cells were washed , resuspended in 5 ml of fresh yapd at the concentration of 3 10 cells / ml and exposed to mms for 4 hours at 30. then cells were washed twice , counted , and plated in ypad at the concentration of 200 cells per plate . to determine the frequency of spontaneous intrachromosomal recombination , single colonies were inoculated into 5 ml of sc - leu , so that recombinants can not grow , and incubated at 25 or 30 for 24 hours . thereafter , cultures were washed twice and counted and appropriate numbers were plated onto sc and sc - his plates to determine the surviving fraction and the frequency of intrachromosomal recombination events , respectively . intrachromosomal recombination was measured following uv , -ray , and mms exposure . the gene targeting events were determined in the rsy12 strain and its derivatives y50 - 12 , agy38 , and agy39 which carry the complete deletion of the ura3 gene . the frequency of homologous gene replacement was calculated as number of total ura3lys2 colonies 10 transformed cells per g dna . the rate of dna damage - induced recombination was extraplotated as follows : for each strain , we measured the number of recombination events induced for a range of increasing dosages in single experiment . 3mea is mainly repaired by base excision repair ( ber ) , but some lesions can be converted to dsbs which are repaired by nonhomologous end joining or homologous recombination [ 3538 ] . it has previously been shown that pol3 mutant cells are sensitive to mms which is taken as evidence for involvement of dna polymerase in the base excision repair pathway . the 3mea dna glycosylase , encoded by the mag1 gene , has been shown to be very important for 3mea removal from dna [ 39 , 40 ] . the pol3-t mutant is sensitive to the alkylating agent mms as reported for other pol3 mutants [ 6 , 14 ] . the deletion of the rad50 gene also confers high sensitivity to mms . in the present study , we determined the epistasis of the mms sensitivity of the pol3-t mutant with the base excision repair mutation mag1 and the double strand break repair gene rad50 . previously , it has been shown that the mag1 and the rad50 mutations show a synergistic interaction with respect to mms sensitivity implying that mag1 and rad50 act in distinct repair pathways . here the pol3-t mutant was more sensitive to mms than wild type ( figure 1 ) , and the double mutant mag1pol3-t was more sensitive to mms than each single mutant indicating that mag1 and pol3 belong to different repair pathways ( figure 1(a ) ) . this suggests that rad50 and pol3 may belong to the same pathway for repairing mms - induced lesions . we previously have shown that the pol3-t mutation causes a hyperrecombination phenotype in yeast that is partially dependent on rad52 and rad1 . moreover , the deletion of the rad50 gene in the pol3-t background decreases the frequency of excision of tn5 . the pol3-t mutation confers a temperature - sensitive phenotype and growth arrest at 37c ; therefore we measured the effect of the rad50 deletion mutation after growth at 25c and 30c . in the pol3-t strain , intrachromosomal recombination increased 14-fold at 25c and 32-fold at 30c confirming that pol3-t confers a hyperrecombination phenotype ( table 2 , ) . in the rad50 background strain , the pol3-t mutation did not increase intrachromosomal recombination at either 25c or 30c ( table 2 ) demonstrating that the rad50 deletion completely abolished the pol3-t - mediated hyperrecombination phenotype . the homologous integration is reduced in the rad1 , rad51 , rad52 , and rad57 deletion mutant while it is unaffected in the rad50 . we , therefore , measured the homologous integration in the pol3-t and in the rad50pol3-t mutant . as an homologous integration event leads to the replacement of chromosomal lys2 gene with the ura3 fragment , the frequency of homologous integration was determined as number of ura3lys2 colonies 10 transformed cells per g dna . in the pol3-t mutant , the frequency of homologous integration increased 11-fold as compared to the wild type ( table 3 ) . in the double mutant rad50pol3-t , the frequency has increased 10.5-fold as compared to the wild type indicating that the elevated level of integration is not rad50 dependent ( table 3 ) . as previously reported , the rad50 mutation did not affect the frequency of homologous integration as compared to the wild type ( table 3 ) . mms , uv , and -rays induced intrachromosomal recombination in the pol3-t mutant ( tables 4 , 5 , and 6 ) . to further characterize the pol3-t phenotype , we looked at whether the rad50 deletion could also suppress mutagen - induced intrachromosomal recombination events in the pol3-t mutant . single colonies of both yr50 - 1 ( rad50 ) and agy34 ( rad50pol3-t ) strains were grown at 25c for 17 hours and then incubated at 30c for 4 hours before mms , uv , and -ray exposure after which survival and intrachromosomal events were scored for a range of doses of each mutagen . to see if pol3-t strains are defective in dna damage - induced intrachromosomal recombination , we compared the rate of recombination induction among strains . survival and intrachromosomal recombination events were measured in wild type and pol3-t strains to 0 , 10 , 100 , 200 , and 500 g / ml mms and in rad50 and rad50pol3-t strains to 0 , 1 , 10 , 50 , 100 , and 200 g / ml . 10 g / ml mms induced a significant increase of intrachromosomal recombination in the wild type but none in each of the mutants ( table 4 ) . yet 100 g / ml mms was the lowest dose used that significantly increased intrachromosomal recombination in each of the strains . the rate of intrachromosomal recombination induction was 19.3 1.9 ( 10 per g / ml mms ) in the wild type and 20.1 1.3 ( 10 per g / ml mms ) in the pol3-t . the rad50 mutation resulted in a significantly lower induction rates of 1.3 0.3 ( 10 per g / ml mms ) ( p < .005 ) and was partially restored to the wild type level in the double mutant rad50pol3-t with 5.0 1.1 ( 10 per g / ml mms ) ( p < .005 when compared to rad50 ) . this suggests that rad50 is required for mms - induced intrachromosomal recombination more so in the wild type than in the pol3-t mutant background . the intrachromosomal recombination induction rate was 42.1 5.5 ( 10 per j / m uv ) in the wild type and 17.3 3.9 ( 10 per j / m uv ) in the pol3-t strain . this rate was significantly lower in both the rad50 and rad50pol3-t strains which exhibited induction rates of 2.1 0.4 and 3.8 1.0 ( 10 per j / m uv ) , respectively ( p this suggests that rad50 had a reducing effect in the pol3 as well as in the pol3-t mutant to uv - induced intrachromosomal recombination events ( table 5 ) . the rad50 and the rad50pol3-t strains are much more sensitive to -rays than the wild type and the pol3-t single mutant strains , respectively , yet little difference in -ray sensitivity was found among the rad50 and rad50pol3-t strains ( table 6 ) . the rad50 phenotypic sensitivity to ionizing radiation is severe ; the dose corresponding to 5%10% survival , 50 gy , is approximately 20x less than an equitoxic dose in the wild type and pol3-t strains . because rad50 and rad50pol3-t strains exhibit extremely low survival to -rays at doses > 50 gy , the rate of intrachromosomal recombination from 0 and 50 gy was compared between each of the four strains , a dose range at which recombination was found to sharply increase with dose . within this dose range , the rate of intrachromosomal recombination in each of the strains was as follows : 79.3 35.5 ( 10 per gy ) in wild type , 101.6 46.9 ( 10 per gy ) in pol3-t 69.5 20.1 ( 10per gy ) in rad50 , and 77.8 22.6 ( 10 per gy ) in the rad50pol3-t double mutant . thus between the range 050 gy , all strains exhibited a similar rate of intrachromosomal recombination . the pol3-t toxicity to ionizing radiation is similar to wild - type , and thus the rate of -ray - induced recombination was compared between these strains between 50 and 1000 gy , a range at which recombination is induced at a lower rate ( table 6 ) . here , the pol3-t strain showed a trend of a lower induction rate than that of wild type : 19.8 4.3 versus 35.6 13.2 ( 10 per gy ) ( p = .06 ) . we found that pol3-t was synergistic with mag1 for mms toxicity but epistatic with rad50 . this suggests that pol3 may participate in the rad50 pathway for repair of mms damage . the mechanism by which the mms - induced lesions are converted to dsbs is not completely understood except that dsb repair - deficient mutants are also sensitive to mms . in theory , it is possible that mms damaged sites are converted into dsbs , and pol3 is involved in their repair even though there is no published evidence for that . however , in our experiments the pol3-t mutant was not more sensitive to ionizing radiation that causes dsbs arguing against pol3 involvement in dsb repair . recently , it has been shown that mms does not induce dsbs in both yeast and mammalian cells . the number of alkylated sites converted to single - strand breaks and dsbs , however , could be too few to be detected by their assay , but enough to require the involvement of the dsb repair pathway . this may explain the reason why the dsb repair mutants that are also deficient in recombination are sensitive to mms . this would imply some involvement of rad50 in resolution of stalled replication forks , which may involve recombination [ 21 , 22 ] . in fact , the gene products 46/47 , the bacteriophage t4 homologs of mre11/rad50 , are required for recombination - induced replication [ 2325 ] . both pol3 as well as rad50 may be involved in replication on the mms - damaged template explaining their epistasis for mms sensitivity . we also found that the elevated level of intrachromosomal deletion recombination events in the pol3-t mutant is dependent on the rad50 gene . the rad50 protein is part of a complex that plays a major role in processing of dna dsb ends ; therefore dna dsb processing may be necessary for conferring the hyperrecombination phenotype of pol3-t . on the other hand , as discussed above for mms toxicity , rad50 may be involved in recombinational resolution of replication forks stalled by dna damage . in the presence of the pol3-t mutation , when replication is stalled at the restrictive temperature , such recombinational resolution may become important . if the second copy of the his3 repeat is accidentally used as template for such resolution rather than the copy at which replication has stalled , the pol3-t - caused hyperrecombination phenotype may be mediated by rad50 . in a similar way for the involvement of rad50 in the pol3-t - mediated replication slippage at direct repeats within lys2 a possible replication function of rad50 has been proposed , rather than dsbs being involved in the slippage events . we , indeed , found that the elevated frequency of homologous integration conferred by the pol3-t mutation was not affected by rad50 . this may indicate that the slow replication rate of the pol3-t mutant may favor homologous recombination events between the chromosomal dna and an exogenous dna fragment . moreover , rad50 that is primarly involved in dsb processing did not affect this phenotype . we found that the rad50 mutant is almost completely deficient in mms , and uv - induced recombination but no difference was observed in -rays - induced recombination . it has also previously been found that rad50 is involved in mms but not uv - induced recombination between homologs in a diploid . we have previously shown that intrachromosomal recombination between repeats is induced by site specific dsbs in g1 , g2 , and dividing cells . a site - specific single strand break , however , induced recombination only in dividing but not in g1 or g2 arrested cells . in a similar fashion , mms , ems , 4-nqo , and most but not all ionizing radiation - induced dna damage were dependent on dna replication for induction of intrachromosomal recombination this indicates that either replication turns the various dna damages into dsbs , which then induces intrachromosomal recombination by single - strand annealing , or that recombination is induced by resolution of dna - damaged replication forks as mentioned above . the latter explanation would be in agreement with rad50 being involved in dna damage - induced resolution of stalled replication forks since most of the damage - induced recombination was rad50-dependent similar to pol3-t induced recombination . at greater levels of dna damage some dsbs may be formed accounting for the low level of recombination induction in the rad50 mutant . interestingly , no difference was observed in -ray - induced recombination events . in the wild type the rate of -ray - induced recombination was observed to be biphasic with the number of events increasing sharply between 0 and 50 gy , and at doses beyond 50 gy the rate of recombination was substantially lower . because the rad50 mutation is extra sensitive to ionizing radiation , the rate of recombination events was not scored at doses above 50 gy . between 0 and 50 gy neither rad50 nor pol3-t mutations had an effect on the rate of -ray - induced recombination . it is possible that rad50 is involved in a second phase of -ray induced recombination at doses above 50 gy or that the types of damage primarily produced by ionizing radiation are not repaired though the rad50 pathway . either of such would explain why no difference in rate of intrachromosomal recombination was observed between 0 and 50 gy . in summary , furthermore , the epistasis for mms sensitivity and the deficiency in dna damage induced as well as complete block in pol3-t induced intrachromosomal recombination by rad50 is in agreement with involvement of rad50 in repair by recombination resolution of stalled replication forks .

both pol and pol can extend the primers formed by pol [ 3 , 4 ] and are proposed to be involved in nucleotide excision repair and base excision repair [ 6 , 7 ] . in addition , the dna polymerase exonuclease is involved in postreplication repair [ 8 , 9 ] . yeast strains lacking the proofreading exonuclease activity of the polymerase have a strong mutator phenotype . the pol3-t mutation is located near the catalytic domain outside the exonuclease domain in a region probably involved in nucleotide binding . the pol3-t mutant allele , initially isolated as tex1 mutant because it increased the rate of excision of a bacterial transposon within the yeast lys2 gene , also enhances intrachromosomal deletion recombination between short repeats of several base pairs separated by long inverted repeats . the molecular analysis of the transposon excision events indicates that dna replication slippage is most likely responsible for these excision events [ 11 , 12 ] . finally , it has been shown that the same mutator phenotype as observed in the pol3-t mutation exists after repression of the pol3 gene , indicating that the mutator phenotype may be due to low levels of pol3 rather than to faulty effects of the pol3 mutant proteins . rad50 is involved in dna double strand break repair by nonhomologous end joining and homologous recombination such as sister chromatid recombination and double strand break ( dsb ) processing . rad50 together with xrs2 and mre11 is part of the mrx complex which localizes to dsbs [ 18 , 19 ] . whereas wild type cells are much more radiation - sensitive in g1 ( 1.5% survival at 150 gy ) compared to g2 ( 70% survival ) , mre11 mutant cells show about the same survival rate in both phases ( 0.6% versus 1% ) , indicating preferential mrx - mediated repair when sister chromatids are present in g2 . in addition the gene products 46/47 of the bacteriophage t4 , homologs of mre11/rad50 , are required for recombination - induced replication [ 2325 ] . recombination - induced replication also may be involved in dsb repair during g2 by synthesis dependent strand annealing , possibly explaining the mrx - mediated preferential repair in g2 cells . among them , a mutant allele of cdc2/pol3 , which encodes the catalytic subunit of the dna polymerase , increases deletion events . intrachromosomal deletion events between duplicated sequences may occur by several mechanisms such as intrachromatid exchange , single - strand annealing , one - sided invasion , unequal sister chromatid exchange or , sister chromatid conversion [ 13 , 2931 ] . here , we report a further characterization of the pol3-t mutant . we investigated the effect of the rad50 gene involved in dsb repair , on the pol3-t phenotype by measuring methyl methanesulfonate ( mms ) sensitivity , the spontaneous as well as mms- , uv- , and -ray - induced intrachromosomal recombination and , finally , the effect on homologous integration . complete media ( ypad ) , synthetic complete ( sc ) , and drop - out ( sd ) media were prepared according to standard procedures . the names and the genotypes of the strains used are listed in table 1 . because pol3-t confers a temperature sensitive phenotype , strains agy34 , agy38 , agy39 , and agy40 were constructed by introducing the pol3-t mutation into strains yr50 - 1 , rsy12 , yr50 - 12 , and ymg1 , respectively . this was done by transformation of the cells with plasmid p171 ( a gift from michael resnick , national institute of environmental health sciences , research triangle park , nc ) , which contains a 2.2-kb ecorv - hindiii fragment containing the pol3-t allele . temperature - sensitive ura colonies that contained the full - length pol3-t allele and a truncated pol3 allele flanking the ura3 gene were isolated . thereafter , cells were washed , resuspended in 5 ml of fresh yapd at the concentration of 3 10 cells / ml and exposed to mms for 4 hours at 30. then cells were washed twice , counted , and plated in ypad at the concentration of 200 cells per plate . this substrate consists of two his3 alleles , one with a deletion at the 3 end and the other with a deletion at the 5 end , which share 400 bp of homology . to determine the frequency of spontaneous intrachromosomal recombination , single colonies were inoculated into 5 ml of sc - leu , so that recombinants can not grow , and incubated at 25 or 30 for 24 hours . thereafter , cultures were washed twice and counted and appropriate numbers were plated onto sc and sc - his plates to determine the surviving fraction and the frequency of intrachromosomal recombination events , respectively . 10 ml aliquots containing 3 10 cells / ml were irradiated in distilled water using a uv source at the dose rate of 3.5 ergs / m / sec . the same number of cells were exposed to -rays using a co -ray source at 9.1 cgy per second [ 30 , 34 ] . following irradiation , thereafter , cells were washed , resuspended in 5 ml of fresh sc - leu at the concentration of 3 10 cells / ml and exposed to mms for 4 hours at 30. then cells were washed , counted and plated as described . at the highest mms dose only the wild type strain grew for one generation ; at low doses all strains grew an average of 2 - 3 generations . the gene targeting events were determined in the rsy12 strain and its derivatives y50 - 12 , agy38 , and agy39 which carry the complete deletion of the ura3 gene . the ecori - hindiii fragment from plasmid pjz102 carrying the lys2 gene disrupted by ura3 insertion was transformed in all the rsy12 derivative strains as previously described . the frequency of homologous gene replacement was calculated as number of total ura3lys2 colonies 10 transformed cells per g dna . the number of transformed cells per g dna was determined using the episomal plasmid yeplac195 as previously described results were statistically analysed using the student 's t - test . probabilities are shown as * p < .05 , * * p < .01 , * * * p < .001 . the rate of dna damage - induced recombination was extraplotated as follows : for each strain , we measured the number of recombination events induced for a range of increasing dosages in single experiment . from one experiment , we fitted the best - fit line to the data and took the slope of this line as the rate of induction . we used a student 's t test to compare between individually extrapolated rate of induction values between strains ( for the same dna damaging agent ) . it has previously been shown that pol3 mutant cells are sensitive to mms which is taken as evidence for involvement of dna polymerase in the base excision repair pathway . in the present study , we determined the epistasis of the mms sensitivity of the pol3-t mutant with the base excision repair mutation mag1 and the double strand break repair gene rad50 . previously , it has been shown that the mag1 and the rad50 mutations show a synergistic interaction with respect to mms sensitivity implying that mag1 and rad50 act in distinct repair pathways . here the pol3-t mutant was more sensitive to mms than wild type ( figure 1 ) , and the double mutant mag1pol3-t was more sensitive to mms than each single mutant indicating that mag1 and pol3 belong to different repair pathways ( figure 1(a ) ) . moreover , the double mutant rad50pol3-t showed the same sensitivity to mms as the rad50 single mutant . this suggests that rad50 and pol3 may belong to the same pathway for repairing mms - induced lesions . we previously have shown that the pol3-t mutation causes a hyperrecombination phenotype in yeast that is partially dependent on rad52 and rad1 . this suggests that replication slippage or a single - strand annealing pathway that is rad52 and rad1 independent could be responsible for the hyperrecombination phenotype . moreover , the deletion of the rad50 gene in the pol3-t background decreases the frequency of excision of tn5 . to investigate the effect of rad50 on pol3-t - mediated recombination , we constructed the haploid strain agy34 which contains an intrachromosomal recombination substrate ( see materials and methods ) . the pol3-t mutation confers a temperature - sensitive phenotype and growth arrest at 37c ; therefore we measured the effect of the rad50 deletion mutation after growth at 25c and 30c . during this period rsy6 ( wild type ) and yr50 - 1 ( rad50 ) underwent 4 to 5 cell divisions at both temperatures . in the pol3-t strain , intrachromosomal recombination increased 14-fold at 25c and 32-fold at 30c confirming that pol3-t confers a hyperrecombination phenotype ( table 2 , ) . in the rad50 background strain , the pol3-t mutation did not increase intrachromosomal recombination at either 25c or 30c ( table 2 ) demonstrating that the rad50 deletion completely abolished the pol3-t - mediated hyperrecombination phenotype . integration of linear dna fragment by homologous recombination into a chromosomal gene is thought to occur by two independent strand invasion events leading to the replacement of the chromosomal target with the dna fragment [ 43 , 44 ] . the homologous integration is reduced in the rad1 , rad51 , rad52 , and rad57 deletion mutant while it is unaffected in the rad50 . we , therefore , measured the homologous integration in the pol3-t and in the rad50pol3-t mutant . as an homologous integration event leads to the replacement of chromosomal lys2 gene with the ura3 fragment , the frequency of homologous integration was determined as number of ura3lys2 colonies 10 transformed cells per g dna . in the pol3-t mutant , the frequency of homologous integration increased 11-fold as compared to the wild type ( table 3 ) . in the double mutant rad50pol3-t , the frequency has increased 10.5-fold as compared to the wild type indicating that the elevated level of integration is not rad50 dependent ( table 3 ) . as previously reported , the rad50 mutation did not affect the frequency of homologous integration as compared to the wild type ( table 3 ) . mms , uv , and -rays induced intrachromosomal recombination in the pol3-t mutant ( tables 4 , 5 , and 6 ) . to further characterize the pol3-t phenotype , we looked at whether the rad50 deletion could also suppress mutagen - induced intrachromosomal recombination events in the pol3-t mutant . single colonies of both yr50 - 1 ( rad50 ) and agy34 ( rad50pol3-t ) strains were grown at 25c for 17 hours and then incubated at 30c for 4 hours before mms , uv , and -ray exposure after which survival and intrachromosomal events were scored for a range of doses of each mutagen . the rate of intrachromosomal recombination induction with dose for each experiment was found by linear regression ; comparison of the induction rate between strains was made using student 's t - test . to see if pol3-t strains are defective in dna damage - induced intrachromosomal recombination , we compared the rate of recombination induction among strains . then , we measured the number of recombination events induced for a range of increasing dosages in single experiment . from one experiment , we fitted the best - fit line to the data and took the slope of this line as the rate of induction . survival and intrachromosomal recombination events were measured in wild type and pol3-t strains to 0 , 10 , 100 , 200 , and 500 g / ml mms and in rad50 and rad50pol3-t strains to 0 , 1 , 10 , 50 , 100 , and 200 g / ml . 10 g / ml mms induced a significant increase of intrachromosomal recombination in the wild type but none in each of the mutants ( table 4 ) . yet 100 g / ml mms was the lowest dose used that significantly increased intrachromosomal recombination in each of the strains . the rate of intrachromosomal recombination induction was 19.3 1.9 ( 10 per g / ml mms ) in the wild type and 20.1 1.3 ( 10 per g / ml mms ) in the pol3-t . the rad50 mutation resulted in a significantly lower induction rates of 1.3 0.3 ( 10 per g / ml mms ) ( p < .005 ) and was partially restored to the wild type level in the double mutant rad50pol3-t with 5.0 1.1 ( 10 per g / ml mms ) ( p < .005 when compared to rad50 ) . this suggests that rad50 is required for mms - induced intrachromosomal recombination more so in the wild type than in the pol3-t mutant background . a fluence of 100 j / m induced a significant increase in each of the strains except the rad50 mutant for which a significant increase was not observed below 500 j / m ( table 5 ) . the intrachromosomal recombination induction rate was 42.1 5.5 ( 10 per j / m uv ) in the wild type and 17.3 3.9 ( 10 per j / m uv ) in the pol3-t strain . this rate was significantly lower in both the rad50 and rad50pol3-t strains which exhibited induction rates of 2.1 0.4 and 3.8 1.0 ( 10 per j / m uv ) , respectively ( p this suggests that rad50 had a reducing effect in the pol3 as well as in the pol3-t mutant to uv - induced intrachromosomal recombination events ( table 5 ) . the rad50 and the rad50pol3-t strains are much more sensitive to -rays than the wild type and the pol3-t single mutant strains , respectively , yet little difference in -ray sensitivity was found among the rad50 and rad50pol3-t strains ( table 6 ) . because rad50 and rad50pol3-t strains exhibit extremely low survival to -rays at doses > 50 gy , the rate of intrachromosomal recombination from 0 and 50 gy was compared between each of the four strains , a dose range at which recombination was found to sharply increase with dose . within this dose range , the rate of intrachromosomal recombination in each of the strains was as follows : 79.3 35.5 ( 10 per gy ) in wild type , 101.6 46.9 ( 10 per gy ) in pol3-t 69.5 20.1 ( 10per gy ) in rad50 , and 77.8 22.6 ( 10 per gy ) in the rad50pol3-t double mutant . the pol3-t toxicity to ionizing radiation is similar to wild - type , and thus the rate of -ray - induced recombination was compared between these strains between 50 and 1000 gy , a range at which recombination is induced at a lower rate ( table 6 ) . here , the pol3-t strain showed a trend of a lower induction rate than that of wild type : 19.8 4.3 versus 35.6 13.2 ( 10 per gy ) ( p = .06 ) . the number of alkylated sites converted to single - strand breaks and dsbs , however , could be too few to be detected by their assay , but enough to require the involvement of the dsb repair pathway . in fact , the gene products 46/47 , the bacteriophage t4 homologs of mre11/rad50 , are required for recombination - induced replication [ 2325 ] . we also found that the elevated level of intrachromosomal deletion recombination events in the pol3-t mutant is dependent on the rad50 gene . the rad50 protein is part of a complex that plays a major role in processing of dna dsb ends ; therefore dna dsb processing may be necessary for conferring the hyperrecombination phenotype of pol3-t . on the other hand , as discussed above for mms toxicity , rad50 may be involved in recombinational resolution of replication forks stalled by dna damage . in the presence of the pol3-t mutation , when replication is stalled at the restrictive temperature , such recombinational resolution may become important . in a similar way for the involvement of rad50 in the pol3-t - mediated replication slippage at direct repeats within lys2 a possible replication function of rad50 has been proposed , rather than dsbs being involved in the slippage events . we , indeed , found that the elevated frequency of homologous integration conferred by the pol3-t mutation was not affected by rad50 . this may indicate that the slow replication rate of the pol3-t mutant may favor homologous recombination events between the chromosomal dna and an exogenous dna fragment . we found that the rad50 mutant is almost completely deficient in mms , and uv - induced recombination but no difference was observed in -rays - induced recombination . in a similar fashion , mms , ems , 4-nqo , and most but not all ionizing radiation - induced dna damage were dependent on dna replication for induction of intrachromosomal recombination this indicates that either replication turns the various dna damages into dsbs , which then induces intrachromosomal recombination by single - strand annealing , or that recombination is induced by resolution of dna - damaged replication forks as mentioned above . the latter explanation would be in agreement with rad50 being involved in dna damage - induced resolution of stalled replication forks since most of the damage - induced recombination was rad50-dependent similar to pol3-t induced recombination . in the wild type the rate of -ray - induced recombination was observed to be biphasic with the number of events increasing sharply between 0 and 50 gy , and at doses beyond 50 gy the rate of recombination was substantially lower . in summary , furthermore , the epistasis for mms sensitivity and the deficiency in dna damage induced as well as complete block in pol3-t induced intrachromosomal recombination by rad50 is in agreement with involvement of rad50 in repair by recombination resolution of stalled replication forks .

the incidence of type 2 diabetes ( t2d ) has increased significantly over the last several decades ( 24 , 42 ) , and management of the disease presents a significant health challenge . t2d is characterized by elevated fasting blood glucose and insulin insensitivity , and can lead to many health complications , including the development of cardiovascular disease ( 8) . exercise is recommended as a therapy for the prevention and management of t2d ( 26 ) . the american college of sports medicine ( acsm ) recommends 150 minutes / week of moderate intensity aerobic exercise conducted over 5 days / wk or more , along with moderate to vigorous - intensity resistance training at least 23 days / wk ( 8) . acutely and chronically , resistance and aerobic exercise have been shown to improve glucose uptake and insulin action ( 2 , 13 , 17 , 18 , 20 , 23 , 27 , 3133 ) . in spite of the known health benefits of exercise , most americans do not meet the recommended amounts of exercise , in part due to a lack of time ( 5 ) . recent studies have examined the effects of high intensity interval training in an attempt to reduce the necessary time commitment while simultaneously improving health ( 3 , 7 , 21 ) . for example , sprint interval training ( sit ) in young volunteers results in significant improvements in muscle oxidative capacity ( 6 , 7 ) , exercise performance , fat oxidation ( 28 , 30 ) , endothelial function ( 29 ) , and glucose tolerance ( i.e. , ability to maintain normal blood glucose ) ( 3 ) . although use of sit induces significant physiological and functional improvements from only 23 minutes of exercise per workout , the total workout time commitment remains close to 30 minutes due to the prolonged rest period between each work interval . thus , in terms of time commitment , sit is no more effective than traditional aerobic training . perhaps more important , sit employing the wingate protocol is conducted at supramaximal intensity and is potentially unsafe for sedentary middle - aged and older adults , especially if they have not undergone proper medical evaluation for determination of exercise safety ( 1 ) . consequently , other hit protocols have been developed that utilize a lower intensity than sit and also requires less time . ( 21 ) utilized a protocol consisting of ten 1-minute exercise intervals at a workload ~ 90100% vo2max interspersed with 60 recovery periods with a total workout time ( excluding warm - up and cool - down ) of ~ 20 minutes . use of this training strategy has been shown to significantly increase skeletal muscle glut-4 protein content ( 21 ) and lower 24-hour glucose in type 2 diabetics ( 21 ) . moreover , because of the 1-minute recovery periods , the metabolic cost , heart rate response , and rating of perceived exertion are similar to continuous steady state running at 70% vo2max ( 11 ) . in spite of these recent findings , hit may not be feasible for many individuals due to various reasons including , but not limited to , the costs associated with exercise testing , prescription , and supervision . kettlebell training has existed for several years and is known to result in significant improvements in muscular strength ( e.g. back squat ) and power output ( e.g. vertical jump ) ( 25 ) . it can differ considerably from traditional resistance training in that exercises may be performed at a significantly lower percentage of 1 repetition maximum , and with less recovery time between exercises . in many respects , kettlebell training is similar to circuit training and represents a hybrid of traditional endurance and resistance training . most kettlebell exercise routines require the use of only one or two kettlebells and can be performed in the home . it is considered a more cost - effective means of exercise , as it does not require expensive equipment or monthly fitness membership . in spite of the significant functional improvements associated with kettlebell training , the effects of kettlebell exercise on selected health markers such as glucose tolerance have yet to be investigated . the purpose of this preliminary study was to examine whether or not a single session of kettlebell exercise impacts glucose tolerance in healthy , but sedentary individuals . a secondary objective was to evaluate the average oxygen cost of the kettlebell workout employed in this study . we hypothesized that immediately following a bout of kettlebell exercise , glucose tolerance would be significantly improved compared to control . we also hypothesized that a bout of kettlebell exercise would be as effective at improving glucose tolerance compared with a bout of hit similar in duration . inclusion criteria included healthy individuals who were sedentary ( exercised 2 day / week ) , had a body mass index < 30 , and had a normal blood glucose response during a two - hour oral glucose tolerance test ( ogtt ) ( i.e. , did not display impaired glucose tolerance or type 2 diabetes ) . exclusion criteria included individuals who were active ( exercised > 2 times / week ) , had a bmi 30 , took glucose or lipid lowering medication , smoked , or had any medical condition where exercise was contraindicated . six healthy sedentary males with a mean age of 23.8 ( 1.8 ) years and a mean bmi of 26.5 ( 0.5 ) kgm completed the study . all subjects provided informed consent prior to participation in the study . to test our hypothesis , a repeated measures design was used . following a screening ogtt and determination of maximal oxygen consumption ( vo2max ) , subjects underwent three different experimental conditions . each visit was separated by a minimum of 5 and maximum of 7 days . all procedures except the graded treadmill test were performed between 06000900 hours following an overnight fast ( > 8 hours ) . subjects were asked to refrain from any vigorous physical activity and to maintain their normal diet throughout the study . the independent variable was the experimental condition whereas the major dependent variables included blood glucose and insulin concentrations . blood was obtained from an ear stick before ( 0 minutes ) , 30 , 60 , 90 , and 120 minutes after rapidly consuming 75 gm of glucose ( 100 ml volume , glucose orange drink 075 , azer scientific , morgantown , pa ) . blood glucose was measured using a commercially available glucose meter ( accu - chek glucose meter , aviva , roche diagnostics , indianapolis , in ) that meets the international organization for standardization requirements ( 35 ) . any subject with a fasting blood glucose > 100 mgdl or with a two - hr ogtt blood glucose concentration > 140 mgdl was excluded from the study . after completing the ogtt , subjects were familiarized with the equipment and procedures to be used throughout the study . on the second visit , body composition was assessed using volume plethysmography ( bodpod , cosmed , chicago , il ) . subjects then underwent a graded exercise test on a treadmill for determination of vo2max . during the treadmill test , subjects ran at an initial self - selected speed between 8.0 12.9 kmh at 0% grade . the grade remained constant throughout the test , but the speed was increased 0.8 kmh every two minutes . subjects were given a three - minute warm - up period ( 4.8 kmh at 0% grade ) prior to the test and a three - minute cool - down period ( 3.2 kmh at 0% grade ) following the test . the exercise test was terminated when the subject could no longer continue ( volitional exhaustion ) . prior to the test , an ear stick was performed to obtain baseline blood lactate ( lactate plus , nova biomedical , waltham , ma ) levels . oxygen consumption was measured breath by breath throughout the test ( viasys vmax , carefusion , san diego , ca ) . heart rate was measured at each stage using a polar heart rate monitor ( polar usa , lake success , ny ) via a chest strap , whereas perceived exertion was measured using the standard 620 borg scale . heart rhythm was also monitored using an electrocardiograph ( mac 550 , ge healthcare , united kingdom ) . was separated by ~ one week ( 57 days ) , and the order of conditions was randomized for each subject . condition 1 was a control visit ( quiet sitting for 2 hours during the ogtt ) where no prior exercise was performed . subjects used either a 9 or 11.3-kg kettlebell for the workout depending on their ability . kettlebell ability was based on whether a subject could complete an exercise with an 11.3 kg kettlebell . if the subject deemed the kettlebell too heavy and was unable to complete the 15 repetitions , a 9-kg kettlebell was provided for that exercise . the workout consisted of seven exercises ( squat and press , chest press , one - arm kettlebell swing , bent over row , front squat , sit - up press - up , and kettlebell swing ) in sequence targeting the muscles of the legs , arms and core . fifteen repetitions per exercise were performed with 30 seconds of standing recovery between each set . after completion of the seven exercises , subjects were given one - minute standing recovery followed by a second circuit of the aforementioned exercises . the workout was preceded by a warm - up consisting of five sets of 20 kettlebell swings . heart rate was measured at the end of each exercise whereas rating of perceived exertion ( rpe ) was measured following each circuit . condition 3 consisted of high - intensity interval running ( hit ) on a motor driven treadmill with a 1:1 work - to - rest ratio . following a three - minute warm - up period at 4.8 kmh , subjects performed 10 one - minute intervals at a running speed corresponding to 90% vo2max . each exercise interval was interspersed with a one - minute active rest period ( 5.6 kmh ) . . heart rate and rpe were recorded throughout the condition . to determine the effect of the hit and kettlebell workouts on blood glucose and lactate , blood was obtained via ear stick before and immediately following the exercise protocols . approximately five minutes following each condition , 7ml of blood was withdrawn from the antecubital vein into edta lined vaccutainer tubes ( 16x100 lavendar , bd , franklin lakes , nj ) . blood glucose was also measured at each draw using the portable glucose meter as previously described . plasma insulin was measured in duplicate using an enzyme - linked immunosorbent assay ( elisa ) kit ( ab100578 , abcam , cambridge , england ) per the manufacturer s instructions . the elisa kit was read at 450 nm wavelength using a microplate reader ( imark , biorad , hercules , ca ) . during a separate visit after completion of the three experimental conditions , a subset of five subjects came into the lab for measurement of the oxygen cost of the kettlebell workout . subjects performed the same kettlebell workout previously described while oxygen consumption and respiratory exchange ratio ( rer ) was continuously monitored ( trueone , parvo medics , sandy , ut ) . glucose and insulin data were analyzed using a two - way ( group * time ) repeated measures anova ( sigma stat ) . the holm - sidak method was used for post - hoc multiple pair - wise comparisons . all data were normally distributed , and values are expressed as mean se ( n = 6 ) . inclusion criteria included healthy individuals who were sedentary ( exercised 2 day / week ) , had a body mass index < 30 , and had a normal blood glucose response during a two - hour oral glucose tolerance test ( ogtt ) ( i.e. , did not display impaired glucose tolerance or type 2 diabetes ) . exclusion criteria included individuals who were active ( exercised > 2 times / week ) , had a bmi 30 , took glucose or lipid lowering medication , smoked , or had any medical condition where exercise was contraindicated . six healthy sedentary males with a mean age of 23.8 ( 1.8 ) years and a mean bmi of 26.5 ( 0.5 ) kgm completed the study . to test our hypothesis , a repeated measures design was used . following a screening ogtt and determination of maximal oxygen consumption ( vo2max ) , subjects underwent three different experimental conditions . each visit was separated by a minimum of 5 and maximum of 7 days . all procedures except the graded treadmill test subjects were asked to refrain from any vigorous physical activity and to maintain their normal diet throughout the study . the independent variable was the experimental condition whereas the major dependent variables included blood glucose and insulin concentrations . blood was obtained from an ear stick before ( 0 minutes ) , 30 , 60 , 90 , and 120 minutes after rapidly consuming 75 gm of glucose ( 100 ml volume , glucose orange drink 075 , azer scientific , morgantown , pa ) . blood glucose was measured using a commercially available glucose meter ( accu - chek glucose meter , aviva , roche diagnostics , indianapolis , in ) that meets the international organization for standardization requirements ( 35 ) . any subject with a fasting blood glucose > 100 mgdl or with a two - hr ogtt blood glucose concentration > 140 mgdl was excluded from the study . after completing the ogtt , subjects were familiarized with the equipment and procedures to be used throughout the study . on the second visit , body composition was assessed using volume plethysmography ( bodpod , cosmed , chicago , il ) . subjects then underwent a graded exercise test on a treadmill for determination of vo2max . during the treadmill test , subjects ran at an initial self - selected speed between 8.0 12.9 kmh at 0% grade . the grade remained constant throughout the test , but the speed was increased 0.8 kmh every two minutes . subjects were given a three - minute warm - up period ( 4.8 kmh at 0% grade ) prior to the test and a three - minute cool - down period ( 3.2 kmh at 0% grade ) following the test . the exercise test was terminated when the subject could no longer continue ( volitional exhaustion ) . prior to the test , an ear stick was performed to obtain baseline blood lactate ( lactate plus , nova biomedical , waltham , ma ) levels . oxygen consumption was measured breath by breath throughout the test ( viasys vmax , carefusion , san diego , ca ) . heart rate was measured at each stage using a polar heart rate monitor ( polar usa , lake success , ny ) via a chest strap , whereas perceived exertion was measured using the standard 620 borg scale . heart rhythm was also monitored using an electrocardiograph ( mac 550 , ge healthcare , united kingdom ) . was separated by ~ one week ( 57 days ) , and the order of conditions was randomized for each subject . condition 1 was a control visit ( quiet sitting for 2 hours during the ogtt ) where no prior exercise was performed . subjects used either a 9 or 11.3-kg kettlebell for the workout depending on their ability . kettlebell ability was based on whether a subject could complete an exercise with an 11.3 kg kettlebell . if the subject deemed the kettlebell too heavy and was unable to complete the 15 repetitions , a 9-kg kettlebell was provided for that exercise . the workout consisted of seven exercises ( squat and press , chest press , one - arm kettlebell swing , bent over row , front squat , sit - up press - up , and kettlebell swing ) in sequence targeting the muscles of the legs , arms and core . fifteen repetitions per exercise were performed with 30 seconds of standing recovery between each set . after completion of the seven exercises , subjects were given one - minute standing recovery followed by a second circuit of the aforementioned exercises . the workout was preceded by a warm - up consisting of five sets of 20 kettlebell swings . heart rate was measured at the end of each exercise whereas rating of perceived exertion ( rpe ) was measured following each circuit . condition 3 consisted of high - intensity interval running ( hit ) on a motor driven treadmill with a 1:1 work - to - rest ratio . following a three - minute warm - up period at 4.8 kmh , subjects performed 10 one - minute intervals at a running speed corresponding to 90% vo2max . each exercise interval was interspersed with a one - minute active rest period ( 5.6 kmh ) . heart rate and rpe were recorded throughout the condition . to determine the effect of the hit and kettlebell workouts on blood glucose and lactate , approximately five minutes following each condition , 7ml of blood was withdrawn from the antecubital vein into edta lined vaccutainer tubes ( 16x100 lavendar , bd , franklin lakes , nj ) . blood glucose was also measured at each draw using the portable glucose meter as previously described . plasma insulin was measured in duplicate using an enzyme - linked immunosorbent assay ( elisa ) kit ( ab100578 , abcam , cambridge , england ) per the manufacturer s instructions . the elisa kit was read at 450 nm wavelength using a microplate reader ( imark , biorad , hercules , ca ) . during a separate visit after completion of the three experimental conditions , a subset of five subjects came into the lab for measurement of the oxygen cost of the kettlebell workout . subjects performed the same kettlebell workout previously described while oxygen consumption and respiratory exchange ratio ( rer ) was continuously monitored ( trueone , parvo medics , sandy , ut ) . glucose and insulin data were analyzed using a two - way ( group * time ) repeated measures anova ( sigma stat ) . the holm - sidak method was used for post - hoc multiple pair - wise comparisons . all data were normally distributed , and values are expressed as mean se ( n = 6 ) . one subject withdrew from the study because they did not wish to have any further venipuncture . a second subject was eliminated from the study because of nausea following ingestion of the glucose drink . another subject was eliminated due to a display of reactive hypoglycemia following the hit workout . values obtained for blood lactate ( > 8.0 mmoll ) or rer ( 1.15 ) at the end of the test indicated that all subjects achieved vo2max . glucose values obtained during a two - hour ogtt after the three experimental conditions appear in figure 2 . there was a significant effect for time ( p < 0.001 ) and interaction ( p = .028 ) . there were no significant differences in blood glucose levels at 0 or 120 minutes between all three conditions . however , 60 minutes after glucose ingestion , blood glucose was significantly lower following hit ( p < 0.001 ) and kettlebell ( p = 0.003 ) exercise when compared to control . figure 4a illustrates the mean heart rate response following each interval throughout the entire hit workout . heart rate gradually increased with each successive interval throughout the workout . at the end of the 10 interval , figure 4b illustrates the mean heart rate response after each exercise throughout the entire kettlebell workout . the lowest heart rate was observed following the chest press exercise ( 2 and 9 ) , whereas the highest heart rate was observed after the one - arm kettlebell swing exercise ( 3 and 10 ) . heart rate averaged ~ 81% and ~ 84% of hrmax after the 1 and 2 circuit , respectively . during the hit workout , rpe gradually increased during the workout and averaged 15.7 ( 0.9 ) after the last interval . during the kettlebell workout , rpe averaged 12.7 ( 1.4 ) and 14.7 ( 1.7 ) after the first and second circuits , respectively . blood glucose values were obtained immediately before and after the kettlebell and hit workouts ( figure 5a ) . pre- and post - exercise glucose values were not significantly different from one another for either condition . moreover , there was no difference between the two exercise conditions . blood lactate was also measured before and immediately following the kettlebell and hit workouts ( figure 5b ) . there was no difference in resting blood lactate levels before the hit and kettlebell workouts . however , after exercise , blood lactate was significantly higher after the kettlebell workout ( 9.2 0.9 mmoll ) compared with hit ( 5.7 0.3 mmoll ) ( figure 5b ) . the blood lactate values were ~ 86% ( kettlebell ) and ~ 53% of that reached during the vo2max test . the oxygen cost of the kettlebell workout was measured in a subset of five subjects . vo2 averaged 20.4 ( 2.2 ) mlkgmin ( 46.5% vo2max ) during the first circuit , and 21.1 ( 2.0 ) mlkgmin ( 48.1% vo2max ) during the second circuit . rer averaged 1.2 ( 0.04 ) and 1.1 ( 0.04 ) during the first and second circuit , respectively . in the present preliminary study , we investigated the effect of a single bout of kettlebell exercise on glucose tolerance in sedentary , but healthy male subjects . the kettlebell and hit workouts ( including warm - up and cool down ) were similar in duration and averaged ~25 and 26 minutes , respectively . we found that immediately following a single session of kettlebell or hit exercise , glucose tolerance during an ogtt was significantly improved at 60 minutes with no concomitant change in insulin concentration . in addition , there was no difference between the two modes of exercise . these findings indicate that kettlebell exercise may serve as an alternative training strategy to hit for improving glucose tolerance . the mechanisms for improved glucose clearance post - exercise are complex and have not been completely elucidated . immediately post exercise , increased glucose clearance can be due to both insulin - dependent and insulin - independent pathways . in a rat study by hamada et al . ( 12 ) , insulin - independent glucose uptake was increased when measured 40 minutes after a single 60-minute session of treadmill exercise and was associated with increased ampk threonine phosphorylation . however , by 85-minutes post exercise , this ampk effect had dissipated and the increased glucose uptake that persisted was likely due to insulin - dependent mechanisms . in human skeletal muscle post - exercise , akt phosphorylation has been shown to increase ( 37 ) or not change ( 36 , 39 ) during a euglycemic clamp with physiological insulin levels . in several other acute exercise studies , no changes were observed in insulin receptor binding or phosphorylation , sub - maximal insulin stimulation of the tyrosine phosphorylation of irs-1 or sub - maximal insulin - stimulation of p13k ( 4 , 14 , 3941 ) . regardless of the mechanism for the improved glucose uptake , glut-4 protein is known to increase after aerobic ( 9 , 16 , 32 , 34 ) , resistance ( 15 , 19 ) , and high - intensity interval ( 22 ) exercise . results from these previous studies suggest that glut-4 is also increased after kettlebell exercise , though definitive data are currently lacking . although the two exercise conditions were qualitatively different from one another , the heart rate response was similar . in addition , the end - workout perceived exertion was also similar , with rpe averaging ~16 and 15 for the hit and kettlebell workout , respectively . in spite of these similarities , however , the blood lactate concentration was significantly higher following the kettlebell workout ( 9.2 mmoll ) when compared to the hit workout ( 5.7 mmoll ) . we hypothesize that the increased blood lactate concentration after the kettlebell workout is due to a higher reliance on glycolysis . exercise order was constructed to alternate between different muscle groups during the workout , with each exercise lasting approximately 30 seconds followed by 30 seconds rest . in contrast , during the hit workout , the same musculature was recruited during each interval , and the work interval was 60-sec in duration . we did not measure the oxygen cost of the hit workout in the present study . however , our previous work indicates that oxygen consumption for the workout employed reaches approximately 70% of vo2max ( 11 ) . during the kettlebell workout in the current study , vo2 averaged ~ 21.1 ( 4.9 ) mlkgmin , or 48% of vo2max . farrar et al . ( 10 ) reported that the oxygen cost of kettlebell exercise was ~ 34.3 ( 5.7 ) mlkgmin . in this latter study , subjects employed a significantly heavier kettlebell and were required to complete as many two - handed swings as possible in 12 minutes ( 10 ) . the contrasting findings are therefore likely due to the different weight and workout characteristics employed in the two studies . utilizing a 10-exercise , 3 circuit design ( 30 seconds exercise , 15-sec recovery ) , wilmore et al . reported that the oxygen cost of circuit weight training in men was approximately 41% of vo2max ( 38 ) . although blood lactate following the kettlebell workout approached that recorded at the end of the graded maximal exercise test and was significantly higher than that observed after hit exercise , all subjects were able to complete this workout . moreover , the subject s perception of effort at the end of kettlebell exercise was similar ( ~ 15 , hard ) to the hit workout . the workout may require modification for older , higher risk individuals who do not tolerate such a high perception of effort . for example , the work - to - rest ratio during interval exercise is known to affect heart rate , lactate , and rpe responses ( 11 ) . thus , when prescribing kettlebell exercise to higher risk individuals , it may be desirable to lower the weight and/or increase the recovery period between exercises to attenuate these variables . high intensity interval training has been studied extensively the last several years , and has been proposed as a time - efficient alternative to traditional aerobic training for the improvement of metabolic and cardiovascular health . in spite of the established health benefits , incorporation of a safe and effective hit program into the daily routine of lay individuals will require laboratory testing and professional consultation . the cost associated with implementation of such a program , as well as other factors ( e.g. , availability of facilities , time and cost of driving , weather , etc . ) may deter many individuals from commencing a hit exercise program . in contrast , the cost associated with kettlebell exercise is quite low , the exercises can easily be learned from web - based videos , and the workouts can be performed in the home or office . limitations to the current study include the small sample size ( n = 6 ) , the use of sedentary but healthy volunteers , exclusion of female subjects , age of the volunteers , as well as the use of only one exercise workout . regardless , this preliminary study suggests that kettlebell exercise acutely improves glucose tolerance in young sedentary subjects , and that the changes are similar to that observed following a bout of high - intensity interval running . thus , kettlebell exercise might serve as a suitable alternative to hit or traditional aerobic training . however , future studies are required to determine the acute and long - term effectiveness of kettlebell training at improving metabolic health in pre - diabetic and diabetic individuals . moreover , because individuals often cite lack of time as an excuse for not exercising regularly , further work is required to determine the minimal kettlebell exercise dose to improve glucose tolerance .

impaired glucose tolerance can have significant health consequences . the purposes of this preliminary study were to examine whether a single session of kettlebell exercise improves acute post - exercise glucose tolerance in sedentary individuals , and whether it was as effective as high - intensity interval running . six sedentary male subjects underwent a two - hour oral glucose tolerance test following three different conditions : 1 ) control ( no exercise ) ; 2 ) kettlebell exercise ( 2 sets of 7 exercises , 15 repetitions per exercise with 30 seconds rest between each exercise ) ; or 3 ) high - intensity interval running ( 10 one - minute intervals at a workload corresponding to 90% vo2max interspersed with one - minute active recovery periods ) . blood glucose and insulin levels were measured before ( 0 minutes ) , and 60 and 120 minutes after glucose ingestion . both kettlebell and high - intensity interval running exercise significantly lowered blood glucose 60 minutes after glucose ingestion compared with control . however , there was no significant difference in blood glucose between the two exercise conditions at any time point . in addition , there were no significant differences in insulin concentration between high intensity interval running , kettlebell , and control conditions at all time points . results indicate that an acute bout of kettlebell exercise is as effective as high intensity interval running at improving glucose tolerance in sedentary young men .

INTRODUCTION METHODS Participants Protocol Statistical Analysis RESULTS DISCUSSION

the incidence of type 2 diabetes ( t2d ) has increased significantly over the last several decades ( 24 , 42 ) , and management of the disease presents a significant health challenge . t2d is characterized by elevated fasting blood glucose and insulin insensitivity , and can lead to many health complications , including the development of cardiovascular disease ( 8) . exercise is recommended as a therapy for the prevention and management of t2d ( 26 ) . the american college of sports medicine ( acsm ) recommends 150 minutes / week of moderate intensity aerobic exercise conducted over 5 days / wk or more , along with moderate to vigorous - intensity resistance training at least 23 days / wk ( 8) . acutely and chronically , resistance and aerobic exercise have been shown to improve glucose uptake and insulin action ( 2 , 13 , 17 , 18 , 20 , 23 , 27 , 3133 ) . recent studies have examined the effects of high intensity interval training in an attempt to reduce the necessary time commitment while simultaneously improving health ( 3 , 7 , 21 ) . for example , sprint interval training ( sit ) in young volunteers results in significant improvements in muscle oxidative capacity ( 6 , 7 ) , exercise performance , fat oxidation ( 28 , 30 ) , endothelial function ( 29 ) , and glucose tolerance ( i.e. , ability to maintain normal blood glucose ) ( 3 ) . although use of sit induces significant physiological and functional improvements from only 23 minutes of exercise per workout , the total workout time commitment remains close to 30 minutes due to the prolonged rest period between each work interval . perhaps more important , sit employing the wingate protocol is conducted at supramaximal intensity and is potentially unsafe for sedentary middle - aged and older adults , especially if they have not undergone proper medical evaluation for determination of exercise safety ( 1 ) . ( 21 ) utilized a protocol consisting of ten 1-minute exercise intervals at a workload ~ 90100% vo2max interspersed with 60 recovery periods with a total workout time ( excluding warm - up and cool - down ) of ~ 20 minutes . use of this training strategy has been shown to significantly increase skeletal muscle glut-4 protein content ( 21 ) and lower 24-hour glucose in type 2 diabetics ( 21 ) . moreover , because of the 1-minute recovery periods , the metabolic cost , heart rate response , and rating of perceived exertion are similar to continuous steady state running at 70% vo2max ( 11 ) . in spite of these recent findings , hit may not be feasible for many individuals due to various reasons including , but not limited to , the costs associated with exercise testing , prescription , and supervision . it can differ considerably from traditional resistance training in that exercises may be performed at a significantly lower percentage of 1 repetition maximum , and with less recovery time between exercises . in many respects , kettlebell training is similar to circuit training and represents a hybrid of traditional endurance and resistance training . most kettlebell exercise routines require the use of only one or two kettlebells and can be performed in the home . in spite of the significant functional improvements associated with kettlebell training , the effects of kettlebell exercise on selected health markers such as glucose tolerance have yet to be investigated . the purpose of this preliminary study was to examine whether or not a single session of kettlebell exercise impacts glucose tolerance in healthy , but sedentary individuals . we hypothesized that immediately following a bout of kettlebell exercise , glucose tolerance would be significantly improved compared to control . we also hypothesized that a bout of kettlebell exercise would be as effective at improving glucose tolerance compared with a bout of hit similar in duration . inclusion criteria included healthy individuals who were sedentary ( exercised 2 day / week ) , had a body mass index < 30 , and had a normal blood glucose response during a two - hour oral glucose tolerance test ( ogtt ) ( i.e. , did not display impaired glucose tolerance or type 2 diabetes ) . exclusion criteria included individuals who were active ( exercised > 2 times / week ) , had a bmi 30 , took glucose or lipid lowering medication , smoked , or had any medical condition where exercise was contraindicated . six healthy sedentary males with a mean age of 23.8 ( 1.8 ) years and a mean bmi of 26.5 ( 0.5 ) kgm completed the study . following a screening ogtt and determination of maximal oxygen consumption ( vo2max ) , subjects underwent three different experimental conditions . each visit was separated by a minimum of 5 and maximum of 7 days . the independent variable was the experimental condition whereas the major dependent variables included blood glucose and insulin concentrations . blood was obtained from an ear stick before ( 0 minutes ) , 30 , 60 , 90 , and 120 minutes after rapidly consuming 75 gm of glucose ( 100 ml volume , glucose orange drink 075 , azer scientific , morgantown , pa ) . blood glucose was measured using a commercially available glucose meter ( accu - chek glucose meter , aviva , roche diagnostics , indianapolis , in ) that meets the international organization for standardization requirements ( 35 ) . any subject with a fasting blood glucose > 100 mgdl or with a two - hr ogtt blood glucose concentration > 140 mgdl was excluded from the study . subjects then underwent a graded exercise test on a treadmill for determination of vo2max . subjects were given a three - minute warm - up period ( 4.8 kmh at 0% grade ) prior to the test and a three - minute cool - down period ( 3.2 kmh at 0% grade ) following the test . was separated by ~ one week ( 57 days ) , and the order of conditions was randomized for each subject . subjects used either a 9 or 11.3-kg kettlebell for the workout depending on their ability . kettlebell ability was based on whether a subject could complete an exercise with an 11.3 kg kettlebell . if the subject deemed the kettlebell too heavy and was unable to complete the 15 repetitions , a 9-kg kettlebell was provided for that exercise . the workout consisted of seven exercises ( squat and press , chest press , one - arm kettlebell swing , bent over row , front squat , sit - up press - up , and kettlebell swing ) in sequence targeting the muscles of the legs , arms and core . fifteen repetitions per exercise were performed with 30 seconds of standing recovery between each set . after completion of the seven exercises , subjects were given one - minute standing recovery followed by a second circuit of the aforementioned exercises . the workout was preceded by a warm - up consisting of five sets of 20 kettlebell swings . heart rate was measured at the end of each exercise whereas rating of perceived exertion ( rpe ) was measured following each circuit . condition 3 consisted of high - intensity interval running ( hit ) on a motor driven treadmill with a 1:1 work - to - rest ratio . following a three - minute warm - up period at 4.8 kmh , subjects performed 10 one - minute intervals at a running speed corresponding to 90% vo2max . each exercise interval was interspersed with a one - minute active rest period ( 5.6 kmh ) . heart rate and rpe were recorded throughout the condition . to determine the effect of the hit and kettlebell workouts on blood glucose and lactate , blood was obtained via ear stick before and immediately following the exercise protocols . blood glucose was also measured at each draw using the portable glucose meter as previously described . glucose and insulin data were analyzed using a two - way ( group * time ) repeated measures anova ( sigma stat ) . the holm - sidak method was used for post - hoc multiple pair - wise comparisons . all data were normally distributed , and values are expressed as mean se ( n = 6 ) . inclusion criteria included healthy individuals who were sedentary ( exercised 2 day / week ) , had a body mass index < 30 , and had a normal blood glucose response during a two - hour oral glucose tolerance test ( ogtt ) ( i.e. , did not display impaired glucose tolerance or type 2 diabetes ) . exclusion criteria included individuals who were active ( exercised > 2 times / week ) , had a bmi 30 , took glucose or lipid lowering medication , smoked , or had any medical condition where exercise was contraindicated . six healthy sedentary males with a mean age of 23.8 ( 1.8 ) years and a mean bmi of 26.5 ( 0.5 ) kgm completed the study . following a screening ogtt and determination of maximal oxygen consumption ( vo2max ) , subjects underwent three different experimental conditions . each visit was separated by a minimum of 5 and maximum of 7 days . the independent variable was the experimental condition whereas the major dependent variables included blood glucose and insulin concentrations . blood was obtained from an ear stick before ( 0 minutes ) , 30 , 60 , 90 , and 120 minutes after rapidly consuming 75 gm of glucose ( 100 ml volume , glucose orange drink 075 , azer scientific , morgantown , pa ) . blood glucose was measured using a commercially available glucose meter ( accu - chek glucose meter , aviva , roche diagnostics , indianapolis , in ) that meets the international organization for standardization requirements ( 35 ) . any subject with a fasting blood glucose > 100 mgdl or with a two - hr ogtt blood glucose concentration > 140 mgdl was excluded from the study . after completing the ogtt , subjects were familiarized with the equipment and procedures to be used throughout the study . subjects then underwent a graded exercise test on a treadmill for determination of vo2max . subjects were given a three - minute warm - up period ( 4.8 kmh at 0% grade ) prior to the test and a three - minute cool - down period ( 3.2 kmh at 0% grade ) following the test . heart rhythm was also monitored using an electrocardiograph ( mac 550 , ge healthcare , united kingdom ) . was separated by ~ one week ( 57 days ) , and the order of conditions was randomized for each subject . kettlebell ability was based on whether a subject could complete an exercise with an 11.3 kg kettlebell . if the subject deemed the kettlebell too heavy and was unable to complete the 15 repetitions , a 9-kg kettlebell was provided for that exercise . the workout consisted of seven exercises ( squat and press , chest press , one - arm kettlebell swing , bent over row , front squat , sit - up press - up , and kettlebell swing ) in sequence targeting the muscles of the legs , arms and core . fifteen repetitions per exercise were performed with 30 seconds of standing recovery between each set . after completion of the seven exercises , subjects were given one - minute standing recovery followed by a second circuit of the aforementioned exercises . the workout was preceded by a warm - up consisting of five sets of 20 kettlebell swings . heart rate was measured at the end of each exercise whereas rating of perceived exertion ( rpe ) was measured following each circuit . condition 3 consisted of high - intensity interval running ( hit ) on a motor driven treadmill with a 1:1 work - to - rest ratio . following a three - minute warm - up period at 4.8 kmh , subjects performed 10 one - minute intervals at a running speed corresponding to 90% vo2max . each exercise interval was interspersed with a one - minute active rest period ( 5.6 kmh ) . to determine the effect of the hit and kettlebell workouts on blood glucose and lactate , approximately five minutes following each condition , 7ml of blood was withdrawn from the antecubital vein into edta lined vaccutainer tubes ( 16x100 lavendar , bd , franklin lakes , nj ) . blood glucose was also measured at each draw using the portable glucose meter as previously described . during a separate visit after completion of the three experimental conditions , a subset of five subjects came into the lab for measurement of the oxygen cost of the kettlebell workout . glucose and insulin data were analyzed using a two - way ( group * time ) repeated measures anova ( sigma stat ) . the holm - sidak method was used for post - hoc multiple pair - wise comparisons . all data were normally distributed , and values are expressed as mean se ( n = 6 ) . glucose values obtained during a two - hour ogtt after the three experimental conditions appear in figure 2 . there was a significant effect for time ( p < 0.001 ) and interaction ( p = .028 ) . there were no significant differences in blood glucose levels at 0 or 120 minutes between all three conditions . however , 60 minutes after glucose ingestion , blood glucose was significantly lower following hit ( p < 0.001 ) and kettlebell ( p = 0.003 ) exercise when compared to control . at the end of the 10 interval , figure 4b illustrates the mean heart rate response after each exercise throughout the entire kettlebell workout . the lowest heart rate was observed following the chest press exercise ( 2 and 9 ) , whereas the highest heart rate was observed after the one - arm kettlebell swing exercise ( 3 and 10 ) . during the hit workout , rpe gradually increased during the workout and averaged 15.7 ( 0.9 ) after the last interval . blood glucose values were obtained immediately before and after the kettlebell and hit workouts ( figure 5a ) . pre- and post - exercise glucose values were not significantly different from one another for either condition . moreover , there was no difference between the two exercise conditions . blood lactate was also measured before and immediately following the kettlebell and hit workouts ( figure 5b ) . there was no difference in resting blood lactate levels before the hit and kettlebell workouts . however , after exercise , blood lactate was significantly higher after the kettlebell workout ( 9.2 0.9 mmoll ) compared with hit ( 5.7 0.3 mmoll ) ( figure 5b ) . vo2 averaged 20.4 ( 2.2 ) mlkgmin ( 46.5% vo2max ) during the first circuit , and 21.1 ( 2.0 ) mlkgmin ( 48.1% vo2max ) during the second circuit . rer averaged 1.2 ( 0.04 ) and 1.1 ( 0.04 ) during the first and second circuit , respectively . in the present preliminary study , we investigated the effect of a single bout of kettlebell exercise on glucose tolerance in sedentary , but healthy male subjects . the kettlebell and hit workouts ( including warm - up and cool down ) were similar in duration and averaged ~25 and 26 minutes , respectively . we found that immediately following a single session of kettlebell or hit exercise , glucose tolerance during an ogtt was significantly improved at 60 minutes with no concomitant change in insulin concentration . in addition , there was no difference between the two modes of exercise . these findings indicate that kettlebell exercise may serve as an alternative training strategy to hit for improving glucose tolerance . the mechanisms for improved glucose clearance post - exercise are complex and have not been completely elucidated . immediately post exercise , increased glucose clearance can be due to both insulin - dependent and insulin - independent pathways . in a rat study by hamada et al . ( 12 ) , insulin - independent glucose uptake was increased when measured 40 minutes after a single 60-minute session of treadmill exercise and was associated with increased ampk threonine phosphorylation . however , by 85-minutes post exercise , this ampk effect had dissipated and the increased glucose uptake that persisted was likely due to insulin - dependent mechanisms . in human skeletal muscle post - exercise , akt phosphorylation has been shown to increase ( 37 ) or not change ( 36 , 39 ) during a euglycemic clamp with physiological insulin levels . in several other acute exercise studies , no changes were observed in insulin receptor binding or phosphorylation , sub - maximal insulin stimulation of the tyrosine phosphorylation of irs-1 or sub - maximal insulin - stimulation of p13k ( 4 , 14 , 3941 ) . regardless of the mechanism for the improved glucose uptake , glut-4 protein is known to increase after aerobic ( 9 , 16 , 32 , 34 ) , resistance ( 15 , 19 ) , and high - intensity interval ( 22 ) exercise . results from these previous studies suggest that glut-4 is also increased after kettlebell exercise , though definitive data are currently lacking . although the two exercise conditions were qualitatively different from one another , the heart rate response was similar . in addition , the end - workout perceived exertion was also similar , with rpe averaging ~16 and 15 for the hit and kettlebell workout , respectively . in spite of these similarities , however , the blood lactate concentration was significantly higher following the kettlebell workout ( 9.2 mmoll ) when compared to the hit workout ( 5.7 mmoll ) . exercise order was constructed to alternate between different muscle groups during the workout , with each exercise lasting approximately 30 seconds followed by 30 seconds rest . in contrast , during the hit workout , the same musculature was recruited during each interval , and the work interval was 60-sec in duration . however , our previous work indicates that oxygen consumption for the workout employed reaches approximately 70% of vo2max ( 11 ) . ( 10 ) reported that the oxygen cost of kettlebell exercise was ~ 34.3 ( 5.7 ) mlkgmin . in this latter study , subjects employed a significantly heavier kettlebell and were required to complete as many two - handed swings as possible in 12 minutes ( 10 ) . the contrasting findings are therefore likely due to the different weight and workout characteristics employed in the two studies . utilizing a 10-exercise , 3 circuit design ( 30 seconds exercise , 15-sec recovery ) , wilmore et al . although blood lactate following the kettlebell workout approached that recorded at the end of the graded maximal exercise test and was significantly higher than that observed after hit exercise , all subjects were able to complete this workout . moreover , the subject s perception of effort at the end of kettlebell exercise was similar ( ~ 15 , hard ) to the hit workout . for example , the work - to - rest ratio during interval exercise is known to affect heart rate , lactate , and rpe responses ( 11 ) . thus , when prescribing kettlebell exercise to higher risk individuals , it may be desirable to lower the weight and/or increase the recovery period between exercises to attenuate these variables . high intensity interval training has been studied extensively the last several years , and has been proposed as a time - efficient alternative to traditional aerobic training for the improvement of metabolic and cardiovascular health . , availability of facilities , time and cost of driving , weather , etc . ) may deter many individuals from commencing a hit exercise program . in contrast , the cost associated with kettlebell exercise is quite low , the exercises can easily be learned from web - based videos , and the workouts can be performed in the home or office . limitations to the current study include the small sample size ( n = 6 ) , the use of sedentary but healthy volunteers , exclusion of female subjects , age of the volunteers , as well as the use of only one exercise workout . regardless , this preliminary study suggests that kettlebell exercise acutely improves glucose tolerance in young sedentary subjects , and that the changes are similar to that observed following a bout of high - intensity interval running . thus , kettlebell exercise might serve as a suitable alternative to hit or traditional aerobic training . however , future studies are required to determine the acute and long - term effectiveness of kettlebell training at improving metabolic health in pre - diabetic and diabetic individuals . moreover , because individuals often cite lack of time as an excuse for not exercising regularly , further work is required to determine the minimal kettlebell exercise dose to improve glucose tolerance .

the incidence of type 2 diabetes ( t2d ) has increased significantly over the last several decades ( 24 , 42 ) , and management of the disease presents a significant health challenge . t2d is characterized by elevated fasting blood glucose and insulin insensitivity , and can lead to many health complications , including the development of cardiovascular disease ( 8) . exercise is recommended as a therapy for the prevention and management of t2d ( 26 ) . the american college of sports medicine ( acsm ) recommends 150 minutes / week of moderate intensity aerobic exercise conducted over 5 days / wk or more , along with moderate to vigorous - intensity resistance training at least 23 days / wk ( 8) . acutely and chronically , resistance and aerobic exercise have been shown to improve glucose uptake and insulin action ( 2 , 13 , 17 , 18 , 20 , 23 , 27 , 3133 ) . in spite of the known health benefits of exercise , most americans do not meet the recommended amounts of exercise , in part due to a lack of time ( 5 ) . recent studies have examined the effects of high intensity interval training in an attempt to reduce the necessary time commitment while simultaneously improving health ( 3 , 7 , 21 ) . for example , sprint interval training ( sit ) in young volunteers results in significant improvements in muscle oxidative capacity ( 6 , 7 ) , exercise performance , fat oxidation ( 28 , 30 ) , endothelial function ( 29 ) , and glucose tolerance ( i.e. although use of sit induces significant physiological and functional improvements from only 23 minutes of exercise per workout , the total workout time commitment remains close to 30 minutes due to the prolonged rest period between each work interval . thus , in terms of time commitment , sit is no more effective than traditional aerobic training . perhaps more important , sit employing the wingate protocol is conducted at supramaximal intensity and is potentially unsafe for sedentary middle - aged and older adults , especially if they have not undergone proper medical evaluation for determination of exercise safety ( 1 ) . ( 21 ) utilized a protocol consisting of ten 1-minute exercise intervals at a workload ~ 90100% vo2max interspersed with 60 recovery periods with a total workout time ( excluding warm - up and cool - down ) of ~ 20 minutes . use of this training strategy has been shown to significantly increase skeletal muscle glut-4 protein content ( 21 ) and lower 24-hour glucose in type 2 diabetics ( 21 ) . moreover , because of the 1-minute recovery periods , the metabolic cost , heart rate response , and rating of perceived exertion are similar to continuous steady state running at 70% vo2max ( 11 ) . in spite of these recent findings , hit may not be feasible for many individuals due to various reasons including , but not limited to , the costs associated with exercise testing , prescription , and supervision . kettlebell training has existed for several years and is known to result in significant improvements in muscular strength ( e.g. it can differ considerably from traditional resistance training in that exercises may be performed at a significantly lower percentage of 1 repetition maximum , and with less recovery time between exercises . in many respects , kettlebell training is similar to circuit training and represents a hybrid of traditional endurance and resistance training . most kettlebell exercise routines require the use of only one or two kettlebells and can be performed in the home . it is considered a more cost - effective means of exercise , as it does not require expensive equipment or monthly fitness membership . in spite of the significant functional improvements associated with kettlebell training , the effects of kettlebell exercise on selected health markers such as glucose tolerance have yet to be investigated . the purpose of this preliminary study was to examine whether or not a single session of kettlebell exercise impacts glucose tolerance in healthy , but sedentary individuals . a secondary objective was to evaluate the average oxygen cost of the kettlebell workout employed in this study . we hypothesized that immediately following a bout of kettlebell exercise , glucose tolerance would be significantly improved compared to control . we also hypothesized that a bout of kettlebell exercise would be as effective at improving glucose tolerance compared with a bout of hit similar in duration . inclusion criteria included healthy individuals who were sedentary ( exercised 2 day / week ) , had a body mass index < 30 , and had a normal blood glucose response during a two - hour oral glucose tolerance test ( ogtt ) ( i.e. six healthy sedentary males with a mean age of 23.8 ( 1.8 ) years and a mean bmi of 26.5 ( 0.5 ) kgm completed the study . following a screening ogtt and determination of maximal oxygen consumption ( vo2max ) , subjects underwent three different experimental conditions . subjects were asked to refrain from any vigorous physical activity and to maintain their normal diet throughout the study . the independent variable was the experimental condition whereas the major dependent variables included blood glucose and insulin concentrations . blood was obtained from an ear stick before ( 0 minutes ) , 30 , 60 , 90 , and 120 minutes after rapidly consuming 75 gm of glucose ( 100 ml volume , glucose orange drink 075 , azer scientific , morgantown , pa ) . blood glucose was measured using a commercially available glucose meter ( accu - chek glucose meter , aviva , roche diagnostics , indianapolis , in ) that meets the international organization for standardization requirements ( 35 ) . any subject with a fasting blood glucose > 100 mgdl or with a two - hr ogtt blood glucose concentration > 140 mgdl was excluded from the study . after completing the ogtt , subjects were familiarized with the equipment and procedures to be used throughout the study . on the second visit , body composition was assessed using volume plethysmography ( bodpod , cosmed , chicago , il ) . during the treadmill test , subjects ran at an initial self - selected speed between 8.0 12.9 kmh at 0% grade . the grade remained constant throughout the test , but the speed was increased 0.8 kmh every two minutes . subjects were given a three - minute warm - up period ( 4.8 kmh at 0% grade ) prior to the test and a three - minute cool - down period ( 3.2 kmh at 0% grade ) following the test . prior to the test , an ear stick was performed to obtain baseline blood lactate ( lactate plus , nova biomedical , waltham , ma ) levels . heart rate was measured at each stage using a polar heart rate monitor ( polar usa , lake success , ny ) via a chest strap , whereas perceived exertion was measured using the standard 620 borg scale . was separated by ~ one week ( 57 days ) , and the order of conditions was randomized for each subject . condition 1 was a control visit ( quiet sitting for 2 hours during the ogtt ) where no prior exercise was performed . kettlebell ability was based on whether a subject could complete an exercise with an 11.3 kg kettlebell . if the subject deemed the kettlebell too heavy and was unable to complete the 15 repetitions , a 9-kg kettlebell was provided for that exercise . the workout consisted of seven exercises ( squat and press , chest press , one - arm kettlebell swing , bent over row , front squat , sit - up press - up , and kettlebell swing ) in sequence targeting the muscles of the legs , arms and core . after completion of the seven exercises , subjects were given one - minute standing recovery followed by a second circuit of the aforementioned exercises . the workout was preceded by a warm - up consisting of five sets of 20 kettlebell swings . heart rate was measured at the end of each exercise whereas rating of perceived exertion ( rpe ) was measured following each circuit . condition 3 consisted of high - intensity interval running ( hit ) on a motor driven treadmill with a 1:1 work - to - rest ratio . following a three - minute warm - up period at 4.8 kmh , subjects performed 10 one - minute intervals at a running speed corresponding to 90% vo2max . to determine the effect of the hit and kettlebell workouts on blood glucose and lactate , blood was obtained via ear stick before and immediately following the exercise protocols . during a separate visit after completion of the three experimental conditions , a subset of five subjects came into the lab for measurement of the oxygen cost of the kettlebell workout . subjects performed the same kettlebell workout previously described while oxygen consumption and respiratory exchange ratio ( rer ) was continuously monitored ( trueone , parvo medics , sandy , ut ) . glucose and insulin data were analyzed using a two - way ( group * time ) repeated measures anova ( sigma stat ) . the holm - sidak method was used for post - hoc multiple pair - wise comparisons . inclusion criteria included healthy individuals who were sedentary ( exercised 2 day / week ) , had a body mass index < 30 , and had a normal blood glucose response during a two - hour oral glucose tolerance test ( ogtt ) ( i.e. six healthy sedentary males with a mean age of 23.8 ( 1.8 ) years and a mean bmi of 26.5 ( 0.5 ) kgm completed the study . following a screening ogtt and determination of maximal oxygen consumption ( vo2max ) , subjects underwent three different experimental conditions . all procedures except the graded treadmill test subjects were asked to refrain from any vigorous physical activity and to maintain their normal diet throughout the study . the independent variable was the experimental condition whereas the major dependent variables included blood glucose and insulin concentrations . blood was obtained from an ear stick before ( 0 minutes ) , 30 , 60 , 90 , and 120 minutes after rapidly consuming 75 gm of glucose ( 100 ml volume , glucose orange drink 075 , azer scientific , morgantown , pa ) . blood glucose was measured using a commercially available glucose meter ( accu - chek glucose meter , aviva , roche diagnostics , indianapolis , in ) that meets the international organization for standardization requirements ( 35 ) . any subject with a fasting blood glucose > 100 mgdl or with a two - hr ogtt blood glucose concentration > 140 mgdl was excluded from the study . after completing the ogtt , subjects were familiarized with the equipment and procedures to be used throughout the study . on the second visit , body composition was assessed using volume plethysmography ( bodpod , cosmed , chicago , il ) . during the treadmill test , subjects ran at an initial self - selected speed between 8.0 12.9 kmh at 0% grade . the grade remained constant throughout the test , but the speed was increased 0.8 kmh every two minutes . subjects were given a three - minute warm - up period ( 4.8 kmh at 0% grade ) prior to the test and a three - minute cool - down period ( 3.2 kmh at 0% grade ) following the test . prior to the test , an ear stick was performed to obtain baseline blood lactate ( lactate plus , nova biomedical , waltham , ma ) levels . heart rate was measured at each stage using a polar heart rate monitor ( polar usa , lake success , ny ) via a chest strap , whereas perceived exertion was measured using the standard 620 borg scale . was separated by ~ one week ( 57 days ) , and the order of conditions was randomized for each subject . condition 1 was a control visit ( quiet sitting for 2 hours during the ogtt ) where no prior exercise was performed . if the subject deemed the kettlebell too heavy and was unable to complete the 15 repetitions , a 9-kg kettlebell was provided for that exercise . the workout consisted of seven exercises ( squat and press , chest press , one - arm kettlebell swing , bent over row , front squat , sit - up press - up , and kettlebell swing ) in sequence targeting the muscles of the legs , arms and core . after completion of the seven exercises , subjects were given one - minute standing recovery followed by a second circuit of the aforementioned exercises . heart rate was measured at the end of each exercise whereas rating of perceived exertion ( rpe ) was measured following each circuit . condition 3 consisted of high - intensity interval running ( hit ) on a motor driven treadmill with a 1:1 work - to - rest ratio . following a three - minute warm - up period at 4.8 kmh , subjects performed 10 one - minute intervals at a running speed corresponding to 90% vo2max . to determine the effect of the hit and kettlebell workouts on blood glucose and lactate , approximately five minutes following each condition , 7ml of blood was withdrawn from the antecubital vein into edta lined vaccutainer tubes ( 16x100 lavendar , bd , franklin lakes , nj ) . during a separate visit after completion of the three experimental conditions , a subset of five subjects came into the lab for measurement of the oxygen cost of the kettlebell workout . subjects performed the same kettlebell workout previously described while oxygen consumption and respiratory exchange ratio ( rer ) was continuously monitored ( trueone , parvo medics , sandy , ut ) . the holm - sidak method was used for post - hoc multiple pair - wise comparisons . a second subject was eliminated from the study because of nausea following ingestion of the glucose drink . values obtained for blood lactate ( > 8.0 mmoll ) or rer ( 1.15 ) at the end of the test indicated that all subjects achieved vo2max . glucose values obtained during a two - hour ogtt after the three experimental conditions appear in figure 2 . there was a significant effect for time ( p < 0.001 ) and interaction ( p = .028 ) . however , 60 minutes after glucose ingestion , blood glucose was significantly lower following hit ( p < 0.001 ) and kettlebell ( p = 0.003 ) exercise when compared to control . figure 4a illustrates the mean heart rate response following each interval throughout the entire hit workout . at the end of the 10 interval , figure 4b illustrates the mean heart rate response after each exercise throughout the entire kettlebell workout . the lowest heart rate was observed following the chest press exercise ( 2 and 9 ) , whereas the highest heart rate was observed after the one - arm kettlebell swing exercise ( 3 and 10 ) . heart rate averaged ~ 81% and ~ 84% of hrmax after the 1 and 2 circuit , respectively . during the hit workout , rpe gradually increased during the workout and averaged 15.7 ( 0.9 ) after the last interval . during the kettlebell workout , rpe averaged 12.7 ( 1.4 ) and 14.7 ( 1.7 ) after the first and second circuits , respectively . pre- and post - exercise glucose values were not significantly different from one another for either condition . however , after exercise , blood lactate was significantly higher after the kettlebell workout ( 9.2 0.9 mmoll ) compared with hit ( 5.7 0.3 mmoll ) ( figure 5b ) . the blood lactate values were ~ 86% ( kettlebell ) and ~ 53% of that reached during the vo2max test . the oxygen cost of the kettlebell workout was measured in a subset of five subjects . vo2 averaged 20.4 ( 2.2 ) mlkgmin ( 46.5% vo2max ) during the first circuit , and 21.1 ( 2.0 ) mlkgmin ( 48.1% vo2max ) during the second circuit . rer averaged 1.2 ( 0.04 ) and 1.1 ( 0.04 ) during the first and second circuit , respectively . in the present preliminary study , we investigated the effect of a single bout of kettlebell exercise on glucose tolerance in sedentary , but healthy male subjects . we found that immediately following a single session of kettlebell or hit exercise , glucose tolerance during an ogtt was significantly improved at 60 minutes with no concomitant change in insulin concentration . these findings indicate that kettlebell exercise may serve as an alternative training strategy to hit for improving glucose tolerance . the mechanisms for improved glucose clearance post - exercise are complex and have not been completely elucidated . immediately post exercise , increased glucose clearance can be due to both insulin - dependent and insulin - independent pathways . ( 12 ) , insulin - independent glucose uptake was increased when measured 40 minutes after a single 60-minute session of treadmill exercise and was associated with increased ampk threonine phosphorylation . however , by 85-minutes post exercise , this ampk effect had dissipated and the increased glucose uptake that persisted was likely due to insulin - dependent mechanisms . in human skeletal muscle post - exercise , akt phosphorylation has been shown to increase ( 37 ) or not change ( 36 , 39 ) during a euglycemic clamp with physiological insulin levels . in several other acute exercise studies , no changes were observed in insulin receptor binding or phosphorylation , sub - maximal insulin stimulation of the tyrosine phosphorylation of irs-1 or sub - maximal insulin - stimulation of p13k ( 4 , 14 , 3941 ) . regardless of the mechanism for the improved glucose uptake , glut-4 protein is known to increase after aerobic ( 9 , 16 , 32 , 34 ) , resistance ( 15 , 19 ) , and high - intensity interval ( 22 ) exercise . results from these previous studies suggest that glut-4 is also increased after kettlebell exercise , though definitive data are currently lacking . in spite of these similarities , however , the blood lactate concentration was significantly higher following the kettlebell workout ( 9.2 mmoll ) when compared to the hit workout ( 5.7 mmoll ) . we hypothesize that the increased blood lactate concentration after the kettlebell workout is due to a higher reliance on glycolysis . we did not measure the oxygen cost of the hit workout in the present study . during the kettlebell workout in the current study , vo2 averaged ~ 21.1 ( 4.9 ) mlkgmin , or 48% of vo2max . although blood lactate following the kettlebell workout approached that recorded at the end of the graded maximal exercise test and was significantly higher than that observed after hit exercise , all subjects were able to complete this workout . moreover , the subject s perception of effort at the end of kettlebell exercise was similar ( ~ 15 , hard ) to the hit workout . for example , the work - to - rest ratio during interval exercise is known to affect heart rate , lactate , and rpe responses ( 11 ) . thus , when prescribing kettlebell exercise to higher risk individuals , it may be desirable to lower the weight and/or increase the recovery period between exercises to attenuate these variables . high intensity interval training has been studied extensively the last several years , and has been proposed as a time - efficient alternative to traditional aerobic training for the improvement of metabolic and cardiovascular health . in spite of the established health benefits , incorporation of a safe and effective hit program into the daily routine of lay individuals will require laboratory testing and professional consultation . in contrast , the cost associated with kettlebell exercise is quite low , the exercises can easily be learned from web - based videos , and the workouts can be performed in the home or office . limitations to the current study include the small sample size ( n = 6 ) , the use of sedentary but healthy volunteers , exclusion of female subjects , age of the volunteers , as well as the use of only one exercise workout . regardless , this preliminary study suggests that kettlebell exercise acutely improves glucose tolerance in young sedentary subjects , and that the changes are similar to that observed following a bout of high - intensity interval running . however , future studies are required to determine the acute and long - term effectiveness of kettlebell training at improving metabolic health in pre - diabetic and diabetic individuals .

image bioinformatics as a field is characterized by an odd mixture of tremendous advances in its computational statistics aspects while suffering from a persistent resistance to their use by biomedical domain experts . the image analysis platform with the deepest domain penetration in the research community is probably national institutes of health ( nih 's ) imagej in large part because of its free open source and pluggable implementation . over the years , an extensive community of image analysis developers and researchers has developed a comprehensive environment for image analysis software development using java . the development cycle can be roughly described as follows : when a promising new algorithm is identified , it might be first prototyped in a specialized environment such as mathwork 's matlab , but to reach a broader non - programming audience , it is then laboriously ported as an imagej plugin . illustrating the domain penetration challenge , a regular stream of new and updated plugins feeds a comprehensive repository that now has several hundred entries ( http://rsbweb.nih.gov/ij/plugins/ ) . however , a visit to the laboratory will show that those who could benefit from an add - on are typically not aware of them . even more striking is the situation that the few researchers using image analysis , who know how to find and install plugins , often also need image analysis applications that are slight modifications or combinations of existing functionalities . paradoxically , the computational statisticians with the quantitative expertise to develop those modifications often do not have the substantial software development skills needed to deliver the novel application as a stand - alone application or as a plugin module . a common solution to for example , the quantitative image analysis of estrogen receptor , progesterone receptor , and more generically , the ki-67 immunohistochemistry staining results in breast cancer tissue sections can be conveniently performed by submitting the image to a public web service . users benefit from improvements in the code automatically and no application needs to installed , therefore overcoming concerns with compromising privacy or degrading the performance of existing applications in the client machine . submitting to a remote service exposes the image to a third party outside the reach of institutional it ; updates in the web service may cause results not to be reproducible and the server side application is typically not exposed to public scrutiny , limiting the opportunities for other quantitative researchers to advance or even fully understand the underlying algorithms ; bioinformatics web services are also notorious for relatively short lifetimes , reflecting a sustainability model that can only be met in the long term by institutional commitment or by commercial products . a third solution might be to develop web services that deliver the software rather than process the data , the software - as - a - service ( saas ) model . the imagejs webapp ecosystem described in this report advances along those exact lines without crossing the lines set by requirements of privacy , scalability , reproducibility , and sustainable deployment associated with the biomedical environment . it also pays particular attention to the need for producing and delivering reproducible research , a topic that is taking center stage as bioinformatics increasingly seeks to deliver data - intensive solutions with translational usage . specifically , if an image analysis result can be retrieved directly from the published image , with both the data and the analytical environment that generated the result , then one could indeed claim to be reporting reproducible research . accordingly , imagejs seeks to deliver image analysis results meeting the gold standard in that report 's description of the spectrum of reproducibility [ figure 1 ] . imagejs migrates through script tag loading to the browser ( by pointing a script.src to http://imagejs.github.com/imagejs.js ) , where it creates an object with a method , imagejs . loadmodule , that will import and register additional components onto imagejs.modules , while keeping data meant to be shared between modules in imagejs.data . the corresponding uml representation is appended to the lower left side of the browser box . the solid lines represent programmatic exchanges and dashed line represents an exchange that requires user intervention , such as the loading or saving an image / analysis the possibility of approaching software development as an open organic process , which is explored by the imagejs webapp environment described here , has been proposed before as a requirement for medicine 2.0 . as discussed in that report , such an approach would blur the separation between software development and software deployment , turning coding into a conduit for workflow assembly that is more akin to social networking mechanisms . this is a departure from conventional bioinformatics infrastructure that can also be construed as a response to the mounting knowledge re - engineering bottleneck . it would provide a beyond the data deluge solution to image analysis where the computation is pushed to where the data is , rather than the customary approach of shuffling the data among a myriad of analytical services . imagejs was entirely developed in javascript ( ecmascript 5th ed . ) , the assembler language of the web . to illustrate the distributed and collaborative nature of the proposed webapp ecosystem , its component modules were developed in multiple versioned code hosting services , github ( http://github.com ) and google code ( http://googlecode.com ) . it can also be started directly from http://imagejs.github.com/ , which serves code hosted at https://github.com/imagejs/imagejs.github.com . the segmentation , feature extraction , and filtering modules were developed , and are hosted at http://imagejs.googlecode.com/. the list of modules can be accessed directly through http://module.imagejs.googlecode.com/git/ , and the development history can be inspected at http://code.google.com/p/imagejs/source/list?repo=module . in some examples , a web read - write resource is used to provide persistent image storage as a web service . a number of alternatives exist ; in this report , we used webrw http - rest api , which is supported by two independent implementations , both open source : one is written in nodejs ( https://github.com/jonasalmeida/webrw ) and another in php ( https://github.com/ebadedude/webrw ) . important note : although the core components and most modules will work in different web browsers , this study makes use of recent html5 features such as the file api ( http://www.w3.org/tr/fileapi/ ) that are still being adopted . to avoid problems with the syntax or support of recent developments in html5 , the reader is advised to use the latest version of google chrome ( version 18 at the time of this reporting ) . the imagejs browser based computational ecosystem is loaded by pointing it ( preferably using google chrome , see availability in materials and methods ) to http://imagejs.github.com . this action will cause imagejs 's document object , imagejs , to migrate to the web browser . a quick inspection of this object will reveal that it includes only a handful of methods , including the one that will load external code , loadmodule . a closer inspection of this method will show that a ) it imports external code by pointing a browser 's document object model ( dom ) script tag to the url of the external code ( module ) and b ) it registers the imported module in imagejs.module(url ) . as is the rest of this section , this inspection is also provided as a webcast video , publicly available at http://www.youtube.com/watch?v=qbkbgb4eche . the features of the imagejs environment as a vehicle to deliver computation were studied by developing and deploying an image analysis workflow that included standard modules for feature segmentation , analysis , and filtering . each of these modules was configured not only to deliver a desirable image analysis functionality and in the process test if the browser is a suitable platform to do so but also to test different models of dependency between modules . the goal of testing different models of articulation between analytical modules is to explore the balance between flexibility and robustness of browser - based computational ecosystems as platforms for collaborative software development . each module represents an independent development exercise , hosted and versioned in its own externally hosted service . as stated in the materials and methods section , to reinforce the perception of independent development , the multiple modules and imagejs core code are scattered between distinct versioned hosting services , namely , github and google code . examples of interface triggers for loading of modules illustrated in this section : menu driven , dependency driven , and programmatically associated with action by another module . the analytical workflow can therefore be assembled by loading modules in response to actions by the user or it can be initiated by a particular result of the analysis the image analysis procedure developed starts with the selection by the user of a chromomarker . a chromomarker , rgbmarker , is defined as the three - element vector with the intensities in the red , green , and blue channels that one uses to retrieve image features with similar chromometric characteristics . to this purpose , the user selects a pixel in the image , i , of size n m as a marker , with [ r , g , b ] intensities , and sets a weighted threshold , t , for a dissimilarity metric , dist , that will be resolved for all pixels in the image , each with its own intensity values [ r , g , b ] : the first module , chromomarkers , will segment all pixels in the image that are at a distance smaller than t. segmentetpixelcoordinates = [ i , j]|dist([r , g , b](i , j ) ) < t ( 2 ) both operations , distance calculation ( equation 1 ) and segmentation ( equation 2 ) , represent a first test of the browser as an efficient environment for image processing . the javascript engine of modern web browsers includes support for map and reduce operations , which we recently found to offer an efficient solution to the parallelization / vectorization of code execution in the context of sequence analysis . for example , the n m distance matrix , d , defined by equation 1 , can be resolved efficiently by nesting two map operations on the image rgb intensities , i : d = i.map(function(x , i){return x.map(function(y , j){returni[r , g , b](i , j ) } ) } ) ( 3 ) the efficiency of this implementation in imagejs relies on the javascript compiler mapping the explicit parallelization of the code to local opportunities for distribution . for example , by possibly using the machine 's graphics processing units ( gpus ) . as can be verified by operating imagejs ( or by viewing the accompanying webcast at http://www.youtube.com/watch?v=qbkbgb4eche ) , chromomarkers will display the edges of segmented image superimposed on the original image . the edge detection is performed by another mapping operation which is worth inspecting in the native javascript syntax to recognize the nested mapping pattern used in equation 3 , and is highlighted below in the code for equation 4 . this time , the nested mapping is being used to inspect the neighborhood of each pixel to find out if it corresponds to a segmented pixel with a non - segmented non - pixel , in which case it is classified as an edge : as can be verified by using imagejs or by viewing the accompanying webcast , the image segmentation operations are executed in real time ( under 1 or 2 sec ) , on par with other image analysis environments . feature extraction represents a tougher challenge . here , the goal is to identify which sets of segmented pixels define a contiguous feature . this module 's interface now separates ( they are triggered by distinct events ) feature analysis operations that can be performed in real time , such as the determination of segmentation density or the ratio between rgb intensities between segmented and non - segmented regions , from those that actually require the extraction of features , such as counting them . the description of this feature extraction procedure provides the opportunity to demonstrate how code can be invoked in a publication without concern that subsequent updates or renaming will cause this information not to be available . for example , at the time of this writing , the code for feature extraction can be found in line # 171 of version 4ec7931d17ccc17d27e724d51ae0 of jmat.js , which can be following that link will not only return the code used , but also expose comments made by co - developers . it is again worth noting that although the imagejs core is hosted and versioned at imagejs.github.com , this procedure happens to be hosted and versioned by a distinct environment , this time at google . both hosting services , which is to say , the hosting services resolved by the links , are likely to last longer ( forever ) than the interest or even the memory of its authors . the ability to simultaneously host versioned development and serve executable javascript code by exactly the same mechanism is a unique feature of the web environment . in the previous section , the feature extraction procedure was described by reference to the specific version and line of code deployment at the time of this writing . the ability to reference specific versions of the code therefore extends to the ability to execute them as well . this feature is enhanced by imagejs by allowing the submission of modules by passing them as arguments to the url call . for example , http://imagejs.github.com/?https://raw.github.com/imagejs/imagejs.github.com/27b596ed08d41e79c6c96e86f0318694ca98cc9e/mainmenu.js and http://module.imagejs.googlecode.com/git-history/ef105347cc13dcb24cdfae621522b5891ff11ab7/mathbiol.chromomarkers.js and http://module.imagejs.googlecode.com/git-history/ef105347cc13dcb24cdfae621522b5891ff11ab7/mathbiol.countshapes.js specifies a very particular combination of versions of the multiple modules involved in the assembly of imagejs . this is of course not a convenient url to exchange , so a shorter reference could be generated , for example , http://bit.ly/h3hvbz . it is worth repeating the note that none of the resources used to enable the assembly of a specific version of the imagejs tool depends on the commitment of the authors of this report to maintain those resources . the imagejs computational environment will natively allow for three mechanisms to incorporate code generated by others . the easiest to distribute is to simply add the address of the modules to the url of imagejs core application , as illustrated above . a more interactive possibility is to use an input box as exemplified by the main menu / load option . the filtering of segmented images offers an illustration of the usefulness of this mechanism in facilitating the interaction with researchers focused on the computational statistics aspects of the analysis . this module , mathbiol.filtershapes , is strictly dedicated to specifying a number of matrix manipulation procedures erode and round at the time of this writing . the association of these procedures with the user interface buttons that trigger them is dealt by the feature extraction module . adding a new filtering procedure to this object we found this style of module development to be particularly appealing to the biostatistics minded researcher because it does not require web programming skills beyond a basic understanding of javascript syntax , which is reminiscent of the c language . in summary , the primary goal of the modules described in this report is to illustrate how the image analysis can be orchestrated to involve multiple contributions , with different dependencies , using different styles , different modes of interaction with the user , hosted in multiple locations , while delivering reproducible results available in the pervasive web browser platform . figure 2 summarizes the orchestration of modules described here , highlighting the salient features and dependencies they were designed to illustrate . the conventional approach of manually loading images from the local file system , by file select or by drag and drop , is supported . manually saving the image and its analysis results back to the files system is also supported and consists of saving the contents of imagejs.data [ figure 1 ] as a json structure . this behavior relies on a relatively new development in web computing standards , the file api ( http://www.w3.org/tr/fileapi ) . another possibility that is particularly useful in collaborative and distributed environments is to rely on the read - write capabilities anticipated for web 3.0 apps . in a nutshell , this is a cloud computing option that treats web services as both remote storage and remote devices , allowing for exchange of data simply by reference . one of the modules , countshapes , illustrates this functionality by using the webrw service described in the materials and methods section . this was adopted as a generic solution of reading and writing back to the web such that we would not have to choose among a slew of excellent commercial resources such as dropbox.com , amazon 's s3 , or google 's fusion tables , which would have required user authentication . regardless of the web read - write cloud service chosen , the attraction of this option is that programmatically , the web is closer to the web browser that to the browser 's machine own file system [ figure 1 ] . accordingly , the same module loading mechanism described in the previous sections will now be used to deal with and saving and loading of data . as an example , see either https://bitly.com/withdata or point a qr reader to the qr code in figure 3 http://bit.ly/withdata.qrcode ; both link to the same data with analysis , as depicted in figures 3 and 4 . quick response ( qr ) code , generated automatically by http://bit.ly/withdata.qrcode for the link http://bit.ly/withdata , which in turn points to http://imagejs.github.com/?http://module . js and http://165.225.128.64/?doc=uid5716226333752275 and http:// module.imagejs.googlecode.com/git/mathbiol.showdata.js , which corresponds to a particular image analysis result that is assembled , re - analyzed , reproducing the display depicted in figure 4 image analysis corresponding to http://bit.ly/withdata . see figure 3 for equivalent qr code for optical reading by tablet devices the proposition that image js0 is particularly adequate to the development of very specific image analysis applications was assessed by developing a new module to assist with the determination of cellular proliferation index from ki67-labeled transmission microscopy images . as proposed , this was pursued by building not only on top of the imagejs abstraction [ figure 1 ] but also integrated with one of the generic modules described earlier , chromomarkers . as a consequence , this exercise was conveniently reduced to the writing of a few image analysis filters that rely on the chromomarkers segmentation utilities to select segmentation thresholds . the ki67 index , a cellular proliferation index , is the ratio of nuclei undergoing division , often estimated from transmission microscopy images of tissue treated with a generic nuclear stain , such as hematoxylin , and an antibody that targets ki67 , a protein present only in the active stages of the cell cycle . to address the vagaries of manual estimation of proliferation from such images , the ki67 imagejs module was designed to assist with the calculation and , more importantly , to report the final value with the graphic display of the corresponding segmentations . a snapshot of this application is presented in figure 5 , and can be invoked by a short link such as http://bit.ly/ki67js or http://uab.mathbiol.org/ki67 in a modern web browser , preferably google chrome . snapshot of the result of using imagejs 's ki67 module to determine proliferation index in a labeled glioblastoma sample imaged by transmission microscopy . a webcast illustrating its usage is available at http://www.youtube.com/watch?v=ncpngrxwwdq and is also provided with the help option of this imagejs application the image analysis procedure developed starts with the selection by the user of a chromomarker . a chromomarker , rgbmarker , is defined as the three - element vector with the intensities in the red , green , and blue channels that one uses to retrieve image features with similar chromometric characteristics . to this purpose , the user selects a pixel in the image , i , of size n m as a marker , with [ r , g , b ] intensities , and sets a weighted threshold , t , for a dissimilarity metric , dist , that will be resolved for all pixels in the image , each with its own intensity values [ r , g , b ] : the first module , chromomarkers , will segment all pixels in the image that are at a distance smaller than t. segmentetpixelcoordinates = [ i , j]|dist([r , g , b](i , j ) ) < t ( 2 ) both operations , distance calculation ( equation 1 ) and segmentation ( equation 2 ) , represent a first test of the browser as an efficient environment for image processing . the javascript engine of modern web browsers includes support for map and reduce operations , which we recently found to offer an efficient solution to the parallelization / vectorization of code execution in the context of sequence analysis . for example , the n m distance matrix , d , defined by equation 1 , can be resolved efficiently by nesting two map operations on the image rgb intensities , i : d = i.map(function(x , i){return x.map(function(y , j){returni[r , g , b](i , j ) } ) } ) ( 3 ) the efficiency of this implementation in imagejs relies on the javascript compiler mapping the explicit parallelization of the code to local opportunities for distribution . for example , by possibly using the machine 's graphics processing units ( gpus ) . as can be verified by operating imagejs ( or by viewing the accompanying webcast at http://www.youtube.com/watch?v=qbkbgb4eche ) , chromomarkers will display the edges of segmented image superimposed on the original image . the edge detection is performed by another mapping operation which is worth inspecting in the native javascript syntax to recognize the nested mapping pattern used in equation 3 , and is highlighted below in the code for equation 4 . this time , the nested mapping is being used to inspect the neighborhood of each pixel to find out if it corresponds to a segmented pixel with a non - segmented non - pixel , in which case it is classified as an edge : as can be verified by using imagejs or by viewing the accompanying webcast , the image segmentation operations are executed in real time ( under 1 or 2 sec ) , on par with other image analysis environments . feature extraction represents a tougher challenge . here , the goal is to identify which sets of segmented pixels define a contiguous feature . this module 's interface now separates ( they are triggered by distinct events ) feature analysis operations that can be performed in real time , such as the determination of segmentation density or the ratio between rgb intensities between segmented and non - segmented regions , from those that actually require the extraction of features , such as counting them . the description of this feature extraction procedure provides the opportunity to demonstrate how code can be invoked in a publication without concern that subsequent updates or renaming will cause this information not to be available . for example , at the time of this writing , the code for feature extraction can be found in line # 171 of version 4ec7931d17ccc17d27e724d51ae0 of jmat.js , which can be forever available at http://code.google.com/p/jmat/source/browse/jmat.js?r=4ec7931d17ccc17d27e724d51ae0342b871bc98a#171 . following that link will not only return the code used , but also expose comments made by co - developers . it is again worth noting that although the imagejs core is hosted and versioned at imagejs.github.com , this procedure happens to be hosted and versioned by a distinct environment , this time at google . both hosting services , which is to say , the hosting services resolved by the links , are likely to last longer ( forever ) than the interest or even the memory of its authors the ability to simultaneously host versioned development and serve executable javascript code by exactly the same mechanism is a unique feature of the web environment . in the previous section , the feature extraction procedure was described by reference to the specific version and line of code deployment at the time of this writing . the ability to reference specific versions of the code therefore extends to the ability to execute them as well . this feature is enhanced by imagejs by allowing the submission of modules by passing them as arguments to the url call . for example , http://imagejs.github.com/?https://raw.github.com/imagejs/imagejs.github.com/27b596ed08d41e79c6c96e86f0318694ca98cc9e/mainmenu.js and http://module.imagejs.googlecode.com/git-history/ef105347cc13dcb24cdfae621522b5891ff11ab7/mathbiol.chromomarkers.js and http://module.imagejs.googlecode.com/git-history/ef105347cc13dcb24cdfae621522b5891ff11ab7/mathbiol.countshapes.js specifies a very particular combination of versions of the multiple modules involved in the assembly of imagejs . this is of course not a convenient url to exchange , so a shorter reference could be generated , for example , http://bit.ly/h3hvbz . it is worth repeating the note that none of the resources used to enable the assembly of a specific version of the imagejs tool depends on the commitment of the authors of this report to maintain those resources . the imagejs computational environment will natively allow for three mechanisms to incorporate code generated by others . the easiest to distribute is to simply add the address of the modules to the url of imagejs core application , as illustrated above . a more interactive possibility is to use an input box as exemplified by the main menu / load option . the filtering of segmented images offers an illustration of the usefulness of this mechanism in facilitating the interaction with researchers focused on the computational statistics aspects of the analysis . this module , mathbiol.filtershapes , is strictly dedicated to specifying a number of matrix manipulation procedures erode and round at the time of this writing . the association of these procedures with the user interface buttons that trigger them is dealt by the feature extraction module . adding a new filtering procedure to this object we found this style of module development to be particularly appealing to the biostatistics minded researcher because it does not require web programming skills beyond a basic understanding of javascript syntax , which is reminiscent of the c language . in summary , the primary goal of the modules described in this report is to illustrate how the image analysis can be orchestrated to involve multiple contributions , with different dependencies , using different styles , different modes of interaction with the user , hosted in multiple locations , while delivering reproducible results available in the pervasive web browser platform . figure 2 summarizes the orchestration of modules described here , highlighting the salient features and dependencies they were designed to illustrate . the conventional approach of manually loading images from the local file system , by file select or by drag and drop , is supported . manually saving the image and its analysis results back to the files system is also supported and consists of saving the contents of imagejs.data [ figure 1 ] as a json structure . this behavior relies on a relatively new development in web computing standards , the file api ( http://www.w3.org/tr/fileapi ) . another possibility that is particularly useful in collaborative and distributed environments is to rely on the read - write capabilities anticipated for web 3.0 apps . in a nutshell , this is a cloud computing option that treats web services as both remote storage and remote devices , allowing for exchange of data simply by reference . one of the modules , countshapes , illustrates this functionality by using the webrw service described in the materials and methods section . this was adopted as a generic solution of reading and writing back to the web such that we would not have to choose among a slew of excellent commercial resources such as dropbox.com , amazon 's s3 , or google 's fusion tables , which would have required user authentication . regardless of the web read - write cloud service chosen , the attraction of this option is that programmatically , the web is closer to the web browser that to the browser 's machine own file system [ figure 1 ] . accordingly , the same module loading mechanism described in the previous sections will now be used to deal with and saving and loading of data . as an example , see either https://bitly.com/withdata or point a qr reader to the qr code in figure 3 http://bit.ly/withdata.qrcode ; both link to the same data with analysis , as depicted in figures 3 and 4 . quick response ( qr ) code , generated automatically by http://bit.ly/withdata.qrcode for the link http://bit.ly/withdata , which in turn points to http://imagejs.github.com/?http://module . js and http://165.225.128.64/?doc=uid5716226333752275 and http:// module.imagejs.googlecode.com/git/mathbiol.showdata.js , which corresponds to a particular image analysis result that is assembled , re - analyzed , reproducing the display depicted in figure 4 image analysis corresponding to http://bit.ly/withdata . see figure 3 for equivalent qr code for optical reading by tablet devices the proposition that image js0 is particularly adequate to the development of very specific image analysis applications was assessed by developing a new module to assist with the determination of cellular proliferation index from ki67-labeled transmission microscopy images . as proposed , this was pursued by building not only on top of the imagejs abstraction [ figure 1 ] but also integrated with one of the generic modules described earlier , chromomarkers . as a consequence , this exercise was conveniently reduced to the writing of a few image analysis filters that rely on the chromomarkers segmentation utilities to select segmentation thresholds . the ki67 index , a cellular proliferation index , is the ratio of nuclei undergoing division , often estimated from transmission microscopy images of tissue treated with a generic nuclear stain , such as hematoxylin , and an antibody that targets ki67 , a protein present only in the active stages of the cell cycle . to address the vagaries of manual estimation of proliferation from such images , the ki67 imagejs module was designed to assist with the calculation and , more importantly , to report the final value with the graphic display of the corresponding segmentations . a snapshot of this application is presented in figure 5 , and can be invoked by a short link such as http://bit.ly/ki67js or http://uab.mathbiol.org/ki67 in a modern web browser , preferably google chrome . snapshot of the result of using imagejs 's ki67 module to determine proliferation index in a labeled glioblastoma sample imaged by transmission microscopy . a webcast illustrating its usage is available at http://www.youtube.com/watch?v=ncpngrxwwdq and is also provided with the help option of this imagejs application because of the increasing volume and heterogeneity of data , the organic development of software that delivers computational solutions to translational environments is not only a challenge of increasing proportions , but also one with increasing rewards . accordingly , advances in biomolecular methodology , coupled with the benefits of an integrated environment in which data science in a biomedical context may be applied , are increasingly seen as a key new frontier for pathology . the opportunity to develop encompassing integrative infrastructure for image analysis has not been missed by the research community , with very comprehensive initiatives such as omero advancing open source software that pathologists with access to informatics research resources and partnerships can use . the advantages of these partnerships are also increasingly compelling as the amount of annotated image data in the public domain increases . furthermore , these are also increasingly object of large - scale modeling exercises such as the recent morphological analysis of the cancer genome atlas ( tcga ) slide images to identify classifiers of tumor subtypes . these advances make the prospect of pushing image analysis to the lab medicine or to the point of care all the more enticing and the inability to do so all the more frustrating . in principle , applications such as nih imagej provide a good solution to the last mile problem of delivering computational solutions to the environments and machines that have access to the image data that need to be analyzed . the use of the java language ( the j in imagej ) to deliver such a solution is in line with its 1990s motto of write once , run anywhere . it was then widely anticipated that this would be the language of the web and that not only would applications be delivered to the browser as applets ( http://java.sun.com/applets/ ) but also eventually the browser would mature to such a high level of integration with the java language ( it would have a native jvm ) that even individual procedures would be delivered into a communal computational pool where an ecosystem of workflows might emerge in reaction to the data and user interaction context . in a nutshell , the web browser was , correctly , anticipated to become the ultimate saas platform endowed with efficient processors and with a protective sandbox such that the machine 's own file system would not be exposed to the foreign code injection . this should allow for the promise of personalizing the analysis of an image in that private file system to benefit from the wealth of reference data being annotated elsewhere . in fact , this promise has since been delivered , not by object - oriented java which still requires a virtual machine to be first loaded by the browser before being interpreted , but by its little functional helper , javascript , which went on to provide the basis for the second generation of web technologies a decade later . another key variation on the original prediction was the emergence of cloud computing infrastructure as a logical extension of the machine 's file system . this is the modern scenario that the imagejs prototype , described in the results section , explores : we now have all the features needed for pervasive image analysis that we can port all the way to the point of care . specifically , a ) we have an efficient computational environment and b ) we can deliver saas in a manner that is both safe and reproducible . the main argument for delivering image analysis using the browser 's native interpreter is that it is the route best aligned with the sandboxed browser , and is therefore least likely to face objections by no download and installation policies in care delivery environments . initially , it was not clear that this environment is sufficiently performant for image analysis . the world wide web consortium ( w3c ) has recognized the need for metrics for different computational operations , but has only recently standardized their acquisition . we have also recently found out when functionally decomposing sequence analysis into mapreduce operations that these improvements may actually justify rather than deter the use of the browser to deploy advanced computational statistics procedures . as described in the results section and documented in the accompanying webcast , the ability to perform image segmentation using weighted euclidean distance , edge detection , and then overlaying the edges on top of the original image , all in real time , suggest that a similar argument may be valid for image analysis . this argument will be on even more solid ground if efficiency refers to power consumption , since that is the very argument why web browsers in mobile devices are increasingly not supporting graphics rendering using adobe 's flash , microsoft 's silverlight , and oracle - sun 's jvm . the argument that the browser offers a safer delivery of applications is the same that leads it departments to disable the installation of applications by researchers and clinicians in biomedical facilities . there certainly are opportunities to make webapps unsafe , but the fact that they are executed within the browser 's sandbox raises effective barriers to accessing the file system or devices attached to the machine where the webapp is being executed . the argument for a more reproducible delivery of image analysis solutions is also not an absolute claim . the first is that the script tag loading mechanism enables the use of hosted code versioning services for versioned application hosting , as illustrated in the results given in the section the second reason for this claim is that scientific publication also relies on the web browser as the primary dissemination mechanism , which signifies that an image analysis result represented as a figure in a published manuscript can include a link to the data and its versioned analysis code such that the result is automatically reproduced by the machine of the reader . as illustrated in figure 4 , the reproduction in the client machine can even include the assembly of the analytical environment where alternative analysis can be attempted . the main argument for delivering image analysis using the browser 's native interpreter is that it is the route best aligned with the sandboxed browser , and is therefore least likely to face objections by no download and installation policies in care delivery environments . initially , it was not clear that this environment is sufficiently performant for image analysis . the world wide web consortium ( w3c ) has recognized the need for metrics for different computational operations , but has only recently standardized their acquisition . we have also recently found out when functionally decomposing sequence analysis into mapreduce operations that these improvements may actually justify rather than deter the use of the browser to deploy advanced computational statistics procedures . as described in the results section and documented in the accompanying webcast , the ability to perform image segmentation using weighted euclidean distance , edge detection , and then overlaying the edges on top of the original image , all in real time , suggest that a similar argument may be valid for image analysis . this argument will be on even more solid ground if efficiency refers to power consumption , since that is the very argument why web browsers in mobile devices are increasingly not supporting graphics rendering using adobe 's flash , microsoft 's silverlight , and oracle - sun 's jvm . the argument that the browser offers a safer delivery of applications is the same that leads it departments to disable the installation of applications by researchers and clinicians in biomedical facilities . there certainly are opportunities to make webapps unsafe , but the fact that they are executed within the browser 's sandbox raises effective barriers to accessing the file system or devices attached to the machine where the webapp is being executed . the argument for a more reproducible delivery of image analysis solutions is also not an absolute claim . the first is that the script tag loading mechanism enables the use of hosted code versioning services for versioned application hosting , as illustrated in the results given in the section the second reason for this claim is that scientific publication also relies on the web browser as the primary dissemination mechanism , which signifies that an image analysis result represented as a figure in a published manuscript can include a link to the data and its versioned analysis code such that the result is automatically reproduced by the machine of the reader . as illustrated in figure 4 , the reproduction in the client machine can even include the assembly of the analytical environment where alternative analysis can be attempted . imagejs provides a pervasive mechanism to deliver image analysis workflows and interactive interfaces to where the data are . it includes a core object model equipped with data - specific attributes and methods , such as a module loader that registers the source urls of the components loaded . the ability to define workflows by chaining the addresses of the modules with imagejs 's own the use of versioned code hosting as a mechanism for deployment is also a stark departure from conventional bioinformatics infrastructure , where the ensuing code injection is normally avoided rather than promoted . however compelling this mechanism for distributed development and deployment may be , and no matter how convenient and scalable the delivery of computation to image repositories may be , imagejs webapp ecosystem is primarily an experiment in informatics . the ability to gather data about the population of this computational webapp ecosystem by others and about collaborative data processing to inform further development of distributed image analysis infrastructure may well be its most valuable purpose .

background : image bioinformatics infrastructure typically relies on a combination of server - side high - performance computing and client desktop applications tailored for graphic rendering . on the server side , matrix manipulation environments are often used as the back - end where deployment of specialized analytical workflows takes place . however , neither the server - side nor the client - side desktop solution , by themselves or combined , is conducive to the emergence of open , collaborative , computational ecosystems for image analysis that are both self - sustained and user driven.materials and methods : imagejs was developed as a browser - based webapp , untethered from a server - side backend , by making use of recent advances in the modern web browser such as a very efficient compiler , high - end graphical rendering capabilities , and i / o tailored for code migration.results:multiple versioned code hosting services were used to develop distinct imagejs modules to illustrate its amenability to collaborative deployment without compromise of reproducibility or provenance . the illustrative examples include modules for image segmentation , feature extraction , and filtering . the deployment of image analysis by code migration is in sharp contrast with the more conventional , heavier , and less safe reliance on data transfer . accordingly , code and data are loaded into the browser by exactly the same script tag loading mechanism , which offers a number of interesting applications that would be hard to attain with more conventional platforms , such as nih 's popular imagej application.conclusions:the modern web browser was found to be advantageous for image bioinformatics in both the research and clinical environments . this conclusion reflects advantages in deployment scalability and analysis reproducibility , as well as the critical ability to deliver advanced computational statistical procedures machines where access to sensitive data is controlled , that is , without local download and installation .

INTRODUCTION MATERIALS AND METHODS RESULTS Dissimilarity Metric Module Module Modular Orchestration Data Management A Pathology Application: KI67 Labeling for Cellular Proliferation DISCUSSION The Web Browser for Computational Efficient Image Analysis Safe and Reproducible SaaS? CONCLUSION

image bioinformatics as a field is characterized by an odd mixture of tremendous advances in its computational statistics aspects while suffering from a persistent resistance to their use by biomedical domain experts . the image analysis platform with the deepest domain penetration in the research community is probably national institutes of health ( nih 's ) imagej in large part because of its free open source and pluggable implementation . over the years , an extensive community of image analysis developers and researchers has developed a comprehensive environment for image analysis software development using java . even more striking is the situation that the few researchers using image analysis , who know how to find and install plugins , often also need image analysis applications that are slight modifications or combinations of existing functionalities . paradoxically , the computational statisticians with the quantitative expertise to develop those modifications often do not have the substantial software development skills needed to deliver the novel application as a stand - alone application or as a plugin module . submitting to a remote service exposes the image to a third party outside the reach of institutional it ; updates in the web service may cause results not to be reproducible and the server side application is typically not exposed to public scrutiny , limiting the opportunities for other quantitative researchers to advance or even fully understand the underlying algorithms ; bioinformatics web services are also notorious for relatively short lifetimes , reflecting a sustainability model that can only be met in the long term by institutional commitment or by commercial products . the imagejs webapp ecosystem described in this report advances along those exact lines without crossing the lines set by requirements of privacy , scalability , reproducibility , and sustainable deployment associated with the biomedical environment . specifically , if an image analysis result can be retrieved directly from the published image , with both the data and the analytical environment that generated the result , then one could indeed claim to be reporting reproducible research . accordingly , imagejs seeks to deliver image analysis results meeting the gold standard in that report 's description of the spectrum of reproducibility [ figure 1 ] . imagejs migrates through script tag loading to the browser ( by pointing a script.src to http://imagejs.github.com/imagejs.js ) , where it creates an object with a method , imagejs . loadmodule , that will import and register additional components onto imagejs.modules , while keeping data meant to be shared between modules in imagejs.data . the solid lines represent programmatic exchanges and dashed line represents an exchange that requires user intervention , such as the loading or saving an image / analysis the possibility of approaching software development as an open organic process , which is explored by the imagejs webapp environment described here , has been proposed before as a requirement for medicine 2.0 . this is a departure from conventional bioinformatics infrastructure that can also be construed as a response to the mounting knowledge re - engineering bottleneck . it would provide a beyond the data deluge solution to image analysis where the computation is pushed to where the data is , rather than the customary approach of shuffling the data among a myriad of analytical services . to illustrate the distributed and collaborative nature of the proposed webapp ecosystem , its component modules were developed in multiple versioned code hosting services , github ( http://github.com ) and google code ( http://googlecode.com ) . the segmentation , feature extraction , and filtering modules were developed , and are hosted at http://imagejs.googlecode.com/. a number of alternatives exist ; in this report , we used webrw http - rest api , which is supported by two independent implementations , both open source : one is written in nodejs ( https://github.com/jonasalmeida/webrw ) and another in php ( https://github.com/ebadedude/webrw ) . important note : although the core components and most modules will work in different web browsers , this study makes use of recent html5 features such as the file api ( http://www.w3.org/tr/fileapi/ ) that are still being adopted . a closer inspection of this method will show that a ) it imports external code by pointing a browser 's document object model ( dom ) script tag to the url of the external code ( module ) and b ) it registers the imported module in imagejs.module(url ) . the features of the imagejs environment as a vehicle to deliver computation were studied by developing and deploying an image analysis workflow that included standard modules for feature segmentation , analysis , and filtering . each of these modules was configured not only to deliver a desirable image analysis functionality and in the process test if the browser is a suitable platform to do so but also to test different models of dependency between modules . the goal of testing different models of articulation between analytical modules is to explore the balance between flexibility and robustness of browser - based computational ecosystems as platforms for collaborative software development . as stated in the materials and methods section , to reinforce the perception of independent development , the multiple modules and imagejs core code are scattered between distinct versioned hosting services , namely , github and google code . a chromomarker , rgbmarker , is defined as the three - element vector with the intensities in the red , green , and blue channels that one uses to retrieve image features with similar chromometric characteristics . to this purpose , the user selects a pixel in the image , i , of size n m as a marker , with [ r , g , b ] intensities , and sets a weighted threshold , t , for a dissimilarity metric , dist , that will be resolved for all pixels in the image , each with its own intensity values [ r , g , b ] : the first module , chromomarkers , will segment all pixels in the image that are at a distance smaller than t. segmentetpixelcoordinates = [ i , j]|dist([r , g , b](i , j ) ) < t ( 2 ) both operations , distance calculation ( equation 1 ) and segmentation ( equation 2 ) , represent a first test of the browser as an efficient environment for image processing . the javascript engine of modern web browsers includes support for map and reduce operations , which we recently found to offer an efficient solution to the parallelization / vectorization of code execution in the context of sequence analysis . this time , the nested mapping is being used to inspect the neighborhood of each pixel to find out if it corresponds to a segmented pixel with a non - segmented non - pixel , in which case it is classified as an edge : as can be verified by using imagejs or by viewing the accompanying webcast , the image segmentation operations are executed in real time ( under 1 or 2 sec ) , on par with other image analysis environments . the ability to simultaneously host versioned development and serve executable javascript code by exactly the same mechanism is a unique feature of the web environment . in the previous section , the feature extraction procedure was described by reference to the specific version and line of code deployment at the time of this writing . for example , http://imagejs.github.com/?https://raw.github.com/imagejs/imagejs.github.com/27b596ed08d41e79c6c96e86f0318694ca98cc9e/mainmenu.js and http://module.imagejs.googlecode.com/git-history/ef105347cc13dcb24cdfae621522b5891ff11ab7/mathbiol.chromomarkers.js and http://module.imagejs.googlecode.com/git-history/ef105347cc13dcb24cdfae621522b5891ff11ab7/mathbiol.countshapes.js specifies a very particular combination of versions of the multiple modules involved in the assembly of imagejs . this module , mathbiol.filtershapes , is strictly dedicated to specifying a number of matrix manipulation procedures erode and round at the time of this writing . in summary , the primary goal of the modules described in this report is to illustrate how the image analysis can be orchestrated to involve multiple contributions , with different dependencies , using different styles , different modes of interaction with the user , hosted in multiple locations , while delivering reproducible results available in the pervasive web browser platform . one of the modules , countshapes , illustrates this functionality by using the webrw service described in the materials and methods section . this was adopted as a generic solution of reading and writing back to the web such that we would not have to choose among a slew of excellent commercial resources such as dropbox.com , amazon 's s3 , or google 's fusion tables , which would have required user authentication . accordingly , the same module loading mechanism described in the previous sections will now be used to deal with and saving and loading of data . as an example , see either https://bitly.com/withdata or point a qr reader to the qr code in figure 3 http://bit.ly/withdata.qrcode ; both link to the same data with analysis , as depicted in figures 3 and 4 . as a consequence , this exercise was conveniently reduced to the writing of a few image analysis filters that rely on the chromomarkers segmentation utilities to select segmentation thresholds . the ki67 index , a cellular proliferation index , is the ratio of nuclei undergoing division , often estimated from transmission microscopy images of tissue treated with a generic nuclear stain , such as hematoxylin , and an antibody that targets ki67 , a protein present only in the active stages of the cell cycle . a snapshot of this application is presented in figure 5 , and can be invoked by a short link such as http://bit.ly/ki67js or http://uab.mathbiol.org/ki67 in a modern web browser , preferably google chrome . a chromomarker , rgbmarker , is defined as the three - element vector with the intensities in the red , green , and blue channels that one uses to retrieve image features with similar chromometric characteristics . to this purpose , the user selects a pixel in the image , i , of size n m as a marker , with [ r , g , b ] intensities , and sets a weighted threshold , t , for a dissimilarity metric , dist , that will be resolved for all pixels in the image , each with its own intensity values [ r , g , b ] : the first module , chromomarkers , will segment all pixels in the image that are at a distance smaller than t. segmentetpixelcoordinates = [ i , j]|dist([r , g , b](i , j ) ) < t ( 2 ) both operations , distance calculation ( equation 1 ) and segmentation ( equation 2 ) , represent a first test of the browser as an efficient environment for image processing . the javascript engine of modern web browsers includes support for map and reduce operations , which we recently found to offer an efficient solution to the parallelization / vectorization of code execution in the context of sequence analysis . this module 's interface now separates ( they are triggered by distinct events ) feature analysis operations that can be performed in real time , such as the determination of segmentation density or the ratio between rgb intensities between segmented and non - segmented regions , from those that actually require the extraction of features , such as counting them . both hosting services , which is to say , the hosting services resolved by the links , are likely to last longer ( forever ) than the interest or even the memory of its authors the ability to simultaneously host versioned development and serve executable javascript code by exactly the same mechanism is a unique feature of the web environment . for example , http://imagejs.github.com/?https://raw.github.com/imagejs/imagejs.github.com/27b596ed08d41e79c6c96e86f0318694ca98cc9e/mainmenu.js and http://module.imagejs.googlecode.com/git-history/ef105347cc13dcb24cdfae621522b5891ff11ab7/mathbiol.chromomarkers.js and http://module.imagejs.googlecode.com/git-history/ef105347cc13dcb24cdfae621522b5891ff11ab7/mathbiol.countshapes.js specifies a very particular combination of versions of the multiple modules involved in the assembly of imagejs . the easiest to distribute is to simply add the address of the modules to the url of imagejs core application , as illustrated above . this module , mathbiol.filtershapes , is strictly dedicated to specifying a number of matrix manipulation procedures erode and round at the time of this writing . adding a new filtering procedure to this object we found this style of module development to be particularly appealing to the biostatistics minded researcher because it does not require web programming skills beyond a basic understanding of javascript syntax , which is reminiscent of the c language . in summary , the primary goal of the modules described in this report is to illustrate how the image analysis can be orchestrated to involve multiple contributions , with different dependencies , using different styles , different modes of interaction with the user , hosted in multiple locations , while delivering reproducible results available in the pervasive web browser platform . this was adopted as a generic solution of reading and writing back to the web such that we would not have to choose among a slew of excellent commercial resources such as dropbox.com , amazon 's s3 , or google 's fusion tables , which would have required user authentication . regardless of the web read - write cloud service chosen , the attraction of this option is that programmatically , the web is closer to the web browser that to the browser 's machine own file system [ figure 1 ] . accordingly , the same module loading mechanism described in the previous sections will now be used to deal with and saving and loading of data . js and http://165.225.128.64/?doc=uid5716226333752275 and http:// module.imagejs.googlecode.com/git/mathbiol.showdata.js , which corresponds to a particular image analysis result that is assembled , re - analyzed , reproducing the display depicted in figure 4 image analysis corresponding to http://bit.ly/withdata . see figure 3 for equivalent qr code for optical reading by tablet devices the proposition that image js0 is particularly adequate to the development of very specific image analysis applications was assessed by developing a new module to assist with the determination of cellular proliferation index from ki67-labeled transmission microscopy images . as a consequence , this exercise was conveniently reduced to the writing of a few image analysis filters that rely on the chromomarkers segmentation utilities to select segmentation thresholds . the ki67 index , a cellular proliferation index , is the ratio of nuclei undergoing division , often estimated from transmission microscopy images of tissue treated with a generic nuclear stain , such as hematoxylin , and an antibody that targets ki67 , a protein present only in the active stages of the cell cycle . a snapshot of this application is presented in figure 5 , and can be invoked by a short link such as http://bit.ly/ki67js or http://uab.mathbiol.org/ki67 in a modern web browser , preferably google chrome . accordingly , advances in biomolecular methodology , coupled with the benefits of an integrated environment in which data science in a biomedical context may be applied , are increasingly seen as a key new frontier for pathology . the opportunity to develop encompassing integrative infrastructure for image analysis has not been missed by the research community , with very comprehensive initiatives such as omero advancing open source software that pathologists with access to informatics research resources and partnerships can use . in principle , applications such as nih imagej provide a good solution to the last mile problem of delivering computational solutions to the environments and machines that have access to the image data that need to be analyzed . it was then widely anticipated that this would be the language of the web and that not only would applications be delivered to the browser as applets ( http://java.sun.com/applets/ ) but also eventually the browser would mature to such a high level of integration with the java language ( it would have a native jvm ) that even individual procedures would be delivered into a communal computational pool where an ecosystem of workflows might emerge in reaction to the data and user interaction context . in a nutshell , the web browser was , correctly , anticipated to become the ultimate saas platform endowed with efficient processors and with a protective sandbox such that the machine 's own file system would not be exposed to the foreign code injection . another key variation on the original prediction was the emergence of cloud computing infrastructure as a logical extension of the machine 's file system . this is the modern scenario that the imagejs prototype , described in the results section , explores : we now have all the features needed for pervasive image analysis that we can port all the way to the point of care . the main argument for delivering image analysis using the browser 's native interpreter is that it is the route best aligned with the sandboxed browser , and is therefore least likely to face objections by no download and installation policies in care delivery environments . we have also recently found out when functionally decomposing sequence analysis into mapreduce operations that these improvements may actually justify rather than deter the use of the browser to deploy advanced computational statistics procedures . as described in the results section and documented in the accompanying webcast , the ability to perform image segmentation using weighted euclidean distance , edge detection , and then overlaying the edges on top of the original image , all in real time , suggest that a similar argument may be valid for image analysis . the argument that the browser offers a safer delivery of applications is the same that leads it departments to disable the installation of applications by researchers and clinicians in biomedical facilities . the first is that the script tag loading mechanism enables the use of hosted code versioning services for versioned application hosting , as illustrated in the results given in the section the second reason for this claim is that scientific publication also relies on the web browser as the primary dissemination mechanism , which signifies that an image analysis result represented as a figure in a published manuscript can include a link to the data and its versioned analysis code such that the result is automatically reproduced by the machine of the reader . the main argument for delivering image analysis using the browser 's native interpreter is that it is the route best aligned with the sandboxed browser , and is therefore least likely to face objections by no download and installation policies in care delivery environments . we have also recently found out when functionally decomposing sequence analysis into mapreduce operations that these improvements may actually justify rather than deter the use of the browser to deploy advanced computational statistics procedures . as described in the results section and documented in the accompanying webcast , the ability to perform image segmentation using weighted euclidean distance , edge detection , and then overlaying the edges on top of the original image , all in real time , suggest that a similar argument may be valid for image analysis . the first is that the script tag loading mechanism enables the use of hosted code versioning services for versioned application hosting , as illustrated in the results given in the section the second reason for this claim is that scientific publication also relies on the web browser as the primary dissemination mechanism , which signifies that an image analysis result represented as a figure in a published manuscript can include a link to the data and its versioned analysis code such that the result is automatically reproduced by the machine of the reader . it includes a core object model equipped with data - specific attributes and methods , such as a module loader that registers the source urls of the components loaded . the ability to define workflows by chaining the addresses of the modules with imagejs 's own the use of versioned code hosting as a mechanism for deployment is also a stark departure from conventional bioinformatics infrastructure , where the ensuing code injection is normally avoided rather than promoted .

the development cycle can be roughly described as follows : when a promising new algorithm is identified , it might be first prototyped in a specialized environment such as mathwork 's matlab , but to reach a broader non - programming audience , it is then laboriously ported as an imagej plugin . paradoxically , the computational statisticians with the quantitative expertise to develop those modifications often do not have the substantial software development skills needed to deliver the novel application as a stand - alone application or as a plugin module . a common solution to for example , the quantitative image analysis of estrogen receptor , progesterone receptor , and more generically , the ki-67 immunohistochemistry staining results in breast cancer tissue sections can be conveniently performed by submitting the image to a public web service . users benefit from improvements in the code automatically and no application needs to installed , therefore overcoming concerns with compromising privacy or degrading the performance of existing applications in the client machine . submitting to a remote service exposes the image to a third party outside the reach of institutional it ; updates in the web service may cause results not to be reproducible and the server side application is typically not exposed to public scrutiny , limiting the opportunities for other quantitative researchers to advance or even fully understand the underlying algorithms ; bioinformatics web services are also notorious for relatively short lifetimes , reflecting a sustainability model that can only be met in the long term by institutional commitment or by commercial products . the imagejs webapp ecosystem described in this report advances along those exact lines without crossing the lines set by requirements of privacy , scalability , reproducibility , and sustainable deployment associated with the biomedical environment . specifically , if an image analysis result can be retrieved directly from the published image , with both the data and the analytical environment that generated the result , then one could indeed claim to be reporting reproducible research . the solid lines represent programmatic exchanges and dashed line represents an exchange that requires user intervention , such as the loading or saving an image / analysis the possibility of approaching software development as an open organic process , which is explored by the imagejs webapp environment described here , has been proposed before as a requirement for medicine 2.0 . to illustrate the distributed and collaborative nature of the proposed webapp ecosystem , its component modules were developed in multiple versioned code hosting services , github ( http://github.com ) and google code ( http://googlecode.com ) . a number of alternatives exist ; in this report , we used webrw http - rest api , which is supported by two independent implementations , both open source : one is written in nodejs ( https://github.com/jonasalmeida/webrw ) and another in php ( https://github.com/ebadedude/webrw ) . important note : although the core components and most modules will work in different web browsers , this study makes use of recent html5 features such as the file api ( http://www.w3.org/tr/fileapi/ ) that are still being adopted . a closer inspection of this method will show that a ) it imports external code by pointing a browser 's document object model ( dom ) script tag to the url of the external code ( module ) and b ) it registers the imported module in imagejs.module(url ) . the features of the imagejs environment as a vehicle to deliver computation were studied by developing and deploying an image analysis workflow that included standard modules for feature segmentation , analysis , and filtering . the goal of testing different models of articulation between analytical modules is to explore the balance between flexibility and robustness of browser - based computational ecosystems as platforms for collaborative software development . as stated in the materials and methods section , to reinforce the perception of independent development , the multiple modules and imagejs core code are scattered between distinct versioned hosting services , namely , github and google code . the analytical workflow can therefore be assembled by loading modules in response to actions by the user or it can be initiated by a particular result of the analysis the image analysis procedure developed starts with the selection by the user of a chromomarker . a chromomarker , rgbmarker , is defined as the three - element vector with the intensities in the red , green , and blue channels that one uses to retrieve image features with similar chromometric characteristics . to this purpose , the user selects a pixel in the image , i , of size n m as a marker , with [ r , g , b ] intensities , and sets a weighted threshold , t , for a dissimilarity metric , dist , that will be resolved for all pixels in the image , each with its own intensity values [ r , g , b ] : the first module , chromomarkers , will segment all pixels in the image that are at a distance smaller than t. segmentetpixelcoordinates = [ i , j]|dist([r , g , b](i , j ) ) < t ( 2 ) both operations , distance calculation ( equation 1 ) and segmentation ( equation 2 ) , represent a first test of the browser as an efficient environment for image processing . the javascript engine of modern web browsers includes support for map and reduce operations , which we recently found to offer an efficient solution to the parallelization / vectorization of code execution in the context of sequence analysis . for example , the n m distance matrix , d , defined by equation 1 , can be resolved efficiently by nesting two map operations on the image rgb intensities , i : d = i.map(function(x , i){return x.map(function(y , j){returni[r , g , b](i , j ) } ) } ) ( 3 ) the efficiency of this implementation in imagejs relies on the javascript compiler mapping the explicit parallelization of the code to local opportunities for distribution . this time , the nested mapping is being used to inspect the neighborhood of each pixel to find out if it corresponds to a segmented pixel with a non - segmented non - pixel , in which case it is classified as an edge : as can be verified by using imagejs or by viewing the accompanying webcast , the image segmentation operations are executed in real time ( under 1 or 2 sec ) , on par with other image analysis environments . this module 's interface now separates ( they are triggered by distinct events ) feature analysis operations that can be performed in real time , such as the determination of segmentation density or the ratio between rgb intensities between segmented and non - segmented regions , from those that actually require the extraction of features , such as counting them . the description of this feature extraction procedure provides the opportunity to demonstrate how code can be invoked in a publication without concern that subsequent updates or renaming will cause this information not to be available . for example , at the time of this writing , the code for feature extraction can be found in line # 171 of version 4ec7931d17ccc17d27e724d51ae0 of jmat.js , which can be following that link will not only return the code used , but also expose comments made by co - developers . it is worth repeating the note that none of the resources used to enable the assembly of a specific version of the imagejs tool depends on the commitment of the authors of this report to maintain those resources . the filtering of segmented images offers an illustration of the usefulness of this mechanism in facilitating the interaction with researchers focused on the computational statistics aspects of the analysis . adding a new filtering procedure to this object we found this style of module development to be particularly appealing to the biostatistics minded researcher because it does not require web programming skills beyond a basic understanding of javascript syntax , which is reminiscent of the c language . in summary , the primary goal of the modules described in this report is to illustrate how the image analysis can be orchestrated to involve multiple contributions , with different dependencies , using different styles , different modes of interaction with the user , hosted in multiple locations , while delivering reproducible results available in the pervasive web browser platform . figure 2 summarizes the orchestration of modules described here , highlighting the salient features and dependencies they were designed to illustrate . this was adopted as a generic solution of reading and writing back to the web such that we would not have to choose among a slew of excellent commercial resources such as dropbox.com , amazon 's s3 , or google 's fusion tables , which would have required user authentication . regardless of the web read - write cloud service chosen , the attraction of this option is that programmatically , the web is closer to the web browser that to the browser 's machine own file system [ figure 1 ] . see figure 3 for equivalent qr code for optical reading by tablet devices the proposition that image js0 is particularly adequate to the development of very specific image analysis applications was assessed by developing a new module to assist with the determination of cellular proliferation index from ki67-labeled transmission microscopy images . the ki67 index , a cellular proliferation index , is the ratio of nuclei undergoing division , often estimated from transmission microscopy images of tissue treated with a generic nuclear stain , such as hematoxylin , and an antibody that targets ki67 , a protein present only in the active stages of the cell cycle . to address the vagaries of manual estimation of proliferation from such images , the ki67 imagejs module was designed to assist with the calculation and , more importantly , to report the final value with the graphic display of the corresponding segmentations . a webcast illustrating its usage is available at http://www.youtube.com/watch?v=ncpngrxwwdq and is also provided with the help option of this imagejs application the image analysis procedure developed starts with the selection by the user of a chromomarker . to this purpose , the user selects a pixel in the image , i , of size n m as a marker , with [ r , g , b ] intensities , and sets a weighted threshold , t , for a dissimilarity metric , dist , that will be resolved for all pixels in the image , each with its own intensity values [ r , g , b ] : the first module , chromomarkers , will segment all pixels in the image that are at a distance smaller than t. segmentetpixelcoordinates = [ i , j]|dist([r , g , b](i , j ) ) < t ( 2 ) both operations , distance calculation ( equation 1 ) and segmentation ( equation 2 ) , represent a first test of the browser as an efficient environment for image processing . the javascript engine of modern web browsers includes support for map and reduce operations , which we recently found to offer an efficient solution to the parallelization / vectorization of code execution in the context of sequence analysis . for example , the n m distance matrix , d , defined by equation 1 , can be resolved efficiently by nesting two map operations on the image rgb intensities , i : d = i.map(function(x , i){return x.map(function(y , j){returni[r , g , b](i , j ) } ) } ) ( 3 ) the efficiency of this implementation in imagejs relies on the javascript compiler mapping the explicit parallelization of the code to local opportunities for distribution . this time , the nested mapping is being used to inspect the neighborhood of each pixel to find out if it corresponds to a segmented pixel with a non - segmented non - pixel , in which case it is classified as an edge : as can be verified by using imagejs or by viewing the accompanying webcast , the image segmentation operations are executed in real time ( under 1 or 2 sec ) , on par with other image analysis environments . this module 's interface now separates ( they are triggered by distinct events ) feature analysis operations that can be performed in real time , such as the determination of segmentation density or the ratio between rgb intensities between segmented and non - segmented regions , from those that actually require the extraction of features , such as counting them . the description of this feature extraction procedure provides the opportunity to demonstrate how code can be invoked in a publication without concern that subsequent updates or renaming will cause this information not to be available . for example , at the time of this writing , the code for feature extraction can be found in line # 171 of version 4ec7931d17ccc17d27e724d51ae0 of jmat.js , which can be forever available at http://code.google.com/p/jmat/source/browse/jmat.js?r=4ec7931d17ccc17d27e724d51ae0342b871bc98a#171 . both hosting services , which is to say , the hosting services resolved by the links , are likely to last longer ( forever ) than the interest or even the memory of its authors the ability to simultaneously host versioned development and serve executable javascript code by exactly the same mechanism is a unique feature of the web environment . it is worth repeating the note that none of the resources used to enable the assembly of a specific version of the imagejs tool depends on the commitment of the authors of this report to maintain those resources . the filtering of segmented images offers an illustration of the usefulness of this mechanism in facilitating the interaction with researchers focused on the computational statistics aspects of the analysis . adding a new filtering procedure to this object we found this style of module development to be particularly appealing to the biostatistics minded researcher because it does not require web programming skills beyond a basic understanding of javascript syntax , which is reminiscent of the c language . in summary , the primary goal of the modules described in this report is to illustrate how the image analysis can be orchestrated to involve multiple contributions , with different dependencies , using different styles , different modes of interaction with the user , hosted in multiple locations , while delivering reproducible results available in the pervasive web browser platform . figure 2 summarizes the orchestration of modules described here , highlighting the salient features and dependencies they were designed to illustrate . this was adopted as a generic solution of reading and writing back to the web such that we would not have to choose among a slew of excellent commercial resources such as dropbox.com , amazon 's s3 , or google 's fusion tables , which would have required user authentication . regardless of the web read - write cloud service chosen , the attraction of this option is that programmatically , the web is closer to the web browser that to the browser 's machine own file system [ figure 1 ] . see figure 3 for equivalent qr code for optical reading by tablet devices the proposition that image js0 is particularly adequate to the development of very specific image analysis applications was assessed by developing a new module to assist with the determination of cellular proliferation index from ki67-labeled transmission microscopy images . the ki67 index , a cellular proliferation index , is the ratio of nuclei undergoing division , often estimated from transmission microscopy images of tissue treated with a generic nuclear stain , such as hematoxylin , and an antibody that targets ki67 , a protein present only in the active stages of the cell cycle . to address the vagaries of manual estimation of proliferation from such images , the ki67 imagejs module was designed to assist with the calculation and , more importantly , to report the final value with the graphic display of the corresponding segmentations . a webcast illustrating its usage is available at http://www.youtube.com/watch?v=ncpngrxwwdq and is also provided with the help option of this imagejs application because of the increasing volume and heterogeneity of data , the organic development of software that delivers computational solutions to translational environments is not only a challenge of increasing proportions , but also one with increasing rewards . accordingly , advances in biomolecular methodology , coupled with the benefits of an integrated environment in which data science in a biomedical context may be applied , are increasingly seen as a key new frontier for pathology . furthermore , these are also increasingly object of large - scale modeling exercises such as the recent morphological analysis of the cancer genome atlas ( tcga ) slide images to identify classifiers of tumor subtypes . it was then widely anticipated that this would be the language of the web and that not only would applications be delivered to the browser as applets ( http://java.sun.com/applets/ ) but also eventually the browser would mature to such a high level of integration with the java language ( it would have a native jvm ) that even individual procedures would be delivered into a communal computational pool where an ecosystem of workflows might emerge in reaction to the data and user interaction context . in a nutshell , the web browser was , correctly , anticipated to become the ultimate saas platform endowed with efficient processors and with a protective sandbox such that the machine 's own file system would not be exposed to the foreign code injection . in fact , this promise has since been delivered , not by object - oriented java which still requires a virtual machine to be first loaded by the browser before being interpreted , but by its little functional helper , javascript , which went on to provide the basis for the second generation of web technologies a decade later . this is the modern scenario that the imagejs prototype , described in the results section , explores : we now have all the features needed for pervasive image analysis that we can port all the way to the point of care . the main argument for delivering image analysis using the browser 's native interpreter is that it is the route best aligned with the sandboxed browser , and is therefore least likely to face objections by no download and installation policies in care delivery environments . as described in the results section and documented in the accompanying webcast , the ability to perform image segmentation using weighted euclidean distance , edge detection , and then overlaying the edges on top of the original image , all in real time , suggest that a similar argument may be valid for image analysis . the first is that the script tag loading mechanism enables the use of hosted code versioning services for versioned application hosting , as illustrated in the results given in the section the second reason for this claim is that scientific publication also relies on the web browser as the primary dissemination mechanism , which signifies that an image analysis result represented as a figure in a published manuscript can include a link to the data and its versioned analysis code such that the result is automatically reproduced by the machine of the reader . the main argument for delivering image analysis using the browser 's native interpreter is that it is the route best aligned with the sandboxed browser , and is therefore least likely to face objections by no download and installation policies in care delivery environments . as described in the results section and documented in the accompanying webcast , the ability to perform image segmentation using weighted euclidean distance , edge detection , and then overlaying the edges on top of the original image , all in real time , suggest that a similar argument may be valid for image analysis . the first is that the script tag loading mechanism enables the use of hosted code versioning services for versioned application hosting , as illustrated in the results given in the section the second reason for this claim is that scientific publication also relies on the web browser as the primary dissemination mechanism , which signifies that an image analysis result represented as a figure in a published manuscript can include a link to the data and its versioned analysis code such that the result is automatically reproduced by the machine of the reader . the ability to define workflows by chaining the addresses of the modules with imagejs 's own the use of versioned code hosting as a mechanism for deployment is also a stark departure from conventional bioinformatics infrastructure , where the ensuing code injection is normally avoided rather than promoted . however compelling this mechanism for distributed development and deployment may be , and no matter how convenient and scalable the delivery of computation to image repositories may be , imagejs webapp ecosystem is primarily an experiment in informatics .

the goals of total knee arthroplasty ( tka ) are to relieve pain , maintain knee function and kinematics , and restore stability and range of motion ( rom ) to facilitate daily activities . of these , restoration of rom has been considered important especially in eastern countries where high flexion is required for religious and social activities . factors that can be associated with rom include the cause of disease , preoperative joint deformity and rom , age , gender , surgical technique , implant fixation strength , postoperative rehabilitation , and implant design1 ) . since first introduced in tka for optimal component positioning and lower limb alignment , navigation systems have been improved with rapid development of computer technology to allow for soft tissue balancing and flexion - extension gap balancing , and accordingly have contributed to the accuracy of tka2 ) . in this study , we analyzed the results of high flexion tka using one of the most recently developed navigation systems , the electromagnetic navigation system , to compare the influence of two different implant designs ( mobile - bearing and fixed bearing designs ) on rom including maximal flexion angle and clinical and radiographic results . the mobile - bearing implant used was nexgen lps - flex mobile knee ( nexgen , legacy posterior - stabilized flexion mobile , zimmer , warsaw , in , usa ) whereas the fixed - bearing implant was nexgen lps - flex fixed knee ( nexgen , legacy posterior - stabilized flexion fixed , zimmer ) . of the patients that had undergone tka by the same surgeon between january 2010 and june 2010 at our institution , 67 patients with a minimum follow - up of one year were randomly selected for this study . the implants used were the lps - flex mobile knee in 32 patients and lps - flex fixed knee in 34 patients . in six of the patients with bilateral tka , the mobile - bearing prosthesis was used on one side and the fixed - bearing on the other side . in all patients , an electromagnetic navigation system ( zimmer computer assisted solutions electromagnetic quad - sparing ) was used . the mean follow - up period was 14.5 months ( range , 12 to 18 months ) and the mean age of the patients was 67.5 years ( range , 60 to 78 years ) in the mobile - bearing group and 68.5 years ( range , 61 to 77 years ) in the fixed - bearing group . the mean preoperative rom was 118 in both groups and no notable intergroup difference could be found regarding flexion contracture ( table 1 ) . considering that the preoperative rom could affect the postoperative rom3,4 ) , we excluded patients who had 90 of rom preoperatively . there were no particular instructions for knee flexion exercises and the same rehabilitation protocol was prescribed in both groups . the clinical results were graded according to the knee society knee score ( ksks ) and knee society functional score ( ksfs ) . patient satisfaction and preference were assessed according to the western ontario and mcmaster universities arthritis index ( womac ) score5 ) , a questionnaire for measuring pain , stiffness , and function . postoperative pain was evaluated using the visual analog scale ( vas ) . a patient 's ability to kneel and sit cross - legged was assessed through interview or observation during follow - up . physical examination and goniometric measurement however , we used a radiographic measurement method devised by edwards et al.6 ) to reduce measurement errors and increase reproducibility . we measured the angle formed by lines drawn down the mid - shafts of the tibia and femur on the lateral radiographs taken with the knee in full extension and full flexion preoperatively , as well as at the final follow - up ( fig . radiographic assessment was done by the same surgeon three times per patient and the mean value was used for analysis . the mechanical axis deviation was measured as the difference in the angle between the femoral mechanical axis and the tibial mechanical axis on the weight bearing long leg anteroposterior radiographs taken preoperatively and postoperatively . the femoral and tibial component positions in the sagittal and coronal planes were assessed using the method of seon and song7 ) . the coronal inclination of the femoral component was measured as the medial angle between the mechanical axis and the bottom of the component on the anteroposterior radiographs and the sagittal inclination as the angle with the femoral shaft on the lateral views . the desired sagittal inclination was defined as 90. the coronal and sagittal tibial component positions were assessed in a similar manner . the bisecting line of the tibial shaft was used as a reference for sagittal tibial component positioning . the desired tibial component inclination was defined as 90 in the coronal plane and 86o in the sagittal plane ( fig . all the operations were performed by the same surgeon using a medial parapatella approach . to increase articulation curvature during flexion , an additional 2 mm posterior femoral bone cut was performed . the anterior portion of the tibial component was removed to avoid interference with the patella and the posterior cam mechanism was modified to facilitate high flexion . the preoperative and postoperative mechanical axes and extent of bone resection could be evaluated using an electromagnetic navigation system in all cases . in particular , varus / valgus femoral resection , degree of flexion / extension , varus / valgus tibial resection , and posterior tilt could be assessed in real time during bone resection of the distal femur and the proximal tibia by placing a paddle on the resection surface ( fig . 3 ) . after bone resection , lower limb alignment was assessed using the navigation system with a temporary implant or an inserted spacer . after implant fixation , the alignment was evaluated and corrected intraoperatively if needed . for the lps - flex mobile bearing knee , any osteophytes that could cause soft tissue tension were thoroughly removed and subperiosteal release of the medial and lateral collateral ligaments at their insertion sites was performed . flexion / extension gaps were measured intraoperatively using a gapper in several trial tests to confirm mediolateral stability prior to soft tissue balancing . for the lps - flex mobile bearing knee , tibial resection preceded femoral resection , whereas it was vice versa for the lps - flex fixed bearing knee . , active joint movement exercises were performed in tandem with passive exercises using cpm devices to facilitate rapid rom recovery . ksks , ksfs , womac score , vas for pain , and rom and maximal flexion angle before surgery and at the last follow - up were analyzed using an independent sample t - test . patient ability to kneel and sit cross - legged was assessed using the chi - square test . of the patients that had undergone tka by the same surgeon between january 2010 and june 2010 at our institution , 67 patients with a minimum follow - up of one year were randomly selected for this study . the implants used were the lps - flex mobile knee in 32 patients and lps - flex fixed knee in 34 patients . in six of the patients with bilateral tka , the mobile - bearing prosthesis was used on one side and the fixed - bearing on the other side . in all patients , an electromagnetic navigation system ( zimmer computer assisted solutions electromagnetic quad - sparing ) was used . the mean follow - up period was 14.5 months ( range , 12 to 18 months ) and the mean age of the patients was 67.5 years ( range , 60 to 78 years ) in the mobile - bearing group and 68.5 years ( range , 61 to 77 years ) in the fixed - bearing group . the mean preoperative rom was 118 in both groups and no notable intergroup difference could be found regarding flexion contracture ( table 1 ) . considering that the preoperative rom could affect the postoperative rom3,4 ) , we excluded patients who had 90 of rom preoperatively . there were no particular instructions for knee flexion exercises and the same rehabilitation protocol was prescribed in both groups . the clinical results were graded according to the knee society knee score ( ksks ) and knee society functional score ( ksfs ) . patient satisfaction and preference were assessed according to the western ontario and mcmaster universities arthritis index ( womac ) score5 ) , a questionnaire for measuring pain , stiffness , and function . a patient 's ability to kneel and sit cross - legged was assessed through interview or observation during follow - up . physical examination and goniometric measurement however , we used a radiographic measurement method devised by edwards et al.6 ) to reduce measurement errors and increase reproducibility . we measured the angle formed by lines drawn down the mid - shafts of the tibia and femur on the lateral radiographs taken with the knee in full extension and full flexion preoperatively , as well as at the final follow - up ( fig . radiographic assessment was done by the same surgeon three times per patient and the mean value was used for analysis . the mechanical axis deviation was measured as the difference in the angle between the femoral mechanical axis and the tibial mechanical axis on the weight bearing long leg anteroposterior radiographs taken preoperatively and postoperatively . the femoral and tibial component positions in the sagittal and coronal planes were assessed using the method of seon and song7 ) . the coronal inclination of the femoral component was measured as the medial angle between the mechanical axis and the bottom of the component on the anteroposterior radiographs and the sagittal inclination as the angle with the femoral shaft on the lateral views . the desired sagittal inclination was defined as 90. the coronal and sagittal tibial component positions were assessed in a similar manner . the bisecting line of the tibial shaft was used as a reference for sagittal tibial component positioning . the desired tibial component inclination was defined as 90 in the coronal plane and 86o in the sagittal plane ( fig . all the operations were performed by the same surgeon using a medial parapatella approach . to increase articulation curvature during flexion the anterior portion of the tibial component was removed to avoid interference with the patella and the posterior cam mechanism was modified to facilitate high flexion . the preoperative and postoperative mechanical axes and extent of bone resection could be evaluated using an electromagnetic navigation system in all cases . in particular , varus / valgus femoral resection , degree of flexion / extension , varus / valgus tibial resection , and posterior tilt could be assessed in real time during bone resection of the distal femur and the proximal tibia by placing a paddle on the resection surface ( fig . lower limb alignment was assessed using the navigation system with a temporary implant or an inserted spacer . after implant fixation , the alignment was evaluated and corrected intraoperatively if needed . for the lps - flex mobile bearing knee , any osteophytes that could cause soft tissue tension were thoroughly removed and subperiosteal release of the medial and lateral collateral ligaments at their insertion sites was performed . flexion / extension gaps were measured intraoperatively using a gapper in several trial tests to confirm mediolateral stability prior to soft tissue balancing . for the lps - flex mobile bearing knee , tibial resection preceded femoral resection , whereas it was vice versa for the lps - flex fixed bearing knee . , active joint movement exercises were performed in tandem with passive exercises using cpm devices to facilitate rapid rom recovery . ksks , ksfs , womac score , vas for pain , and rom and maximal flexion angle before surgery and at the last follow - up were analyzed using an independent sample t - test . patient ability to kneel and sit cross - legged was assessed using the chi - square test . there were no statistically significant intergroup differences in the ksks and ksfs ( p>0.05 ) . in the mobile - bearing group , the mean ksks and ksfs increased from 48.2 ( 48.26.4 ) preoperatively to 94.5 ( 94.53.2 ) at the last follow - up and from 45.3 ( 45.35.8 ) preoperatively to 93.8 ( 93.82.8 ) at the last follow - up , respectively . in the fixed - bearing group , the mean ksks and ksfs increased from 49.5 ( 49.55.6 ) preoperatively to 95.1 ( 95.12.8 ) at the last follow - up and from 46.9 ( 46.95.9 ) preoperatively to 94.2 ( 94.23.0 ) at the last follow - up ( p>0.05 ) . the mean womac score decreased in both groups , from 81.0 ( 81.05.5 ) preoperatively to 14.5 ( 14.51.6 ) at the last follow - up in the mobile - bearing group and from 82.5 ( 82.56.0 ) preoperatively to 15.2 ( 15.21.8 ) at the last follow - up in the fixed - bearing group ( p>0.05 ) . there were no notable intergroup differences in the mean vas pain score ( p>0.05 ) ( table 2 ) . the mean vas pain score improved from 7.8 ( 7.81.6 ) preoperatively to 1.5 ( 1.50.6 ) at the last follow - up in the mobile - bearing group and from 7.6 ( 7.61.5 ) preoperatively to 1.4 ( 1.40.5 ) at the last follow - up in the fixed - bearing group . significant intergroup differences were not identified in the ability to kneel and sit cross - legged through observation or interview during the follow - up ( p>0.05 ) ( table 3 ) . kneeling and cross - legged sitting was possible in 22 and 28 cases , respectively , in the mobile bearing group ( n=32 ) and in 24 and 29 cases , respectively , in the fixed - bearing group ( n=34 ) . no remarkable intergroup differences were noted in patients with 130 maximal flexion angle ( high flexion ) ( p>0.05 ) ( table 4 ) . the number of patients with 130 maximal flexion angle was 18 in the mobile - bearing group and 19 in the fixed - bearing group . kneeling and cross - legged sitting was possible in 15 and 18 cases , respectively , in patients with high flexion in the mobile - bearing group and in 16 and 18 cases , respectively , in patients with high flexion in the fixed - bearing group . on the lower limb alignment , the mechanical axis deviation was more varus in the fixed - bearing group ( 0.91.1 ) than in the mobile - bearing group ( 0.71.0 ) , but there was no significant difference between the groups . the coronal inclination of femoral component coronal and tibial component was 89.32.8 and 89.81.0 , respectively , in the fixed - bearing group and 89.63.2 and 89.31.2 , respectively , in the mobile - bearing group . the sagittal inclination of femoral component and tibial component was 88.51.5 and 86.61.4 in the fixed - bearing group and 88.51.8 and 86.22.0 in the mobile - bearing group . thus , the coronal and sagittal implant positioning was satisfactory in both groups , which showed no significant intergroup differences ( tables 5 , 6 ) . there were no statistically significant intergroup differences in rom and maximal flexion angle ( p>0.05 ) . the mean rom increased from 118.5 ( 118.55.8 ) preoperatively to 130.0 ( 130.03.8 ) at the last follow - up in the mobile - bearing group and from 118.0 ( 118.05.2 ) preoperatively to 129.5 ( 129.54.0 ) at the last follow - up in the fixed - bearing group . the mean maximal flexion angle increased from 122.0 ( 122.05.2 ) preoperatively to 131.1 ( 131.13.6 ) at the last follow - up in the mobile - bearing group and from 121.8 ( 121.85.0 ) preoperatively to 130.8 ( 130.83.3 ) at the last follow - up in the fixed - bearing group ( p>0.05 ) ( table 7 ) ( figs . 4 , 5 ) . polyethylene component wear , posterior instability , component loosening , or infection was not observed in any of the patients during the 1 year follow - up period . complications requiring revision surgery were not noted during the short - term follow - up period . there were no statistically significant intergroup differences in the ksks and ksfs ( p>0.05 ) . in the mobile - bearing group , the mean ksks and ksfs increased from 48.2 ( 48.26.4 ) preoperatively to 94.5 ( 94.53.2 ) at the last follow - up and from 45.3 ( 45.35.8 ) preoperatively to 93.8 ( 93.82.8 ) at the last follow - up , respectively . in the fixed - bearing group , the mean ksks and ksfs increased from 49.5 ( 49.55.6 ) preoperatively to 95.1 ( 95.12.8 ) at the last follow - up and from 46.9 ( 46.95.9 ) preoperatively to 94.2 ( 94.23.0 ) at the last follow - up ( p>0.05 ) . the mean womac score decreased in both groups , from 81.0 ( 81.05.5 ) preoperatively to 14.5 ( 14.51.6 ) at the last follow - up in the mobile - bearing group and from 82.5 ( 82.56.0 ) preoperatively to 15.2 ( 15.21.8 ) at the last follow - up in the fixed - bearing group ( p>0.05 ) . there were no notable intergroup differences in the mean vas pain score ( p>0.05 ) ( table 2 ) . the mean vas pain score improved from 7.8 ( 7.81.6 ) preoperatively to 1.5 ( 1.50.6 ) at the last follow - up in the mobile - bearing group and from 7.6 ( 7.61.5 ) preoperatively to 1.4 ( 1.40.5 ) at the last follow - up in the fixed - bearing group . significant intergroup differences were not identified in the ability to kneel and sit cross - legged through observation or interview during the follow - up ( p>0.05 ) ( table 3 ) . kneeling and cross - legged sitting was possible in 22 and 28 cases , respectively , in the mobile bearing group ( n=32 ) and in 24 and 29 cases , respectively , in the fixed - bearing group ( n=34 ) . no remarkable intergroup differences were noted in patients with 130 maximal flexion angle ( high flexion ) ( p>0.05 ) ( table 4 ) . the number of patients with 130 maximal flexion angle was 18 in the mobile - bearing group and 19 in the fixed - bearing group . kneeling and cross - legged sitting was possible in 15 and 18 cases , respectively , in patients with high flexion in the mobile - bearing group and in 16 and 18 cases , respectively , in patients with high flexion in the fixed - bearing group . on the lower limb alignment , the mechanical axis deviation was more varus in the fixed - bearing group ( 0.91.1 ) than in the mobile - bearing group ( 0.71.0 ) , but there was no significant difference between the groups . the coronal inclination of femoral component coronal and tibial component was 89.32.8 and 89.81.0 , respectively , in the fixed - bearing group and 89.63.2 and 89.31.2 , respectively , in the mobile - bearing group . the sagittal inclination of femoral component and tibial component was 88.51.5 and 86.61.4 in the fixed - bearing group and 88.51.8 and 86.22.0 in the mobile - bearing group . thus , the coronal and sagittal implant positioning was satisfactory in both groups , which showed no significant intergroup differences ( tables 5 , 6 ) . there were no statistically significant intergroup differences in rom and maximal flexion angle ( p>0.05 ) . the mean rom increased from 118.5 ( 118.55.8 ) preoperatively to 130.0 ( 130.03.8 ) at the last follow - up in the mobile - bearing group and from 118.0 ( 118.05.2 ) preoperatively to 129.5 ( 129.54.0 ) at the last follow - up in the fixed - bearing group . the mean maximal flexion angle increased from 122.0 ( 122.05.2 ) preoperatively to 131.1 ( 131.13.6 ) at the last follow - up in the mobile - bearing group and from 121.8 ( 121.85.0 ) preoperatively to 130.8 ( 130.83.3 ) at the last follow - up in the fixed - bearing group ( p>0.05 ) ( table 7 ) ( figs . 4 , 5 ) . polyethylene component wear , posterior instability , component loosening , or infection was not observed in any of the patients during the 1 year follow - up period . complications requiring revision surgery were not noted during the short - term follow - up period . the primary goal of tka is to alleviate pain but it is more favorable to restore normal knee function8 ) . postoperative rom is especially important for asian patients who frequently squat or sit in a cross - legged position9 ) . goodfellow and o'connor10 ) introduced a mobile - bearing knee system in 1976 to address component loosening and wear , the two most common issues of tka . the mechanism that hinders knee flexion remains unclear , but the ability to restore posterior femoral translation has been considered crucial for improving range of flexion after tka11,12 ) . a decrease in posterior femoral translation causes impingement of the posterior edge of the tibial component on the femoral shaft , thus constraining flexion motion11 - 13 ) . high flexion knee system was designed to increase posterior femoral translation for high flexion12,13 ) . we used the high - flexion lps - flex total knee system in this study . an additional 2 mm posterior femoral bone resection was performed to extend the posterior condyle of the femoral component by 2 mm and increase the articular contact area and thus posterior femoral translation for high flexion . in addition , the stress on the polyethylene implant was reduced , which contributed to the low incidence of component wearing . the lps - flex mobile system is a mobile bearing implant designed to allow for a total of 50 of axial rotation ( internal rotation , 25 and external rotation , 25 ) of the polyethylene insert in the tibial component during knee flexion . therefore , we used a technique similar to the ' gap technique ' for soft tissue balancing in the mobile - bearing group : after tibial resection , internal / external stability was confirmed by measuring the flexion / extension gaps in several trial tests . in contrast , ' measured resection technique ' where femoral resection precedes tibial resection , was used in the fixed - bearing group . in our opinion , comparisons of the postoperative lower limb alignment and coronal / sagittal implant positions between the groups should be performed in further studies . therefore , a range of errors could be identified at every step of the surgery and the surgeon could give real time feedback based on surgical experience and intraoperative data to achieve ideal values14 ) . there are many studies showing that navigation - assisted tka produces more satisfactory results than the established tka in terms of implant positioning and lower limb alignment that are associated with long - term longevity15 - 18 ) . mobile - bearing knee prostheses are more effective in reducing fatigue wear because the enlarged contact surface causes less stresses compared to fixed - bearing ones19 ) and in lowering stresses on the bone - implant interface and risk of loosening , which potentially increases implant longevity10,20 ) . another advantage is that the motion between the tibial component and the polyethylene insert can be adjusted to minimize backside wear21 ) . clinical results of tka using mobile - bearing and fixed - bearing implants have been addressed in various studies . woolson et al.22 ) reported that there were no significant differences between the mobile - bearing implant group ( 57 cases ) and fixed - bearing implant group ( 45 cases ) in terms of the ksks , ksfs , pain score , and postoperative flexion angle . ranawat et al.23 ) used mobile - bearing and fixed - bearing implants during tka in 26 patients and found no notable differences between the two implants in terms of the ksks and ksfs , postoperative rom and maximal flexion angle , knee pain , and patient satisfaction . lampe et al.24 ) used navigation in 52 cases of mobile - bearing knee replacement and 48 cases of fixed - bearing knee replacement . at 2 years postoperatively , no significant differences were noted in the ksks and ksfs , oxford knee score , and rom . kim et al.25 ) performed 116 cases of bilateral tkas using a mobile - bearing implant and a fixed - bearing implant on each side and found no significant differences between the two types of implants in terms of the rom , ksks , pain score , patient satisfaction , and polyethylene wear . in contrast , price et al.26 ) reported that the ksks and pain score at 1 year after surgery were higher , albeit slightly , in the mobile - bearing group although the rom was similar between the groups with 105.3. luring et al.27 ) reported that the mediolateral stability was higher in the mobile - bearing group after pcl retaining tka . sohn et al.28 ) followed 32 patients that underwent bilateral tka using a mobile - bearing implant and a fixed - bearing implant on each side and noted that the postoperative subjective satisfaction and preference evaluated using the womac score was higher in the mobile - bearing group although the two implants did not result in differences in the ksks , ksfs , and postoperative rom . in this study , significant intergroup differences were not observed in the ksks and ksfs , womac score , rom and maximal flexion angle , and possibility of kneeling and sitting cross - legged ( p>0.05 ) . the radiographic results showed that there were no notable intergroup differences in the postoperative mechanical axis deviation and the femoral and tibial component positions in the sagittal and coronal planes ( p>0.05 ) . however , we think these results should be confirmed by further studies with longer follow - up periods . the clinical results of high - flexion tka were satisfactory in both the mobile - bearing and fixed - bearing group . there were no significant intergroup differences regarding the clinical results ( kss , ksfs , and womac score ) , radiological results ( rom and maximal flexion angle ) , and possibility of kneeling and sitting cross - legged . however , these results should be verified in long - term follow - up studies that address the longevity of the implants .

purposewe compared and analyzed the short term results of high flexion total knee arthroplasty ( tka ) with mobile - bearing and fixed bearing designs.materials and methodswe studied 32 patients that had undergone tka with lps - flex mobile and 34 patients with lps - flex fixed using an electromagnetic navigation system between january 2010 and june 2010 , and were followed up for at least 1 year.resultsknee society functional score ( ksfs ) and knee society knee score ( ksks ) of the mobile - bearing group were 94.5 and 93.8 points , respectively , and were 48.2 and 45.3 points preoperatively , whereas those of the fixed - bearing group were 95.1 and 94.2 points , respectively , and were 49.5 and 46.9 points preoperatively . postoperative mechanical axis deviation and implant position of the femoral and tibial component both on the coronal and sigittal planes showed no significant differences between the two groups . range of motion ( rom ) and maximal flexion angle ( mfa ) of the knee joint also showed no significant differences between the two groups . the possibility of crossed - legged sitting and kneeling position also showed no significant differences between the two groups.conclusionsclinical and radiologic parameters , rom and mfa of knee joints showed no significant differences in both the groups , but long term follow - up results may be necessary , including survival rate .

Introduction Materials and Methods 1. Materials 2. Clinical Assessment 3. Radiographic Assessment 4. Surgical Technique 5. Rehabilitation 6. Statistical Analysis Results 1. Clinical Results 2. Kneeling and Cross-Legged Sitting 3. Radiographic Results 4. ROM and Maximum Knee Flexion 5. Complications Discussion Conclusions

the goals of total knee arthroplasty ( tka ) are to relieve pain , maintain knee function and kinematics , and restore stability and range of motion ( rom ) to facilitate daily activities . in this study , we analyzed the results of high flexion tka using one of the most recently developed navigation systems , the electromagnetic navigation system , to compare the influence of two different implant designs ( mobile - bearing and fixed bearing designs ) on rom including maximal flexion angle and clinical and radiographic results . the mobile - bearing implant used was nexgen lps - flex mobile knee ( nexgen , legacy posterior - stabilized flexion mobile , zimmer , warsaw , in , usa ) whereas the fixed - bearing implant was nexgen lps - flex fixed knee ( nexgen , legacy posterior - stabilized flexion fixed , zimmer ) . of the patients that had undergone tka by the same surgeon between january 2010 and june 2010 at our institution , 67 patients with a minimum follow - up of one year were randomly selected for this study . the implants used were the lps - flex mobile knee in 32 patients and lps - flex fixed knee in 34 patients . in six of the patients with bilateral tka , the mobile - bearing prosthesis was used on one side and the fixed - bearing on the other side . the mean follow - up period was 14.5 months ( range , 12 to 18 months ) and the mean age of the patients was 67.5 years ( range , 60 to 78 years ) in the mobile - bearing group and 68.5 years ( range , 61 to 77 years ) in the fixed - bearing group . the clinical results were graded according to the knee society knee score ( ksks ) and knee society functional score ( ksfs ) . we measured the angle formed by lines drawn down the mid - shafts of the tibia and femur on the lateral radiographs taken with the knee in full extension and full flexion preoperatively , as well as at the final follow - up ( fig . the mechanical axis deviation was measured as the difference in the angle between the femoral mechanical axis and the tibial mechanical axis on the weight bearing long leg anteroposterior radiographs taken preoperatively and postoperatively . the femoral and tibial component positions in the sagittal and coronal planes were assessed using the method of seon and song7 ) . the coronal inclination of the femoral component was measured as the medial angle between the mechanical axis and the bottom of the component on the anteroposterior radiographs and the sagittal inclination as the angle with the femoral shaft on the lateral views . the preoperative and postoperative mechanical axes and extent of bone resection could be evaluated using an electromagnetic navigation system in all cases . for the lps - flex mobile bearing knee , any osteophytes that could cause soft tissue tension were thoroughly removed and subperiosteal release of the medial and lateral collateral ligaments at their insertion sites was performed . for the lps - flex mobile bearing knee , tibial resection preceded femoral resection , whereas it was vice versa for the lps - flex fixed bearing knee . ksks , ksfs , womac score , vas for pain , and rom and maximal flexion angle before surgery and at the last follow - up were analyzed using an independent sample t - test . of the patients that had undergone tka by the same surgeon between january 2010 and june 2010 at our institution , 67 patients with a minimum follow - up of one year were randomly selected for this study . the implants used were the lps - flex mobile knee in 32 patients and lps - flex fixed knee in 34 patients . in six of the patients with bilateral tka , the mobile - bearing prosthesis was used on one side and the fixed - bearing on the other side . the mean follow - up period was 14.5 months ( range , 12 to 18 months ) and the mean age of the patients was 67.5 years ( range , 60 to 78 years ) in the mobile - bearing group and 68.5 years ( range , 61 to 77 years ) in the fixed - bearing group . the clinical results were graded according to the knee society knee score ( ksks ) and knee society functional score ( ksfs ) . we measured the angle formed by lines drawn down the mid - shafts of the tibia and femur on the lateral radiographs taken with the knee in full extension and full flexion preoperatively , as well as at the final follow - up ( fig . the mechanical axis deviation was measured as the difference in the angle between the femoral mechanical axis and the tibial mechanical axis on the weight bearing long leg anteroposterior radiographs taken preoperatively and postoperatively . the femoral and tibial component positions in the sagittal and coronal planes were assessed using the method of seon and song7 ) . the coronal inclination of the femoral component was measured as the medial angle between the mechanical axis and the bottom of the component on the anteroposterior radiographs and the sagittal inclination as the angle with the femoral shaft on the lateral views . the desired sagittal inclination was defined as 90. the coronal and sagittal tibial component positions were assessed in a similar manner . the preoperative and postoperative mechanical axes and extent of bone resection could be evaluated using an electromagnetic navigation system in all cases . for the lps - flex mobile bearing knee , any osteophytes that could cause soft tissue tension were thoroughly removed and subperiosteal release of the medial and lateral collateral ligaments at their insertion sites was performed . for the lps - flex mobile bearing knee , tibial resection preceded femoral resection , whereas it was vice versa for the lps - flex fixed bearing knee . ksks , ksfs , womac score , vas for pain , and rom and maximal flexion angle before surgery and at the last follow - up were analyzed using an independent sample t - test . in the mobile - bearing group , the mean ksks and ksfs increased from 48.2 ( 48.26.4 ) preoperatively to 94.5 ( 94.53.2 ) at the last follow - up and from 45.3 ( 45.35.8 ) preoperatively to 93.8 ( 93.82.8 ) at the last follow - up , respectively . in the fixed - bearing group , the mean ksks and ksfs increased from 49.5 ( 49.55.6 ) preoperatively to 95.1 ( 95.12.8 ) at the last follow - up and from 46.9 ( 46.95.9 ) preoperatively to 94.2 ( 94.23.0 ) at the last follow - up ( p>0.05 ) . the mean womac score decreased in both groups , from 81.0 ( 81.05.5 ) preoperatively to 14.5 ( 14.51.6 ) at the last follow - up in the mobile - bearing group and from 82.5 ( 82.56.0 ) preoperatively to 15.2 ( 15.21.8 ) at the last follow - up in the fixed - bearing group ( p>0.05 ) . the mean vas pain score improved from 7.8 ( 7.81.6 ) preoperatively to 1.5 ( 1.50.6 ) at the last follow - up in the mobile - bearing group and from 7.6 ( 7.61.5 ) preoperatively to 1.4 ( 1.40.5 ) at the last follow - up in the fixed - bearing group . significant intergroup differences were not identified in the ability to kneel and sit cross - legged through observation or interview during the follow - up ( p>0.05 ) ( table 3 ) . kneeling and cross - legged sitting was possible in 22 and 28 cases , respectively , in the mobile bearing group ( n=32 ) and in 24 and 29 cases , respectively , in the fixed - bearing group ( n=34 ) . no remarkable intergroup differences were noted in patients with 130 maximal flexion angle ( high flexion ) ( p>0.05 ) ( table 4 ) . the number of patients with 130 maximal flexion angle was 18 in the mobile - bearing group and 19 in the fixed - bearing group . kneeling and cross - legged sitting was possible in 15 and 18 cases , respectively , in patients with high flexion in the mobile - bearing group and in 16 and 18 cases , respectively , in patients with high flexion in the fixed - bearing group . on the lower limb alignment , the mechanical axis deviation was more varus in the fixed - bearing group ( 0.91.1 ) than in the mobile - bearing group ( 0.71.0 ) , but there was no significant difference between the groups . the coronal inclination of femoral component coronal and tibial component was 89.32.8 and 89.81.0 , respectively , in the fixed - bearing group and 89.63.2 and 89.31.2 , respectively , in the mobile - bearing group . the sagittal inclination of femoral component and tibial component was 88.51.5 and 86.61.4 in the fixed - bearing group and 88.51.8 and 86.22.0 in the mobile - bearing group . thus , the coronal and sagittal implant positioning was satisfactory in both groups , which showed no significant intergroup differences ( tables 5 , 6 ) . there were no statistically significant intergroup differences in rom and maximal flexion angle ( p>0.05 ) . the mean rom increased from 118.5 ( 118.55.8 ) preoperatively to 130.0 ( 130.03.8 ) at the last follow - up in the mobile - bearing group and from 118.0 ( 118.05.2 ) preoperatively to 129.5 ( 129.54.0 ) at the last follow - up in the fixed - bearing group . the mean maximal flexion angle increased from 122.0 ( 122.05.2 ) preoperatively to 131.1 ( 131.13.6 ) at the last follow - up in the mobile - bearing group and from 121.8 ( 121.85.0 ) preoperatively to 130.8 ( 130.83.3 ) at the last follow - up in the fixed - bearing group ( p>0.05 ) ( table 7 ) ( figs . polyethylene component wear , posterior instability , component loosening , or infection was not observed in any of the patients during the 1 year follow - up period . complications requiring revision surgery were not noted during the short - term follow - up period . in the mobile - bearing group , the mean ksks and ksfs increased from 48.2 ( 48.26.4 ) preoperatively to 94.5 ( 94.53.2 ) at the last follow - up and from 45.3 ( 45.35.8 ) preoperatively to 93.8 ( 93.82.8 ) at the last follow - up , respectively . in the fixed - bearing group , the mean ksks and ksfs increased from 49.5 ( 49.55.6 ) preoperatively to 95.1 ( 95.12.8 ) at the last follow - up and from 46.9 ( 46.95.9 ) preoperatively to 94.2 ( 94.23.0 ) at the last follow - up ( p>0.05 ) . the mean womac score decreased in both groups , from 81.0 ( 81.05.5 ) preoperatively to 14.5 ( 14.51.6 ) at the last follow - up in the mobile - bearing group and from 82.5 ( 82.56.0 ) preoperatively to 15.2 ( 15.21.8 ) at the last follow - up in the fixed - bearing group ( p>0.05 ) . the mean vas pain score improved from 7.8 ( 7.81.6 ) preoperatively to 1.5 ( 1.50.6 ) at the last follow - up in the mobile - bearing group and from 7.6 ( 7.61.5 ) preoperatively to 1.4 ( 1.40.5 ) at the last follow - up in the fixed - bearing group . significant intergroup differences were not identified in the ability to kneel and sit cross - legged through observation or interview during the follow - up ( p>0.05 ) ( table 3 ) . kneeling and cross - legged sitting was possible in 22 and 28 cases , respectively , in the mobile bearing group ( n=32 ) and in 24 and 29 cases , respectively , in the fixed - bearing group ( n=34 ) . no remarkable intergroup differences were noted in patients with 130 maximal flexion angle ( high flexion ) ( p>0.05 ) ( table 4 ) . the number of patients with 130 maximal flexion angle was 18 in the mobile - bearing group and 19 in the fixed - bearing group . kneeling and cross - legged sitting was possible in 15 and 18 cases , respectively , in patients with high flexion in the mobile - bearing group and in 16 and 18 cases , respectively , in patients with high flexion in the fixed - bearing group . on the lower limb alignment , the mechanical axis deviation was more varus in the fixed - bearing group ( 0.91.1 ) than in the mobile - bearing group ( 0.71.0 ) , but there was no significant difference between the groups . the coronal inclination of femoral component coronal and tibial component was 89.32.8 and 89.81.0 , respectively , in the fixed - bearing group and 89.63.2 and 89.31.2 , respectively , in the mobile - bearing group . the sagittal inclination of femoral component and tibial component was 88.51.5 and 86.61.4 in the fixed - bearing group and 88.51.8 and 86.22.0 in the mobile - bearing group . thus , the coronal and sagittal implant positioning was satisfactory in both groups , which showed no significant intergroup differences ( tables 5 , 6 ) . there were no statistically significant intergroup differences in rom and maximal flexion angle ( p>0.05 ) . the mean rom increased from 118.5 ( 118.55.8 ) preoperatively to 130.0 ( 130.03.8 ) at the last follow - up in the mobile - bearing group and from 118.0 ( 118.05.2 ) preoperatively to 129.5 ( 129.54.0 ) at the last follow - up in the fixed - bearing group . the mean maximal flexion angle increased from 122.0 ( 122.05.2 ) preoperatively to 131.1 ( 131.13.6 ) at the last follow - up in the mobile - bearing group and from 121.8 ( 121.85.0 ) preoperatively to 130.8 ( 130.83.3 ) at the last follow - up in the fixed - bearing group ( p>0.05 ) ( table 7 ) ( figs . polyethylene component wear , posterior instability , component loosening , or infection was not observed in any of the patients during the 1 year follow - up period . complications requiring revision surgery were not noted during the short - term follow - up period . goodfellow and o'connor10 ) introduced a mobile - bearing knee system in 1976 to address component loosening and wear , the two most common issues of tka . a decrease in posterior femoral translation causes impingement of the posterior edge of the tibial component on the femoral shaft , thus constraining flexion motion11 - 13 ) . the lps - flex mobile system is a mobile bearing implant designed to allow for a total of 50 of axial rotation ( internal rotation , 25 and external rotation , 25 ) of the polyethylene insert in the tibial component during knee flexion . therefore , we used a technique similar to the ' gap technique ' for soft tissue balancing in the mobile - bearing group : after tibial resection , internal / external stability was confirmed by measuring the flexion / extension gaps in several trial tests . in contrast , ' measured resection technique ' where femoral resection precedes tibial resection , was used in the fixed - bearing group . in our opinion , comparisons of the postoperative lower limb alignment and coronal / sagittal implant positions between the groups should be performed in further studies . mobile - bearing knee prostheses are more effective in reducing fatigue wear because the enlarged contact surface causes less stresses compared to fixed - bearing ones19 ) and in lowering stresses on the bone - implant interface and risk of loosening , which potentially increases implant longevity10,20 ) . clinical results of tka using mobile - bearing and fixed - bearing implants have been addressed in various studies . woolson et al.22 ) reported that there were no significant differences between the mobile - bearing implant group ( 57 cases ) and fixed - bearing implant group ( 45 cases ) in terms of the ksks , ksfs , pain score , and postoperative flexion angle . ranawat et al.23 ) used mobile - bearing and fixed - bearing implants during tka in 26 patients and found no notable differences between the two implants in terms of the ksks and ksfs , postoperative rom and maximal flexion angle , knee pain , and patient satisfaction . at 2 years postoperatively , no significant differences were noted in the ksks and ksfs , oxford knee score , and rom . kim et al.25 ) performed 116 cases of bilateral tkas using a mobile - bearing implant and a fixed - bearing implant on each side and found no significant differences between the two types of implants in terms of the rom , ksks , pain score , patient satisfaction , and polyethylene wear . in contrast , price et al.26 ) reported that the ksks and pain score at 1 year after surgery were higher , albeit slightly , in the mobile - bearing group although the rom was similar between the groups with 105.3. luring et al.27 ) reported that the mediolateral stability was higher in the mobile - bearing group after pcl retaining tka . sohn et al.28 ) followed 32 patients that underwent bilateral tka using a mobile - bearing implant and a fixed - bearing implant on each side and noted that the postoperative subjective satisfaction and preference evaluated using the womac score was higher in the mobile - bearing group although the two implants did not result in differences in the ksks , ksfs , and postoperative rom . in this study , significant intergroup differences were not observed in the ksks and ksfs , womac score , rom and maximal flexion angle , and possibility of kneeling and sitting cross - legged ( p>0.05 ) . the radiographic results showed that there were no notable intergroup differences in the postoperative mechanical axis deviation and the femoral and tibial component positions in the sagittal and coronal planes ( p>0.05 ) . the clinical results of high - flexion tka were satisfactory in both the mobile - bearing and fixed - bearing group . there were no significant intergroup differences regarding the clinical results ( kss , ksfs , and womac score ) , radiological results ( rom and maximal flexion angle ) , and possibility of kneeling and sitting cross - legged . however , these results should be verified in long - term follow - up studies that address the longevity of the implants .

the goals of total knee arthroplasty ( tka ) are to relieve pain , maintain knee function and kinematics , and restore stability and range of motion ( rom ) to facilitate daily activities . factors that can be associated with rom include the cause of disease , preoperative joint deformity and rom , age , gender , surgical technique , implant fixation strength , postoperative rehabilitation , and implant design1 ) . since first introduced in tka for optimal component positioning and lower limb alignment , navigation systems have been improved with rapid development of computer technology to allow for soft tissue balancing and flexion - extension gap balancing , and accordingly have contributed to the accuracy of tka2 ) . in this study , we analyzed the results of high flexion tka using one of the most recently developed navigation systems , the electromagnetic navigation system , to compare the influence of two different implant designs ( mobile - bearing and fixed bearing designs ) on rom including maximal flexion angle and clinical and radiographic results . the mobile - bearing implant used was nexgen lps - flex mobile knee ( nexgen , legacy posterior - stabilized flexion mobile , zimmer , warsaw , in , usa ) whereas the fixed - bearing implant was nexgen lps - flex fixed knee ( nexgen , legacy posterior - stabilized flexion fixed , zimmer ) . of the patients that had undergone tka by the same surgeon between january 2010 and june 2010 at our institution , 67 patients with a minimum follow - up of one year were randomly selected for this study . the implants used were the lps - flex mobile knee in 32 patients and lps - flex fixed knee in 34 patients . in six of the patients with bilateral tka , the mobile - bearing prosthesis was used on one side and the fixed - bearing on the other side . the mean follow - up period was 14.5 months ( range , 12 to 18 months ) and the mean age of the patients was 67.5 years ( range , 60 to 78 years ) in the mobile - bearing group and 68.5 years ( range , 61 to 77 years ) in the fixed - bearing group . the mean preoperative rom was 118 in both groups and no notable intergroup difference could be found regarding flexion contracture ( table 1 ) . considering that the preoperative rom could affect the postoperative rom3,4 ) , we excluded patients who had 90 of rom preoperatively . the clinical results were graded according to the knee society knee score ( ksks ) and knee society functional score ( ksfs ) . patient satisfaction and preference were assessed according to the western ontario and mcmaster universities arthritis index ( womac ) score5 ) , a questionnaire for measuring pain , stiffness , and function . we measured the angle formed by lines drawn down the mid - shafts of the tibia and femur on the lateral radiographs taken with the knee in full extension and full flexion preoperatively , as well as at the final follow - up ( fig . the mechanical axis deviation was measured as the difference in the angle between the femoral mechanical axis and the tibial mechanical axis on the weight bearing long leg anteroposterior radiographs taken preoperatively and postoperatively . the femoral and tibial component positions in the sagittal and coronal planes were assessed using the method of seon and song7 ) . the coronal inclination of the femoral component was measured as the medial angle between the mechanical axis and the bottom of the component on the anteroposterior radiographs and the sagittal inclination as the angle with the femoral shaft on the lateral views . the anterior portion of the tibial component was removed to avoid interference with the patella and the posterior cam mechanism was modified to facilitate high flexion . in particular , varus / valgus femoral resection , degree of flexion / extension , varus / valgus tibial resection , and posterior tilt could be assessed in real time during bone resection of the distal femur and the proximal tibia by placing a paddle on the resection surface ( fig . for the lps - flex mobile bearing knee , any osteophytes that could cause soft tissue tension were thoroughly removed and subperiosteal release of the medial and lateral collateral ligaments at their insertion sites was performed . for the lps - flex mobile bearing knee , tibial resection preceded femoral resection , whereas it was vice versa for the lps - flex fixed bearing knee . ksks , ksfs , womac score , vas for pain , and rom and maximal flexion angle before surgery and at the last follow - up were analyzed using an independent sample t - test . of the patients that had undergone tka by the same surgeon between january 2010 and june 2010 at our institution , 67 patients with a minimum follow - up of one year were randomly selected for this study . the implants used were the lps - flex mobile knee in 32 patients and lps - flex fixed knee in 34 patients . in six of the patients with bilateral tka , the mobile - bearing prosthesis was used on one side and the fixed - bearing on the other side . the mean follow - up period was 14.5 months ( range , 12 to 18 months ) and the mean age of the patients was 67.5 years ( range , 60 to 78 years ) in the mobile - bearing group and 68.5 years ( range , 61 to 77 years ) in the fixed - bearing group . the mean preoperative rom was 118 in both groups and no notable intergroup difference could be found regarding flexion contracture ( table 1 ) . patient satisfaction and preference were assessed according to the western ontario and mcmaster universities arthritis index ( womac ) score5 ) , a questionnaire for measuring pain , stiffness , and function . we measured the angle formed by lines drawn down the mid - shafts of the tibia and femur on the lateral radiographs taken with the knee in full extension and full flexion preoperatively , as well as at the final follow - up ( fig . the mechanical axis deviation was measured as the difference in the angle between the femoral mechanical axis and the tibial mechanical axis on the weight bearing long leg anteroposterior radiographs taken preoperatively and postoperatively . the femoral and tibial component positions in the sagittal and coronal planes were assessed using the method of seon and song7 ) . the coronal inclination of the femoral component was measured as the medial angle between the mechanical axis and the bottom of the component on the anteroposterior radiographs and the sagittal inclination as the angle with the femoral shaft on the lateral views . to increase articulation curvature during flexion the anterior portion of the tibial component was removed to avoid interference with the patella and the posterior cam mechanism was modified to facilitate high flexion . in particular , varus / valgus femoral resection , degree of flexion / extension , varus / valgus tibial resection , and posterior tilt could be assessed in real time during bone resection of the distal femur and the proximal tibia by placing a paddle on the resection surface ( fig . for the lps - flex mobile bearing knee , any osteophytes that could cause soft tissue tension were thoroughly removed and subperiosteal release of the medial and lateral collateral ligaments at their insertion sites was performed . for the lps - flex mobile bearing knee , tibial resection preceded femoral resection , whereas it was vice versa for the lps - flex fixed bearing knee . ksks , ksfs , womac score , vas for pain , and rom and maximal flexion angle before surgery and at the last follow - up were analyzed using an independent sample t - test . there were no statistically significant intergroup differences in the ksks and ksfs ( p>0.05 ) . in the mobile - bearing group , the mean ksks and ksfs increased from 48.2 ( 48.26.4 ) preoperatively to 94.5 ( 94.53.2 ) at the last follow - up and from 45.3 ( 45.35.8 ) preoperatively to 93.8 ( 93.82.8 ) at the last follow - up , respectively . in the fixed - bearing group , the mean ksks and ksfs increased from 49.5 ( 49.55.6 ) preoperatively to 95.1 ( 95.12.8 ) at the last follow - up and from 46.9 ( 46.95.9 ) preoperatively to 94.2 ( 94.23.0 ) at the last follow - up ( p>0.05 ) . the mean womac score decreased in both groups , from 81.0 ( 81.05.5 ) preoperatively to 14.5 ( 14.51.6 ) at the last follow - up in the mobile - bearing group and from 82.5 ( 82.56.0 ) preoperatively to 15.2 ( 15.21.8 ) at the last follow - up in the fixed - bearing group ( p>0.05 ) . there were no notable intergroup differences in the mean vas pain score ( p>0.05 ) ( table 2 ) . the mean vas pain score improved from 7.8 ( 7.81.6 ) preoperatively to 1.5 ( 1.50.6 ) at the last follow - up in the mobile - bearing group and from 7.6 ( 7.61.5 ) preoperatively to 1.4 ( 1.40.5 ) at the last follow - up in the fixed - bearing group . significant intergroup differences were not identified in the ability to kneel and sit cross - legged through observation or interview during the follow - up ( p>0.05 ) ( table 3 ) . kneeling and cross - legged sitting was possible in 22 and 28 cases , respectively , in the mobile bearing group ( n=32 ) and in 24 and 29 cases , respectively , in the fixed - bearing group ( n=34 ) . kneeling and cross - legged sitting was possible in 15 and 18 cases , respectively , in patients with high flexion in the mobile - bearing group and in 16 and 18 cases , respectively , in patients with high flexion in the fixed - bearing group . on the lower limb alignment , the mechanical axis deviation was more varus in the fixed - bearing group ( 0.91.1 ) than in the mobile - bearing group ( 0.71.0 ) , but there was no significant difference between the groups . the coronal inclination of femoral component coronal and tibial component was 89.32.8 and 89.81.0 , respectively , in the fixed - bearing group and 89.63.2 and 89.31.2 , respectively , in the mobile - bearing group . the sagittal inclination of femoral component and tibial component was 88.51.5 and 86.61.4 in the fixed - bearing group and 88.51.8 and 86.22.0 in the mobile - bearing group . thus , the coronal and sagittal implant positioning was satisfactory in both groups , which showed no significant intergroup differences ( tables 5 , 6 ) . the mean rom increased from 118.5 ( 118.55.8 ) preoperatively to 130.0 ( 130.03.8 ) at the last follow - up in the mobile - bearing group and from 118.0 ( 118.05.2 ) preoperatively to 129.5 ( 129.54.0 ) at the last follow - up in the fixed - bearing group . the mean maximal flexion angle increased from 122.0 ( 122.05.2 ) preoperatively to 131.1 ( 131.13.6 ) at the last follow - up in the mobile - bearing group and from 121.8 ( 121.85.0 ) preoperatively to 130.8 ( 130.83.3 ) at the last follow - up in the fixed - bearing group ( p>0.05 ) ( table 7 ) ( figs . polyethylene component wear , posterior instability , component loosening , or infection was not observed in any of the patients during the 1 year follow - up period . in the mobile - bearing group , the mean ksks and ksfs increased from 48.2 ( 48.26.4 ) preoperatively to 94.5 ( 94.53.2 ) at the last follow - up and from 45.3 ( 45.35.8 ) preoperatively to 93.8 ( 93.82.8 ) at the last follow - up , respectively . in the fixed - bearing group , the mean ksks and ksfs increased from 49.5 ( 49.55.6 ) preoperatively to 95.1 ( 95.12.8 ) at the last follow - up and from 46.9 ( 46.95.9 ) preoperatively to 94.2 ( 94.23.0 ) at the last follow - up ( p>0.05 ) . the mean womac score decreased in both groups , from 81.0 ( 81.05.5 ) preoperatively to 14.5 ( 14.51.6 ) at the last follow - up in the mobile - bearing group and from 82.5 ( 82.56.0 ) preoperatively to 15.2 ( 15.21.8 ) at the last follow - up in the fixed - bearing group ( p>0.05 ) . there were no notable intergroup differences in the mean vas pain score ( p>0.05 ) ( table 2 ) . the mean vas pain score improved from 7.8 ( 7.81.6 ) preoperatively to 1.5 ( 1.50.6 ) at the last follow - up in the mobile - bearing group and from 7.6 ( 7.61.5 ) preoperatively to 1.4 ( 1.40.5 ) at the last follow - up in the fixed - bearing group . significant intergroup differences were not identified in the ability to kneel and sit cross - legged through observation or interview during the follow - up ( p>0.05 ) ( table 3 ) . kneeling and cross - legged sitting was possible in 22 and 28 cases , respectively , in the mobile bearing group ( n=32 ) and in 24 and 29 cases , respectively , in the fixed - bearing group ( n=34 ) . kneeling and cross - legged sitting was possible in 15 and 18 cases , respectively , in patients with high flexion in the mobile - bearing group and in 16 and 18 cases , respectively , in patients with high flexion in the fixed - bearing group . on the lower limb alignment , the mechanical axis deviation was more varus in the fixed - bearing group ( 0.91.1 ) than in the mobile - bearing group ( 0.71.0 ) , but there was no significant difference between the groups . the coronal inclination of femoral component coronal and tibial component was 89.32.8 and 89.81.0 , respectively , in the fixed - bearing group and 89.63.2 and 89.31.2 , respectively , in the mobile - bearing group . the sagittal inclination of femoral component and tibial component was 88.51.5 and 86.61.4 in the fixed - bearing group and 88.51.8 and 86.22.0 in the mobile - bearing group . the mean rom increased from 118.5 ( 118.55.8 ) preoperatively to 130.0 ( 130.03.8 ) at the last follow - up in the mobile - bearing group and from 118.0 ( 118.05.2 ) preoperatively to 129.5 ( 129.54.0 ) at the last follow - up in the fixed - bearing group . the mean maximal flexion angle increased from 122.0 ( 122.05.2 ) preoperatively to 131.1 ( 131.13.6 ) at the last follow - up in the mobile - bearing group and from 121.8 ( 121.85.0 ) preoperatively to 130.8 ( 130.83.3 ) at the last follow - up in the fixed - bearing group ( p>0.05 ) ( table 7 ) ( figs . polyethylene component wear , posterior instability , component loosening , or infection was not observed in any of the patients during the 1 year follow - up period . the lps - flex mobile system is a mobile bearing implant designed to allow for a total of 50 of axial rotation ( internal rotation , 25 and external rotation , 25 ) of the polyethylene insert in the tibial component during knee flexion . therefore , we used a technique similar to the ' gap technique ' for soft tissue balancing in the mobile - bearing group : after tibial resection , internal / external stability was confirmed by measuring the flexion / extension gaps in several trial tests . there are many studies showing that navigation - assisted tka produces more satisfactory results than the established tka in terms of implant positioning and lower limb alignment that are associated with long - term longevity15 - 18 ) . mobile - bearing knee prostheses are more effective in reducing fatigue wear because the enlarged contact surface causes less stresses compared to fixed - bearing ones19 ) and in lowering stresses on the bone - implant interface and risk of loosening , which potentially increases implant longevity10,20 ) . woolson et al.22 ) reported that there were no significant differences between the mobile - bearing implant group ( 57 cases ) and fixed - bearing implant group ( 45 cases ) in terms of the ksks , ksfs , pain score , and postoperative flexion angle . ranawat et al.23 ) used mobile - bearing and fixed - bearing implants during tka in 26 patients and found no notable differences between the two implants in terms of the ksks and ksfs , postoperative rom and maximal flexion angle , knee pain , and patient satisfaction . kim et al.25 ) performed 116 cases of bilateral tkas using a mobile - bearing implant and a fixed - bearing implant on each side and found no significant differences between the two types of implants in terms of the rom , ksks , pain score , patient satisfaction , and polyethylene wear . in contrast , price et al.26 ) reported that the ksks and pain score at 1 year after surgery were higher , albeit slightly , in the mobile - bearing group although the rom was similar between the groups with 105.3. luring et al.27 ) reported that the mediolateral stability was higher in the mobile - bearing group after pcl retaining tka . sohn et al.28 ) followed 32 patients that underwent bilateral tka using a mobile - bearing implant and a fixed - bearing implant on each side and noted that the postoperative subjective satisfaction and preference evaluated using the womac score was higher in the mobile - bearing group although the two implants did not result in differences in the ksks , ksfs , and postoperative rom . in this study , significant intergroup differences were not observed in the ksks and ksfs , womac score , rom and maximal flexion angle , and possibility of kneeling and sitting cross - legged ( p>0.05 ) . the radiographic results showed that there were no notable intergroup differences in the postoperative mechanical axis deviation and the femoral and tibial component positions in the sagittal and coronal planes ( p>0.05 ) . there were no significant intergroup differences regarding the clinical results ( kss , ksfs , and womac score ) , radiological results ( rom and maximal flexion angle ) , and possibility of kneeling and sitting cross - legged .

autophagy , or the process of degradation of intracellular components in lysosomes , has been traditionally linked to cellular energy balance and to the cellular nutritional status [ 1 , 2 ] . in fact , although during the recent revival of the autophagic process , most of the emphasis has been placed on its role in other cellular functions such as cellular quality control , remodeling , or cell defense , the first descriptions of the autophagic process in the early 1960s already stated that conditions such as starvation lead to its activation [ 35 ] . these early studies proposed that autophagic activation during starvation was necessary to maintain the cellular energetic balance . later studies in yeast , in fact confirmed that activation of autophagy was essential to preserve cellular viability during nutritional starvation ( nitrogen depletion in yeast ) , and that mutants defective in autophagy were lethal [ 6 , 7 ] . in most of these studies emphasis was placed on the ability of autophagy to supply through degradation of protein products the amino acids required to maintain protein synthesis under the extreme nutritional conditions . however , the contribution of autophagy to the cellular energetic balance may not be solely dependent on this capacity to provide free amino acids , which in fact , are a relatively inefficient source of energy when oxidized to urea and carbon dioxide . recent studies support that autophagy can also provide energetically more efficient essential components , such as free fatty acids ( ffas ) and sugars . in this paper , we focus on the contribution of autophagy to lipid catabolism and the consequences of this novel autophagic function in the cellular energetic balance as well as in specific lipid - mediated regulatory functions . lastly , we also discuss the possible implications of alterations in the autophagic breakdown of lipids in human health and disease , with emphasis on common metabolic disorders . cells store fat in the form of lipid droplets ( lds)intracellular deposits of lipid esters surrounded by a monolayer of phospholipids and separated from the hydrophilic cytosolic environment by a coat of structural proteins , known generically as perilipins ( figure 1 ) [ 8 , 9 ] . despite their misleading appearance of inert stores , studies during recent years have revealed that ld are sites of high activity and that their functions are not limited to passive store of lipids . in fact , their dynamic nature , multifunctionality , and defined identity have now conferred upon them the category of intracellular organelles [ 8 , 9 ] . furthermore , as many other organelles , lds have been shown to interact in a regulated manner with other intracellular compartments ( likely , to provide them with specific lipids for their membranes ) and to adapt to changes in the cellular environment [ 10 , 11 ] . a growing theme in the field of ld research is now the identification of functions for the ld beyond those related to lipid metabolism or supply of membrane lipids . pioneering among those has been the finding that the hydrophobic matrix of the ld can become a sequestering surface for misfolded proteins that if left free in the cytosol could organize into oligomeric and aggregated products highly toxic for cells [ 12 , 13 ] . ld sequestration of proteins does not only apply to pathogenic proteins destined for degradation , but also may have a regulatory role in the availability of some fully functional proteins . for example , certain histones elude nuclear translocation through dynamic and reversible interactions with ld . interestingly , pathogens such as some types of viruses , have found in the ld ideal platforms for assembly [ 15 , 16 ] . although lipid droplets are particularly prominent in the adipose tissue where they organize as a single large droplet ( up to 100 m diameter ) that occupies almost the totality of the cytoplasm all cells contain lipid droplets to variable extents that range from 0.1 to 10 m . in addition to size differences , the adipose tissue ld has a core predominantly formed by triglycerides ( tgs ) whereas in most cells cholesterol and tg share the nuclear core of the ld [ 8 , 9 ] . ld originate from the er and maintain a close connection with this organelle , which facilitates exchange of lipids and proteins between both compartments to accommodate to the metabolic requirements of the cell the interaction of lipases present at the surface of the ld with the structural proteins that surround ld and with inhibitory proteins in the cytosol contributes to modulate the rate of lipolysis . cells activate lipolysis not only when they need energy but also in response to a large affluence of lipids to prevent stores from becoming compromisingly enlarged for the cell . although , traditionally , mobilization of ld by lipolysis has been solely attributed to the ld - associated lipases , recent studies have revealed a role for autophagy in ld breakdown ( figure 1 ) . the presence of lipases in the lysosomal lumen , along with a large variety of hydrolases such as proteases , glycases and nucleases , has been acknowledged since the early days of the discovery of this organelle . however , lysosomal lipases , also known as acid lipases because of their optimal acidic pka , were thought to serve mainly in the degradation of lipids contributed by the diet through endocytosis or those present in the membranes of the organelles digested during the autophagic process . the elevation of ld to the category of cytosolic organelles was in part a motivation to address their turnover by autophagy . this catabolic process is capable of sequestering whole cytosolic organelles inside double - membrane vesicles known as autophagosomes , which deliver this cargo to lysosomes upon heterotypic fusion with their membrane [ 18 , 19 ] . each of the events of the autophagic process is coordinated by a complex network of more than 32 genes and their protein products ( autophagy - related genes ( atgs ) and proteins ( atgs ) ) . atgs participate at the level of : ( 1 ) activation , ( 2 ) nucleation of the autophagosome membrane that forms de novo through conjugation of proteins and lipids from different cellular compartments , ( 3 ) elongation of the membrane and sealing to form the autophagosome , ( 4 ) trafficking toward the lysosomes , and ( 5 ) fusion of the two membranes . the first hint that ld could become substrates of the autophagic process originated from studies in cultured hepatocytes knocked down for atg5 , one of the genes essential for the formation of autophagosomes . hepatocytes respond to an acute oleic challenge by increasing lipolysis , which would prevent massive enlargement of the ld compartment . oleic challenge resulted in a marked increase in the number and size of ld in cells with compromised macroautophagy . the same was true in vivo , when knockout in liver of another essential autophagy gene ( atg7 ) led to an accelerated development of liver steatosis ( fatty liver ) in the autophagy compromised animals , when compared to control animals . detailed biochemical and functional analyses helped in establishing that the observed lipid accumulation did not result from increased formation of ld or reduced lipid secretion from hepatocytes , but that , instead , it could be explained almost exclusively on the basis of reduced lipolysis . it is possible that , through mechanisms yet to be identified , changes in autophagic activity may modulate the ld - associated lipases and contribute to the observed changes in lipolysis . however , independent of this possibility , there is now evidence that the autophagic system contributes directly to the mobilization of lipids from ld to lysosomes , wherein luminal lipases mediate their lipolysis . in fact , neutralization of the lysosomal ph , that would have a marked effect on the lysosome - resident lipases but does not modify the activity of the cytosolic lipases , was enough to almost completely block the lipolysis activated in response to a lipid challenge . sequestration of cytosolic components inside the forming autophagosome was considered for a long time a nonselective in - bulk process by which cytosolic material was randomly delivered for lysosomal degradation . however , recent years have revealed the existence of a growing number of proteins dedicated to the tagging and recognition of cytosolic components for autophagic degradation . in the case of intracellular protein aggregates , the presence of polyubiquitinated chains formed through specific types of linkage ( the best characterized uses the lysine 63 in ubiquitin to link one ubiquitin moiety to another in the polyubiquitin chains ) is the tag identified by the cargo - recognition machinery . similarly , ubiquitination of proteins on the surface of peroxisomes and of different pathogens contribute to their segregation towards the autophagic system [ 23 , 24 ] . however , ubiquitin is not the only signal identified as marker for autophagic degradation . selection of mitochondria for mitophagy ( selective degradation of mitochondria by autophagy ) has been shown to occur through different mechanisms , which likely coexist in most cells . in most cases , changes in structural components of the mitochondrial membrane are identified by partner cytosolic proteins ( such as parkin or nix ) , that once bound at the surface of this organelle , tag it for degradation . all cargo recognition molecules or autophagy receptors share their ability to bind to the tagging molecule in the organelle to be degraded as well as to specific components of the autophagic machinery ( in almost all cases the light chain protein 3 or lc3 ) [ 21 , 26 ] . this recruitment of autophagic components toward the cytosolic material to be degraded is proposed to initiate the in situ formation of the autophagosome around this material and to mediate selectivity . some levels of lipophagy may always occur even during the random sequestration of cytosolic material by in - bulk autophagy . in fact , analysis of the components inside autophagosomes in cells maintained in basal conditions revealed the presence of lipid material and ld structural proteins inside these vesicles , along with other cytosolic material . however , as described in the previous section , when lipophagy is activated in response to a lipid challenge or prolonged starvation , there seems to be a switch toward the preferential sequestration of ld , supporting some level of selectivity in this process . an intriguing observation in the studies of hepatic lipophagy was the fact that ld do not always seem to be sequestered as a whole by the autophagosomes , but , on the contrary , in many instances , only fractions of the ld underwent autophagy ( figure 1 ) . thus , membranous structures enriched in lc3 , in support of their autophagic origin , appear to grow from the surface of the droplet towards the inner core . often these membranes curve to finally seal , giving rise to double - membrane vesicles of a slightly smaller size than a conventional autophagosome ( 50100 nm ) and contain only components of the ld in their lumen . interestingly , formation of the membranes seems to be polarized in only one site on the surface of the ld . other components of the autophagic machinery , such as atg5 and atg7 , also localize to these areas of the ld in further support that formation of the limiting membrane occurs at the surface of the ld ( figure 1 ) . in the process of de novo formation of the autophagic membrane , atg7 acts as the enzyme regulating conjugation of atg12 to atg5 ( to serve as scaffold for assembling other components of the forming membrane ) , as well as the conjugation of lc3 to a lipid ( phosphatidylethanolamine , pe ) to generate lc3-ii that is one of the interestingly , atg7 is not required for the recruitment of lc3 to the ld , since this protein , although in its nonconjugated form , is still found associated to ld in cells defective in atg7 . pe , the only lipid known to conjugate to lc3 , is among the phospholipids that contribute to form the delimiting phospholipid monolayer of ld . the specific mechanism by which the limiting autophagosome membrane grows from the ld surface is still poorly characterized , but the presence of membrane - like structures in the ld core has been previously described . in addition , although the core is predominantly composed of lipids , some proteins can also be detected in this region . early studies have suggested that these proteins may form complexes with phospholipids to form structures compatible with the hydrophobic environment inside the ld . in this respect , conjugation of lc3 to pe on the surface of the ld may provide the right conformation for the forming membrane to advance towards the inside of the ld . polyubiquitination has been detected in polarized areas of ld in part resulting from the accumulation of clusters of polyubiquitinated apolipoprotein b ( apob ) on their surface . the fate of apob in this location seems to be to undergo lysosomal degradation . whether or not the lysosomal degradation of apob occurs as a result of the activation of lipophagy and how the accumulation of this protein contributes to the initiation of the process requires future investigation . recent studies have shown now the integration into the ld surface of the ancient ubiquitous protein 1 ( aup1 ) , which bears a c - terminus able to bind enzymes involved in ubiquitination . whether or not the presence of aup1 in ld is necessary or precedes the arrival of the autophagic machinery requires future investigation . of particular interest is the fact that lds have been shown to dynamically interact with two of the organelles that have been proposed as sites of formation of the limiting membrane of the autophagosomes the er and the mitochondria ( figure 1 ) [ 11 , 31 ] . interactions with the er may be related to ld biogenesis , as this is the compartment from where these organelles originate , but may also favor the distribution of lipids from the ld towards other organelles through the endosecretory pathway . in the case of mitochondria , the close interaction between ld and the outer - membrane of this organelle could facilitate delivery of the ffa released by lipolysis for mitochondrial -oxidation . however , considering the described association of the autophagic initiation complex to punctual areas in the membrane of the er and the mitochondria , and the formation of cup - like precursors of the limiting membrane of the autophagosomes from these regions [ 32 , 33 ] , it is tantalizing to at least propose that the previously described interactions of ld with these organelles , could also contribute to the initiation of their autophagic degradation . as described in previous sections , mobilization of ld by autophagy was first observed both in cultured hepatocytes in response to fatty acid exposure , and in liver of mice maintained on a diet enriched in fat for prolonged periods of time ( 4 months ) . the liver responds to the massive influx of lipids from the blood by upregulating ld biogenesis , as a mechanism of defense against the toxicity of fa , which upon esterification get converted into tg and stored into ld . however , in order to prevent uncontrolled expansion of ld , activation of lipolysis also occurs under these conditions and contributes to maintain ld size . failure to regulate lipid accumulation in hepatocytes may be the basis of pathogenic conditions such as liver steatosis and steatohepatitis . autophagy has now been added to the mechanisms that control the growth of the hepatic ld under these conditions ( figure 2 ) . besides lipid challenges , other stimuli such as starvation also engage the lipolytic contribution of the autophagic system . classic measurement of protein catabolism in liver during starvation , revealed that most of the protein degradation in this organ occurs during the 46 h that follow starvation , and that protein breakdown , even of autophagic origin , decreases markedly once the 810 h of starvation are reached . however , this decrease in autophagic degradation of proteins by autophagy is not equivalent to a decrease in overall autophagic activity . formation and clearance of autophagosomes seems to be maintained throughout the starvation period , but there is a consistent change in the type of cytosolic components sequestered in these vesicles . whereas cytosolic proteins and some organelles are the main cargo of the autophagic process during the early hours of starvation , as lack of nutrients persists , there is a gradual change towards preferential sequestration of lipid droplets inside autophagosomes ( figure 2 ) . this selective autophagy of lipid stores , known now as lipophagy , accounts for a high percentage of the lipolysis occurring during prolonged starvation in liver . interestingly , although lipophagy is markedly upregulated in response to lipid challenges and during prolonged starvation , it is possible that a certain percentage of degradation of lipid stores in lysosomes occurs continuously in many cell types . thus , blockage of autophagy through knockdown of any of the essential atg in hepatocytes in culture , leads to a significant increase in the number of lipid droplets in these cells even when maintained under normal nutritional conditions and in the absence of any additional challenge . similar basal lipophagy has also been observed in cell types not typically known to store fat such as fibroblasts , macrophages , t cells , dendritic cells , lymphoblasts , glia , striatal cell lines , and even primary neurons [ 20 , 3436 ] , although the relative contribution of autophagy to basal lipolysis may vary depending on the cell type . further studies are necessary to determine the reasons behind the coexistence of the two different mechanisms for lipolysis the one mediated by the cytosolic lipases and the one occurring through the autophagic system . it is possible that activation of one or the other may mainly lead to quantitative differences ( i.e. , lipophagy may be able to provide large amounts of ffa in shorter time ) . however , because the lysosomal lipases have been poorly characterized , it is also possible that the quality and type of the resulting lipolytic products differs between cytosolic and lysosomal lipases . lastly , in light of the growing evidence in support of the heterogeneity of the cellular ld , it is also plausible that the two lipolytic systems target different subpopulations of ld . as detailed earlier , lipophagy appears to contribute significantly to the mobilization of cellular lipids for provision of energy . however , the identification of lipophagy in cell types other than those involved in lipid storage , such as in immune or neuronal cells , suggests that autophagic turnover of lipids is perhaps a generic mechanism for the utilization of cellular fat stores in diverse cell types . in fact , recent reports have now included two additional cell types ; hypothalamic neurons and macrophage foam cells , to the increasing list of cells where lipophagy has been shown to be present and functionally important . the neurons within the mediobasal hypothalamus ( mbh ) form part of a focal neural network that integrates nutritional and hormonal information from two main cellular kinases , the mammalian target of rapamycin ( mtor ) and the phosphoinositol-3-kinase ( pi3k ) to control food intake and energy balance . hypothalamic fatty acid metabolism , amongst other neuronal mechanisms , has been linked to the regulation of appetite [ 41 , 42 ] . although recent work suggests that neuronal ffa availability and oxidation provide the energetic requirements for activation and firing of orexigenic agouti - related peptide ( agrp ) neurons , the lipolytic mechanisms that generate neuron - intrinsic ffa have remained poorly elucidated . since autophagy is activated by starvation in most cells , it was plausible that autophagic mobilization of lipids in the hypothalamus could contribute to the generation of neuronal ffa during starvation that , in turn , trigger mechanisms driving food intake . in fact , a recent study in mice knockout for atg7 in agrp neurons shows that the hypothalamus , indeed , needs autophagy to upregulate expression of agrp in response to nutritional depletion . this indicates the distinctive characteristic of hypothalamic neurons in their ability to activate autophagy , quite unlike other regions of the brain in which autophagy does not seem to be under this type of nutritional regulation . the activation of autophagy occurred in parallel to starvation - induced increases in hypothalamic ffa uptake ( figure 3 ) , suggesting that , as in the case of the liver , acute ffa stimulus might be a mechanism for activation of hypothalamic autophagy during starvation . indeed , the exposure of hypothalamic cells to ffa or ffa - rich serum from starved rodents increased autophagy ( figure 3 ) . the activation of autophagy in hypothalamic neurons upon acute lipid stimulus associated with increases in the levels of phosphorylated ampk and ulk1 , a kinase involved in the regulation of the autophagic process and recently described to be an ampk substrate . these findings indicate that hypothalamic ampk and ulk1 may contribute to a ffa sensing mechanism that modulates autophagy in response to changes in nutrient signals . however , the mechanisms connecting ffa release and agrp expression remain unknown for the most part . although it is possible that ffa modulate some of the signaling cascades involved in agrp regulation , a direct effect of the autophagic process on secretion of agrp - containing vesicles can not be ruled out . the immediate fate of the ffa taken up by hypothalamic cells is esterification into neuronal ld , which underscored the requirement of a lipolytic mechanism to liberate neuronal ffa during starvation . indeed , the activation of hypothalamic autophagy observed under these conditions , leads to increased mobilization of neuronal lipids to lysosomes . the physiological consequence of these interactions is the generation of neuronal ffa , since inhibiting lysosomal hydrolysis or interfering with the autophagic process significantly decreases hypothalamic ffa levels . lipophagy - generated hypothalamic ffa directly regulate the increase in orexigenic agrp expression that occurs in agrp hypothalamic neurons in response to starvation or to exposure to extracellular ffa . in fact , selective blockage of autophagy in agrp neurons has been shown to reduce fasting - induced increases in hypothalamic agrp levels , food intake , and body weights . interestingly , mice deficient in autophagy in agrp neurons also displayed higher levels of the anorexigenic peptide -melanocyte stimulating hormone ( msh ) , which is produced within the adjacent proopiomelanocortin ( pomc ) neuronal population , which could contribute to the reduced adiposity in these mice . in this respect , it is interesting to note that the effect of autophagy in lipid mobilization and the downstream consequences of neuronal lipophagy may be very neuronal type - specific . for example , a recent study using acute intrahypothalamic injection of sirna against atg7 revealed increased adiposity and glucose intolerance in the injected mice , in contrast to the lean phenotype observed when only agrp autophagy is compromised . it is possible that adiposity in this model occurred from concurrent reduction of atg7 in both agrp and pomc neuronal populations , or from autophagic deficiency in additional cell types , for instance hypothalamic glial cells , also shown to regulate glucose homeostasis . however , as the actual impact of the sirna injection in the autophagic flux in this model is not known , is not possible to discard that differences in efficiency of the autophagic compromise between pomc and agrp neurons are the real reason behind the different phenotype , or even that autophagy was not affected in the agrp neurons and the phenotype only resulted from reduced pomc autophagy . although future investigation is required to clarify the cell type differences and to identify additional stimuli that may also modulate hypothalamic autophagy , the current findings highlight an exciting new role for lipophagy in control of food intake and whole body energy balance by modulating the controlled production of neuronal ffa that regulate agrp levels ( figure 3 ) . in this way , the contribution of autophagy to the cellular and organismal energetic balance is no longer merely limited to its role in active breakdown of macromolecules or cellular stores to obtain energetic products , but has been raised to a more global regulatory function that includes the modulation of food intake . forthcoming studies should help in addressing the possible implications of these findings for metabolic diseases such as obesity and the impact that these metabolic changes could have on hypothalamic autophagy . the presence of lipophagy in diverse cell types raises the question whether lipophagy might also serve to mobilize lipids within the principal fat storing organ in the body , the adipose tissue . surprisingly , recent reports by two independent groups demonstrate a completely new and unexpected function of autophagy in regulating adipose physiology [ 48 , 49 ] , which is quite distinct from the role of autophagy in mobilizing lipids observed in other cell types [ 20 , 3436 ] . in fact , adipose - selective knockout of essential autophagy genes in mouse significantly reduced adipocyte lipid droplet content and fat tissue mass [ 48 , 49 ] . likewise , blocking autophagy in cultured preadipocytes decreased cellular triglyceride content and levels of key adipogenic transcription factors cebp- ( ccaat / enhancer - binding protein alpha ) , cebp- ( ccaat / enhancer - binding protein beta ) and ppar- . conceivably , reduced expression of adipocyte - specific genes led to the formation of an adipose tissue that predominantly consisted of immature fat - deficient preadipocytes judging by their reduced levels of terminal differentiation markers ( fatty acid synthase , fatty acid - binding protein-4 ( fabp-4/ap-2 ) , glucose transporter 4 ( glut4 ) , or stearoyl coa desaturase 1 ) . intriguingly , selective inhibition of autophagy in white adipose tissue ( wat ) in vivo not only impaired wat differentiation but also introduced brown adipose tissue- ( bat- ) like features in autophagy - deficient wat . the autophagy - deficient white adipocytes exhibited a cellular morphology that resembled closely that of brown adipocytes . for instance , atg7-deficient white adipocytes displayed increased number of smaller multiloculated lipid droplets and mitochondria , rounded nuclei , and larger cytoplasmic size [ 48 , 49 ] . the molecular characteristics of the adipose tissue in the autophagy - deficient mice also mimicked those of bat as reflected by increased levels of brown adipogenic factors , ppar- transcriptional coactivator ( pgc-1 ) , and uncoupling protein-1 ( ucp-1 ) . acquisition of bat - like properties resulted in higher adipose tissue -oxidation rates in knock - out animals than in controls [ 48 , 49 ] . the physiological consequences of this shift in wat phenotype were a decrease in body weight , reduced adiposity and resistance against high fat diet - induced alterations in glucose homeostasis [ 48 , 49 ] . although , the mechanism by which autophagy controls adipocyte differentiation or modulates the phenotypic switch from wat to bat - like is unclear , a likely possibility is that autophagy modulates levels of key regulatory proteins to control adipocyte cell fate , differentiation and fat storage . however , it is still plausible that specific autophagy - related components may be directly involved in the process of adipogenesis and that this function is not only limited to the adipose tissue . this concept is supported by studies performed at ages before adulthood in the same mouse model null for autophagy in liver used in the discovery of hepatic lipophagy . in contrast to the massive accumulation of lipids observed in the adult animals , very young animals , at least when unchallenged , had consistently lower hepatocyte content of ld . the previously described association of lc3-ii to ld , also confirmed in this latter work , was proposed to be required for ld formation . since expression of the enzyme used to flox out the autophagic gene does not start in this mouse model until 3 - 4 months after birth , it is possible that if autophagy is involved in both formation and mobilization of ld , the partial blockage of autophagy was initially sufficient to cope with the lipophagic requirements of the young animals and prevent the accumulation of ld . however , as the animals reached adulthood , the complete and persistent blockage of the autophagic system tilted the balance between lipogenesis and lipolysis toward the former one , leading to ld accumulation . this dual involvement of autophagy in lipogenesis ( ld formation ) and lipolysis proposed in the liver now raises the question of whether a similar dual role could also occur in the adipose tissue . in fact , recent studies with a mouse model deficient in caveolin 1 have shown lipoatrophy of the adipose tissue mediated by massive upregulation of autophagy in this tissue . future studies with inducible autophagy knockouts in the adipose tissue of adult mice are needed to determine if autophagy does contribute to lipid mobilization also from fully formed adipose tissue . after the first observations demonstrating the existence of lipophagy and the upregulation of this process in response to a lipid challenge , numerous studies have confirmed the stimulatory effect of dietary lipids on the autophagic process . upregulation of autophagy in response to increased ffa has been demonstrated in neurons , muscle , pancreas , mammary epithelial cells , liver - derived cells , and even in colon cancer cells [ 5256 ] . although the mechanisms that modulate the activation of the autophagic process under these conditions are still poorly elucidated , at least in the case of the pancreatic beta cell , autophagic activation has been proposed to occur through activation of the c - jun n - terminal kinase 1 pathway in a manner independent of er or oxidative stress . in contrast to this stimulatory effect of a lipid challenge on the autophagic system , an equal number of studies have started to report inhibition of autophagy in response to exposure to high concentrations or particular type of lipids ( figure 4 ) . for example unsaturated ffa such as oleic acid has a marked stimulatory effect on autophagy in many cells , at least up to some concentrations [ 20 , 57 , 58 ] . in contrast , saturated ffa such as palmitic acid maybe due in part to its lower incorporation into ld remains in the cytosol at higher concentrations and suppresses autophagy . likewise , in animals exposed to a high - fat diet for prolonged periods of time it is possible to detect an increase in autophagic activity during the first weeks of treatment , which is progressively followed by a gradual decrease in autophagy . this decrease further contributes to the expansion of the ld compartment , eventually leading to hepatotoxicity and steatosis [ 20 , 57 , 58 ] . interestingly , the switch from activation to inhibition of autophagy in response to lipogenic stimuli can also be cell type dependent . thus , same amounts of oxidized low - density lipoprotein that stimulate autophagic activity in schwannoma cells have been shown to be toxic for neuroblastoma cells . although many mechanisms could contribute to the inhibitory effect of ffa on autophagy , a systematic analysis of the different steps of the autophagic process has revealed a primary defect in the fusion between autophagosomes and lysosomes in cells exposed to high concentrations of ffa or in animals subjected to prolonged high - fat diet . interestingly , this failure to deliver autophagosome cargo directly to lysosomes is initially compensated for by increasing fusion of autophagic compartments with late endosomes ( to generate what is known as an amphisome ) . however , as the high levels of intracellular lipids persist , defective intracellular turnover becomes evident , either because of further compromise of the pathway or maybe because of an additional failure in the endocytic system as autophagic cargo builds up in these compartments . analysis of autophagic vacuoles from animals exposed to a high - fat diet revealed that changes in the lipid composition of the membrane of these vesicles are behind their compromised fusogenicity . this - dual effect of dietary lipids on autophagy and lipophagy should be taken into consideration when contemplating manipulations of the autophagic system as a therapeutic strategy for metabolic disorders . the fast development of steatosis and fatty liver observed in mice defective for autophagy in this organ strongly supported the contribution of altered autophagy to the pathogenesis of this common disease . in fact , a compromise in hepatic autophagy has been proposed to underline also the basis for the accumulation of ld upon exposure to toxic concentrations of ethanol . furthermore , recent studies have demonstrated that pharmacological upregulation of autophagy reduces hepatotoxicity and steatosis in an alcohol - induced model of fatty liver . however , future studies are needed before autophagy activation can be used as a generalized treatment against this disease , because , for example , upregulation of the autophagic process in hepatic stellate cells has been shown to favor their activation and consequently initiate liver fibrosis . the liver responds to some stressors through global activation of autophagy , including degradation of lipids , proteins , and organelles . however , in other instances upregulation of autophagy as a protective mechanism against liver injury can be specific for lipophagy . for example , the autophagy upregulated as a first line of defense against alcohol - induced toxicity in liver , selectively targets mitochondria , and lipid droplets , while excluding soluble cytosolic proteins and other organelles . future efforts should focus in understanding how selective forms of autophagy can be individually modulated for therapeutic purposes . the recently discovered capability of the autophagic system to mobilize hepatic lipids is also utilized by viruses to favor their replication . earlier studies have shown that although autophagy is usually an efficient mechanism in the defense against most viral infections , upregulation of autophagy could also favor replication of some viruses such as the dengue virus . recent studies have demonstrated that dengue virus - dependent induction of autophagy mediates ld breakdown and release of ffa necessary to maintain the high levels of intracellular atp required for dengue viral replication . future studies are needed to determine which subset of hepatotropic virus makes use of lipophagy for their own replication and whether blockage of the autophagic system can be performed in this organ in a selective way to preferentially affect virogenesis but not normal liver metabolism . the finding that autophagy is required for adipogenesis has elicited a considerable interest in the interplay between autophagy and metabolic disorders such as obesity . studies performed in human subjects with different types and degrees of obesity have revealed a direct correlation between autophagic activity and the sizes of various fat depots . interestingly , autophagy was found to be inappropriately active in omental fat tissues extracted from obese individuals , and , in fact , autophagic activity was remarkably raised in insulin - resistant obese subjects . this indicates that although functional autophagy may be a requirement for adipose differentiation during development , autophagy might also be involved in the maintenance of adipose tissue size and lipid storage in adults . the fact that autophagic upregulation occurs before obesity - associated morbidity becomes manifested , still leaves open the possibility that this system could be activated as a defensive mechanism against the increase in intracellular lipids . however , the final outcome and autophagy effect may be very different depending on the metabolic status . for example , in adipocytes from type 2 diabetes patients characterized by unresponsiveness to insulin , maintained attenuation of mtor has been recently described and proposed as the main mechanism responsible for the upregulation of autophagy in these cells . the concomitant increase in ld formation under these conditions along with their enhanced autophagy favors cellular toxicity due to the excessive release of ffa from ld . it is anticipated that blockage of autophagy , or at least downregulation to a normal level , may be better under these conditions . lipophagy has been recently proposed as a possible defensive mechanism against atherosclerosis , or the thickening of the artery walls due to abnormal accumulation of lipid deposits in macrophage foam cells . the recent finding that autophagy contributes to lipolytic mobilization of ld in macrophages also , provides now a new possible mechanism for the pathogenesis of atherosclerosis . this study has revealed that macrophage lipophagy is upregulated both in vitro and in vivo in response to lipid loading and that failure to upregulate the autophagic system , in mice defective for this pathway , results in inefficient clearance of cholesterol in macrophages . in light of the inhibitory effect that high concentrations of intracellular lipids can have on autophagy , it is reasonable to propose that chronic exposure to high levels of circulating lipids may compromise the autophagic system of the artery wall macrophages and lead to their transformation into foam cells as lipids accumulate in their cytoplasm . this massive accumulation of lipids is the seeding for the subsequent formation of the atherosclerotic plaque . current therapeutic strategies in this disease are aimed at promoting cholesterol efflux from these macrophages to reduce the size of the lipid - enriched plaque that they form beneath the endothelial cells . consequently , manipulations aimed at enhancing macrophage autophagy and thus favoring cholesterol efflux from these cells , may have therapeutic potential in the atherosclerotic artery walls . interestingly , the contribution of changes in autophagy to atherosclerotic plaque development may go beyond macrophages and involve also the smooth muscle cells of the arterial wall . recent studies have shown compromised autophagy in these cells as a consequence of the inflammatory response associated to the plaques . the fact that a decline in autophagic activity , and in particular in lipophagy , would contribute to intracellular accumulation of ld and that , as described in previous sections , these abnormally expanded lipid stores would further reduce autophagic activity , makes this an attractive feedback loop for the perpetuation of the metabolic syndrome of aging ( characterized by hypercholesterolemia , accumulation of lipid deposits in organs , and insulin resistance ) ( figure 4 ) . interestingly , and in some way contra intuitively , treatment with antilipolytic agents has been shown to improve the age - related hypercholesterolemic phenotype and overall health - span in old mouse models . however , recent studies support that most of the beneficial effect observed with these agents is dependent on their ability to induce autophagy , likely as a response to the increase in intracellular lipid stores . genetic connections among autophagy , lipid metabolism , and longevity have also been recently highlighted in studies in c. elegans . functional autophagy is necessary in this model to maintain the activation of a cellular lipase ( lipl-4 ) and conversely , this lipase is required for induction of autophagy . interestingly , both the activity of this lipase and autophagy are required to attain the extension in life span observed upon germline removal in worms . although the specific lipid targets of this lipase and the way in which it participates in autophagy remain unknown , it is tempting to propose that part of the effect in life - span could be due to better intracellular lipid handling by lipophagy . the recent discovery of lipophagy has contributed to link two major intracellular catabolic pathways autophagy and lipolysis . this new function of autophagy in lipid metabolism expands the physiological relevance of the autophagic process by making its contribution to the energetic balance more relevant ( when considering the higher energetic value of lipids versus proteins ) , but also including now under the list of autophagic functions the control of many of the regulatory activities that lipids exert inside cells . does lipophagy regulation occur through similar signaling pathways to those described for other types of autophagy ? are there differences between the types of lipid byproducts generated by lipophagy when compared to cytosolic lipolysis ? what determines the threshold for the switch from a stimulatory to an inhibitory effect of ffa on lipophagy ? and also , in a more general context , what are the possible effects of lipophagy on the combinations of metabolic defects that often coexist in our population such as obesity , diabetes , and hyperlipidemia ? does defective hypothalamic lipophagy with age contribute to the reduced food intake observed in advanced aging ? although these are still early days for lipophagy , there is now ample evidence that organ - specific targeting of this process may have implications for development of novel therapeutic interventions against common human metabolic disorders such as obesity and insulin resistance .

lipid droplets ( lds ) , initially considered inert lipid deposits , have gained during the last decade the classification of cytosolic organelles due to their defined composition and the multiplicity of specific cellular functions in which they are involved . the classification of ld as organelles brings along the need for their regulated turnover and recent findings support the direct contribution of autophagy to this turnover through a process now described as lipophagy . this paper focuses on the characteristics of this new type of selective autophagy and the cellular consequences of the mobilization of intracellular lipids through this process . lipophagy impacts the cellular energetic balance directly , through lipid breakdown and , indirectly , by regulating food intake . defective lipophagy has been already linked to important metabolic disorders such as fatty liver , obesity and atherosclerosis , and the age - dependent decrease in autophagy could underline the basis for the metabolic syndrome of aging .

1. Introduction 2. Mobilization of Lipid Droplets by Autophagy 3. Physiology of Lipophagy 4. Pathology of Lipophagy 5. Concluding Remarks

autophagy , or the process of degradation of intracellular components in lysosomes , has been traditionally linked to cellular energy balance and to the cellular nutritional status [ 1 , 2 ] . in fact , although during the recent revival of the autophagic process , most of the emphasis has been placed on its role in other cellular functions such as cellular quality control , remodeling , or cell defense , the first descriptions of the autophagic process in the early 1960s already stated that conditions such as starvation lead to its activation [ 35 ] . these early studies proposed that autophagic activation during starvation was necessary to maintain the cellular energetic balance . later studies in yeast , in fact confirmed that activation of autophagy was essential to preserve cellular viability during nutritional starvation ( nitrogen depletion in yeast ) , and that mutants defective in autophagy were lethal [ 6 , 7 ] . in most of these studies emphasis was placed on the ability of autophagy to supply through degradation of protein products the amino acids required to maintain protein synthesis under the extreme nutritional conditions . however , the contribution of autophagy to the cellular energetic balance may not be solely dependent on this capacity to provide free amino acids , which in fact , are a relatively inefficient source of energy when oxidized to urea and carbon dioxide . in this paper , we focus on the contribution of autophagy to lipid catabolism and the consequences of this novel autophagic function in the cellular energetic balance as well as in specific lipid - mediated regulatory functions . cells store fat in the form of lipid droplets ( lds)intracellular deposits of lipid esters surrounded by a monolayer of phospholipids and separated from the hydrophilic cytosolic environment by a coat of structural proteins , known generically as perilipins ( figure 1 ) [ 8 , 9 ] . furthermore , as many other organelles , lds have been shown to interact in a regulated manner with other intracellular compartments ( likely , to provide them with specific lipids for their membranes ) and to adapt to changes in the cellular environment [ 10 , 11 ] . interestingly , pathogens such as some types of viruses , have found in the ld ideal platforms for assembly [ 15 , 16 ] . ld originate from the er and maintain a close connection with this organelle , which facilitates exchange of lipids and proteins between both compartments to accommodate to the metabolic requirements of the cell the interaction of lipases present at the surface of the ld with the structural proteins that surround ld and with inhibitory proteins in the cytosol contributes to modulate the rate of lipolysis . although , traditionally , mobilization of ld by lipolysis has been solely attributed to the ld - associated lipases , recent studies have revealed a role for autophagy in ld breakdown ( figure 1 ) . the presence of lipases in the lysosomal lumen , along with a large variety of hydrolases such as proteases , glycases and nucleases , has been acknowledged since the early days of the discovery of this organelle . however , lysosomal lipases , also known as acid lipases because of their optimal acidic pka , were thought to serve mainly in the degradation of lipids contributed by the diet through endocytosis or those present in the membranes of the organelles digested during the autophagic process . the elevation of ld to the category of cytosolic organelles was in part a motivation to address their turnover by autophagy . the first hint that ld could become substrates of the autophagic process originated from studies in cultured hepatocytes knocked down for atg5 , one of the genes essential for the formation of autophagosomes . detailed biochemical and functional analyses helped in establishing that the observed lipid accumulation did not result from increased formation of ld or reduced lipid secretion from hepatocytes , but that , instead , it could be explained almost exclusively on the basis of reduced lipolysis . however , independent of this possibility , there is now evidence that the autophagic system contributes directly to the mobilization of lipids from ld to lysosomes , wherein luminal lipases mediate their lipolysis . in most cases , changes in structural components of the mitochondrial membrane are identified by partner cytosolic proteins ( such as parkin or nix ) , that once bound at the surface of this organelle , tag it for degradation . some levels of lipophagy may always occur even during the random sequestration of cytosolic material by in - bulk autophagy . however , as described in the previous section , when lipophagy is activated in response to a lipid challenge or prolonged starvation , there seems to be a switch toward the preferential sequestration of ld , supporting some level of selectivity in this process . an intriguing observation in the studies of hepatic lipophagy was the fact that ld do not always seem to be sequestered as a whole by the autophagosomes , but , on the contrary , in many instances , only fractions of the ld underwent autophagy ( figure 1 ) . interestingly , formation of the membranes seems to be polarized in only one site on the surface of the ld . in the process of de novo formation of the autophagic membrane , atg7 acts as the enzyme regulating conjugation of atg12 to atg5 ( to serve as scaffold for assembling other components of the forming membrane ) , as well as the conjugation of lc3 to a lipid ( phosphatidylethanolamine , pe ) to generate lc3-ii that is one of the interestingly , atg7 is not required for the recruitment of lc3 to the ld , since this protein , although in its nonconjugated form , is still found associated to ld in cells defective in atg7 . in this respect , conjugation of lc3 to pe on the surface of the ld may provide the right conformation for the forming membrane to advance towards the inside of the ld . recent studies have shown now the integration into the ld surface of the ancient ubiquitous protein 1 ( aup1 ) , which bears a c - terminus able to bind enzymes involved in ubiquitination . of particular interest is the fact that lds have been shown to dynamically interact with two of the organelles that have been proposed as sites of formation of the limiting membrane of the autophagosomes the er and the mitochondria ( figure 1 ) [ 11 , 31 ] . in the case of mitochondria , the close interaction between ld and the outer - membrane of this organelle could facilitate delivery of the ffa released by lipolysis for mitochondrial -oxidation . however , considering the described association of the autophagic initiation complex to punctual areas in the membrane of the er and the mitochondria , and the formation of cup - like precursors of the limiting membrane of the autophagosomes from these regions [ 32 , 33 ] , it is tantalizing to at least propose that the previously described interactions of ld with these organelles , could also contribute to the initiation of their autophagic degradation . as described in previous sections , mobilization of ld by autophagy was first observed both in cultured hepatocytes in response to fatty acid exposure , and in liver of mice maintained on a diet enriched in fat for prolonged periods of time ( 4 months ) . besides lipid challenges , other stimuli such as starvation also engage the lipolytic contribution of the autophagic system . classic measurement of protein catabolism in liver during starvation , revealed that most of the protein degradation in this organ occurs during the 46 h that follow starvation , and that protein breakdown , even of autophagic origin , decreases markedly once the 810 h of starvation are reached . formation and clearance of autophagosomes seems to be maintained throughout the starvation period , but there is a consistent change in the type of cytosolic components sequestered in these vesicles . whereas cytosolic proteins and some organelles are the main cargo of the autophagic process during the early hours of starvation , as lack of nutrients persists , there is a gradual change towards preferential sequestration of lipid droplets inside autophagosomes ( figure 2 ) . this selective autophagy of lipid stores , known now as lipophagy , accounts for a high percentage of the lipolysis occurring during prolonged starvation in liver . thus , blockage of autophagy through knockdown of any of the essential atg in hepatocytes in culture , leads to a significant increase in the number of lipid droplets in these cells even when maintained under normal nutritional conditions and in the absence of any additional challenge . similar basal lipophagy has also been observed in cell types not typically known to store fat such as fibroblasts , macrophages , t cells , dendritic cells , lymphoblasts , glia , striatal cell lines , and even primary neurons [ 20 , 3436 ] , although the relative contribution of autophagy to basal lipolysis may vary depending on the cell type . further studies are necessary to determine the reasons behind the coexistence of the two different mechanisms for lipolysis the one mediated by the cytosolic lipases and the one occurring through the autophagic system . however , because the lysosomal lipases have been poorly characterized , it is also possible that the quality and type of the resulting lipolytic products differs between cytosolic and lysosomal lipases . lastly , in light of the growing evidence in support of the heterogeneity of the cellular ld , it is also plausible that the two lipolytic systems target different subpopulations of ld . as detailed earlier , lipophagy appears to contribute significantly to the mobilization of cellular lipids for provision of energy . in fact , recent reports have now included two additional cell types ; hypothalamic neurons and macrophage foam cells , to the increasing list of cells where lipophagy has been shown to be present and functionally important . since autophagy is activated by starvation in most cells , it was plausible that autophagic mobilization of lipids in the hypothalamus could contribute to the generation of neuronal ffa during starvation that , in turn , trigger mechanisms driving food intake . this indicates the distinctive characteristic of hypothalamic neurons in their ability to activate autophagy , quite unlike other regions of the brain in which autophagy does not seem to be under this type of nutritional regulation . the activation of autophagy occurred in parallel to starvation - induced increases in hypothalamic ffa uptake ( figure 3 ) , suggesting that , as in the case of the liver , acute ffa stimulus might be a mechanism for activation of hypothalamic autophagy during starvation . the activation of autophagy in hypothalamic neurons upon acute lipid stimulus associated with increases in the levels of phosphorylated ampk and ulk1 , a kinase involved in the regulation of the autophagic process and recently described to be an ampk substrate . in fact , selective blockage of autophagy in agrp neurons has been shown to reduce fasting - induced increases in hypothalamic agrp levels , food intake , and body weights . interestingly , mice deficient in autophagy in agrp neurons also displayed higher levels of the anorexigenic peptide -melanocyte stimulating hormone ( msh ) , which is produced within the adjacent proopiomelanocortin ( pomc ) neuronal population , which could contribute to the reduced adiposity in these mice . in this respect , it is interesting to note that the effect of autophagy in lipid mobilization and the downstream consequences of neuronal lipophagy may be very neuronal type - specific . however , as the actual impact of the sirna injection in the autophagic flux in this model is not known , is not possible to discard that differences in efficiency of the autophagic compromise between pomc and agrp neurons are the real reason behind the different phenotype , or even that autophagy was not affected in the agrp neurons and the phenotype only resulted from reduced pomc autophagy . in this way , the contribution of autophagy to the cellular and organismal energetic balance is no longer merely limited to its role in active breakdown of macromolecules or cellular stores to obtain energetic products , but has been raised to a more global regulatory function that includes the modulation of food intake . forthcoming studies should help in addressing the possible implications of these findings for metabolic diseases such as obesity and the impact that these metabolic changes could have on hypothalamic autophagy . the molecular characteristics of the adipose tissue in the autophagy - deficient mice also mimicked those of bat as reflected by increased levels of brown adipogenic factors , ppar- transcriptional coactivator ( pgc-1 ) , and uncoupling protein-1 ( ucp-1 ) . the physiological consequences of this shift in wat phenotype were a decrease in body weight , reduced adiposity and resistance against high fat diet - induced alterations in glucose homeostasis [ 48 , 49 ] . since expression of the enzyme used to flox out the autophagic gene does not start in this mouse model until 3 - 4 months after birth , it is possible that if autophagy is involved in both formation and mobilization of ld , the partial blockage of autophagy was initially sufficient to cope with the lipophagic requirements of the young animals and prevent the accumulation of ld . in fact , recent studies with a mouse model deficient in caveolin 1 have shown lipoatrophy of the adipose tissue mediated by massive upregulation of autophagy in this tissue . after the first observations demonstrating the existence of lipophagy and the upregulation of this process in response to a lipid challenge , numerous studies have confirmed the stimulatory effect of dietary lipids on the autophagic process . upregulation of autophagy in response to increased ffa has been demonstrated in neurons , muscle , pancreas , mammary epithelial cells , liver - derived cells , and even in colon cancer cells [ 5256 ] . in contrast to this stimulatory effect of a lipid challenge on the autophagic system , an equal number of studies have started to report inhibition of autophagy in response to exposure to high concentrations or particular type of lipids ( figure 4 ) . likewise , in animals exposed to a high - fat diet for prolonged periods of time it is possible to detect an increase in autophagic activity during the first weeks of treatment , which is progressively followed by a gradual decrease in autophagy . however , as the high levels of intracellular lipids persist , defective intracellular turnover becomes evident , either because of further compromise of the pathway or maybe because of an additional failure in the endocytic system as autophagic cargo builds up in these compartments . this - dual effect of dietary lipids on autophagy and lipophagy should be taken into consideration when contemplating manipulations of the autophagic system as a therapeutic strategy for metabolic disorders . the fast development of steatosis and fatty liver observed in mice defective for autophagy in this organ strongly supported the contribution of altered autophagy to the pathogenesis of this common disease . in fact , a compromise in hepatic autophagy has been proposed to underline also the basis for the accumulation of ld upon exposure to toxic concentrations of ethanol . however , future studies are needed before autophagy activation can be used as a generalized treatment against this disease , because , for example , upregulation of the autophagic process in hepatic stellate cells has been shown to favor their activation and consequently initiate liver fibrosis . for example , the autophagy upregulated as a first line of defense against alcohol - induced toxicity in liver , selectively targets mitochondria , and lipid droplets , while excluding soluble cytosolic proteins and other organelles . earlier studies have shown that although autophagy is usually an efficient mechanism in the defense against most viral infections , upregulation of autophagy could also favor replication of some viruses such as the dengue virus . recent studies have demonstrated that dengue virus - dependent induction of autophagy mediates ld breakdown and release of ffa necessary to maintain the high levels of intracellular atp required for dengue viral replication . the finding that autophagy is required for adipogenesis has elicited a considerable interest in the interplay between autophagy and metabolic disorders such as obesity . interestingly , autophagy was found to be inappropriately active in omental fat tissues extracted from obese individuals , and , in fact , autophagic activity was remarkably raised in insulin - resistant obese subjects . however , the final outcome and autophagy effect may be very different depending on the metabolic status . for example , in adipocytes from type 2 diabetes patients characterized by unresponsiveness to insulin , maintained attenuation of mtor has been recently described and proposed as the main mechanism responsible for the upregulation of autophagy in these cells . lipophagy has been recently proposed as a possible defensive mechanism against atherosclerosis , or the thickening of the artery walls due to abnormal accumulation of lipid deposits in macrophage foam cells . the recent finding that autophagy contributes to lipolytic mobilization of ld in macrophages also , provides now a new possible mechanism for the pathogenesis of atherosclerosis . in light of the inhibitory effect that high concentrations of intracellular lipids can have on autophagy , it is reasonable to propose that chronic exposure to high levels of circulating lipids may compromise the autophagic system of the artery wall macrophages and lead to their transformation into foam cells as lipids accumulate in their cytoplasm . this massive accumulation of lipids is the seeding for the subsequent formation of the atherosclerotic plaque . interestingly , the contribution of changes in autophagy to atherosclerotic plaque development may go beyond macrophages and involve also the smooth muscle cells of the arterial wall . the fact that a decline in autophagic activity , and in particular in lipophagy , would contribute to intracellular accumulation of ld and that , as described in previous sections , these abnormally expanded lipid stores would further reduce autophagic activity , makes this an attractive feedback loop for the perpetuation of the metabolic syndrome of aging ( characterized by hypercholesterolemia , accumulation of lipid deposits in organs , and insulin resistance ) ( figure 4 ) . interestingly , and in some way contra intuitively , treatment with antilipolytic agents has been shown to improve the age - related hypercholesterolemic phenotype and overall health - span in old mouse models . although the specific lipid targets of this lipase and the way in which it participates in autophagy remain unknown , it is tempting to propose that part of the effect in life - span could be due to better intracellular lipid handling by lipophagy . the recent discovery of lipophagy has contributed to link two major intracellular catabolic pathways autophagy and lipolysis . this new function of autophagy in lipid metabolism expands the physiological relevance of the autophagic process by making its contribution to the energetic balance more relevant ( when considering the higher energetic value of lipids versus proteins ) , but also including now under the list of autophagic functions the control of many of the regulatory activities that lipids exert inside cells . and also , in a more general context , what are the possible effects of lipophagy on the combinations of metabolic defects that often coexist in our population such as obesity , diabetes , and hyperlipidemia ? although these are still early days for lipophagy , there is now ample evidence that organ - specific targeting of this process may have implications for development of novel therapeutic interventions against common human metabolic disorders such as obesity and insulin resistance .

in fact , although during the recent revival of the autophagic process , most of the emphasis has been placed on its role in other cellular functions such as cellular quality control , remodeling , or cell defense , the first descriptions of the autophagic process in the early 1960s already stated that conditions such as starvation lead to its activation [ 35 ] . however , the contribution of autophagy to the cellular energetic balance may not be solely dependent on this capacity to provide free amino acids , which in fact , are a relatively inefficient source of energy when oxidized to urea and carbon dioxide . in this paper , we focus on the contribution of autophagy to lipid catabolism and the consequences of this novel autophagic function in the cellular energetic balance as well as in specific lipid - mediated regulatory functions . lastly , we also discuss the possible implications of alterations in the autophagic breakdown of lipids in human health and disease , with emphasis on common metabolic disorders . cells store fat in the form of lipid droplets ( lds)intracellular deposits of lipid esters surrounded by a monolayer of phospholipids and separated from the hydrophilic cytosolic environment by a coat of structural proteins , known generically as perilipins ( figure 1 ) [ 8 , 9 ] . pioneering among those has been the finding that the hydrophobic matrix of the ld can become a sequestering surface for misfolded proteins that if left free in the cytosol could organize into oligomeric and aggregated products highly toxic for cells [ 12 , 13 ] . although lipid droplets are particularly prominent in the adipose tissue where they organize as a single large droplet ( up to 100 m diameter ) that occupies almost the totality of the cytoplasm all cells contain lipid droplets to variable extents that range from 0.1 to 10 m . ld originate from the er and maintain a close connection with this organelle , which facilitates exchange of lipids and proteins between both compartments to accommodate to the metabolic requirements of the cell the interaction of lipases present at the surface of the ld with the structural proteins that surround ld and with inhibitory proteins in the cytosol contributes to modulate the rate of lipolysis . the presence of lipases in the lysosomal lumen , along with a large variety of hydrolases such as proteases , glycases and nucleases , has been acknowledged since the early days of the discovery of this organelle . however , lysosomal lipases , also known as acid lipases because of their optimal acidic pka , were thought to serve mainly in the degradation of lipids contributed by the diet through endocytosis or those present in the membranes of the organelles digested during the autophagic process . each of the events of the autophagic process is coordinated by a complex network of more than 32 genes and their protein products ( autophagy - related genes ( atgs ) and proteins ( atgs ) ) . atgs participate at the level of : ( 1 ) activation , ( 2 ) nucleation of the autophagosome membrane that forms de novo through conjugation of proteins and lipids from different cellular compartments , ( 3 ) elongation of the membrane and sealing to form the autophagosome , ( 4 ) trafficking toward the lysosomes , and ( 5 ) fusion of the two membranes . the same was true in vivo , when knockout in liver of another essential autophagy gene ( atg7 ) led to an accelerated development of liver steatosis ( fatty liver ) in the autophagy compromised animals , when compared to control animals . however , independent of this possibility , there is now evidence that the autophagic system contributes directly to the mobilization of lipids from ld to lysosomes , wherein luminal lipases mediate their lipolysis . in fact , neutralization of the lysosomal ph , that would have a marked effect on the lysosome - resident lipases but does not modify the activity of the cytosolic lipases , was enough to almost completely block the lipolysis activated in response to a lipid challenge . in the case of intracellular protein aggregates , the presence of polyubiquitinated chains formed through specific types of linkage ( the best characterized uses the lysine 63 in ubiquitin to link one ubiquitin moiety to another in the polyubiquitin chains ) is the tag identified by the cargo - recognition machinery . in most cases , changes in structural components of the mitochondrial membrane are identified by partner cytosolic proteins ( such as parkin or nix ) , that once bound at the surface of this organelle , tag it for degradation . all cargo recognition molecules or autophagy receptors share their ability to bind to the tagging molecule in the organelle to be degraded as well as to specific components of the autophagic machinery ( in almost all cases the light chain protein 3 or lc3 ) [ 21 , 26 ] . in fact , analysis of the components inside autophagosomes in cells maintained in basal conditions revealed the presence of lipid material and ld structural proteins inside these vesicles , along with other cytosolic material . however , as described in the previous section , when lipophagy is activated in response to a lipid challenge or prolonged starvation , there seems to be a switch toward the preferential sequestration of ld , supporting some level of selectivity in this process . an intriguing observation in the studies of hepatic lipophagy was the fact that ld do not always seem to be sequestered as a whole by the autophagosomes , but , on the contrary , in many instances , only fractions of the ld underwent autophagy ( figure 1 ) . other components of the autophagic machinery , such as atg5 and atg7 , also localize to these areas of the ld in further support that formation of the limiting membrane occurs at the surface of the ld ( figure 1 ) . in the process of de novo formation of the autophagic membrane , atg7 acts as the enzyme regulating conjugation of atg12 to atg5 ( to serve as scaffold for assembling other components of the forming membrane ) , as well as the conjugation of lc3 to a lipid ( phosphatidylethanolamine , pe ) to generate lc3-ii that is one of the interestingly , atg7 is not required for the recruitment of lc3 to the ld , since this protein , although in its nonconjugated form , is still found associated to ld in cells defective in atg7 . in this respect , conjugation of lc3 to pe on the surface of the ld may provide the right conformation for the forming membrane to advance towards the inside of the ld . of particular interest is the fact that lds have been shown to dynamically interact with two of the organelles that have been proposed as sites of formation of the limiting membrane of the autophagosomes the er and the mitochondria ( figure 1 ) [ 11 , 31 ] . interactions with the er may be related to ld biogenesis , as this is the compartment from where these organelles originate , but may also favor the distribution of lipids from the ld towards other organelles through the endosecretory pathway . in the case of mitochondria , the close interaction between ld and the outer - membrane of this organelle could facilitate delivery of the ffa released by lipolysis for mitochondrial -oxidation . however , considering the described association of the autophagic initiation complex to punctual areas in the membrane of the er and the mitochondria , and the formation of cup - like precursors of the limiting membrane of the autophagosomes from these regions [ 32 , 33 ] , it is tantalizing to at least propose that the previously described interactions of ld with these organelles , could also contribute to the initiation of their autophagic degradation . as described in previous sections , mobilization of ld by autophagy was first observed both in cultured hepatocytes in response to fatty acid exposure , and in liver of mice maintained on a diet enriched in fat for prolonged periods of time ( 4 months ) . the liver responds to the massive influx of lipids from the blood by upregulating ld biogenesis , as a mechanism of defense against the toxicity of fa , which upon esterification get converted into tg and stored into ld . classic measurement of protein catabolism in liver during starvation , revealed that most of the protein degradation in this organ occurs during the 46 h that follow starvation , and that protein breakdown , even of autophagic origin , decreases markedly once the 810 h of starvation are reached . whereas cytosolic proteins and some organelles are the main cargo of the autophagic process during the early hours of starvation , as lack of nutrients persists , there is a gradual change towards preferential sequestration of lipid droplets inside autophagosomes ( figure 2 ) . thus , blockage of autophagy through knockdown of any of the essential atg in hepatocytes in culture , leads to a significant increase in the number of lipid droplets in these cells even when maintained under normal nutritional conditions and in the absence of any additional challenge . similar basal lipophagy has also been observed in cell types not typically known to store fat such as fibroblasts , macrophages , t cells , dendritic cells , lymphoblasts , glia , striatal cell lines , and even primary neurons [ 20 , 3436 ] , although the relative contribution of autophagy to basal lipolysis may vary depending on the cell type . further studies are necessary to determine the reasons behind the coexistence of the two different mechanisms for lipolysis the one mediated by the cytosolic lipases and the one occurring through the autophagic system . lastly , in light of the growing evidence in support of the heterogeneity of the cellular ld , it is also plausible that the two lipolytic systems target different subpopulations of ld . however , the identification of lipophagy in cell types other than those involved in lipid storage , such as in immune or neuronal cells , suggests that autophagic turnover of lipids is perhaps a generic mechanism for the utilization of cellular fat stores in diverse cell types . the neurons within the mediobasal hypothalamus ( mbh ) form part of a focal neural network that integrates nutritional and hormonal information from two main cellular kinases , the mammalian target of rapamycin ( mtor ) and the phosphoinositol-3-kinase ( pi3k ) to control food intake and energy balance . although recent work suggests that neuronal ffa availability and oxidation provide the energetic requirements for activation and firing of orexigenic agouti - related peptide ( agrp ) neurons , the lipolytic mechanisms that generate neuron - intrinsic ffa have remained poorly elucidated . since autophagy is activated by starvation in most cells , it was plausible that autophagic mobilization of lipids in the hypothalamus could contribute to the generation of neuronal ffa during starvation that , in turn , trigger mechanisms driving food intake . this indicates the distinctive characteristic of hypothalamic neurons in their ability to activate autophagy , quite unlike other regions of the brain in which autophagy does not seem to be under this type of nutritional regulation . the activation of autophagy occurred in parallel to starvation - induced increases in hypothalamic ffa uptake ( figure 3 ) , suggesting that , as in the case of the liver , acute ffa stimulus might be a mechanism for activation of hypothalamic autophagy during starvation . the activation of autophagy in hypothalamic neurons upon acute lipid stimulus associated with increases in the levels of phosphorylated ampk and ulk1 , a kinase involved in the regulation of the autophagic process and recently described to be an ampk substrate . although it is possible that ffa modulate some of the signaling cascades involved in agrp regulation , a direct effect of the autophagic process on secretion of agrp - containing vesicles can not be ruled out . interestingly , mice deficient in autophagy in agrp neurons also displayed higher levels of the anorexigenic peptide -melanocyte stimulating hormone ( msh ) , which is produced within the adjacent proopiomelanocortin ( pomc ) neuronal population , which could contribute to the reduced adiposity in these mice . it is possible that adiposity in this model occurred from concurrent reduction of atg7 in both agrp and pomc neuronal populations , or from autophagic deficiency in additional cell types , for instance hypothalamic glial cells , also shown to regulate glucose homeostasis . however , as the actual impact of the sirna injection in the autophagic flux in this model is not known , is not possible to discard that differences in efficiency of the autophagic compromise between pomc and agrp neurons are the real reason behind the different phenotype , or even that autophagy was not affected in the agrp neurons and the phenotype only resulted from reduced pomc autophagy . although future investigation is required to clarify the cell type differences and to identify additional stimuli that may also modulate hypothalamic autophagy , the current findings highlight an exciting new role for lipophagy in control of food intake and whole body energy balance by modulating the controlled production of neuronal ffa that regulate agrp levels ( figure 3 ) . in this way , the contribution of autophagy to the cellular and organismal energetic balance is no longer merely limited to its role in active breakdown of macromolecules or cellular stores to obtain energetic products , but has been raised to a more global regulatory function that includes the modulation of food intake . surprisingly , recent reports by two independent groups demonstrate a completely new and unexpected function of autophagy in regulating adipose physiology [ 48 , 49 ] , which is quite distinct from the role of autophagy in mobilizing lipids observed in other cell types [ 20 , 3436 ] . likewise , blocking autophagy in cultured preadipocytes decreased cellular triglyceride content and levels of key adipogenic transcription factors cebp- ( ccaat / enhancer - binding protein alpha ) , cebp- ( ccaat / enhancer - binding protein beta ) and ppar- . conceivably , reduced expression of adipocyte - specific genes led to the formation of an adipose tissue that predominantly consisted of immature fat - deficient preadipocytes judging by their reduced levels of terminal differentiation markers ( fatty acid synthase , fatty acid - binding protein-4 ( fabp-4/ap-2 ) , glucose transporter 4 ( glut4 ) , or stearoyl coa desaturase 1 ) . the molecular characteristics of the adipose tissue in the autophagy - deficient mice also mimicked those of bat as reflected by increased levels of brown adipogenic factors , ppar- transcriptional coactivator ( pgc-1 ) , and uncoupling protein-1 ( ucp-1 ) . although , the mechanism by which autophagy controls adipocyte differentiation or modulates the phenotypic switch from wat to bat - like is unclear , a likely possibility is that autophagy modulates levels of key regulatory proteins to control adipocyte cell fate , differentiation and fat storage . since expression of the enzyme used to flox out the autophagic gene does not start in this mouse model until 3 - 4 months after birth , it is possible that if autophagy is involved in both formation and mobilization of ld , the partial blockage of autophagy was initially sufficient to cope with the lipophagic requirements of the young animals and prevent the accumulation of ld . this dual involvement of autophagy in lipogenesis ( ld formation ) and lipolysis proposed in the liver now raises the question of whether a similar dual role could also occur in the adipose tissue . after the first observations demonstrating the existence of lipophagy and the upregulation of this process in response to a lipid challenge , numerous studies have confirmed the stimulatory effect of dietary lipids on the autophagic process . upregulation of autophagy in response to increased ffa has been demonstrated in neurons , muscle , pancreas , mammary epithelial cells , liver - derived cells , and even in colon cancer cells [ 5256 ] . although the mechanisms that modulate the activation of the autophagic process under these conditions are still poorly elucidated , at least in the case of the pancreatic beta cell , autophagic activation has been proposed to occur through activation of the c - jun n - terminal kinase 1 pathway in a manner independent of er or oxidative stress . in contrast to this stimulatory effect of a lipid challenge on the autophagic system , an equal number of studies have started to report inhibition of autophagy in response to exposure to high concentrations or particular type of lipids ( figure 4 ) . likewise , in animals exposed to a high - fat diet for prolonged periods of time it is possible to detect an increase in autophagic activity during the first weeks of treatment , which is progressively followed by a gradual decrease in autophagy . although many mechanisms could contribute to the inhibitory effect of ffa on autophagy , a systematic analysis of the different steps of the autophagic process has revealed a primary defect in the fusion between autophagosomes and lysosomes in cells exposed to high concentrations of ffa or in animals subjected to prolonged high - fat diet . future studies are needed to determine which subset of hepatotropic virus makes use of lipophagy for their own replication and whether blockage of the autophagic system can be performed in this organ in a selective way to preferentially affect virogenesis but not normal liver metabolism . this study has revealed that macrophage lipophagy is upregulated both in vitro and in vivo in response to lipid loading and that failure to upregulate the autophagic system , in mice defective for this pathway , results in inefficient clearance of cholesterol in macrophages . in light of the inhibitory effect that high concentrations of intracellular lipids can have on autophagy , it is reasonable to propose that chronic exposure to high levels of circulating lipids may compromise the autophagic system of the artery wall macrophages and lead to their transformation into foam cells as lipids accumulate in their cytoplasm . the fact that a decline in autophagic activity , and in particular in lipophagy , would contribute to intracellular accumulation of ld and that , as described in previous sections , these abnormally expanded lipid stores would further reduce autophagic activity , makes this an attractive feedback loop for the perpetuation of the metabolic syndrome of aging ( characterized by hypercholesterolemia , accumulation of lipid deposits in organs , and insulin resistance ) ( figure 4 ) . although the specific lipid targets of this lipase and the way in which it participates in autophagy remain unknown , it is tempting to propose that part of the effect in life - span could be due to better intracellular lipid handling by lipophagy . this new function of autophagy in lipid metabolism expands the physiological relevance of the autophagic process by making its contribution to the energetic balance more relevant ( when considering the higher energetic value of lipids versus proteins ) , but also including now under the list of autophagic functions the control of many of the regulatory activities that lipids exert inside cells . and also , in a more general context , what are the possible effects of lipophagy on the combinations of metabolic defects that often coexist in our population such as obesity , diabetes , and hyperlipidemia ?

acute lung injury ( ali ) and acute respiratory distress syndrome ( ards ) are life - threatening syndromes that cause high morbidity and mortality . epidemiologic data have revealed that the incidence of ali / ards varies widely by geographic location . the incidence ranges from 64.2 to 78.9 cases/100.000 person - years in the usa , and in northern europe , it is of 17 cases/100.000 person - years . the subsequent neutrophil activation leads to tissue damage via the release of proteases , oxidants , and cationic peptides . through the recent use of molecular and cellular assays and knockout animals , considerable progress has been made towards the understanding of the genetic , tissue - specific , and immunological factors that contribute to the development of ali pathophysiology . however , no specific therapies are available , and the disease outcome has yet to be improved by pharmacologic treatments . ventilation with lower tidal volumes is the only method that has shown a level of benefit . thus , the identification of new molecules that can modulate ali - associated inflammation is highly desirable and is a significant goal of pharmaceutical companies . recent evidence has suggested that certain compounds are effective in inhibiting neutrophil function and infiltration , and such inhibition is desirable for the treatment of patients with ali and sepsis . natural products and their derivatives ( secondary metabolites ) are the most common sources of these drugs . many plant - derived secondary metabolites are capable of directly modulating inflammation by altering the production and activity of second messengers , the expression of transcription factors , and the expression of key proinflammatory molecules [ 68 ] . in addition , they can provide relief from symptoms that is comparable to that obtained from allopathic medicines . ellagic acid , a polyphenol , is present in several fruits , such as grapes , strawberries , pomegranates , and walnuts , as well as several medicinal plants [ 911 ] . ellagic acid has exhibited antioxidant , anticancer , antiallergic , and anti - inflammatory [ 11 , 15 ] activities , among others . interestingly , ellagic acid , as well as extracts that are rich in ellagic acid , has demonstrated significant effects on the airways . in this context , hilaire ( lythraceae ) extracts and ellagic acid reduced most of the phenotypes of experimental ovalbumin - induced allergic airway inflammation , and punica granatum ( lythraceae ) fruit extract reduced the airway inflammation during lps - induced ali in mice . here , we used the nonlethal acid - initiated experimental model of ali to determine whether preventive or therapeutic administration of ellagic acid could interfere with the development and establishment of ali - associated inflammation . in this study , ellagic acid demonstrated potent anti - inflammatory effects , accelerated the resolution of inflammation , and decreased the exacerbation of the inflammation process caused by selective cox-2 inhibition . taken together , these results suggest the potential of ellagic acid as a candidate for the treatment of ali - associated inflammation . all animal care and procedures used in this study were in compliance with the guidelines on the use of animals of the uftm ethics committee ( protocol no . 162 ) , which follow the nih principles of laboratory animal care publication no . 8523 . the experiments were conducted using female balb / c mice ( 57 weeks old and weighing 2025 g ) that were kept in controlled temperature ( 22 2c ) and humidity ( 45%55% ) under a 12:12 h light - dark cycle ( lights on 07:00 h ) . the mice were anesthetised with ketamine ( 50 mg / kg ) and xylazine ( 8 mg / kg ) , and hydrochloric acid ( 0.1 n hcl , ph 1.5 , 50 l ) was intratracheally instilled into the left lung via a 24-gauge angiocatheter . at 12 , 48 , and/or 72 h following acid - initiated acute lung injury bronchoalveolar lavage fluid ( balf ) and lung tissue were harvested . one group of animals was instilled with saline into the left lung ( the control group ) . the treatment of animals with ellagic acid was carried out as described by rogerio et al . . once ellagic acid demonstrates poor solubility in water , the treatment in each animal was carried out with a suspension of ellagic in water at dose 10 mg / kg . the suspension was homogenized with the syringe used in the oral administration before each animal treatment . to study the preventive anti - inflammatory effects , ellagic acid ( 10 mg / kg ) or a vehicle control ( water ) was given daily by oral gavage ( 30 minutes prior and 24 , 47 , and/or 71 h following the intratracheal acid instillation ) . in a second cohort , the mice were therapeutically treated with ellagic acid or the vehicle by oral gavage ( 2 , 24 , 47 , and/or 71 h following the acid - initiated injury ) . in a third cohort , the ellagic acid or the vehicle was administered during the resolution phase after the peak of inflammation ( 12 h ) at 24 , 47 , and/or 71 h following acid - initiated acute lung injury . in a fourth cohort , the animals were pretreated with a selective inhibitor of cyclooxygenase-2 ( cox-2 ) ( celebra , 10 mg / kg ; intraperitoneal route ) 30 min prior to the intratracheal acid instillation [ 18 , 19 ] and then treated with the ellagic acid or the vehicle ( 2 , 24 , and 47 h after the acid - initiated injury ) . as a positive control , the mice were treated with dexamethasone as described earlier ( 1 mg / kg , s.c . injection ) . at 12 , 48 , and/or 72 h following the acid - initiated acute lung injury , the mice were euthanised by sodium pentobarbital overdose ( 70 mg / kg , intraperitoneal ) , and the balf and lung were collected . balf was performed with 1 ml of phosphate - buffered saline ( pbs ) plus 0.6 mm ethylenediamine tetraacetic acid ( edta ) and placed on ice . the total cell and differential leukocyte counts were made according to rogerio et al . . following centrifugation ( 400 g , 5 min , 4c ) , the supernatants of the balf were collected and stored at 80c for subsequent cytokine determination . the animals were injected with evans blue dye ( 30 mg / kg ) via the left retro - orbital plexus 2 h prior to euthanasia . the balf was collected at 12 h following the acid - initiated acute lung injury , and the extravasation of the dye was quantified by spectrophotometry ( absorbance at 650 nm ) [ 20 , 21 ] . il-6 , kc , il-1 , and il-10 levels were assayed by elisa according to the manufacturer 's instructions ( r&d systems , minneapolis , mn , usa ) . when significant differences were identified , the individual comparisons were subsequently made with tukey 's test . ellagic acid ( sigma - aldrich , mo , usa ) , dexamethasone ( decadron , laboratrio teuto brasileiro , go , bra ) , celecoxib ( celebra , pfizer pharmaceuticals llc , sp , bra ) , evans blue ( vetec qumica fina ltda , rj , bra ) , hcl ( vetec qumica fina ltda , rj , bra ) , xylazine ( hertape calier saude animal s / a , mg , bra ) , and ketamine ( cristlia - produtos qumicos farmacuticos ltda , sp , bra ) were purchased . we first evaluated the preventive effect of ellagic acid ( 10 mg / kg , p.o . ) on the acid - initiated acute lung inflammation . the total cells , neutrophils , macrophages , and lymphocytes were quantified from the balf . in nearly all of the time points analysed , the numbers of balf total cells , neutrophils , macrophages , and lymphocytes from the vehicle - treated group were significantly increased compared to the saline control group ( figure 1(a ) ) . the neutrophil numbers significantly increased after lung injury with maximal numbers at 12 h. ellagic acid or dexamethasone treatment significantly reduced the total number of cells and neutrophils in the balf compared to the vehicle - treated mice at 12 , 48 , and 72 h after injury ( figures 1(a ) and 1(b ) ) . the balf neutrophil numbers of mice treated with ellagic acid were reduced by approximately 60% , 67% , and 73% , while the dexamethasone treatment reduced them by 78% , 50% , and 57% at 12 , 48 , and 72 h , respectively . moreover , the ellagic acid significantly reduced the macrophages ( 45% ) and the lymphocytes ( 87% ) numbers at 48 h , while the dexamethasone only reduced the macrophages ( 38% ) and the lymphocytes ( 60% ) numbers at 72 h ( figure 1(d ) ) . the lungs of the vehicle - treated mice demonstrated increased edema , thickening of the alveolar septum and interstitium , and leukocyte infiltration compared to the control group . ellagic acid and dexamethasone treatment reduced all of the aforementioned inflammatory parameters compared to the vehicle - treated mice ( figure 1(e ) ) . to investigate the therapeutic anti - inflammatory effect of ellagic acid on acid - induced ali , the mice were treated with ellagic acid or the vehicle control at 2 , 24 , 47 , or 71 h after lung injury . the numbers of total cells , neutrophils , lymphocytes , and macrophages in the vehicle - treated mice significantly increased compared to the saline control group in nearly all of the time points ( figure 2 ) . the ellagic acid significantly reduced the numbers of total cells and neutrophils in the balf compared to the vehicle - treated mice at all of the time points ( 12 , 48 , and 72 h ) ( figures 2(a ) and 2(b ) ) . the dexamethasone treatment reduced the total cell numbers at 12 and 72 h and reduced the neutrophil numbers at 12 h ( figures 2(a ) and 2(b ) ) . the number of neutrophils in the balf of animals that were treated with ellagic acid was reduced by approximately 52% , 71% , and 70% at 12 , 48 , and 72 h , respectively , while the dexamethasone treatment reduced the neutrophils by 87% at 12 h. the number of macrophages in the balf was reduced by both ellagic acid ( 33% ) and dexamethasone ( 51% ) at 72 h ( figure 2(c ) ) . no significant alteration was observed in the lymphocytes numbers by both ellagic acid and dexamethasone at all of the time points analyzed ( figure 2(d ) ) . additionally , ellagic acid and dexamethasone treatment reduced edema , thickening of the alveolar septum and interstitium , and leukocyte infiltration compared to the vehicle - treated mice ( figure 2(e ) ) . we next evaluated the therapeutic effect of ellagic acid on the balf cytokine levels at 12 h after intratracheal administration of acid . the concentrations of il-6 , kc , and il-1 were increased in the vehicle - treated mice compared to the saline control mice ( figures 3(a)3(c ) ) . ellagic acid and dexamethasone treatment significantly reduced the il-6 levels in the balf ( figure 3(a ) ) . il-6 levels were reduced from 34.4 7.4 pg / ml ( vehicle control ) to 11.369 3.6 pg / ml ( ellagic acid ) and 13.3 15.3 pg / ml ( dexamethasone ) ( mean sem ) . treatment with dexamethasone , but not ellagic acid , reduced the balf il-1 concentration compared to the vehicle - treated mice ( figure 3(b ) ) . in addition , neither ellagic acid nor dexamethasone reduced the kc levels compared to the vehicle control ( figure 3(c ) ) . no significant alteration in il-10 levels was observed in the vehicle - treated mice when compared to the saline control group ( figure 3(d ) ) . ellagic acid and dexamethasone treatment increased the levels of il-10 by approximately eight- and elevenfold , respectively , compared to the saline control group ( figure 3(d ) ) . the vascular permeability changes were increased in vehicle - treated mice compared to the saline control group . ellagic acid and dexamethasone treatment also reduced the vascular permeability changes associated with ali compared to the vehicle - treated mice ( figure 3(e ) ) . we used immunohistochemistry to assess the effects of therapeutic ellagic acid treatment on p - selectin expression and on the phosphorylation state of ap-1 and nf-b at the peak of lung inflammation ( 12 h ) . p65 nf-b staining was observed in the nucleus of the lung cells from the saline control mice . a discrete activation of c - p65 nf-b and c - jun activation was observed in the bronchial epithelium of the vehicle - treated mice ( figures 4 and 5 ) compared to the control group . treatment with dexamethasone , but not ellagic acid , decreased the activation of p65 nf-b and c - jun compared with the vehicle - treated mice ( figures 4 and 5 ) . p - selectin staining was observed in all groups ; however , no significant alteration in staining was observed among the groups ( data not shown ) . we next assessed the therapeutic effect of ellagic acid on the exacerbated inflammatory response of mice exposed to experimental acid - induced ali and treated with the selective cox-2 inhibitor ( celecoxib , 10 mg / kg ; i.p . ) at 48 h. compared to the saline control group , the vehicle- or the celecoxib - treated mice displayed increased numbers of total cells , neutrophils , macrophages , and lymphocytes in the balf ( figure 6 ) . in addition , celecoxib - treated mice had increased total cells , neutrophils , and lymphocytes in the balf when compared to the vehicle - treated mice ( figure 6 ) . ellagic acid ( 10 mg / kg ) and dexamethasone ( 1 mg / kg ) reduced the exacerbation of ali inflammation induced by cox-2 inhibition . both treatments reduced the recruitment of total cells , neutrophils , macrophages , and lymphocytes into the balf . ellagic acid and dexamethasone treatment reduced the number of total cells from 178.3 3.2 ( selective inhibitor of cox-2 ) ( mean 10 cells / ml sem ) by approximately 69% and 77% , respectively , to 55.0 3.2 ( mean 10 cells / ml sem ) and 40.0 1.8 ( mean 10 cells / ml sem ) ( figure 6(a ) ) . similarly , ellagic acid and dexamethasone treatment reduced the number of neutrophils from 73.7 6.4 ( selective inhibitor of cox-2 ) ( mean 10 cells / ml sem ) by approximately 82% and 83% , respectively , to 12.7 1.6 ( mean 10 cells / ml sem ) and 12.3 1.0 ( mean 10 cells / ml sem ) ( figure 6(b ) ) . the number of macrophages was reduced from 82.0 6.5 ( selective inhibitor of cox-2 ) by approximately 53% and 72% to 37.9 3.3 ( ellagic acid ) ( mean 10 cells / ml sem ) and 22.5 2.8 ( dexamethasone ) ( mean 10 cells / ml sem ) , respectively ( figure 6(c ) ) . the number of lymphocytes was reduced from 21.6 4.0 ( selective inhibitor of cox-2 ) by approximately 75% and 82% to 5.5 13.6 ( ellagic acid ) ( mean 10 cells / ml sem ) and 3.8 2.8 ( dexamethasone ) ( mean 10 cells / ml sem ) , respectively ( figure 6(d ) ) . as our results suggested a protective effect of ellagic acid on the airway , we next determined the influence of ellagic acid on the resolution of established airway inflammation . t50 correspond to the time point to reduce 50% of neutrophils , and resolution interval ( ri ) is defined as the time required for the cell numbers to decrease to 50% of the peak of inflammation ( the interval between peak of inflammation , at 12 h , and t50 ) [ 22 , 23 ] ( figure 7(a ) ) . in the vehicle - exposed mice , the t50 was 43 h , and the endogenous resolution interval for the balf neutrophils was 31 h. the treatment with ellagic acid , vehicle , and dexamethasone was carried out 12 h ( in the resolution phase ) after the peak of inflammation ( 12 h ) . the t50 and ri for the balf neutrophils were markedly decreased with the ellagic acid treatment to 33 h and 21 h ( 67% of the vehicle resolution interval ) , respectively ( figure 7(b ) ) , indicative of more rapid resolution of acute inflammation . the dexamethasone treatment also decreased the t50 and ri to 31 h and 19 h ( 61% of the vehicle resolution interval ) ( figure 7(b ) ) , respectively . in the present study , the ellagic acid displayed anti - inflammatory properties by decreasing the severity of hcl acid - initiated ali , accelerating the resolution of inflammation and decreasing the cox-2 inhibitor - induced exacerbation of inflammation . ellagic acid reduced several inflammatory parameters , including the vascular permeability alterations and the neutrophil recruitment to the balf and the lung . in addition , ellagic acid reduced the proinflammatory cytokine il-6 and increased the anti - inflammatory cytokine il-10 in the balf without downregulating the nf-b and ap-1 signaling pathways ( different from dexamethasone ) . together , these findings demonstrated that ellagic acid has potential anti - inflammatory effects for the resolution of ali inflammation . although inflammation is essential for the maintenance of tissue homeostasis and protection against infection , uncontrolled and persistent inflammation may contribute to tissue damage , a characteristic phenomenon of several inflammatory disorders , including ali . in airway inflammation of ali , the neutrophils are the first cells to be recruited and are the predominant cause of tissue damage . a persisting neutrophilia is associated with a poor outcome of ali . in addition , another hallmark of ali is edema , which is a consequence of the increased permeability of the alveolar - capillary barrier , as well as epithelial damage , which results in the impairment of arterial oxygenation [ 24 , 26 ] . over the past several years , no therapeutic agents have demonstrated a clear benefit during ali treatment [ 4 , 27 ] . in the continued search for bioactive plant - derived products , several groups , including our own , have successfully employed experimental models to screen the pharmacologic activities of plant extracts , as well as isolated compounds , such as ellagic acid . utilising secondary metabolites from medicinal plants that can control leukocyte recruitment , such as neutrophils , could be a strategy to reduce the lung damage during ali . phytochemical and pharmacological studies have identified many potential anti - inflammatory substances , particularly those derived from plants used in folk medicine . lafoensia pacari ( lythraceae ) has been used in traditional medicine to treat gastric ulcers and inflammation in the state of mato grosso ( brazil ) . in a bioassay - guided fractionation of the lafoensia pacari extract , rogerio et al . identified ellagic acid as the compound responsible for the reduction of neutrophil recruitment into the peritoneal cavity of mice induced to injury by histoplasma capsulatum - derived -glucan . this reduction in the recruitment of neutrophils and eosinophils to the balf by ellagic acid has also been observed in an ovalbumin - induced experimental allergic airway inflammation model . in addition , lafoensia pacari extract and ellagic acid demonstrated antiedematous activity in a mouse paw edema model [ 11 , 28 ] . ellagitannins present in the pomegranate ( punica granatum ) fruit , which has been used for centuries for medical purposes . studies with pomegranate extract have demonstrated its anti - inflammatory effects in murine models of collagen - induced arthritis and experimental colitis [ 30 , 31 ] . in an experimental model of ali ( lps - initiated ) , the pomegranate extract also reduced the myeloperoxidase ( a heme enzyme present in the primary granules of neutrophils ) in the lungs of mice . the free ellagic acid is absorbed in the gut , but its bioavailability is low . it binds in the intestinal epithelial cells , and its uptake might occur through monolayer of these cells . in addition , ellagic acid could be conjugated ( sulphate ester , glucuronide , and glutathione conjugates ) [ 33 , 35 ] or metabolized by bacteria in the gut to produce urolithins ( a and b ) . these microbial metabolites are more bioavailable than ellagic acid , and in the human plasma , they were detected after consuming pomegranate extract capsule ( 21.6 mg of free ellagic acid ) at 8 and 24 h . so , these metabolites could be also responsible for the biological activity of ellagic acid such as anti - inflammatory activity [ 29 , 37 ] . interesting , while these metabolites do not accumulate in organ tissues , the free ellagic acid irreversibly binds to macromolecules ( proteins and dna ) and consequently might accumulate in the epithelial cells and others cells . ellagic acid was detected in the lung of mouse after oral administration ( at dose 2.0 mmol ~6 mg / kg ) . in addition , these authors demonstrated that ellagic acid localized preferentially in lung ( 10-fold higher ) when compared to liver . so , if beneficial effects of ellagic acid such as the anti - inflammatory activity observed by us in the acute lung injury are associated with urolithin production is not possible to answer . so , as ellagic acid , urolithins , or both could be responsible by this activity , however , only ellagic acid is detected in the lung , and because of this , probably the ellagic acid is major responsible for reducing the airways inflammation . the key result from our study was that the ellagic acid demonstrated both preventive and therapeutic effects in reducing edema and leukocyte recruitment to the balf , similar to dexamethasone , during ali . in addition , ellagic acid , similar to dexamethasone , accelerated the resolution of inflammation by promoting the recovery from lung injury and reduced the exacerbation of inflammation induced by cox-2 inhibition . these results suggest that ellagic acid has potent anti - inflammatory activity and is able to reduce the airway inflammation , as well as its exacerbation , during ali . selectins , a family of transmembrane molecules that are expressed on the surface of leukocytes and activated endothelial cells , are critically involved in leukocyte recruitment . we evaluated the expression of p - selectin in the lungs , as this molecule is considered to be an important target for modulating neutrophil influx into the inflamed tissue [ 40 , 41 ] . our findings revealed no significant alterations in the expression of p - selectin in the bronchial epithelium among the groups ( data not shown ) . demonstrated that nf-b expression and nuclear translocation increased in airway epithelial cells in an ali model ( acid - initiated ) . unlike dexamethasone treatment , ellagic acid did not reduce nf-b activation during the peak of inflammation ( 12 h ) in acid - initiated ali . these findings suggest that the effect of ellagic acid on acid - induced ali was nf-b and ap-1 independent . in ali , a complex network of cytokines and chemokines , such as il-6 , il-1 , and kc , among others , several studies have demonstrated that ellagic acid can inhibit cytokines and chemokines in vivo and in vitro [ 14 , 44 ] . of particular interest , ellagic acid , similar to dexamethasone , reduced the levels of il-6 in the balf of mice with acid - initiated ali . in mice with lps - initiated ali , il-6 was correlated with a proinflammatory phenotype , and high levels of il-6 in the plasma and balf of humans were associated with an increased risk of developing ali . il-10 is an anti - inflammatory cytokine with a significant role in preventing inflammatory diseases . il-10 demonstrated a protective role in lps - induced ali . in the acid - initiated ali , ellagic acid , similar to dexamethasone , increased the il-10 concentration in the balf . therefore , these results suggest that ellagic acid could improve the outcome , as well as the prognosis , of ali in patients by modulating the cytokines involved in ali physiopathology . however , further studies are needed before this possibility can be examined in humans . in conclusion , ali is a disease with high morbidity and mortality , and currently , the disease outcome has yet to be improved by pharmacologic treatment . ellagic acid has demonstrated antioxidant and anti - inflammatory activity in several in vivo and in vitro experimental inflammation models . in hcl acid - initiated ali , ellagic acid demonstrated potent anti - inflammatory effects , accelerated the resolution of inflammation , and decreased the exacerbation of the inflammation process caused by selective cox-2 inhibition . these effects were due to the modulation of cytokines ( decrease of il-6 and increase of il-10 in the balf ) . the inability of ellagic acid to modulate the nf-b and ap-1 signaling pathways suggests that this molecule may control inflammation without inducing an immunosuppressive response , as is observed during dexamethasone treatment . l of ellagic acid , did not produce toxic effects in humans over a 3-year period . moreover , the l. pacari extract ( which contains ellagic acid ) did not show cytotoxicity in vitro or in vivo . in conclusion , ellagic acid might be a future alternative treatment for the reduction of inflammation during ali and other inflammatory diseases with fewer adverse effects than corticosteroids .

acute lung injury ( ali ) is characterized by alveolar edema and uncontrolled neutrophil migration to the lung , and no specific therapy is still available . ellagic acid , a compound present in several fruits and medicinal plants , has shown anti - inflammatory activity in several experimental disease models . we used the nonlethal acid aspiration model of ali in mice to determine whether preventive or therapeutic administration of ellagic acid ( 10 mg / kg ; oral route ) could interfere with the development or establishment of ali inflammation . dexamethasone ( 1 mg / kg ; subcutaneous route ) was used as a positive control . in both preventive and therapeutic treatments , ellagic acid reduced the vascular permeability changes and neutrophil recruitment to the bronchoalveolar lavage fluid ( balf ) and to lung compared to the vehicle . in addition , the ellagic acid accelerated the resolution for lung neutrophilia . moreover , ellagic acid reduced the cox-2-induced exacerbation of inflammation . these results were similar to the dexamethasone . however , while the anti - inflammatory effects of dexamethasone treatment were due to the reduced activation of nf-b and ap-1 , the ellagic acid treatment led to reduced balf levels of il-6 and increased levels of il-10 . in addition , dexamethasone treatment reduced il-1. together , these findings identify ellagic acid as a potential therapeutic agent for ali - associated inflammation .

1. Introduction 2. Methods 3. Materials 4. Results 5. Discussion

acute lung injury ( ali ) and acute respiratory distress syndrome ( ards ) are life - threatening syndromes that cause high morbidity and mortality . through the recent use of molecular and cellular assays and knockout animals , considerable progress has been made towards the understanding of the genetic , tissue - specific , and immunological factors that contribute to the development of ali pathophysiology . however , no specific therapies are available , and the disease outcome has yet to be improved by pharmacologic treatments . thus , the identification of new molecules that can modulate ali - associated inflammation is highly desirable and is a significant goal of pharmaceutical companies . ellagic acid , a polyphenol , is present in several fruits , such as grapes , strawberries , pomegranates , and walnuts , as well as several medicinal plants [ 911 ] . ellagic acid has exhibited antioxidant , anticancer , antiallergic , and anti - inflammatory [ 11 , 15 ] activities , among others . interestingly , ellagic acid , as well as extracts that are rich in ellagic acid , has demonstrated significant effects on the airways . in this context , hilaire ( lythraceae ) extracts and ellagic acid reduced most of the phenotypes of experimental ovalbumin - induced allergic airway inflammation , and punica granatum ( lythraceae ) fruit extract reduced the airway inflammation during lps - induced ali in mice . here , we used the nonlethal acid - initiated experimental model of ali to determine whether preventive or therapeutic administration of ellagic acid could interfere with the development and establishment of ali - associated inflammation . in this study , ellagic acid demonstrated potent anti - inflammatory effects , accelerated the resolution of inflammation , and decreased the exacerbation of the inflammation process caused by selective cox-2 inhibition . taken together , these results suggest the potential of ellagic acid as a candidate for the treatment of ali - associated inflammation . the mice were anesthetised with ketamine ( 50 mg / kg ) and xylazine ( 8 mg / kg ) , and hydrochloric acid ( 0.1 n hcl , ph 1.5 , 50 l ) was intratracheally instilled into the left lung via a 24-gauge angiocatheter . at 12 , 48 , and/or 72 h following acid - initiated acute lung injury bronchoalveolar lavage fluid ( balf ) and lung tissue were harvested . once ellagic acid demonstrates poor solubility in water , the treatment in each animal was carried out with a suspension of ellagic in water at dose 10 mg / kg . to study the preventive anti - inflammatory effects , ellagic acid ( 10 mg / kg ) or a vehicle control ( water ) was given daily by oral gavage ( 30 minutes prior and 24 , 47 , and/or 71 h following the intratracheal acid instillation ) . in a second cohort , the mice were therapeutically treated with ellagic acid or the vehicle by oral gavage ( 2 , 24 , 47 , and/or 71 h following the acid - initiated injury ) . in a third cohort , the ellagic acid or the vehicle was administered during the resolution phase after the peak of inflammation ( 12 h ) at 24 , 47 , and/or 71 h following acid - initiated acute lung injury . in a fourth cohort , the animals were pretreated with a selective inhibitor of cyclooxygenase-2 ( cox-2 ) ( celebra , 10 mg / kg ; intraperitoneal route ) 30 min prior to the intratracheal acid instillation [ 18 , 19 ] and then treated with the ellagic acid or the vehicle ( 2 , 24 , and 47 h after the acid - initiated injury ) . as a positive control , the mice were treated with dexamethasone as described earlier ( 1 mg / kg , s.c . at 12 , 48 , and/or 72 h following the acid - initiated acute lung injury , the mice were euthanised by sodium pentobarbital overdose ( 70 mg / kg , intraperitoneal ) , and the balf and lung were collected . the balf was collected at 12 h following the acid - initiated acute lung injury , and the extravasation of the dye was quantified by spectrophotometry ( absorbance at 650 nm ) [ 20 , 21 ] . ellagic acid ( sigma - aldrich , mo , usa ) , dexamethasone ( decadron , laboratrio teuto brasileiro , go , bra ) , celecoxib ( celebra , pfizer pharmaceuticals llc , sp , bra ) , evans blue ( vetec qumica fina ltda , rj , bra ) , hcl ( vetec qumica fina ltda , rj , bra ) , xylazine ( hertape calier saude animal s / a , mg , bra ) , and ketamine ( cristlia - produtos qumicos farmacuticos ltda , sp , bra ) were purchased . we first evaluated the preventive effect of ellagic acid ( 10 mg / kg , p.o . ) in nearly all of the time points analysed , the numbers of balf total cells , neutrophils , macrophages , and lymphocytes from the vehicle - treated group were significantly increased compared to the saline control group ( figure 1(a ) ) . the neutrophil numbers significantly increased after lung injury with maximal numbers at 12 h. ellagic acid or dexamethasone treatment significantly reduced the total number of cells and neutrophils in the balf compared to the vehicle - treated mice at 12 , 48 , and 72 h after injury ( figures 1(a ) and 1(b ) ) . the balf neutrophil numbers of mice treated with ellagic acid were reduced by approximately 60% , 67% , and 73% , while the dexamethasone treatment reduced them by 78% , 50% , and 57% at 12 , 48 , and 72 h , respectively . moreover , the ellagic acid significantly reduced the macrophages ( 45% ) and the lymphocytes ( 87% ) numbers at 48 h , while the dexamethasone only reduced the macrophages ( 38% ) and the lymphocytes ( 60% ) numbers at 72 h ( figure 1(d ) ) . the lungs of the vehicle - treated mice demonstrated increased edema , thickening of the alveolar septum and interstitium , and leukocyte infiltration compared to the control group . ellagic acid and dexamethasone treatment reduced all of the aforementioned inflammatory parameters compared to the vehicle - treated mice ( figure 1(e ) ) . to investigate the therapeutic anti - inflammatory effect of ellagic acid on acid - induced ali , the mice were treated with ellagic acid or the vehicle control at 2 , 24 , 47 , or 71 h after lung injury . the numbers of total cells , neutrophils , lymphocytes , and macrophages in the vehicle - treated mice significantly increased compared to the saline control group in nearly all of the time points ( figure 2 ) . the ellagic acid significantly reduced the numbers of total cells and neutrophils in the balf compared to the vehicle - treated mice at all of the time points ( 12 , 48 , and 72 h ) ( figures 2(a ) and 2(b ) ) . the dexamethasone treatment reduced the total cell numbers at 12 and 72 h and reduced the neutrophil numbers at 12 h ( figures 2(a ) and 2(b ) ) . the number of neutrophils in the balf of animals that were treated with ellagic acid was reduced by approximately 52% , 71% , and 70% at 12 , 48 , and 72 h , respectively , while the dexamethasone treatment reduced the neutrophils by 87% at 12 h. the number of macrophages in the balf was reduced by both ellagic acid ( 33% ) and dexamethasone ( 51% ) at 72 h ( figure 2(c ) ) . additionally , ellagic acid and dexamethasone treatment reduced edema , thickening of the alveolar septum and interstitium , and leukocyte infiltration compared to the vehicle - treated mice ( figure 2(e ) ) . we next evaluated the therapeutic effect of ellagic acid on the balf cytokine levels at 12 h after intratracheal administration of acid . the concentrations of il-6 , kc , and il-1 were increased in the vehicle - treated mice compared to the saline control mice ( figures 3(a)3(c ) ) . treatment with dexamethasone , but not ellagic acid , reduced the balf il-1 concentration compared to the vehicle - treated mice ( figure 3(b ) ) . in addition , neither ellagic acid nor dexamethasone reduced the kc levels compared to the vehicle control ( figure 3(c ) ) . no significant alteration in il-10 levels was observed in the vehicle - treated mice when compared to the saline control group ( figure 3(d ) ) . ellagic acid and dexamethasone treatment increased the levels of il-10 by approximately eight- and elevenfold , respectively , compared to the saline control group ( figure 3(d ) ) . the vascular permeability changes were increased in vehicle - treated mice compared to the saline control group . ellagic acid and dexamethasone treatment also reduced the vascular permeability changes associated with ali compared to the vehicle - treated mice ( figure 3(e ) ) . we used immunohistochemistry to assess the effects of therapeutic ellagic acid treatment on p - selectin expression and on the phosphorylation state of ap-1 and nf-b at the peak of lung inflammation ( 12 h ) . a discrete activation of c - p65 nf-b and c - jun activation was observed in the bronchial epithelium of the vehicle - treated mice ( figures 4 and 5 ) compared to the control group . treatment with dexamethasone , but not ellagic acid , decreased the activation of p65 nf-b and c - jun compared with the vehicle - treated mice ( figures 4 and 5 ) . we next assessed the therapeutic effect of ellagic acid on the exacerbated inflammatory response of mice exposed to experimental acid - induced ali and treated with the selective cox-2 inhibitor ( celecoxib , 10 mg / kg ; i.p . ) at 48 h. compared to the saline control group , the vehicle- or the celecoxib - treated mice displayed increased numbers of total cells , neutrophils , macrophages , and lymphocytes in the balf ( figure 6 ) . in addition , celecoxib - treated mice had increased total cells , neutrophils , and lymphocytes in the balf when compared to the vehicle - treated mice ( figure 6 ) . ellagic acid ( 10 mg / kg ) and dexamethasone ( 1 mg / kg ) reduced the exacerbation of ali inflammation induced by cox-2 inhibition . ellagic acid and dexamethasone treatment reduced the number of total cells from 178.3 3.2 ( selective inhibitor of cox-2 ) ( mean 10 cells / ml sem ) by approximately 69% and 77% , respectively , to 55.0 3.2 ( mean 10 cells / ml sem ) and 40.0 1.8 ( mean 10 cells / ml sem ) ( figure 6(a ) ) . similarly , ellagic acid and dexamethasone treatment reduced the number of neutrophils from 73.7 6.4 ( selective inhibitor of cox-2 ) ( mean 10 cells / ml sem ) by approximately 82% and 83% , respectively , to 12.7 1.6 ( mean 10 cells / ml sem ) and 12.3 1.0 ( mean 10 cells / ml sem ) ( figure 6(b ) ) . as our results suggested a protective effect of ellagic acid on the airway , we next determined the influence of ellagic acid on the resolution of established airway inflammation . t50 correspond to the time point to reduce 50% of neutrophils , and resolution interval ( ri ) is defined as the time required for the cell numbers to decrease to 50% of the peak of inflammation ( the interval between peak of inflammation , at 12 h , and t50 ) [ 22 , 23 ] ( figure 7(a ) ) . in the vehicle - exposed mice , the t50 was 43 h , and the endogenous resolution interval for the balf neutrophils was 31 h. the treatment with ellagic acid , vehicle , and dexamethasone was carried out 12 h ( in the resolution phase ) after the peak of inflammation ( 12 h ) . the t50 and ri for the balf neutrophils were markedly decreased with the ellagic acid treatment to 33 h and 21 h ( 67% of the vehicle resolution interval ) , respectively ( figure 7(b ) ) , indicative of more rapid resolution of acute inflammation . the dexamethasone treatment also decreased the t50 and ri to 31 h and 19 h ( 61% of the vehicle resolution interval ) ( figure 7(b ) ) , respectively . in the present study , the ellagic acid displayed anti - inflammatory properties by decreasing the severity of hcl acid - initiated ali , accelerating the resolution of inflammation and decreasing the cox-2 inhibitor - induced exacerbation of inflammation . ellagic acid reduced several inflammatory parameters , including the vascular permeability alterations and the neutrophil recruitment to the balf and the lung . in addition , ellagic acid reduced the proinflammatory cytokine il-6 and increased the anti - inflammatory cytokine il-10 in the balf without downregulating the nf-b and ap-1 signaling pathways ( different from dexamethasone ) . together , these findings demonstrated that ellagic acid has potential anti - inflammatory effects for the resolution of ali inflammation . in addition , another hallmark of ali is edema , which is a consequence of the increased permeability of the alveolar - capillary barrier , as well as epithelial damage , which results in the impairment of arterial oxygenation [ 24 , 26 ] . identified ellagic acid as the compound responsible for the reduction of neutrophil recruitment into the peritoneal cavity of mice induced to injury by histoplasma capsulatum - derived -glucan . in addition , lafoensia pacari extract and ellagic acid demonstrated antiedematous activity in a mouse paw edema model [ 11 , 28 ] . studies with pomegranate extract have demonstrated its anti - inflammatory effects in murine models of collagen - induced arthritis and experimental colitis [ 30 , 31 ] . in an experimental model of ali ( lps - initiated ) , the pomegranate extract also reduced the myeloperoxidase ( a heme enzyme present in the primary granules of neutrophils ) in the lungs of mice . in addition , ellagic acid could be conjugated ( sulphate ester , glucuronide , and glutathione conjugates ) [ 33 , 35 ] or metabolized by bacteria in the gut to produce urolithins ( a and b ) . these microbial metabolites are more bioavailable than ellagic acid , and in the human plasma , they were detected after consuming pomegranate extract capsule ( 21.6 mg of free ellagic acid ) at 8 and 24 h . so , these metabolites could be also responsible for the biological activity of ellagic acid such as anti - inflammatory activity [ 29 , 37 ] . interesting , while these metabolites do not accumulate in organ tissues , the free ellagic acid irreversibly binds to macromolecules ( proteins and dna ) and consequently might accumulate in the epithelial cells and others cells . ellagic acid was detected in the lung of mouse after oral administration ( at dose 2.0 mmol ~6 mg / kg ) . in addition , these authors demonstrated that ellagic acid localized preferentially in lung ( 10-fold higher ) when compared to liver . so , if beneficial effects of ellagic acid such as the anti - inflammatory activity observed by us in the acute lung injury are associated with urolithin production is not possible to answer . so , as ellagic acid , urolithins , or both could be responsible by this activity , however , only ellagic acid is detected in the lung , and because of this , probably the ellagic acid is major responsible for reducing the airways inflammation . the key result from our study was that the ellagic acid demonstrated both preventive and therapeutic effects in reducing edema and leukocyte recruitment to the balf , similar to dexamethasone , during ali . in addition , ellagic acid , similar to dexamethasone , accelerated the resolution of inflammation by promoting the recovery from lung injury and reduced the exacerbation of inflammation induced by cox-2 inhibition . these results suggest that ellagic acid has potent anti - inflammatory activity and is able to reduce the airway inflammation , as well as its exacerbation , during ali . unlike dexamethasone treatment , ellagic acid did not reduce nf-b activation during the peak of inflammation ( 12 h ) in acid - initiated ali . these findings suggest that the effect of ellagic acid on acid - induced ali was nf-b and ap-1 independent . in ali , a complex network of cytokines and chemokines , such as il-6 , il-1 , and kc , among others , several studies have demonstrated that ellagic acid can inhibit cytokines and chemokines in vivo and in vitro [ 14 , 44 ] . of particular interest , ellagic acid , similar to dexamethasone , reduced the levels of il-6 in the balf of mice with acid - initiated ali . in mice with lps - initiated ali , il-6 was correlated with a proinflammatory phenotype , and high levels of il-6 in the plasma and balf of humans were associated with an increased risk of developing ali . in the acid - initiated ali , ellagic acid , similar to dexamethasone , increased the il-10 concentration in the balf . therefore , these results suggest that ellagic acid could improve the outcome , as well as the prognosis , of ali in patients by modulating the cytokines involved in ali physiopathology . ellagic acid has demonstrated antioxidant and anti - inflammatory activity in several in vivo and in vitro experimental inflammation models . in hcl acid - initiated ali , ellagic acid demonstrated potent anti - inflammatory effects , accelerated the resolution of inflammation , and decreased the exacerbation of the inflammation process caused by selective cox-2 inhibition . these effects were due to the modulation of cytokines ( decrease of il-6 and increase of il-10 in the balf ) . the inability of ellagic acid to modulate the nf-b and ap-1 signaling pathways suggests that this molecule may control inflammation without inducing an immunosuppressive response , as is observed during dexamethasone treatment . l of ellagic acid , did not produce toxic effects in humans over a 3-year period . moreover , the l. pacari extract ( which contains ellagic acid ) did not show cytotoxicity in vitro or in vivo .

through the recent use of molecular and cellular assays and knockout animals , considerable progress has been made towards the understanding of the genetic , tissue - specific , and immunological factors that contribute to the development of ali pathophysiology . thus , the identification of new molecules that can modulate ali - associated inflammation is highly desirable and is a significant goal of pharmaceutical companies . recent evidence has suggested that certain compounds are effective in inhibiting neutrophil function and infiltration , and such inhibition is desirable for the treatment of patients with ali and sepsis . many plant - derived secondary metabolites are capable of directly modulating inflammation by altering the production and activity of second messengers , the expression of transcription factors , and the expression of key proinflammatory molecules [ 68 ] . ellagic acid , a polyphenol , is present in several fruits , such as grapes , strawberries , pomegranates , and walnuts , as well as several medicinal plants [ 911 ] . in this context , hilaire ( lythraceae ) extracts and ellagic acid reduced most of the phenotypes of experimental ovalbumin - induced allergic airway inflammation , and punica granatum ( lythraceae ) fruit extract reduced the airway inflammation during lps - induced ali in mice . here , we used the nonlethal acid - initiated experimental model of ali to determine whether preventive or therapeutic administration of ellagic acid could interfere with the development and establishment of ali - associated inflammation . in this study , ellagic acid demonstrated potent anti - inflammatory effects , accelerated the resolution of inflammation , and decreased the exacerbation of the inflammation process caused by selective cox-2 inhibition . taken together , these results suggest the potential of ellagic acid as a candidate for the treatment of ali - associated inflammation . all animal care and procedures used in this study were in compliance with the guidelines on the use of animals of the uftm ethics committee ( protocol no . the mice were anesthetised with ketamine ( 50 mg / kg ) and xylazine ( 8 mg / kg ) , and hydrochloric acid ( 0.1 n hcl , ph 1.5 , 50 l ) was intratracheally instilled into the left lung via a 24-gauge angiocatheter . once ellagic acid demonstrates poor solubility in water , the treatment in each animal was carried out with a suspension of ellagic in water at dose 10 mg / kg . to study the preventive anti - inflammatory effects , ellagic acid ( 10 mg / kg ) or a vehicle control ( water ) was given daily by oral gavage ( 30 minutes prior and 24 , 47 , and/or 71 h following the intratracheal acid instillation ) . in a second cohort , the mice were therapeutically treated with ellagic acid or the vehicle by oral gavage ( 2 , 24 , 47 , and/or 71 h following the acid - initiated injury ) . in a third cohort , the ellagic acid or the vehicle was administered during the resolution phase after the peak of inflammation ( 12 h ) at 24 , 47 , and/or 71 h following acid - initiated acute lung injury . in a fourth cohort , the animals were pretreated with a selective inhibitor of cyclooxygenase-2 ( cox-2 ) ( celebra , 10 mg / kg ; intraperitoneal route ) 30 min prior to the intratracheal acid instillation [ 18 , 19 ] and then treated with the ellagic acid or the vehicle ( 2 , 24 , and 47 h after the acid - initiated injury ) . at 12 , 48 , and/or 72 h following the acid - initiated acute lung injury , the mice were euthanised by sodium pentobarbital overdose ( 70 mg / kg , intraperitoneal ) , and the balf and lung were collected . the balf was collected at 12 h following the acid - initiated acute lung injury , and the extravasation of the dye was quantified by spectrophotometry ( absorbance at 650 nm ) [ 20 , 21 ] . ellagic acid ( sigma - aldrich , mo , usa ) , dexamethasone ( decadron , laboratrio teuto brasileiro , go , bra ) , celecoxib ( celebra , pfizer pharmaceuticals llc , sp , bra ) , evans blue ( vetec qumica fina ltda , rj , bra ) , hcl ( vetec qumica fina ltda , rj , bra ) , xylazine ( hertape calier saude animal s / a , mg , bra ) , and ketamine ( cristlia - produtos qumicos farmacuticos ltda , sp , bra ) were purchased . in nearly all of the time points analysed , the numbers of balf total cells , neutrophils , macrophages , and lymphocytes from the vehicle - treated group were significantly increased compared to the saline control group ( figure 1(a ) ) . the neutrophil numbers significantly increased after lung injury with maximal numbers at 12 h. ellagic acid or dexamethasone treatment significantly reduced the total number of cells and neutrophils in the balf compared to the vehicle - treated mice at 12 , 48 , and 72 h after injury ( figures 1(a ) and 1(b ) ) . the balf neutrophil numbers of mice treated with ellagic acid were reduced by approximately 60% , 67% , and 73% , while the dexamethasone treatment reduced them by 78% , 50% , and 57% at 12 , 48 , and 72 h , respectively . moreover , the ellagic acid significantly reduced the macrophages ( 45% ) and the lymphocytes ( 87% ) numbers at 48 h , while the dexamethasone only reduced the macrophages ( 38% ) and the lymphocytes ( 60% ) numbers at 72 h ( figure 1(d ) ) . the lungs of the vehicle - treated mice demonstrated increased edema , thickening of the alveolar septum and interstitium , and leukocyte infiltration compared to the control group . ellagic acid and dexamethasone treatment reduced all of the aforementioned inflammatory parameters compared to the vehicle - treated mice ( figure 1(e ) ) . to investigate the therapeutic anti - inflammatory effect of ellagic acid on acid - induced ali , the mice were treated with ellagic acid or the vehicle control at 2 , 24 , 47 , or 71 h after lung injury . the numbers of total cells , neutrophils , lymphocytes , and macrophages in the vehicle - treated mice significantly increased compared to the saline control group in nearly all of the time points ( figure 2 ) . the ellagic acid significantly reduced the numbers of total cells and neutrophils in the balf compared to the vehicle - treated mice at all of the time points ( 12 , 48 , and 72 h ) ( figures 2(a ) and 2(b ) ) . the number of neutrophils in the balf of animals that were treated with ellagic acid was reduced by approximately 52% , 71% , and 70% at 12 , 48 , and 72 h , respectively , while the dexamethasone treatment reduced the neutrophils by 87% at 12 h. the number of macrophages in the balf was reduced by both ellagic acid ( 33% ) and dexamethasone ( 51% ) at 72 h ( figure 2(c ) ) . no significant alteration was observed in the lymphocytes numbers by both ellagic acid and dexamethasone at all of the time points analyzed ( figure 2(d ) ) . additionally , ellagic acid and dexamethasone treatment reduced edema , thickening of the alveolar septum and interstitium , and leukocyte infiltration compared to the vehicle - treated mice ( figure 2(e ) ) . we next evaluated the therapeutic effect of ellagic acid on the balf cytokine levels at 12 h after intratracheal administration of acid . the concentrations of il-6 , kc , and il-1 were increased in the vehicle - treated mice compared to the saline control mice ( figures 3(a)3(c ) ) . il-6 levels were reduced from 34.4 7.4 pg / ml ( vehicle control ) to 11.369 3.6 pg / ml ( ellagic acid ) and 13.3 15.3 pg / ml ( dexamethasone ) ( mean sem ) . treatment with dexamethasone , but not ellagic acid , reduced the balf il-1 concentration compared to the vehicle - treated mice ( figure 3(b ) ) . no significant alteration in il-10 levels was observed in the vehicle - treated mice when compared to the saline control group ( figure 3(d ) ) . ellagic acid and dexamethasone treatment increased the levels of il-10 by approximately eight- and elevenfold , respectively , compared to the saline control group ( figure 3(d ) ) . ellagic acid and dexamethasone treatment also reduced the vascular permeability changes associated with ali compared to the vehicle - treated mice ( figure 3(e ) ) . we used immunohistochemistry to assess the effects of therapeutic ellagic acid treatment on p - selectin expression and on the phosphorylation state of ap-1 and nf-b at the peak of lung inflammation ( 12 h ) . a discrete activation of c - p65 nf-b and c - jun activation was observed in the bronchial epithelium of the vehicle - treated mice ( figures 4 and 5 ) compared to the control group . treatment with dexamethasone , but not ellagic acid , decreased the activation of p65 nf-b and c - jun compared with the vehicle - treated mice ( figures 4 and 5 ) . p - selectin staining was observed in all groups ; however , no significant alteration in staining was observed among the groups ( data not shown ) . we next assessed the therapeutic effect of ellagic acid on the exacerbated inflammatory response of mice exposed to experimental acid - induced ali and treated with the selective cox-2 inhibitor ( celecoxib , 10 mg / kg ; i.p . ) at 48 h. compared to the saline control group , the vehicle- or the celecoxib - treated mice displayed increased numbers of total cells , neutrophils , macrophages , and lymphocytes in the balf ( figure 6 ) . in addition , celecoxib - treated mice had increased total cells , neutrophils , and lymphocytes in the balf when compared to the vehicle - treated mice ( figure 6 ) . ellagic acid ( 10 mg / kg ) and dexamethasone ( 1 mg / kg ) reduced the exacerbation of ali inflammation induced by cox-2 inhibition . ellagic acid and dexamethasone treatment reduced the number of total cells from 178.3 3.2 ( selective inhibitor of cox-2 ) ( mean 10 cells / ml sem ) by approximately 69% and 77% , respectively , to 55.0 3.2 ( mean 10 cells / ml sem ) and 40.0 1.8 ( mean 10 cells / ml sem ) ( figure 6(a ) ) . similarly , ellagic acid and dexamethasone treatment reduced the number of neutrophils from 73.7 6.4 ( selective inhibitor of cox-2 ) ( mean 10 cells / ml sem ) by approximately 82% and 83% , respectively , to 12.7 1.6 ( mean 10 cells / ml sem ) and 12.3 1.0 ( mean 10 cells / ml sem ) ( figure 6(b ) ) . the number of macrophages was reduced from 82.0 6.5 ( selective inhibitor of cox-2 ) by approximately 53% and 72% to 37.9 3.3 ( ellagic acid ) ( mean 10 cells / ml sem ) and 22.5 2.8 ( dexamethasone ) ( mean 10 cells / ml sem ) , respectively ( figure 6(c ) ) . the number of lymphocytes was reduced from 21.6 4.0 ( selective inhibitor of cox-2 ) by approximately 75% and 82% to 5.5 13.6 ( ellagic acid ) ( mean 10 cells / ml sem ) and 3.8 2.8 ( dexamethasone ) ( mean 10 cells / ml sem ) , respectively ( figure 6(d ) ) . as our results suggested a protective effect of ellagic acid on the airway , we next determined the influence of ellagic acid on the resolution of established airway inflammation . t50 correspond to the time point to reduce 50% of neutrophils , and resolution interval ( ri ) is defined as the time required for the cell numbers to decrease to 50% of the peak of inflammation ( the interval between peak of inflammation , at 12 h , and t50 ) [ 22 , 23 ] ( figure 7(a ) ) . in the vehicle - exposed mice , the t50 was 43 h , and the endogenous resolution interval for the balf neutrophils was 31 h. the treatment with ellagic acid , vehicle , and dexamethasone was carried out 12 h ( in the resolution phase ) after the peak of inflammation ( 12 h ) . the t50 and ri for the balf neutrophils were markedly decreased with the ellagic acid treatment to 33 h and 21 h ( 67% of the vehicle resolution interval ) , respectively ( figure 7(b ) ) , indicative of more rapid resolution of acute inflammation . in the present study , the ellagic acid displayed anti - inflammatory properties by decreasing the severity of hcl acid - initiated ali , accelerating the resolution of inflammation and decreasing the cox-2 inhibitor - induced exacerbation of inflammation . in addition , ellagic acid reduced the proinflammatory cytokine il-6 and increased the anti - inflammatory cytokine il-10 in the balf without downregulating the nf-b and ap-1 signaling pathways ( different from dexamethasone ) . although inflammation is essential for the maintenance of tissue homeostasis and protection against infection , uncontrolled and persistent inflammation may contribute to tissue damage , a characteristic phenomenon of several inflammatory disorders , including ali . in airway inflammation of ali , the neutrophils are the first cells to be recruited and are the predominant cause of tissue damage . in addition , another hallmark of ali is edema , which is a consequence of the increased permeability of the alveolar - capillary barrier , as well as epithelial damage , which results in the impairment of arterial oxygenation [ 24 , 26 ] . in the continued search for bioactive plant - derived products , several groups , including our own , have successfully employed experimental models to screen the pharmacologic activities of plant extracts , as well as isolated compounds , such as ellagic acid . lafoensia pacari ( lythraceae ) has been used in traditional medicine to treat gastric ulcers and inflammation in the state of mato grosso ( brazil ) . identified ellagic acid as the compound responsible for the reduction of neutrophil recruitment into the peritoneal cavity of mice induced to injury by histoplasma capsulatum - derived -glucan . this reduction in the recruitment of neutrophils and eosinophils to the balf by ellagic acid has also been observed in an ovalbumin - induced experimental allergic airway inflammation model . studies with pomegranate extract have demonstrated its anti - inflammatory effects in murine models of collagen - induced arthritis and experimental colitis [ 30 , 31 ] . in an experimental model of ali ( lps - initiated ) , the pomegranate extract also reduced the myeloperoxidase ( a heme enzyme present in the primary granules of neutrophils ) in the lungs of mice . these microbial metabolites are more bioavailable than ellagic acid , and in the human plasma , they were detected after consuming pomegranate extract capsule ( 21.6 mg of free ellagic acid ) at 8 and 24 h . so , if beneficial effects of ellagic acid such as the anti - inflammatory activity observed by us in the acute lung injury are associated with urolithin production is not possible to answer . so , as ellagic acid , urolithins , or both could be responsible by this activity , however , only ellagic acid is detected in the lung , and because of this , probably the ellagic acid is major responsible for reducing the airways inflammation . the key result from our study was that the ellagic acid demonstrated both preventive and therapeutic effects in reducing edema and leukocyte recruitment to the balf , similar to dexamethasone , during ali . in addition , ellagic acid , similar to dexamethasone , accelerated the resolution of inflammation by promoting the recovery from lung injury and reduced the exacerbation of inflammation induced by cox-2 inhibition . these results suggest that ellagic acid has potent anti - inflammatory activity and is able to reduce the airway inflammation , as well as its exacerbation , during ali . we evaluated the expression of p - selectin in the lungs , as this molecule is considered to be an important target for modulating neutrophil influx into the inflamed tissue [ 40 , 41 ] . our findings revealed no significant alterations in the expression of p - selectin in the bronchial epithelium among the groups ( data not shown ) . these findings suggest that the effect of ellagic acid on acid - induced ali was nf-b and ap-1 independent . in ali , a complex network of cytokines and chemokines , such as il-6 , il-1 , and kc , among others , several studies have demonstrated that ellagic acid can inhibit cytokines and chemokines in vivo and in vitro [ 14 , 44 ] . of particular interest , ellagic acid , similar to dexamethasone , reduced the levels of il-6 in the balf of mice with acid - initiated ali . in mice with lps - initiated ali , il-6 was correlated with a proinflammatory phenotype , and high levels of il-6 in the plasma and balf of humans were associated with an increased risk of developing ali . therefore , these results suggest that ellagic acid could improve the outcome , as well as the prognosis , of ali in patients by modulating the cytokines involved in ali physiopathology . in conclusion , ali is a disease with high morbidity and mortality , and currently , the disease outcome has yet to be improved by pharmacologic treatment . in hcl acid - initiated ali , ellagic acid demonstrated potent anti - inflammatory effects , accelerated the resolution of inflammation , and decreased the exacerbation of the inflammation process caused by selective cox-2 inhibition . these effects were due to the modulation of cytokines ( decrease of il-6 and increase of il-10 in the balf ) . the inability of ellagic acid to modulate the nf-b and ap-1 signaling pathways suggests that this molecule may control inflammation without inducing an immunosuppressive response , as is observed during dexamethasone treatment . in conclusion , ellagic acid might be a future alternative treatment for the reduction of inflammation during ali and other inflammatory diseases with fewer adverse effects than corticosteroids .

the metabolic syndrome ( ms ) consists of multiple and interrelated risk factors of metabolic origin that appear to directly promote the development of atherosclerotic cardiovascular disease . the ms strongly associates with type 2 diabetes mellitus , or the risk for this condition . although the exact etiology of the ms still remains unclear , it is known to involve complex interactions between genetic , metabolic , and environmental factors , where diet is of central importance [ 13 ] . there has been a substantial increase in fructose consumption , in the last decades , which has been associated with some adverse metabolic changes similar to those observed in the ms [ 47 ] . on the other hand , minerals like potassium , calcium , and magnesium , proposed as protective against the ms , are generally deficient in ms - inducing diets [ 3 , 810 ] . natural mineral waters are waters of underground origin , protected from contamination and microbiologically wholesome . they are characterized by their purity at source , content in minerals , trace elements , and other constituents as well as by favorable effects on human health . additionally , bioavailability of minerals from natural mineral waters is high [ 1214 ] . the fructose - fed rat is an interesting and well - validated animal model of diet - induced ms ( predominantly acquired ms model ) that is commonly used in ms research . different rat strains with distinct fructose ingestion protocols are reported in the literature and , in all cases , fructose has been observed to induce ms features such as moderate hypertension , glucose intolerance , hyperinsulinemia , insulin resistance , dyslipidemia ( hypertriglyceridemia , hypercholesterolemia ) , altered cytokine and adipokine status ( altered tumor necrosis factor - alpha ( tnf- ) and leptin levels , e.g. ) , decreased melatonin production , and/or increased body fat and/or body weight [ 1520 ] . both in humans and rats , a strong association has been found between ms and oxidative stress and fructose - feeding associates with modification of the hepatic redox status [ 16 , 2224 ] . beneficial effects of acute or chronic natural mineral - rich waters ingestion on blood pressure ( bp ) [ 2527 ] , metabolic profile ( plasma insulin sensitivity , fasting serum glucose concentration , and fasting serum lipid profile [ 25 , 29 ] ) , and plasma oxidative stress markers ( reactive oxygen species , lipid and protein oxidation product levels , total antioxidant capacity , and total thiol levels ) have been published , but , to our knowledge , not in ms individuals or animal models . the natural mineral waters tested are rich , albeit in different proportions , in bicarbonate , calcium , magnesium , potassium , and/or sodium . we aimed to investigate the possible beneficial effect of natural mineral - rich water on ms induction by fructose - feeding . in the present work , sprague - dawley rats ( sdr ) were fed with 10% fructose in natural mineral - rich water ( pedras salgadas ) for 8 weeks and compared to animals fed 10% fructose in tap water . the natural mineral - rich water tested has high total mineralization content ( 2855 mg / l ) , being mainly rich in sodium and bicarbonate and with higher potassium , calcium , and magnesium content than tap water . the study was carried out in 21 adult male cd sdr ( 388483 g ) , from charles river laboratories ( chatillon / chalaronne , france ) . telemetry transmitters ( ta11pa - c40 , data sciences international ( dsi ) , st . paul , mn , usa ) were implanted in the abdominal cavity , with the catheter in the abdominal aorta , by charles river . rats were individually housed , in an enriched environment , and maintained on a daily photoperiod of 12 h lighting schedule ( 2022c ) with free access to standard laboratory pellet food ( 2014 teklad global 14% protein rodent maintenance diet from harlan interfauna iberica sa , barcelona , spain ) and tap water . acclimatization took place for 10 days before starting the experimental protocol , which was authorized by the veterinary national department of the ministry of agriculture , rural development and fisheries . during acclimatization , the handling and care of the animals were conducted in conformity with the european community council guidelines for the use of experimental animals ( 86/609/eec ) and act 129/92 . animals were randomly divided into 3 groups ( 7 animals each ) with free access to different drinking solutions : ( a ) tap water ( cont ) , ( b ) 10% fructose in tap water ( fruct ) , or ( c ) 10% fructose in natural mineral - rich water ( fructmin ) . all experimental groups were fed ad libitum with the standard laboratory chow diet mentioned above ( 20% of energy derived from protein , 13% from fat , and 67% from carbohydrate ) . a 3-week pretreatment period with the natural mineral - rich water was performed to the fructmin group ( while the other rats were drinking tap water ) to allow adjustment to water flavor and sparkles . this period of time induced no change in the pattern of food intake or increase of animals body weight ( data not shown ; all animals weighed 475597 g at the beginning of the dietary manipulation with fructose ) . body weight and food and fluid ingestion values were registered weekly , from week 0 to week 7 or 8 ( as shown in figures 1(a)1(c ) , resp . ) . on week 0 these parameters were evaluated on the same day , which occurred 24 to 48 h after starting the dietary manipulation , and then on every one week after the first measurement . each week , from week 0 to 7 , all animals spent 24 h inside metabolic cages for evaluation of food and fluid consumption as well as for urine collection ( the latter at 0 , 2 , 4 , and 6 weeks ) . at each occasion , the three groups of rats were represented , with an equal number of animals per group . total energy ingestion was calculated by multiplying food and fluid ingestion values by the corresponding reference energy values and then adding these two results . the chemical characteristics of tap and natural mineral - rich waters are given in table 1 . the latter is classified as a hypersaline sodium - rich naturally sparkling mineral water , in conformity with the european community council guidelines for natural mineral waters ( 2009/54/eec ) , and was kindly provided by unicer bebidas , sa ( lea do balio , matosinhos , portugal ) . dataquest art 4.1 silver telemetry system ( dsi ) with rcp-1 receivers ( and apr-1 for ambient pressure reference ) was used for telemetric measurement of bp ( mm hg ) and heart rate ( hr ; beats / min ) in the animal cage . dataquest art acquisition software ( dsi ) was used to monitor all rats , for 8 weeks . sampling was performed every min and setting segment duration at 20 s ; 3 different animals ( one from each experimental group ) were evaluated per day from 4:00 p.m. to 8:00 a.m. , seven days per week . bp values were not included / considered if pulse pressure ( the difference between systolic and diastolic bp ) was below 20 mm hg and hr values were not used if pulse pressure values were lower than 10 mm hg . data were exported from dataquest art 4.0 analysis program ( dsi ) to microsoft excel 2010 ( redmond , wa , usa ) and then subjected to statistical analysis . all chemical substances used in all the experiments were of analytical grade . at the end of the dietary intervention kg of body weight ) and blood was collected from the left ventricle into heparinized syringes . after perfusion , liver , heart , kidneys , and epididymal adipose tissue were rapidly removed from the thoracic and abdominal cavities , washed in cold saline solution , placed in qualitative filter paper for excess liquid removal , and weighed . the liver was cut into several fragments that were immersed in liquid nitrogen and stored at 80c , until further processing . plasma concentrations of glucose , triacylglycerols , total cholesterol , hdl - cholesterol , ldl - cholesterol , c - reactive protein ( crp ) , glutamic - oxaloacetic transaminase ( got ) , glutamic - pyruvic transaminase ( gpt ) , total bilirubin , uric acid , urea , creatinine , total proteins , albumin , ferritin , sodium , potassium , chloride , magnesium , calcium , and phosphorus were determined . all these quantifications were made at the clinical pathology unit of so joo hospital centre , epe , porto , portugal , using standardized methods for human sample routine hospital measurements . plasma levels of insulin ( mercodia ab , 10 - 1137 - 01 ( uppsala , sweden ) ) , adiponectin ( invitrogen corporation , krp0041 ( camarillo , ca , usa ) ) , aldosterone ( uscn life science inc . , e0911ra ( wuhan , china ) ) , substance p ( r&d systems inc . , kge007/skge007/pkge007 ( minneapolis , mn , usa ) ) , interleukin-6 ( il-6 ; cusabio biotech co. ltd . , csb - e04640r ( wuhan , china ) ) , and tnf- ( cusabio , csb - e11987r ) were evaluated according to the manufacturers ' instructions from the specific elisa kits . ( rsh69 k ) , nuclear factor kappa - b ligand ( rankl ; rbn-31 k-1rankl ) , leptin , and osteoprotegerin ( opg ; rbn1 - 31 k ) were measured with a luminex 200 analyzer ( luminex corporation , austin , tx , usa ) according to protocols ( milliplex map kits ) of millipore corporation ( billerica , ma , usa ) . raw data ( mean fluorescence intensity ) were analyzed using istm 2.3 software ( luminex corporation ) . oxidative damage to lipids , proteins , and dna was evaluated by measuring thiobarbituric acid - reactive substances ( viz . , malondialdehyde ( mda ) ) , carbonyls , and 8-hydroxy-2-deoxyguanosine ( 8-ohdg ) levels , respectively . catalase , total superoxide dismutase ( sod ) , glutathione - s - transferase ( gst ) , glutathione - peroxidase ( gpx ) , and glutathione - reductase ( gr ) activities were quantified . , except for the use of bradford method for protein quantification and the use of a kit for 8-ohdg quantification ( the dna extraction kit ( v - gene ) was purchased from bioron international ( ludwigshafen , germany ) and the 8-ohdg kit from japan institute for the control of aging ( haruoka , fukuroi , shizuoka , japan ) ) . liver tissue samples ( 300450 mg ) were homogenized with a teflon - glass homogenizer in an equal volume of protein extraction buffer ( 50 mm tris - base , 150 mm nacl , ph 7.4 , 1% triton x-100 , 0.5% sodium deoxycholate , 0.1% sodium dodecyl sulfate ( sds ) , 1 mm edta , tablets of protease inhibitors , and phosphatase inhibitors ( 100 mm sodium fluoride and 10 mm sodium orthovanadate ) ) , with subsequent agitation for 30 min at 4c . then , each sample was centrifuged , at 13 000 g for 20 min at 4c , and the protein solution under the lipid layer was collected and kept at 80c , until further analysis . proteins were quantified by using the bicinchoninic acid protein assay kit ( pierce , rockford , il , usa ) . proteins were dissolved ( 1 : 1 ) in loading buffer ( 50 mm tris - hcl , ph 6.8 , 100 mm dithiothreitol , 2% sds , 0.01% bromophenol blue and 10% glycerol ) and denatured , for 5 min at 95c . then , 40 g of each sample was loaded per well , separated by electrophoresis in a 12% sds polyacrylamide gel , and transferred to a nitrocellulose membrane ( hybond c - extra , amersham , ge healthcare , buckinghamshire , uk ) . the membrane was blocked in tris - base - buffered saline with 0.1% tween 20 ( v / v ) ( tbst ) containing 5% bovine serum albumin ( w / v ) and incubated overnight with the primary antibody against sirtuin 3 ( sirt3 ; cell signaling technology inc . , danvers , ma , usa ) diluted 1 : 1500 in tbst , with gentle agitation , at 4c . then , the membrane was washed in tbst and incubated with donkey anti - rabbit polyclonal antibody conjugated to horseradish peroxidase ( santa cruz biotechnology inc . , heidelberg , germany ) , diluted 1 : 5000 in tbst , for 1 h at room temperature . detection was performed with an enhanced chemiluminescence reagent ( amersham , ge healthcare , buckinghamshire , uk ) . band intensity was determined using image lab software ( version 4.0.1 ; bio - rad laboratories , hercules , ca , usa ) and normalized for -actin expression ( 1 : 1000 and 1 : 2000 for primary and secondary antibodies ( santa cruz biotechnology inc . , heidelberg , germany ) , respectively , diluted in 5% ( w / v ) of nonfat dry powdered milk sveltesse ( nestl portugal sa , linda - a - velha , portugal ) in tbst ) . liver magnesium and calcium content were measured by inductively coupled plasma optical emission spectrometry ( icp - oes ; activam , jobinyvon , horiba scientific , edison , nj , usa ) , at 285.213 nm and 422.673 nm , respectively , according to iso 11885 ( water quality determination of selected elements by icp - oes ( https://www.astandis.at/shopv5/preview.action;jsessionid=cf7234fbac2bcfdd35a4593a11bd4700?preview=&dokkey=347061&selectedlocale=en ) ) , after microwave oven ( mars 5 , cem corporation , matthews , nc , usa ) assisted acid digestion of liver fragments according to epa 3052 ( microwave assisted acid digestion of siliceous and organically based matrices ( http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3052.pdf ) ) . the significance of differences of each week , cross - sectional statistical analysis , among groups regarding systolic and diastolic bp , hr , body weight , food and fluid ingestions , urine volume , urinary sodium and creatinine excretions , total energy ingestion , and percentage energy supplied by fluid / total energy ingestion was evaluated using anova followed by bonferroni 's multiple comparison test or by kruskal - wallis followed by dunn 's multiple comparison test , according to their distribution . these statistical methods were also used for evaluation of significance of differences among groups regarding organ weight / body weight , hepatic oxidative stress markers , and mineral content as well as plasma biochemical , metabolic , hormonal , and inflammatory data , at the end of the dietary intervention . the association between the outcomes ( systolic and diastolic bp , hr , body weight , food and fluid ingestions , urine volume , urinary sodium and creatinine excretions , total energy ingestion , and percentage energy supplied by fluid / total energy ingestion ) and the interaction of dietary intervention with time evolution ( evaluated in weeks ) , longitudinal statistical analysis , was measured with the interaction terms ( ) , which were estimated by mixed effects model with random effect in the intercept . the area under the curve ( auc ) was calculated through linear interpolation using the composite trapezoid rule . statistical analysis was performed using r : a language and environment for statistical computing , graphpad prism software ( version 6.00 ; la jolla , ca , usa ) , or ibm spss statistics software ( version 20.0 ; armonk , ny , usa ) . values were presented as mean standard error of the mean and differences considered significant for p < 0.05 . in general , on each separate week ( week by week ) , body weight ( figure 1(a ) ) and food ingestion ( figure 1(b ) ) revealed similar values for the 3 animal groups . with time ( over the dietary intervention period ) , a significantly higher and similar increase of body weight for both fructose groups versus cont group was observed ( see supplementary table 1 available online at http://dx.doi.org/10.1155/2014/384583 and inset in figure 1(a ) ) . with time fructmin rats decreased food ingestion significantly more than cont rats and showed a trend towards a higher decrease with time than fruct rats ( supplementary table 1 ) . week by week , fluid ingestion showed significantly higher values for both fructose - fed groups versus cont group , without any significant difference between fruct and fructmin ( figure 1(c ) ) , which was in accordance with auc values ( supplementary table 2 ) . fluid ingestion increased significantly for fructmin versus cont and fruct with time ( supplementary table 1 ) . week by week , no differences were observed for fructose ingestion ( either from fluid ingestion or from both food and fluid ingestions ( data not shown ) ) neither for percentage energy supplied by fluid / total energy ingestion between the two intervention groups ( the latter being in accordance with auc values ; figure 2(a ) and supplementary table 2 ) , in which there was a substantial proportion of energy ingested from fluid ( 4872% of total energy ingestion ; figure 2(a ) ) . total energy ingestion was significantly higher every week of the protocol for both fructose - fed groups versus cont group , without any significant difference between fruct and fructmin ( figure 2(b ) ) , which was in accordance with auc ( supplementary table 2 ) and body weight ( figure 1(a ) ) results . total energy ingestion decreased similarly with time for all groups ( supplementary table 3 ; data not shown for controls ) . with time , urine volume was significantly higher in fructmin versus cont and fruct ( supplementary table 4 ) . week by week , urinary sodium excretion values were expected when taking into consideration the sodium content of tap and natural mineral - rich waters : fructmin group had significantly higher values than the other two animal groups , without any significant difference between fruct and cont ( figure 3(b ) ) , which also agreed with auc values ( supplementary table 2 ) . between weeks 1 and 5 , fruct rats had a significantly higher hr than cont rats ( figure 4(b ) ) . interestingly , both systolic and diastolic bp and hr evolution over time seemed to be protected from fructose effects by the natural mineral - rich water until approximately half of the dietary intervention period ( figures 4(a)-4(b ) , resp . ) . a significant increase of systolic bp with time for both fructose groups versus cont group was observed ( supplementary table 5 ) . diastolic bp in fructmin group showed a tendency to increase with time versus cont group ( supplementary table 5 ) . a significant increase of hr with time for fructmin versus cont was observed ( supplementary table 5 ) . liver and both kidneys weight to body weight ratios were significantly higher in fruct versus cont ( figures 5(a ) , 5(c ) , and 5(d ) , resp . ) . additionally , the liver showed a strong trend to an increase in fructmin versus cont ( p = 0.053 ) and a significant increase in fruct versus fructmin ( figure 5(a ) ) . natural mineral - rich water ingestion prevented fructose effects on liver and both kidneys weight to body weight ratios . epididymal adipose tissue to body weight ratio was slightly and similarly higher in both fructose - fed animal groups versus cont group ( figure 5(b ) ) . no differences were found among groups regarding heart weight / body weight ( data not shown ) . triacylglycerol levels significantly increased in fruct versus cont and a tendency to an increase in fructmin versus cont ( p = 0.080 ) was observed ( figure 6(b ) ) . insulin significantly increased ( figure 6(c ) ) and leptin variation followed the same pattern in fruct versus cont ( p = 0.057 ) ( figure 6(d ) ) . insulin sensitivity index was also calculated and a strong tendency to a decrease was observed in fruct versus cont ( p and global p = 0.055 ; 0.247 10 0.032 10 , 0.137 10 0.009 10 , and 0.211 10 0.030 10 for cont , fruct , and fructmin , resp . ) . glucose ( figure 6(a ) ) and aldosterone ( figure 6(e ) ) seemed to increase and melatonin ( figure 6(f ) ) seemed to decrease in fruct versus cont . natural mineral - rich water ingestion appeared to counteract these fructose - induced metabolic and hormonal effects . urea ( table 2 ) and magnesium ( table 3 ) levels significantly decreased in the two fructose - fed groups versus the cont group . total proteins and albumin levels significantly increased in both groups of fructose - fed animals versus cont group ( except for total proteins in fructmin versus cont where a strong tendency was observed ) ( table 2 ) . tnf- and il-6 levels seemed to increase and opg to rankl ratio seemed to decrease in fruct versus cont ( table 2 ) , with the natural mineral - rich water improving these parameters . crp and substance p levels slightly increased in fructmin versus the other two animal groups ( table 2 ) . the replacement of food by fructose solution as an energy source could explain the similar decreases in plasma urea , magnesium , got , gpt , ferritin , and uric acid levels in both fructose - fed sdr groups versus cont group ( although significantly only for some parameters ) . catalase and sod activities and gsh to gssg ratio increased ( figures 7(a ) , 7(b ) , and 7(e ) , resp . ) and gpx activity , gssg level and sirt3 protein expression decreased ( figures 7(c ) , 7(d ) , and 7(f ) , resp . ) in fruct versus cont ( significantly for catalase , gpx , and gssg and a strong tendency for gsh / gssg ( p = 0.062 , global p = 0.045 ) ) . regarding catalase and gssg , there was a strong trend to , respectively , a decrease and an increase in fructmin versus fruct ( p = 0.065 and p = 0.055 , resp . ) . no significant modifications were observed for 8-ohdg levels ( data not shown ) neither for other redox parameters ( table 4 ) . a slight decrease was observed in fruct versus cont for both liver magnesium and calcium content that was prevented by natural mineral - rich water ingestion ( figures 8(a ) and 8(b ) , resp . ) , most particularly for magnesium . the fructose - fed sdr model mimics a predominantly environmentally acquired ms model that is commonly used in ms research . similarly , in the present study , many of the alterations observed in different protocols of fructose - induced ms were recapitulated . increased systolic bp , adiposity index and liver and kidney weight to body weight ratios as well as modulation of the hepatic redox status and similar changes in the plasma levels of hormones , except for aldosterone , and/or energy substrates evaluated in this work have been reported in different protocols of fructose - induced ms in sdr [ 1518 , 20 , 2224 , 3638 ] . as previously reported , fructose intervention increases sdr body weight , but besides fructose metabolic effects , two details of our experimental protocol could have contributed to body weight increase : rats were housed individually , which may have reduced their physical activity , and were already adult rats at the beginning of the dietary manipulation ( their age was reflected in the high body weight values 475597 g ) , which may have amplified fructose metabolic effects . additionally to the variations observed in food and fluid ingestions seen in the cont group with aging ( over the 8 weeks of dietary intervention ) , fructose - fed rats adjusted fluid and food ingestions , aiming to maintain the level of energy consumption , as previously described . fructose - fed animals increased their body weight similarly between them and more than control rats ( associated with a small increase of epididymal body - fat ) , reflecting the absence of any major natural mineral - rich water consumption effect on both food and fructose ingestions . accordingly , effects shown below against ms induction in fructmin rats related exclusively to natural mineral - rich water ingestion and , interestingly , natural mineral - rich water ingestion reduced / prevented the majority of the fructose effects and , consequently , protected against ms induction , which , to our knowledge , is described here for the first time . ms represents a risk for cardiovascular disease ( whose prevalence is increasing worldwide ) , which , together with the recent report of luo et al . on the consumption of low - mineral bottled water that increases the significant increase in plasma insulin levels in the fruct versus cont group could have contributed to the significant effects in systolic bp and hr described before ( the effect of natural mineral - rich water with time on hr and diastolic bp decreased after body weight adjustment ) . hyperinsulinemia may increase bp and hr by increasing the sympathetic nervous system activity , through alteration in the neuronal vascular control and/or by enhancement of kidney sodium reabsorption [ 6 , 41 , 42 ] . increased sympathetic modulation of vessels and heart precedes metabolic dysfunction in mice drinking 10% fructose in tap water for up to 2 months . hr , a marker of autonomic dysfunction , associates with ms , particularly with insulin resistance , and interestingly , in japan , the prevalence of ms increases linearly with the increase in hr [ 1 , 44 ] . bp correlates with plasma aldosterone levels and an association between plasma aldosterone levels and hyperinsulinemia has been described in obesity . although both fructose - fed groups in the present study had significantly increased body weight versus controls , the insulin value in the fructmin group ( that after body weight adjustment presented a strong tendency to decrease versus fruct ( data not shown ) ) , along with the later bp increase , was in accordance with the unaltered aldosterone levels in the fructmin group . in fructose - fed sdr , the absence of a significant increase in body weight associates with no increase of aldosterone levels , in spite of hyperinsulinemia . leptin resistance may be an early feature of metabolic dysfunction induced by fructose - feeding , since it may precede increased adiposity , elevated circulating leptin levels , and changes in glucose metabolism in rats . despite higher leptin levels ( with the comparison versus cont significant after adjustment for body weight ( data not shown ) ) , which would anticipate a reduction in food intake and body fat in healthy conditions , fruct rats had a lower decrease of food ingestion with time than fructmin rats ( significant after adjustment for body weight ( data not shown ) ) as well as a similar weight gain and amount of epididymal fat . these results could reflect a phenomenon of selective leptin resistance that , together with the activation of the sympathetic nerves by hyperleptinemia , could have contributed to the earlier development of hypertension in fruct rats [ 5 , 4749 ] . melatonin has anti - inflammatory , antihyperlipidemic , and antihypertensive properties and it is known to influence insulin secretion and to enhance its action ( it increases insulin sensitivity and enhances insulin effects on leptin expression ) [ 19 , 5052 ] . the apparent deregulation of leptin , melatonin , insulin , and aldosterone observed in the fruct group , owing to modifications in the hormone levels , was less evident in the fructmin group . fructose is highly lipogenic as its hepatic metabolism provides great amounts of triose phosphate precursors for fatty acid synthesis [ 5 , 6 ] . the difference in triacylglycerol levels in the two fructose - fed groups could be explained by the improvement of leptin , insulin , and aldosterone levels in fructmin induced by the natural mineral - rich water ingestion ( the magnitude of triacylglycerols increase versus cont was reduced after body weight adjustment ( data not shown ) ) . the increase in plasma albumin and total protein levels has been described in fructose - fed rats , which could reflect a combination of undernutrition ( also because of the decrease in food ingestion ) , some degree of liver disorder ( resulting from ms induction ) , and/or dehydration ( owing to loose stools resulting from incomplete fructose absorption ) [ 56 , 57 ] . nevertheless , the pattern of urine volume mirrored the pattern of fluid ingestion and we did not observe loose stools , which makes dehydration unlikely in the fructose - fed rats . although we found a significant increase in both kidneys weight to body weight ratios in the fruct group , we believe that there was no renal functional alteration in this sdr group taking into consideration the plasma and/or urinary profiles of creatinine , urea , albumin , total proteins , magnesium , sodium , potassium , calcium , chloride , and phosphorous . despite an increase in the kidney weight to body weight ratio , rizkalla et al . reported no glomerular basement membrane thickening in sdr after 10 weeks of 57% fructose - feeding . fructose - feeding accelerates osteoporosis and , accordingly , the opg to rankl ratio ( reflecting the ratio of osteoblast versus osteoclast activities [ 58 , 59 ] ) seemed to decrease in the fruct group . interestingly , and in accordance with fruct group results , tnf- and il-6 are important mediators in the process of osteoclast differentiation and activation and , thus , the changes observed in their levels might have contributed to the lower opg to rankl ratio . high levels of leptin and aldosterone have been linked to proinflammatory and prooxidant actions [ 45 , 47 ] . in the fructmin group , the improvement of the leptin , aldosterone , tnf- , and il-6 values could have contributed to the improvement of the opg to rankl ratio . taking into consideration all the results obtained for both fructose - fed sdr groups , the slightly increased levels of substance p and crp in fructmin rats were unexpected . plasma substance p levels increase under magnesium deficiency and contribute to increase inflammation and protein and lipid oxidation . in fact , plasma magnesium levels of both fructose groups were significantly decreased , but the fructmin group displayed better plasma tnf- and il-6 levels as well as lower hepatic protein oxidation content ( after adjustment for body weight this latter parameter showed a tendency to a decrease in fructmin versus the other two groups , most particularly versus fruct ( data not shown ) ) . the absence of oxidative lesions in lipids , proteins , and dna ( the same as for proteins happened for dna oxidative lesions ( data not shown ) ) could be explained by the significantly increased catalase activity and apparently increased sod activity . describe similar results for lipid oxidative lesions and catalase and sod activities by 10% fructose ingestion in tap water . the significant decrease in gpx activity in fruct rats could be partially compensated by the significant increase in catalase activity , since both enzymes can eliminate hydrogen peroxide ( converting it to water ) . catalase is responsible for the elimination of high concentrations of hydrogen peroxide , while gpx does it when concentrations of hydrogen peroxide are low . the higher levels of hydrogen peroxide could have resulted from the fructose - feeding [ 7 , 63 ] and the small decrease of sirt3 protein expression could have intensified reactive oxygen species production in fruct rats . the significant decrease in hepatic gpx activity could have contributed to the strong tendency for an increase in the gsh / gssg ratio in fruct versus cont , by oxidizing less gsh to gssg . increased gssg efflux from hepatocytes and/or increased hepatic gsh synthesis induced by fructose could also apply . however , we did not observe an increase in gsh level as it might have been expected from a lower gsh oxidation and/or increased gsh synthesis . although cellular atp depletion induced by fructose prevents atp - dependent gssg efflux in freshly isolated rat hepatocytes , this phenomenon should not have a strong impact here since an increase in uric acid formation was not observed . the variations observed for gpx , sod , and catalase activities in the two fructose - fed groups are in accordance with the antioxidant actions of melatonin ( probably its primary function ) and sirt3 . melatonin possesses free radical - scavenging activity , stimulates antioxidant enzymes ( e.g. , gpx ) , and inhibits reactive oxygen / nitrogen species producing enzymes [ 66 , 67 ] . sirt3 has beneficial effects on mitochondrial electron transport chain ( contributing to a reduction in the production of reactive oxygen species ) and mitochondrial antioxidant enzymes ( probably also on gpx , like melatonin ) . reduction of sirt3 associates with an accelerated development of metabolic abnormalities similar to the ms , which is in agreement with our overall results . our results also showed that the natural mineral - rich water could contribute to the preservation of the hepatic intracellular ions , namely the magnesium content . it is well documented that plasma ion levels might not reflect their tissue levels , and here this was evident regarding plasma and hepatic concentrations of magnesium and calcium . for quite some time , it was thought that it could be the cause of insulin resistance , but very recently it was described that type 2 diabetes mellitus and a lower degree of metabolic control are essential in accounting for the lower levels of serum magnesium that occur in obese individuals . figure 9 summarizes the significant effects of fructose - feeding obtained in this research that were reduced / prevented by the natural mineral - rich water ( taking into consideration that when fructose was coingested with the natural mineral - rich water no significant effects were observed versus the control ) . still , although for some of the parameters evaluated in our study the extension of differences among groups did not achieve statistical significance , the variations observed were consistent with the pattern expected , which reinforces their biological relevance and justifies their presentation and discussion . the results here described suggest that this natural mineral - rich water seems to have potential to prevent ms induction . we hypothesize that its regular intake in the context of modern diets , which have a general acidic character interfering with mineral homeostasis and are poor in micronutrients , namely potassium , calcium , and magnesium , could add surplus value and attenuate imbalances , thus contributing to metabolic and redox health and , consequently , decreasing the risk for atherosclerotic cardiovascular disease .

the metabolic syndrome increases the risk for atherosclerotic cardiovascular disease and type 2 diabetes mellitus . increased fructose consumption and/or mineral deficiency have been associated with metabolic syndrome development . this study aimed to investigate the effects of 8 weeks consumption of a hypersaline sodium - rich naturally sparkling mineral water on 10% fructose - fed sprague - dawley rats ( metabolic syndrome animal model ) . the ingestion of the mineral water ( rich in sodium bicarbonate and with higher potassium , calcium , and magnesium content than the tap water used as control ) reduced / prevented not only the fructose - induced increase of heart rate , plasma triacylglycerols , insulin and leptin levels , hepatic catalase activity , and organ weight to body weight ratios ( for liver and both kidneys ) but also the decrease of hepatic glutathione peroxidase activity and oxidized glutathione content . this mineral - rich water seems to have potential to prevent metabolic syndrome induction by fructose . we hypothesize that its regular intake in the context of modern diets , which have a general acidic character interfering with mineral homeostasis and are poor in micronutrients , namely potassium , calcium , and magnesium , could add surplus value and attenuate imbalances , thus contributing to metabolic and redox health and , consequently , decreasing the risk for atherosclerotic cardiovascular disease .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusion

the metabolic syndrome ( ms ) consists of multiple and interrelated risk factors of metabolic origin that appear to directly promote the development of atherosclerotic cardiovascular disease . the ms strongly associates with type 2 diabetes mellitus , or the risk for this condition . although the exact etiology of the ms still remains unclear , it is known to involve complex interactions between genetic , metabolic , and environmental factors , where diet is of central importance [ 13 ] . there has been a substantial increase in fructose consumption , in the last decades , which has been associated with some adverse metabolic changes similar to those observed in the ms [ 47 ] . on the other hand , minerals like potassium , calcium , and magnesium , proposed as protective against the ms , are generally deficient in ms - inducing diets [ 3 , 810 ] . the fructose - fed rat is an interesting and well - validated animal model of diet - induced ms ( predominantly acquired ms model ) that is commonly used in ms research . different rat strains with distinct fructose ingestion protocols are reported in the literature and , in all cases , fructose has been observed to induce ms features such as moderate hypertension , glucose intolerance , hyperinsulinemia , insulin resistance , dyslipidemia ( hypertriglyceridemia , hypercholesterolemia ) , altered cytokine and adipokine status ( altered tumor necrosis factor - alpha ( tnf- ) and leptin levels , e.g. ) both in humans and rats , a strong association has been found between ms and oxidative stress and fructose - feeding associates with modification of the hepatic redox status [ 16 , 2224 ] . beneficial effects of acute or chronic natural mineral - rich waters ingestion on blood pressure ( bp ) [ 2527 ] , metabolic profile ( plasma insulin sensitivity , fasting serum glucose concentration , and fasting serum lipid profile [ 25 , 29 ] ) , and plasma oxidative stress markers ( reactive oxygen species , lipid and protein oxidation product levels , total antioxidant capacity , and total thiol levels ) have been published , but , to our knowledge , not in ms individuals or animal models . the natural mineral waters tested are rich , albeit in different proportions , in bicarbonate , calcium , magnesium , potassium , and/or sodium . we aimed to investigate the possible beneficial effect of natural mineral - rich water on ms induction by fructose - feeding . in the present work , sprague - dawley rats ( sdr ) were fed with 10% fructose in natural mineral - rich water ( pedras salgadas ) for 8 weeks and compared to animals fed 10% fructose in tap water . the natural mineral - rich water tested has high total mineralization content ( 2855 mg / l ) , being mainly rich in sodium and bicarbonate and with higher potassium , calcium , and magnesium content than tap water . rats were individually housed , in an enriched environment , and maintained on a daily photoperiod of 12 h lighting schedule ( 2022c ) with free access to standard laboratory pellet food ( 2014 teklad global 14% protein rodent maintenance diet from harlan interfauna iberica sa , barcelona , spain ) and tap water . acclimatization took place for 10 days before starting the experimental protocol , which was authorized by the veterinary national department of the ministry of agriculture , rural development and fisheries . animals were randomly divided into 3 groups ( 7 animals each ) with free access to different drinking solutions : ( a ) tap water ( cont ) , ( b ) 10% fructose in tap water ( fruct ) , or ( c ) 10% fructose in natural mineral - rich water ( fructmin ) . a 3-week pretreatment period with the natural mineral - rich water was performed to the fructmin group ( while the other rats were drinking tap water ) to allow adjustment to water flavor and sparkles . this period of time induced no change in the pattern of food intake or increase of animals body weight ( data not shown ; all animals weighed 475597 g at the beginning of the dietary manipulation with fructose ) . on week 0 these parameters were evaluated on the same day , which occurred 24 to 48 h after starting the dietary manipulation , and then on every one week after the first measurement . the chemical characteristics of tap and natural mineral - rich waters are given in table 1 . the latter is classified as a hypersaline sodium - rich naturally sparkling mineral water , in conformity with the european community council guidelines for natural mineral waters ( 2009/54/eec ) , and was kindly provided by unicer bebidas , sa ( lea do balio , matosinhos , portugal ) . dataquest art 4.1 silver telemetry system ( dsi ) with rcp-1 receivers ( and apr-1 for ambient pressure reference ) was used for telemetric measurement of bp ( mm hg ) and heart rate ( hr ; beats / min ) in the animal cage . at the end of the dietary intervention kg of body weight ) and blood was collected from the left ventricle into heparinized syringes . plasma concentrations of glucose , triacylglycerols , total cholesterol , hdl - cholesterol , ldl - cholesterol , c - reactive protein ( crp ) , glutamic - oxaloacetic transaminase ( got ) , glutamic - pyruvic transaminase ( gpt ) , total bilirubin , uric acid , urea , creatinine , total proteins , albumin , ferritin , sodium , potassium , chloride , magnesium , calcium , and phosphorus were determined . , malondialdehyde ( mda ) ) , carbonyls , and 8-hydroxy-2-deoxyguanosine ( 8-ohdg ) levels , respectively . the significance of differences of each week , cross - sectional statistical analysis , among groups regarding systolic and diastolic bp , hr , body weight , food and fluid ingestions , urine volume , urinary sodium and creatinine excretions , total energy ingestion , and percentage energy supplied by fluid / total energy ingestion was evaluated using anova followed by bonferroni 's multiple comparison test or by kruskal - wallis followed by dunn 's multiple comparison test , according to their distribution . these statistical methods were also used for evaluation of significance of differences among groups regarding organ weight / body weight , hepatic oxidative stress markers , and mineral content as well as plasma biochemical , metabolic , hormonal , and inflammatory data , at the end of the dietary intervention . the association between the outcomes ( systolic and diastolic bp , hr , body weight , food and fluid ingestions , urine volume , urinary sodium and creatinine excretions , total energy ingestion , and percentage energy supplied by fluid / total energy ingestion ) and the interaction of dietary intervention with time evolution ( evaluated in weeks ) , longitudinal statistical analysis , was measured with the interaction terms ( ) , which were estimated by mixed effects model with random effect in the intercept . with time ( over the dietary intervention period ) , a significantly higher and similar increase of body weight for both fructose groups versus cont group was observed ( see supplementary table 1 available online at http://dx.doi.org/10.1155/2014/384583 and inset in figure 1(a ) ) . week by week , fluid ingestion showed significantly higher values for both fructose - fed groups versus cont group , without any significant difference between fruct and fructmin ( figure 1(c ) ) , which was in accordance with auc values ( supplementary table 2 ) . total energy ingestion was significantly higher every week of the protocol for both fructose - fed groups versus cont group , without any significant difference between fruct and fructmin ( figure 2(b ) ) , which was in accordance with auc ( supplementary table 2 ) and body weight ( figure 1(a ) ) results . week by week , urinary sodium excretion values were expected when taking into consideration the sodium content of tap and natural mineral - rich waters : fructmin group had significantly higher values than the other two animal groups , without any significant difference between fruct and cont ( figure 3(b ) ) , which also agreed with auc values ( supplementary table 2 ) . interestingly , both systolic and diastolic bp and hr evolution over time seemed to be protected from fructose effects by the natural mineral - rich water until approximately half of the dietary intervention period ( figures 4(a)-4(b ) , resp . ) liver and both kidneys weight to body weight ratios were significantly higher in fruct versus cont ( figures 5(a ) , 5(c ) , and 5(d ) , resp . ) natural mineral - rich water ingestion prevented fructose effects on liver and both kidneys weight to body weight ratios . epididymal adipose tissue to body weight ratio was slightly and similarly higher in both fructose - fed animal groups versus cont group ( figure 5(b ) ) . natural mineral - rich water ingestion appeared to counteract these fructose - induced metabolic and hormonal effects . urea ( table 2 ) and magnesium ( table 3 ) levels significantly decreased in the two fructose - fed groups versus the cont group . total proteins and albumin levels significantly increased in both groups of fructose - fed animals versus cont group ( except for total proteins in fructmin versus cont where a strong tendency was observed ) ( table 2 ) . tnf- and il-6 levels seemed to increase and opg to rankl ratio seemed to decrease in fruct versus cont ( table 2 ) , with the natural mineral - rich water improving these parameters . the replacement of food by fructose solution as an energy source could explain the similar decreases in plasma urea , magnesium , got , gpt , ferritin , and uric acid levels in both fructose - fed sdr groups versus cont group ( although significantly only for some parameters ) . and gpx activity , gssg level and sirt3 protein expression decreased ( figures 7(c ) , 7(d ) , and 7(f ) , resp . ) a slight decrease was observed in fruct versus cont for both liver magnesium and calcium content that was prevented by natural mineral - rich water ingestion ( figures 8(a ) and 8(b ) , resp . ) the fructose - fed sdr model mimics a predominantly environmentally acquired ms model that is commonly used in ms research . similarly , in the present study , many of the alterations observed in different protocols of fructose - induced ms were recapitulated . increased systolic bp , adiposity index and liver and kidney weight to body weight ratios as well as modulation of the hepatic redox status and similar changes in the plasma levels of hormones , except for aldosterone , and/or energy substrates evaluated in this work have been reported in different protocols of fructose - induced ms in sdr [ 1518 , 20 , 2224 , 3638 ] . as previously reported , fructose intervention increases sdr body weight , but besides fructose metabolic effects , two details of our experimental protocol could have contributed to body weight increase : rats were housed individually , which may have reduced their physical activity , and were already adult rats at the beginning of the dietary manipulation ( their age was reflected in the high body weight values 475597 g ) , which may have amplified fructose metabolic effects . additionally to the variations observed in food and fluid ingestions seen in the cont group with aging ( over the 8 weeks of dietary intervention ) , fructose - fed rats adjusted fluid and food ingestions , aiming to maintain the level of energy consumption , as previously described . fructose - fed animals increased their body weight similarly between them and more than control rats ( associated with a small increase of epididymal body - fat ) , reflecting the absence of any major natural mineral - rich water consumption effect on both food and fructose ingestions . accordingly , effects shown below against ms induction in fructmin rats related exclusively to natural mineral - rich water ingestion and , interestingly , natural mineral - rich water ingestion reduced / prevented the majority of the fructose effects and , consequently , protected against ms induction , which , to our knowledge , is described here for the first time . ms represents a risk for cardiovascular disease ( whose prevalence is increasing worldwide ) , which , together with the recent report of luo et al . on the consumption of low - mineral bottled water that increases the significant increase in plasma insulin levels in the fruct versus cont group could have contributed to the significant effects in systolic bp and hr described before ( the effect of natural mineral - rich water with time on hr and diastolic bp decreased after body weight adjustment ) . hyperinsulinemia may increase bp and hr by increasing the sympathetic nervous system activity , through alteration in the neuronal vascular control and/or by enhancement of kidney sodium reabsorption [ 6 , 41 , 42 ] . increased sympathetic modulation of vessels and heart precedes metabolic dysfunction in mice drinking 10% fructose in tap water for up to 2 months . although both fructose - fed groups in the present study had significantly increased body weight versus controls , the insulin value in the fructmin group ( that after body weight adjustment presented a strong tendency to decrease versus fruct ( data not shown ) ) , along with the later bp increase , was in accordance with the unaltered aldosterone levels in the fructmin group . in fructose - fed sdr , the absence of a significant increase in body weight associates with no increase of aldosterone levels , in spite of hyperinsulinemia . leptin resistance may be an early feature of metabolic dysfunction induced by fructose - feeding , since it may precede increased adiposity , elevated circulating leptin levels , and changes in glucose metabolism in rats . despite higher leptin levels ( with the comparison versus cont significant after adjustment for body weight ( data not shown ) ) , which would anticipate a reduction in food intake and body fat in healthy conditions , fruct rats had a lower decrease of food ingestion with time than fructmin rats ( significant after adjustment for body weight ( data not shown ) ) as well as a similar weight gain and amount of epididymal fat . these results could reflect a phenomenon of selective leptin resistance that , together with the activation of the sympathetic nerves by hyperleptinemia , could have contributed to the earlier development of hypertension in fruct rats [ 5 , 4749 ] . the apparent deregulation of leptin , melatonin , insulin , and aldosterone observed in the fruct group , owing to modifications in the hormone levels , was less evident in the fructmin group . the difference in triacylglycerol levels in the two fructose - fed groups could be explained by the improvement of leptin , insulin , and aldosterone levels in fructmin induced by the natural mineral - rich water ingestion ( the magnitude of triacylglycerols increase versus cont was reduced after body weight adjustment ( data not shown ) ) . the increase in plasma albumin and total protein levels has been described in fructose - fed rats , which could reflect a combination of undernutrition ( also because of the decrease in food ingestion ) , some degree of liver disorder ( resulting from ms induction ) , and/or dehydration ( owing to loose stools resulting from incomplete fructose absorption ) [ 56 , 57 ] . nevertheless , the pattern of urine volume mirrored the pattern of fluid ingestion and we did not observe loose stools , which makes dehydration unlikely in the fructose - fed rats . although we found a significant increase in both kidneys weight to body weight ratios in the fruct group , we believe that there was no renal functional alteration in this sdr group taking into consideration the plasma and/or urinary profiles of creatinine , urea , albumin , total proteins , magnesium , sodium , potassium , calcium , chloride , and phosphorous . despite an increase in the kidney weight to body weight ratio , rizkalla et al . fructose - feeding accelerates osteoporosis and , accordingly , the opg to rankl ratio ( reflecting the ratio of osteoblast versus osteoclast activities [ 58 , 59 ] ) seemed to decrease in the fruct group . interestingly , and in accordance with fruct group results , tnf- and il-6 are important mediators in the process of osteoclast differentiation and activation and , thus , the changes observed in their levels might have contributed to the lower opg to rankl ratio . in the fructmin group , the improvement of the leptin , aldosterone , tnf- , and il-6 values could have contributed to the improvement of the opg to rankl ratio . taking into consideration all the results obtained for both fructose - fed sdr groups , the slightly increased levels of substance p and crp in fructmin rats were unexpected . in fact , plasma magnesium levels of both fructose groups were significantly decreased , but the fructmin group displayed better plasma tnf- and il-6 levels as well as lower hepatic protein oxidation content ( after adjustment for body weight this latter parameter showed a tendency to a decrease in fructmin versus the other two groups , most particularly versus fruct ( data not shown ) ) . the absence of oxidative lesions in lipids , proteins , and dna ( the same as for proteins happened for dna oxidative lesions ( data not shown ) ) could be explained by the significantly increased catalase activity and apparently increased sod activity . describe similar results for lipid oxidative lesions and catalase and sod activities by 10% fructose ingestion in tap water . the significant decrease in gpx activity in fruct rats could be partially compensated by the significant increase in catalase activity , since both enzymes can eliminate hydrogen peroxide ( converting it to water ) . the higher levels of hydrogen peroxide could have resulted from the fructose - feeding [ 7 , 63 ] and the small decrease of sirt3 protein expression could have intensified reactive oxygen species production in fruct rats . although cellular atp depletion induced by fructose prevents atp - dependent gssg efflux in freshly isolated rat hepatocytes , this phenomenon should not have a strong impact here since an increase in uric acid formation was not observed . the variations observed for gpx , sod , and catalase activities in the two fructose - fed groups are in accordance with the antioxidant actions of melatonin ( probably its primary function ) and sirt3 . sirt3 has beneficial effects on mitochondrial electron transport chain ( contributing to a reduction in the production of reactive oxygen species ) and mitochondrial antioxidant enzymes ( probably also on gpx , like melatonin ) . our results also showed that the natural mineral - rich water could contribute to the preservation of the hepatic intracellular ions , namely the magnesium content . it is well documented that plasma ion levels might not reflect their tissue levels , and here this was evident regarding plasma and hepatic concentrations of magnesium and calcium . for quite some time , it was thought that it could be the cause of insulin resistance , but very recently it was described that type 2 diabetes mellitus and a lower degree of metabolic control are essential in accounting for the lower levels of serum magnesium that occur in obese individuals . figure 9 summarizes the significant effects of fructose - feeding obtained in this research that were reduced / prevented by the natural mineral - rich water ( taking into consideration that when fructose was coingested with the natural mineral - rich water no significant effects were observed versus the control ) . still , although for some of the parameters evaluated in our study the extension of differences among groups did not achieve statistical significance , the variations observed were consistent with the pattern expected , which reinforces their biological relevance and justifies their presentation and discussion . the results here described suggest that this natural mineral - rich water seems to have potential to prevent ms induction . we hypothesize that its regular intake in the context of modern diets , which have a general acidic character interfering with mineral homeostasis and are poor in micronutrients , namely potassium , calcium , and magnesium , could add surplus value and attenuate imbalances , thus contributing to metabolic and redox health and , consequently , decreasing the risk for atherosclerotic cardiovascular disease .

there has been a substantial increase in fructose consumption , in the last decades , which has been associated with some adverse metabolic changes similar to those observed in the ms [ 47 ] . on the other hand , minerals like potassium , calcium , and magnesium , proposed as protective against the ms , are generally deficient in ms - inducing diets [ 3 , 810 ] . different rat strains with distinct fructose ingestion protocols are reported in the literature and , in all cases , fructose has been observed to induce ms features such as moderate hypertension , glucose intolerance , hyperinsulinemia , insulin resistance , dyslipidemia ( hypertriglyceridemia , hypercholesterolemia ) , altered cytokine and adipokine status ( altered tumor necrosis factor - alpha ( tnf- ) and leptin levels , e.g. ) both in humans and rats , a strong association has been found between ms and oxidative stress and fructose - feeding associates with modification of the hepatic redox status [ 16 , 2224 ] . beneficial effects of acute or chronic natural mineral - rich waters ingestion on blood pressure ( bp ) [ 2527 ] , metabolic profile ( plasma insulin sensitivity , fasting serum glucose concentration , and fasting serum lipid profile [ 25 , 29 ] ) , and plasma oxidative stress markers ( reactive oxygen species , lipid and protein oxidation product levels , total antioxidant capacity , and total thiol levels ) have been published , but , to our knowledge , not in ms individuals or animal models . we aimed to investigate the possible beneficial effect of natural mineral - rich water on ms induction by fructose - feeding . in the present work , sprague - dawley rats ( sdr ) were fed with 10% fructose in natural mineral - rich water ( pedras salgadas ) for 8 weeks and compared to animals fed 10% fructose in tap water . the natural mineral - rich water tested has high total mineralization content ( 2855 mg / l ) , being mainly rich in sodium and bicarbonate and with higher potassium , calcium , and magnesium content than tap water . during acclimatization , the handling and care of the animals were conducted in conformity with the european community council guidelines for the use of experimental animals ( 86/609/eec ) and act 129/92 . animals were randomly divided into 3 groups ( 7 animals each ) with free access to different drinking solutions : ( a ) tap water ( cont ) , ( b ) 10% fructose in tap water ( fruct ) , or ( c ) 10% fructose in natural mineral - rich water ( fructmin ) . a 3-week pretreatment period with the natural mineral - rich water was performed to the fructmin group ( while the other rats were drinking tap water ) to allow adjustment to water flavor and sparkles . this period of time induced no change in the pattern of food intake or increase of animals body weight ( data not shown ; all animals weighed 475597 g at the beginning of the dietary manipulation with fructose ) . on week 0 these parameters were evaluated on the same day , which occurred 24 to 48 h after starting the dietary manipulation , and then on every one week after the first measurement . plasma concentrations of glucose , triacylglycerols , total cholesterol , hdl - cholesterol , ldl - cholesterol , c - reactive protein ( crp ) , glutamic - oxaloacetic transaminase ( got ) , glutamic - pyruvic transaminase ( gpt ) , total bilirubin , uric acid , urea , creatinine , total proteins , albumin , ferritin , sodium , potassium , chloride , magnesium , calcium , and phosphorus were determined . , csb - e04640r ( wuhan , china ) ) , and tnf- ( cusabio , csb - e11987r ) were evaluated according to the manufacturers ' instructions from the specific elisa kits . ( rsh69 k ) , nuclear factor kappa - b ligand ( rankl ; rbn-31 k-1rankl ) , leptin , and osteoprotegerin ( opg ; rbn1 - 31 k ) were measured with a luminex 200 analyzer ( luminex corporation , austin , tx , usa ) according to protocols ( milliplex map kits ) of millipore corporation ( billerica , ma , usa ) . catalase , total superoxide dismutase ( sod ) , glutathione - s - transferase ( gst ) , glutathione - peroxidase ( gpx ) , and glutathione - reductase ( gr ) activities were quantified . , except for the use of bradford method for protein quantification and the use of a kit for 8-ohdg quantification ( the dna extraction kit ( v - gene ) was purchased from bioron international ( ludwigshafen , germany ) and the 8-ohdg kit from japan institute for the control of aging ( haruoka , fukuroi , shizuoka , japan ) ) . liver magnesium and calcium content were measured by inductively coupled plasma optical emission spectrometry ( icp - oes ; activam , jobinyvon , horiba scientific , edison , nj , usa ) , at 285.213 nm and 422.673 nm , respectively , according to iso 11885 ( water quality determination of selected elements by icp - oes ( https://www.astandis.at/shopv5/preview.action;jsessionid=cf7234fbac2bcfdd35a4593a11bd4700?preview=&dokkey=347061&selectedlocale=en ) ) , after microwave oven ( mars 5 , cem corporation , matthews , nc , usa ) assisted acid digestion of liver fragments according to epa 3052 ( microwave assisted acid digestion of siliceous and organically based matrices ( http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3052.pdf ) ) . the significance of differences of each week , cross - sectional statistical analysis , among groups regarding systolic and diastolic bp , hr , body weight , food and fluid ingestions , urine volume , urinary sodium and creatinine excretions , total energy ingestion , and percentage energy supplied by fluid / total energy ingestion was evaluated using anova followed by bonferroni 's multiple comparison test or by kruskal - wallis followed by dunn 's multiple comparison test , according to their distribution . these statistical methods were also used for evaluation of significance of differences among groups regarding organ weight / body weight , hepatic oxidative stress markers , and mineral content as well as plasma biochemical , metabolic , hormonal , and inflammatory data , at the end of the dietary intervention . the association between the outcomes ( systolic and diastolic bp , hr , body weight , food and fluid ingestions , urine volume , urinary sodium and creatinine excretions , total energy ingestion , and percentage energy supplied by fluid / total energy ingestion ) and the interaction of dietary intervention with time evolution ( evaluated in weeks ) , longitudinal statistical analysis , was measured with the interaction terms ( ) , which were estimated by mixed effects model with random effect in the intercept . with time ( over the dietary intervention period ) , a significantly higher and similar increase of body weight for both fructose groups versus cont group was observed ( see supplementary table 1 available online at http://dx.doi.org/10.1155/2014/384583 and inset in figure 1(a ) ) . with time fructmin rats decreased food ingestion significantly more than cont rats and showed a trend towards a higher decrease with time than fruct rats ( supplementary table 1 ) . week by week , fluid ingestion showed significantly higher values for both fructose - fed groups versus cont group , without any significant difference between fruct and fructmin ( figure 1(c ) ) , which was in accordance with auc values ( supplementary table 2 ) . week by week , no differences were observed for fructose ingestion ( either from fluid ingestion or from both food and fluid ingestions ( data not shown ) ) neither for percentage energy supplied by fluid / total energy ingestion between the two intervention groups ( the latter being in accordance with auc values ; figure 2(a ) and supplementary table 2 ) , in which there was a substantial proportion of energy ingested from fluid ( 4872% of total energy ingestion ; figure 2(a ) ) . total energy ingestion was significantly higher every week of the protocol for both fructose - fed groups versus cont group , without any significant difference between fruct and fructmin ( figure 2(b ) ) , which was in accordance with auc ( supplementary table 2 ) and body weight ( figure 1(a ) ) results . week by week , urinary sodium excretion values were expected when taking into consideration the sodium content of tap and natural mineral - rich waters : fructmin group had significantly higher values than the other two animal groups , without any significant difference between fruct and cont ( figure 3(b ) ) , which also agreed with auc values ( supplementary table 2 ) . interestingly , both systolic and diastolic bp and hr evolution over time seemed to be protected from fructose effects by the natural mineral - rich water until approximately half of the dietary intervention period ( figures 4(a)-4(b ) , resp . ) additionally , the liver showed a strong trend to an increase in fructmin versus cont ( p = 0.053 ) and a significant increase in fruct versus fructmin ( figure 5(a ) ) . insulin sensitivity index was also calculated and a strong tendency to a decrease was observed in fruct versus cont ( p and global p = 0.055 ; 0.247 10 0.032 10 , 0.137 10 0.009 10 , and 0.211 10 0.030 10 for cont , fruct , and fructmin , resp . ) urea ( table 2 ) and magnesium ( table 3 ) levels significantly decreased in the two fructose - fed groups versus the cont group . total proteins and albumin levels significantly increased in both groups of fructose - fed animals versus cont group ( except for total proteins in fructmin versus cont where a strong tendency was observed ) ( table 2 ) . tnf- and il-6 levels seemed to increase and opg to rankl ratio seemed to decrease in fruct versus cont ( table 2 ) , with the natural mineral - rich water improving these parameters . the replacement of food by fructose solution as an energy source could explain the similar decreases in plasma urea , magnesium , got , gpt , ferritin , and uric acid levels in both fructose - fed sdr groups versus cont group ( although significantly only for some parameters ) . in fruct versus cont ( significantly for catalase , gpx , and gssg and a strong tendency for gsh / gssg ( p = 0.062 , global p = 0.045 ) ) . regarding catalase and gssg , there was a strong trend to , respectively , a decrease and an increase in fructmin versus fruct ( p = 0.065 and p = 0.055 , resp . ) similarly , in the present study , many of the alterations observed in different protocols of fructose - induced ms were recapitulated . increased systolic bp , adiposity index and liver and kidney weight to body weight ratios as well as modulation of the hepatic redox status and similar changes in the plasma levels of hormones , except for aldosterone , and/or energy substrates evaluated in this work have been reported in different protocols of fructose - induced ms in sdr [ 1518 , 20 , 2224 , 3638 ] . as previously reported , fructose intervention increases sdr body weight , but besides fructose metabolic effects , two details of our experimental protocol could have contributed to body weight increase : rats were housed individually , which may have reduced their physical activity , and were already adult rats at the beginning of the dietary manipulation ( their age was reflected in the high body weight values 475597 g ) , which may have amplified fructose metabolic effects . additionally to the variations observed in food and fluid ingestions seen in the cont group with aging ( over the 8 weeks of dietary intervention ) , fructose - fed rats adjusted fluid and food ingestions , aiming to maintain the level of energy consumption , as previously described . fructose - fed animals increased their body weight similarly between them and more than control rats ( associated with a small increase of epididymal body - fat ) , reflecting the absence of any major natural mineral - rich water consumption effect on both food and fructose ingestions . accordingly , effects shown below against ms induction in fructmin rats related exclusively to natural mineral - rich water ingestion and , interestingly , natural mineral - rich water ingestion reduced / prevented the majority of the fructose effects and , consequently , protected against ms induction , which , to our knowledge , is described here for the first time . on the consumption of low - mineral bottled water that increases the significant increase in plasma insulin levels in the fruct versus cont group could have contributed to the significant effects in systolic bp and hr described before ( the effect of natural mineral - rich water with time on hr and diastolic bp decreased after body weight adjustment ) . hr , a marker of autonomic dysfunction , associates with ms , particularly with insulin resistance , and interestingly , in japan , the prevalence of ms increases linearly with the increase in hr [ 1 , 44 ] . although both fructose - fed groups in the present study had significantly increased body weight versus controls , the insulin value in the fructmin group ( that after body weight adjustment presented a strong tendency to decrease versus fruct ( data not shown ) ) , along with the later bp increase , was in accordance with the unaltered aldosterone levels in the fructmin group . in fructose - fed sdr , the absence of a significant increase in body weight associates with no increase of aldosterone levels , in spite of hyperinsulinemia . despite higher leptin levels ( with the comparison versus cont significant after adjustment for body weight ( data not shown ) ) , which would anticipate a reduction in food intake and body fat in healthy conditions , fruct rats had a lower decrease of food ingestion with time than fructmin rats ( significant after adjustment for body weight ( data not shown ) ) as well as a similar weight gain and amount of epididymal fat . these results could reflect a phenomenon of selective leptin resistance that , together with the activation of the sympathetic nerves by hyperleptinemia , could have contributed to the earlier development of hypertension in fruct rats [ 5 , 4749 ] . the apparent deregulation of leptin , melatonin , insulin , and aldosterone observed in the fruct group , owing to modifications in the hormone levels , was less evident in the fructmin group . the difference in triacylglycerol levels in the two fructose - fed groups could be explained by the improvement of leptin , insulin , and aldosterone levels in fructmin induced by the natural mineral - rich water ingestion ( the magnitude of triacylglycerols increase versus cont was reduced after body weight adjustment ( data not shown ) ) . the increase in plasma albumin and total protein levels has been described in fructose - fed rats , which could reflect a combination of undernutrition ( also because of the decrease in food ingestion ) , some degree of liver disorder ( resulting from ms induction ) , and/or dehydration ( owing to loose stools resulting from incomplete fructose absorption ) [ 56 , 57 ] . nevertheless , the pattern of urine volume mirrored the pattern of fluid ingestion and we did not observe loose stools , which makes dehydration unlikely in the fructose - fed rats . although we found a significant increase in both kidneys weight to body weight ratios in the fruct group , we believe that there was no renal functional alteration in this sdr group taking into consideration the plasma and/or urinary profiles of creatinine , urea , albumin , total proteins , magnesium , sodium , potassium , calcium , chloride , and phosphorous . fructose - feeding accelerates osteoporosis and , accordingly , the opg to rankl ratio ( reflecting the ratio of osteoblast versus osteoclast activities [ 58 , 59 ] ) seemed to decrease in the fruct group . interestingly , and in accordance with fruct group results , tnf- and il-6 are important mediators in the process of osteoclast differentiation and activation and , thus , the changes observed in their levels might have contributed to the lower opg to rankl ratio . in the fructmin group , the improvement of the leptin , aldosterone , tnf- , and il-6 values could have contributed to the improvement of the opg to rankl ratio . taking into consideration all the results obtained for both fructose - fed sdr groups , the slightly increased levels of substance p and crp in fructmin rats were unexpected . in fact , plasma magnesium levels of both fructose groups were significantly decreased , but the fructmin group displayed better plasma tnf- and il-6 levels as well as lower hepatic protein oxidation content ( after adjustment for body weight this latter parameter showed a tendency to a decrease in fructmin versus the other two groups , most particularly versus fruct ( data not shown ) ) . the absence of oxidative lesions in lipids , proteins , and dna ( the same as for proteins happened for dna oxidative lesions ( data not shown ) ) could be explained by the significantly increased catalase activity and apparently increased sod activity . the significant decrease in gpx activity in fruct rats could be partially compensated by the significant increase in catalase activity , since both enzymes can eliminate hydrogen peroxide ( converting it to water ) . the significant decrease in hepatic gpx activity could have contributed to the strong tendency for an increase in the gsh / gssg ratio in fruct versus cont , by oxidizing less gsh to gssg . the variations observed for gpx , sod , and catalase activities in the two fructose - fed groups are in accordance with the antioxidant actions of melatonin ( probably its primary function ) and sirt3 . sirt3 has beneficial effects on mitochondrial electron transport chain ( contributing to a reduction in the production of reactive oxygen species ) and mitochondrial antioxidant enzymes ( probably also on gpx , like melatonin ) . our results also showed that the natural mineral - rich water could contribute to the preservation of the hepatic intracellular ions , namely the magnesium content . for quite some time , it was thought that it could be the cause of insulin resistance , but very recently it was described that type 2 diabetes mellitus and a lower degree of metabolic control are essential in accounting for the lower levels of serum magnesium that occur in obese individuals . figure 9 summarizes the significant effects of fructose - feeding obtained in this research that were reduced / prevented by the natural mineral - rich water ( taking into consideration that when fructose was coingested with the natural mineral - rich water no significant effects were observed versus the control ) . still , although for some of the parameters evaluated in our study the extension of differences among groups did not achieve statistical significance , the variations observed were consistent with the pattern expected , which reinforces their biological relevance and justifies their presentation and discussion . we hypothesize that its regular intake in the context of modern diets , which have a general acidic character interfering with mineral homeostasis and are poor in micronutrients , namely potassium , calcium , and magnesium , could add surplus value and attenuate imbalances , thus contributing to metabolic and redox health and , consequently , decreasing the risk for atherosclerotic cardiovascular disease .

the evolution of the nervous system in the animal kingdom is currently under major debate in evolutionary developmental biology and comparative embryology 1 . in this context , the evolutionary path that leads to the morphological complexity of the nervous system passes through the invertebrate / vertebrate transition . at the beginning of the xx century , the cephalochordate amphioxus occupied the most prominent role in illuminating , at the morphological level , such a transition ( see 2 for an historical perspective ) . during the last decade , increasing information on the molecular aspects of amphioxus development has revealed a number of previously hidden features . one prominent example is the molecular regionalisation of the amphioxus nervous system , which is greatly hindered by its anatomical simplicity . the central nervous system of an adult amphioxus is a mere tubular nerve cord lying dorsally over the notochord . the nerve cord and the notochord cover together the entire length of the animal , except in the anterior region , where only the notochord reaches the rostral tip of the body ( fig . 1 ) . this exclusive rostral expansion of the notochord is a hallmark of the taxon cephalochordata ( corda for corda dorsalis , original name of the notochord - in the head ) . the only externally visible landmarks of the amphioxus nerve cord are discrete enlargements at both the anterior and posterior ends , regarded as the anterior vesicle and the caudal ampulla , respectively . transiently during embryonic development , in young larvae , the anterior swelling is commonly known as cerebral vesicle and is much more pronounced than in adults . apart from the serially repeated exit points of the dorsal nerves , the nerve cord of amphioxus is devoid of rhombomeres or other external morphological indications of segmentation . the outgoing dorsal nerves , unsupplied with ganglia , contribute to the peripheral nervous system by innervating most of the organs of the body , shaping an irregular nerve net , where a number of peripheral neurons are also accommodated 3 in contrast with the anatomically non - regionalised central nervous system and the apparently disorganised peripheral nervous system , the discrete expression patterns of distinct genes and the specific distribution of certain proteins outline a completely different chronicle for the nervous system of amphioxus . most of the neural genes characterised to date reveal gapped expression domains within the developing central nervous system , and restricted expression to certain cell subpopulations within the embryonic epidermis . retinoic acid treatments greatly affect these gene expression domains 4 , suggesting a well - controlled pre - patterning that defines distinct territories , not only within the neural ectoderm but also within the epidermis of amphioxus embryos . after two centuries of heated debate , amphioxus is still an epicentre for the controversial evolutionary origins of vertebrates . whatever its actual position , as the sister group of vertebrates , as the earliest chordate , or as ( less likely ) the earliest deuterostome 5 , its privileged phylogenetic position sets amphioxus in the appropriate place to cast light on some of the main transitions that occurred during animal evolution : the origin of deuterostomes , the origin of chordates , or the origin of vertebrates , all of them correlated with novel evolutionary features , and some of them linked to the elaboration of the most complex nervous systems . the relative phylogenetic position of cephalochordates and urochordates has very recently received a lot of attention 6 . both cephalochordates and urochordates are invertebrate chordates that share with vertebrates certain anatomical features : a notochord , pharyngeal slits and a dorsal hollow nerve cord . however , these pre - vertebrate characteristics are lost in tunicates after metamorphosis , while they are maintained for life in cephalochordates . furthermore , cephalochordates but not urochordates possess additional vertebrate - like characters , such as a post - anal tail and segmentally arranged muscles ( myomeres ) ( fig . 1 ) . both clades include the only invertebrates able to form a dorsal nerve cord through neurulation . however , unlike in urochordates , in amphioxus the nervous system seems to be developmentally active beyond metamorphosis . this is supported by modern anatomy 3 , which shows that cytoarchytectural reorganisations should occur within the nerve cord of amphioxus until reaching a definitive internal structure in the adult . the present communion between modern morphology and the use of molecular biology tools is revealing an unexpected variety of cell types , territories and boundaries along the antero - posterior axis of the nerve cord and on the epidermis of amphioxus . here i attempt to summarize distinct patterns of gene expression , giving an overall view of the formation of the central and peripheral nervous systems of amphioxus and suggesting the possible origin of two characteristic neural features in vertebrates : the placodes and the neural crest . in amphioxus , the embryonic anterior - posterior axis is determined early ( by the time of gastrulation ) , as a consequence of the animal - vegetal polarity of the oocyte , induced by the sperm entry point during fertilisation . at the onset of gastrulation , the vegetal pole of the blastula invaginates , leaving a wide - open blastopore , which subsequently closes . as soon as invagination begins , amphiwnt1 turns on around the blastoporal opening in conjunction with ambra , followed by the expression of amphiwnt8 and a down - regulation of -catenin in the invaginating mesendoderm 7,8 . this territorial area homing the expression of these genes is soon segregated into the blastoporal lips , which remain on the posterior side of the embryo and will contribute to the tail bud later in development 9 . other amphioxus wnt genes are also expressed around the blastopore in an overlapping pattern to that of amphinotch and ambra 7 , 10 , 11 . this indicates that both the wnt and notch pathways may act as a posterior signalling centre involved in the specification and maintenance of the posterior identity . whether the notch and wnt signalling systems are involved in the specification of the dorso - ventral axis however , it has been suggested that wnt signals may be involved in restricting expression of genes such as amphisox1/2/3 , amphidral and amphidll to the ectoderm 7 . amphisox1/2/3 and amphineurogenin are the earliest known markers for the presumptive neuroectoderm in amphioxus 12 . the expression of both largely overlaps at the early gastrula , on the outer dorsal epiblast ( ectoderm ) , thus indicating the existence of a dorso - ventral axis already at this stage . opposite amphisox1/2/3 and amphineurogenin , amphidral is expressed in the non - neural ectoderm , suggesting that the neural / non - neural boundary might become delimited early in development 13 . throughout gastrulation , some of the genes involved in neural patterning are expressed in the dorsal blastoporal lip , which has been regarded as the homolog of the amphibian spemann 's organiser 14 , crucially involved in neural induction . in vertebrates , the early nodal expression is involved in the establishment of the spemann 's organiser and induces the expression of organiser - specific genes such as goosecoid . in amphioxus , amphinodal 's strongest expression is in the inner hypoblast ( mesendoderm ) of the dorsal blastoporal lip 15 . therefore , amphinodal might well induce the expression of goosecoid in the dorsal blastoporal lip , where it overlaps with other organiser - related genes such as amhnf3 , ambra and amphifoxd , but not amphiotx 11 , 16 - 19 . this incomplete overlapping expression occurs for a short period of time and , in comparison with vertebrates , it is slightly delayed in development . thus , it is unclear to what extent the dorsal blastoporal lip functions , if it does , as a dorsal - ventral organiser , though it may have some properties related to the spemann 's organiser , such as influencing the formation of the neural plate and the underlying chordamesoderm . as gastrulation proceeds , some genes begin to be restrictively expressed in certain regions of the neuroectoderm , which may be an anticipation of the cryptic regionalisation of the neural tube and the epidermis observed in succeeding stages . islet and amphipax6 are first solely expressed in the anterior neuroectoderm , whereas amphievxa is expressed just in the posterior neuroectoderm and amphimsx in the middle part 20 - 23 . the homogeneous expression of amphisox1/2/3 and amphineurogenin in the dorsal epiblast is gradually restricted only to the neural ectoderm 12 . amphizic and amphivent , which were earlier widely expressed dorsally , become restricted to the lateral regions of the nascent neural plate 14 , 24 . conversely , other genes are expanded to the neuroectoderm , as shown by amphibrn1/2/4 , amphipax3/7 and the various amphihairy genes . as the gastrula elongates to reach the earliest neurula stage , the expression of amphibrn1/2/4 , initially located in the dorsal epiblast , gradually spans from the posterior to the anterior neural plate 25 . the initial expression of amphipax3/7 in the dorsal presumptive axial and paraxial mesoderm is additionally extended to the dorsal ectoderm , where it also appears at the lateral edges of the presumptive neural plate 26 . likewise , the amphihairy genes also label the forming neural plate , in this case accompanied by a complementary striped expression in the presumptive somitic mesoderm 27 . this raises the recurrent question on the effect that mesoderm segmentation might exert on the molecular regionalisation of the neural tube in amphioxus . although not segmental , the mesodermal expression of i d and amphidmbx could be tentatively related to this issue , since in vertebrates they have a well - defined location within the developing central nervous system 28 , 29 . furthermore , the apposed ectodermal - mesodermal expression also observed for the amphioxus snail gene , which is detected in the dorsal presumptive somitic mesoderm and the overlying edges of the forming neural plate , and several other genes ( e.g. amphizic , amphivent ) also suggest a collaborative role between the two germ layers 30 . at the end of gastrulation , as the neural plate starts to form , the amphioxus embryo becomes more vertebrate - like . however , in contrast with vertebrates , neural folds do not form during amphioxus neurulation . instead , the non - neural ectoderm detaches from the edges of the open neural plate , overgrowing laterally over the neural plate and fusing in the dorsal midline . as the non - neural ectoderm covers the neural plate , the lateral edges of the latter curl up dorsally and fuse in the dorsal midline , underneath the non - neural ectoderm , to form the neural tube . once the neural tube is formed , it stills remains anteriorly open through the neuropore . subsequently , as the neurula elongates , the posterior neural tube and the adjacent notochord and somites stem from the tail bud , which probably still maintains some of the organiser properties mentioned above . throughout neurulation , the various gene expression patterns are highly dynamic within the central nervous system ( table 1 ) and reveal discrete molecular boundaries , most of them hidden at a morphological level . the central nervous system of amphioxus neurulae , in its advanced stages , consists of a dorsal neural cord lacking constrictions along its rostrocaudal axis . the only regional difference lies at the anterior end , only regional difference lies at the anterior end only regional difference lies at the anterior end where the neural canal is slightly dilated into a cerebral vesicle , anterior to the level of somite 1 and up to the anterior part of somite 2 . most of the cerebral vesicle is dominated by the expression of amphiotx , yet with a gap of expression halfway along its anterior - posterior axis . this organ is the source of the reissner 's fiber , which fills the central neural canal , and is probably the homologue of the subcommisural organ of the vertebrate diencephalon 16 . in this region similarly , amphidll - positive cells are also embedded in the rostral domain of amphiotx , but located more anteriorly than amphibf-1 , in the preneuroporal region 32 . amphid1 is also expressed in the cerebral vesicle at the level of the presumptive infundibular organ , while amphith is expressed at the level of the presumptive lamellar organ , which in turn is covered by the caudal expression of amphiotx 33,34 . these sharp expressions probably reflect a complex internal morphology within the cerebral vesicle ; this is also observed for amphipax6 , which is expressed in a few cells of the developing frontal eye 20 . the posterior expression of amphiotx overlaps with that of amphidll , amphipax6 , amphisim and the anteriormost expression of amphifoxb . the delimited expression of amphisim in this area is especially interesting , since it is a marker of the posterior diencephalon and midbrain in vertebrates . however , amphioxus orthologues of other vertebrate midbrain and mhb ( midbrain - hindbrain boundary ) markers , such as amphievx , amphien , amphipax2/5/8 and amphiwnt1 , have failed to provide evidence for a true midbrain and mhb homolog in amphioxus 29,35 . both amphibf-1 and amphidll are orthologue genes to those marking the vertebrate telencephalon and , in the case of amphidll , other parts of the vertebrate diencephalon as well . together with the expression of amphiotx , the orthologues of which are markers of the vertebrate telencephalon , diencephalon and midbrain , and the territorial domain occupied by amphisim , combined gene expression suggests that the amphioxus cerebral vesicle is largely homologous to the vertebrate forebrain ( telencephalon plus diencephalon ) , although certain homology with the midbrain can not be completely ruled out . in the hypothetical absence of a midbrain homolog in amphioxus , the territory posterior to the cerebral vesicle , caudal to amphiotx expression at the level of somite 2 , has been widely seen as a homolog of the craniate hindbrain . following the expression of amphiotx , amphioxus hindbrain is subjugated to the expression of different hox genes , partially overlapped along the anterior - posterior axis . spatially , the first hox gene expressed in the anterior hindbrain is amphihox1 ( somites 2 - 4 ) , followed by amphihox3 ( somites 4 - 8 ) and amphihox4 ( somites 6 - 8 ) 36 . amphifoxb is expressed segmentally as well , concomitantly with hox genes , though it shows a prominent gap of expression at the level of somite 5 , where the pigment spot will form and , intriguingly , where the expression of other segmental genes is also disrupted ( e.g. mnx , islet ) 35 . other genes broadly expressed within the neural tube of amphioxus are : amphisox1/2/3 , amphinetrin , amphif - spondin , the different hairy genes and amphibrn1/2/4 . all of them label certain fractions of the hindbrain and some of them include some spots of expression within the cerebral vesicle . while amphisox1/2/3 is excluded along the midline and the anterior part of the neural tube , amphinetrin shows a fairly complementary pattern , being expressed just along the midline 12,37 . notwithstanding , amphinetrin expression in the floor plate lasts longer than amphisox1/2/3 expression does and always overlies its own expression in the dorsal notochord , even below the cerebral vesicle ( from which it is excluded ) , until the larval stages , when amphinetrin is lost at both anterior and posterior ends of the notochord . the long - lasting expression of amphinetrin in the floor plate suggests a role of the amphioxus floor plate in the control of axon path finding , possibly guiding newborn neurons within the nerve cord 37 . amphif - spondin expression by the end of neurulation largely overlaps the expression of amphinetrin , though covering broader areas within the neural tube , including the cerebral vesicle , where it shows a gap of expression just on the ventral side 38 . similar to amphisox1/2/3 , amphibrn1/2/4 is excluded from the midline , but is additionally expressed in the cerebral vesicle with the same gap of expression as the one observed for the amphif - spondin gene 25 . throughout the neural tube , amphioxus hairy genes are expressed in complementary patterns . amphihairyb is expressed in the anterior neural tube , spatially followed by amphihairyc and amphihairyd , being amphihairya the most restricted one to the posterior neural tube . independently , all of them show gapped expression and overlap at certain levels , just in some cases filling the gaps of one another . as mentioned previously , in general the expression of the different amphihairy genes in the neural tube is accompanied by a striped expression in the somitic mesoderm , but in the case of amphihairyc and amphihairyd , it is further escorted by their expression in the notochord 27 . the apposed expression of the notochord and the neural plate is also observable for amphinetrin , amhnf3 and amphihh , at neurula stages 17,37,39 . though the expression of these three genes is undetectable in the ventral cerebral vesicle , it is clearly seen throughout the rest of the neural tube and along the whole length of the notochord , including the portion underlying the cerebral vesicle . in vertebrates , the notochord is a transient embryonic structure that acts as an organiser inducing the floor plate and cooperating in the establishment of the dorso - ventral axis of the neural tube and somites . although in amphioxus this structure is permanent , based on comparable gene expression patterns , it may well have properties similar to those of the vertebrate notochord , yet with a longer period of action that could extend beyond metamorphosis . in this regard , however , the role of the anterior notochord is obscured by the fact that genes such as amphinetrin , amhnf3 or amphihh seem to be unable to induce floor plate fates in the ventral cerebral vesicle , which suggests that the cells lying ventrally in the cerebral vesicle may not be competent to respond to their signals 37 . the cryptic internal segmentation of the amphioxus neural tube has been visualized by iterative gene expression patterns . as illustrative examples , amphikrox , shox , islet and amphimnx share a one somite - wide periodicity of expression throughout the neural tube . all of them are expressed in the hox - delineated hindbrain regions , though not all their patterns are in the same phase , indicating that different sets of cells take part in the iteration . both shox and islet transcripts are located at the level of somite boundaries , while amphikrox transcripts are centralised at the level of somites and amphimnx ones are close to the posterior border of the somites 39 - 41 . the repetitive pattern appears more synchronised adjacent to the first four somites and is altered at the level of somite number 5 , as occurs with the segmental expression of amphifoxb . similarly , amphicoe and amphielav are also iteratively expressed along the nerve cord during neurulation 42 - 44 . the amphicoe - expressing cells within the nerve cord show a periodicity matching the adjacent somites , in a pattern similar to those of amphifoxb and amphineurogenin , at the same developmental stages 12,35 . unlike amphikrox , shox and amphimnx expression , the amphicoe transcripts are not solely confined to the ventral part of the nerve cord , since they are also present in dorsal cells lying in comparable positions to those expressing islet . interestingly , the dorsal expression of both islet and amphicoe only occurs beyond the level of somite 5 , where the dorsal cells expressing them are displaced anteriorly to the accompanying expressing cells in the ventrolateral floor of the nerve cord 41 . however , though serially arranged , amphielav - expressing cells do not clearly match somite boundaries and are located on both the dorsal and ventral sides of the nerve cord , at all levels posterior to the cerebral vesicle 44 . in most cases , the iterative expression of these genes is maintained up to the larval stages , being down - regulated , but not completely missing , at about three days post - fertilisation . similar expression patterns have been described for other genes , such as amphink2 - 1 , amphink2 - 2 , amphifringe or amphimsx , although their transcripts are not detected in the neural tube that far in development 21 , 45 - 47 . the four genes are segmentally expressed in the neural tube at mid - neurula stages , just after mesoderm segmentation has commenced , but fade away from these locations by the end of neurulation . again , this transient expression may correlate mesoderm segmentation with the development of internal metameric neural structures . however , other genes , such as amphidach and amphierr , show an iterative pattern of expression , most obviously at larval stages 48,49 . in both cases , the series of positive cells have a caudal limit of expression at the level of somites 5 - 6 . it would seem that the only genes iteratively expressed beyond the level of somite 5 up to the latest stages of development analysed are amphicoe , amphielav and amphineurogenin , but always excluding the most posterior part of the neural tube 12,43,44 . since these genes are early markers for neuronal fates , this might suggest that posterior neuronal fates are delayed in development until later larval stages , at which their expression has not yet been analysed . the combinatorial iterative expression of all these genes , at different times and in distinct subsets of cells , suggests that the amphioxus nerve cord is internally organised in a variety of neurons , segmentally arranged in a dorso - ventral and an antero - posterior order , which might affect their relative connectivity and consequently their particular functions . although it is difficult to correlate the internal morphology of the embryonic and adult nerve cord , the differentiation events associated with the expression of the above - mentioned genes might only reflect the preliminary organisation of the anterior part of the adult nerve cord , which according to wicht and lacalli spans up to the level of myomere number 11 3 . given that the intermediate and posterior parts of the adult nerve cord show a slightly distinct internal organisation , these regions could be morphogenetically delineated later in development . similar early genetic programs could be acting at advanced larval stages and even beyond metamorphosis , when the embryo is still posteriorly elongating and dramatic morphological changes occur along the body , including the formation of certain structures that will be subsequently innervated by the descending branches of the dorsal nerves . however , this possibility is still to be explored , since the metamorphic and post - metamorphic larvae of amphioxus have rarely been used for gene expression studies . in recent years , the epidermis of amphioxus has received particular attention 50 , as a number of genes are specifically expressed by distinct epidermal cell populations during amphioxus embryogenesis , which indicates that distinct genetic programmes are switched on in particular cells and territories of epidermal tissue , previously thought to be uniform . some of these genes , such as amphicoe , amphielav and amphitrk , spread spottily over the epidermal surface of the embryo 43,44,51 . while amphicoe and amphielav are also expressed in the central nervous system , by the time they do in epidermal cells , amphitrk expression is solely confined to the non - neural ectoderm during neurulation , yet is restrictively expressed in the central nervous system in adult amphioxus . within the neuroectoderm , all three of these genes are expressed in developing neuronal precursors , or putative mature neurons in the case of amphitrk . noticeably , the respective orthologue genes in vertebrates are widely expressed in neuronal precursors during embryogenesis . all amphicoe , amphielav and amphitrk genes are expressed by scattered individual epidermal cells distributed over the ventrolateral flanks of neurulae . these cells show a distinctive elongated morphology that clearly sets them apart from surrounding epidermal cells . the number of cells expressing amphielav is apparently higher than the number of cells expressing amphicoe or amphitrk , which suggests that the embryonic epidermis might contain sub - populations of neuronal cells , which might depend on the combinatorial or differential expression of certain genes , such as amphicoe or amphitrk , to abandon their epidermal condition . thus , the scattered epidermal expression of genes involved in neural specification or differentiation suggests a neurogenic potential for the embryonic epidermis of amphioxus , which would be able to generate neuronal cells from ordinary ectodermal cells , giving rise , among other cells perhaps , to the primary sensory neurons observed by scanning electron microscopy ( sem ) in the epidermis of late neurulae 43 . conversely , other genes show a far more limited axial expression in the epidermis than those mentioned above . in particular , the anterior epidermis appears to concentrate much of the epidermal gene expression studied to date : amphineurogenin , amphipax6 , islet , amphitrk , amphimsx , amphiotx , amphibmp2/4 , amphiwnt11 , amphifoxq2 and amphitob are all expressed in this region , though in a partially overlapping manner 12,16,20,21,41,51,52 - 55 . this could correlate with the concurrence of a variety of specialised sensory cell types morphologically characterised in the anterior part of late larval and adult amphioxus specimens 3 . in this respect , the rostral expression of amphimsx and amphitrk at early larval stages has been related to the development of the corpuscles of de quatrefages , a multi - cellular organ composed of supporting cells and peripheral neurons that remain enclosed within a subepidermal capsule of connective tissue in the rostrum 21,51 . at least some of the peripheral neurons of the corpuscles of de quatrefages are primary neurons that send axonal processes to the central nervous system via the rostral nerves , but they are morphologically different from those primary neurons observed by sem at late neurula stages . the latter probably represent the early steps of differentiation of the type - i receptors described in late larval and adult amphioxus . the most common receptor cell types are called type i and type ii receptors . while type i receptors are primary sensory neurons , type ii receptors are secondary sensory neurons devoid of axons . both types of sensory neurons are widely distributed over the entire epidermis but appear densely grouped in the rostrum , buccal cirri and tail 3 . the oral region of late larvae is also rich in other neuronal cell types exclusively located in this area , such as the oral spines and those of the synaptic complex of the oral nerve 56 . although the origins of all these peripheral neurons are not clear , there is no evidence indicating anything other than a local origin . therefore , the assorted neural - related gene expression in these areas could be associated with distinct steps of neural commitment and differentiation of concrete ectodermal cells . the overlapping expression area of both amphineurogenin and amphipax6 in the ectoderm has been homologised with the vertebrate olfactory epithelia , which expresses the respective orthologous genes 12,20 . the vertebrate pax-6 gene is the most specific maker of the olfactory epithelia , but is also expressed by the lens placode . conversely , sox2 , sox3 , neurogenin1 and neurogenin2 , delta-1 and myt-1 ( neurod ) are expressed in most of the vertebrate placodes 57 . few specific placodal genes have been studied to date in amphioxus . among these , except for amphisox1/2/3 , both amphipax6 and amphineurogenin are broadly expressed in the rostral ectoderm of early larvae , where amphimsx is also expressed , yet covering a narrower area on each side of the larvae . since amphioxus is devoid of placodes , it is very tempting to correlate these gene expression data with the presence of a putative rudimentary placode - like region , which could be defined as a relatively broad ectodermal area of the rostrum able to give rise to distinct sensorial organs and neuronal subpopulations , and located only in the anterior part of the embryonic ectoderm . apart from the scattered expression of certain neural genes ( amphitrk , amphicoe and amphielav ) , the embryonic epidermis of amphioxus expresses a few other genes almost ubiquitously . this is the case of amphievxb , amphidral , amphidll and ap-2 , which are widely expressed in the non - neural ectoderm throughout neurulation 13,32,58,59 . these unexpected expression patterns make interpretation of the function of these genes in amphioxus embryogenesis very difficult . furthermore , amphievxb is a highly derived gene , fruit of a specific cephalochordate duplication and a general , cephalochordate - specific role , in epidermal functions has been suggested for this gene 58 . however , the overall epidermal expression of the genes characterised to date in amphioxus might lead to an alternative hypothesis . if the embryonic epidermis of amphioxus is endowed with the neurogenic potential of generating assorted neuronal cell types , amphidll and ap-2 could participate in the epidermal versus non - epidermal sorting of the different cells in the embryonic skin of amphioxus . given that both genes are homogeneously expressed in this tissue , the decision between epidermal and neural would not rely on the expression of these genes , but on their expression together with other genes such as amphielav or other uncharacterised proneural genes . the neural crest is a vertebrate - specific embryonic cell population defined by its origins at the neural plate border , migratory capacity , pluripotency and characteristic gene expression profile . although the genetic machinery of neural crest appears to be at least partially conserved in both urochordates and cephalochordates 60 , there is no compelling evidence , to date , for any invertebrate embryonic cell population that has all the properties of the vertebrate neural crest . even so , some migratory populations have been described in both urochordate and cephalochordate embryos . in both cases , while these cells express hnk-1 and zic gene markers in the ascidian ectainascidia turbinata , no gene expression profile could be determined for the migratory cells in amphioxus 51,61 . nevertheless , as noted in the previous section , amphioxus gene expression data demonstrate that particular epidermal cells have a distinctive gene expression profile , which would match the distinctive behaviour of these cells that migrate in a ventro - dorsal direction during neurulation . although this is only data correlation , the genetic program of these individually migrating cells is likely to be different from the surrounding epidermal cells lacking these properties . recent studies on brdu incorporation throughout amphioxus development demonstrate that the embryonic epidermis of the neurulae is devoid of cell proliferation 62 . these results , together with the absence of apoptosis at the same stages and in tissue 63 , indicate that the migrated epidermal cells in late neurulae did not relocate by movements caused by the elimination or addition of nearby cells . the absence of epidermal mitosis is consistent with the ciliation of most cells in the embryonic skin of amphioxus neurula . as mitosis and ciliation seem to be mutually excluding in most eukaryotic cells , proliferation and differentiation this is consistent with some epidermal cells being able to differentiate during neurulation , when mitosis arrests occur . as in vertebrates , amphidral may be involved in the cell division shutdown , since its downregulation in the epidermis only happens when proliferation is resumed in this tissue , at advanced larval stages 13,61 . under these circumstances , some cells might be able to activate certain differentiation genetic programs that would lead into the early differentiation of the primary neurons observed at late neurula stages 43 . however , such a spatio - temporal genetic program is currently unknown , though it is possible to establish certain associations between gene expression and epidermal subpopulations . at present , it seems clear that distinct genetic programs are operating in the embryonic skin , distinguishing individual and possibly migrating cells from the rest of the epidermis . as previously mentioned , the most prominent epidermal subpopulations are those labelled by amphicoe , amphielav and amphitrk expression . these are followed by the much smaller subpopulations expressing islet and amphierr , which , unlike those expressing amphicoe , amphielav and amphitrk , appear only in the trunk region 4 , 49 . as a common denominator , all these genes are involved in the specification of neuronal precursors or in neuronal differentiation , thereby strengthening the local neurogenic potential of the amphioxus embryonic epidermis . in tandem with this , one of the most striking features of amphitrk and amphielav expression is the dorsalisation of the signal as neurulation proceeds , which follows the same direction at the same stages of development of those migrating individual cells observed by dii tracing 51 . furthermore , the detailed position of amphitrk- and amphielav - positive cells locates them within the epidermis or just beneath it , suggesting that if these cells are migrating they might be doing it either through the epidermis or through the subepidermal plexus . on taking this independent evidence together with the expression of the vertebrate trk and hu / elav orthologue genes in the neural crest and/or its derivatives , it is very tempting to establish parallelism between these cells and the vertebrate neural crest . therefore , if these cells ( amphitrk- and amphielav - positive cells ) individually detached from the epidermal layer and entered the sub - epidermal plexus , until reaching the definitive ( and unknown ) location at which they would finally differentiate into particular neuronal types , they would conform some of the properties of vertebrate neural crest cells . the particular migration of vertebrate neural crest cells is also characterised by the precise definition of their individual routes , which are crucially dependent on an antero - posterior hox code interestingly , nested hox expression in scattered epidermal cells has been observed in the embryonic epidermis of amphioxus 4 by the time that migration begins and the earliest neural gene expression is already occurring . furthermore , under retinoic acid treatment the location of those individual epidermal cells that express hox genes is altered , as is that of the amphicoe , islet and amphierr - positive cells and , importantly , that of the primary neurons observed at late neurula stages 4 . these results suggest that the embryonic skin of amphioxus is probably pre - patterned following an antero - posterior hox code , which in conjunction with other genes would divide the epidermis into distinct territories with particular gene expression profiles . under this hypothesis , individual cells would migrate vertically through defined routes , which would molecularly , but not morphologically , be delimited into epidermal meridians ( fig . , amphioxus skin possesses a moderate diversity of peripheral sensory neurons , probably born locally from normal epidermal cells through different expression profiles 50 . collation of all the data set out here suggests that the individually migrating cells in amphioxus epidermis may well match cells with a particular gene expression profile that are responsible for the final differentiation into distinct types of peripheral sensory neurons , though these have much less pluripotency than in vertebrates . in summary , based on : i ) the presence of distinct genetic programs in individual embryonic epidermal cells ; ii ) the ability to migrate as individual cells ; iii ) the ability to detach , presumably before migration ; iv ) correlation with the antero - posterior boundaries of hox expression domains ; and v ) the partial pluripotency of these cells , i suggest that amphioxus possesses an embryonic cell population that exhibits most of the properties of vertebrate neural crest cells . thus , it is tempting to speculate that they might represent an intermediate evolutionary step that ended up in the deployment of vertebrate neural crest cells . the main caveats of this thesis are that these cells do not originate from the edges of the neural tube and that migration has the opposite orientation ( ventro - dorsal ) . however these two caveats can be reconciled if , in an evolutionary sense , the amphioxus nerve neural system represents an intermediate state , from a diffuse neural epidermal net all around the body , like that of hemichordates 64 , to full concentration on the dorsal side of the body , where the nerve tube cohabits in vertebrates with the embryonic germ of the neural crest and placodes . accumulating data on gene expression during amphioxus embryogenesis challenges the classic morphological simplicity of the amphioxus nervous system . spottily-expressed genes , neural and non - neural ectodermal specificity , cell migration in the ectoderm and cryptic complexity are all providing us with new insights into the neural genetic programs of this , our old beast , half - way along the path of construction of complex vertebrate nervous systems . in the near post - genomic future , more and more data will be arriving and , hopefully , experimental embryology ( by means of genetic manipulation of living embryos ) will help clarify the numerous question marks on the evolution of the nervous system , for which the friendly little amphioxus may have some answers . lateral view showing the internal anatomy and part of the overlying segmented muscle blocks ( myomeres ) . hypothetical model for neural crest - like cell migration in amphioxus . the expression of characteristic neural genes in scattered individual cells in the epidermis indicates that neuronal precursors are generated from the normal epidermal cells following a particular genetic program switched on during neurulation . these unknown genetic programs should endow these particular cells with additional distinctive properties , which might include the acquisition of individual migratory behaviour . the migration and terminal differentiation of those neuronal precursors , in particular locations , could depend on the pre - patterning of the neurula epidermis . this model summarises and gives an oversimplified view of the epidermal expression of different genes that seem to participate in the epidermal patterning of the amphioxus neurula . as in the neural tube , hox genes are expressed in a nested manner and , together with the expression of amphipax6 , amphien and amphicdx , divide the entire epidermis into meridians of differential expression . all these genes are expressed in a scattered manner , but are delimited along the anteroposterior axis as indicated by colour code . in the case of the hox genes , the meridians were established by the combinatorial expression of amphihox 1 , 3 , 4 and 6 . under this hypothetical model , those individual cells ( red dots ) mentioned above , would migrate vertically ( arrows ) through defined routes , as delimited by each meridian of combinatorial expression .

the elaboration of extremely complex nervous systems is a major success of evolution . however , at the dawn of the post - genomic era , few data have helped yet to unravel how a nervous system develops and evolves to complexity . on the evolutionary road to vertebrates , amphioxus occupies a key position to tackle this exciting issue . its simple nervous system basically consists of a dorsal nerve cord and a diffuse net of peripheral neurons , which contrasts greatly with the complexity of vertebrate nervous systems . notwithstanding , increasing data on gene expression has faced up this simplicity by revealing a mounting level of cryptic complexity , with unexpected levels of neuronal diversity , organisation and regionalisation of the central and peripheral nervous systems . furthermore , recent gene expression data also point to the high neurogenic potential of the epidermis of amphioxus , suggestive of a skin - brain track for the evolution of the vertebrate nervous system . here i attempt to catalogue and synthesise current gene expression data in the amphioxus nervous system . from this global point of view , i suggest scenarios for the evolutionary origin of complex features in the vertebrate nervous system , with special emphasis on the evolutionary origin of placodes and neural crest , and postulate a pre - patterned migratory pathway of cells , which , in the epidermis , may represent an intermediate state towards the deployment of one of the most striking innovative features of vertebrates : the neural crest and its derivatives .

1. Introduction 2. Early gene expression 3. Patterning of the neural tube 4. Iterative gene expression patterns 5. Neural expression in the non-neural ectoderm 6. Caveats on putative neural crest precursors 7. Conclusion Figures and Tables

the evolution of the nervous system in the animal kingdom is currently under major debate in evolutionary developmental biology and comparative embryology 1 . in this context , the evolutionary path that leads to the morphological complexity of the nervous system passes through the invertebrate / vertebrate transition . at the beginning of the xx century , the cephalochordate amphioxus occupied the most prominent role in illuminating , at the morphological level , such a transition ( see 2 for an historical perspective ) . one prominent example is the molecular regionalisation of the amphioxus nervous system , which is greatly hindered by its anatomical simplicity . the central nervous system of an adult amphioxus is a mere tubular nerve cord lying dorsally over the notochord . the nerve cord and the notochord cover together the entire length of the animal , except in the anterior region , where only the notochord reaches the rostral tip of the body ( fig . the only externally visible landmarks of the amphioxus nerve cord are discrete enlargements at both the anterior and posterior ends , regarded as the anterior vesicle and the caudal ampulla , respectively . the outgoing dorsal nerves , unsupplied with ganglia , contribute to the peripheral nervous system by innervating most of the organs of the body , shaping an irregular nerve net , where a number of peripheral neurons are also accommodated 3 in contrast with the anatomically non - regionalised central nervous system and the apparently disorganised peripheral nervous system , the discrete expression patterns of distinct genes and the specific distribution of certain proteins outline a completely different chronicle for the nervous system of amphioxus . most of the neural genes characterised to date reveal gapped expression domains within the developing central nervous system , and restricted expression to certain cell subpopulations within the embryonic epidermis . retinoic acid treatments greatly affect these gene expression domains 4 , suggesting a well - controlled pre - patterning that defines distinct territories , not only within the neural ectoderm but also within the epidermis of amphioxus embryos . whatever its actual position , as the sister group of vertebrates , as the earliest chordate , or as ( less likely ) the earliest deuterostome 5 , its privileged phylogenetic position sets amphioxus in the appropriate place to cast light on some of the main transitions that occurred during animal evolution : the origin of deuterostomes , the origin of chordates , or the origin of vertebrates , all of them correlated with novel evolutionary features , and some of them linked to the elaboration of the most complex nervous systems . both clades include the only invertebrates able to form a dorsal nerve cord through neurulation . this is supported by modern anatomy 3 , which shows that cytoarchytectural reorganisations should occur within the nerve cord of amphioxus until reaching a definitive internal structure in the adult . the present communion between modern morphology and the use of molecular biology tools is revealing an unexpected variety of cell types , territories and boundaries along the antero - posterior axis of the nerve cord and on the epidermis of amphioxus . here i attempt to summarize distinct patterns of gene expression , giving an overall view of the formation of the central and peripheral nervous systems of amphioxus and suggesting the possible origin of two characteristic neural features in vertebrates : the placodes and the neural crest . at the onset of gastrulation , the vegetal pole of the blastula invaginates , leaving a wide - open blastopore , which subsequently closes . this territorial area homing the expression of these genes is soon segregated into the blastoporal lips , which remain on the posterior side of the embryo and will contribute to the tail bud later in development 9 . whether the notch and wnt signalling systems are involved in the specification of the dorso - ventral axis however , it has been suggested that wnt signals may be involved in restricting expression of genes such as amphisox1/2/3 , amphidral and amphidll to the ectoderm 7 . thus , it is unclear to what extent the dorsal blastoporal lip functions , if it does , as a dorsal - ventral organiser , though it may have some properties related to the spemann 's organiser , such as influencing the formation of the neural plate and the underlying chordamesoderm . as gastrulation proceeds , some genes begin to be restrictively expressed in certain regions of the neuroectoderm , which may be an anticipation of the cryptic regionalisation of the neural tube and the epidermis observed in succeeding stages . the homogeneous expression of amphisox1/2/3 and amphineurogenin in the dorsal epiblast is gradually restricted only to the neural ectoderm 12 . the initial expression of amphipax3/7 in the dorsal presumptive axial and paraxial mesoderm is additionally extended to the dorsal ectoderm , where it also appears at the lateral edges of the presumptive neural plate 26 . this raises the recurrent question on the effect that mesoderm segmentation might exert on the molecular regionalisation of the neural tube in amphioxus . furthermore , the apposed ectodermal - mesodermal expression also observed for the amphioxus snail gene , which is detected in the dorsal presumptive somitic mesoderm and the overlying edges of the forming neural plate , and several other genes ( e.g. instead , the non - neural ectoderm detaches from the edges of the open neural plate , overgrowing laterally over the neural plate and fusing in the dorsal midline . the central nervous system of amphioxus neurulae , in its advanced stages , consists of a dorsal neural cord lacking constrictions along its rostrocaudal axis . the only regional difference lies at the anterior end , only regional difference lies at the anterior end only regional difference lies at the anterior end where the neural canal is slightly dilated into a cerebral vesicle , anterior to the level of somite 1 and up to the anterior part of somite 2 . this organ is the source of the reissner 's fiber , which fills the central neural canal , and is probably the homologue of the subcommisural organ of the vertebrate diencephalon 16 . amphid1 is also expressed in the cerebral vesicle at the level of the presumptive infundibular organ , while amphith is expressed at the level of the presumptive lamellar organ , which in turn is covered by the caudal expression of amphiotx 33,34 . both amphibf-1 and amphidll are orthologue genes to those marking the vertebrate telencephalon and , in the case of amphidll , other parts of the vertebrate diencephalon as well . together with the expression of amphiotx , the orthologues of which are markers of the vertebrate telencephalon , diencephalon and midbrain , and the territorial domain occupied by amphisim , combined gene expression suggests that the amphioxus cerebral vesicle is largely homologous to the vertebrate forebrain ( telencephalon plus diencephalon ) , although certain homology with the midbrain can not be completely ruled out . in the hypothetical absence of a midbrain homolog in amphioxus , the territory posterior to the cerebral vesicle , caudal to amphiotx expression at the level of somite 2 , has been widely seen as a homolog of the craniate hindbrain . the long - lasting expression of amphinetrin in the floor plate suggests a role of the amphioxus floor plate in the control of axon path finding , possibly guiding newborn neurons within the nerve cord 37 . similar to amphisox1/2/3 , amphibrn1/2/4 is excluded from the midline , but is additionally expressed in the cerebral vesicle with the same gap of expression as the one observed for the amphif - spondin gene 25 . as mentioned previously , in general the expression of the different amphihairy genes in the neural tube is accompanied by a striped expression in the somitic mesoderm , but in the case of amphihairyc and amphihairyd , it is further escorted by their expression in the notochord 27 . the apposed expression of the notochord and the neural plate is also observable for amphinetrin , amhnf3 and amphihh , at neurula stages 17,37,39 . though the expression of these three genes is undetectable in the ventral cerebral vesicle , it is clearly seen throughout the rest of the neural tube and along the whole length of the notochord , including the portion underlying the cerebral vesicle . in vertebrates , the notochord is a transient embryonic structure that acts as an organiser inducing the floor plate and cooperating in the establishment of the dorso - ventral axis of the neural tube and somites . although in amphioxus this structure is permanent , based on comparable gene expression patterns , it may well have properties similar to those of the vertebrate notochord , yet with a longer period of action that could extend beyond metamorphosis . in this regard , however , the role of the anterior notochord is obscured by the fact that genes such as amphinetrin , amhnf3 or amphihh seem to be unable to induce floor plate fates in the ventral cerebral vesicle , which suggests that the cells lying ventrally in the cerebral vesicle may not be competent to respond to their signals 37 . the cryptic internal segmentation of the amphioxus neural tube has been visualized by iterative gene expression patterns . both shox and islet transcripts are located at the level of somite boundaries , while amphikrox transcripts are centralised at the level of somites and amphimnx ones are close to the posterior border of the somites 39 - 41 . the repetitive pattern appears more synchronised adjacent to the first four somites and is altered at the level of somite number 5 , as occurs with the segmental expression of amphifoxb . the amphicoe - expressing cells within the nerve cord show a periodicity matching the adjacent somites , in a pattern similar to those of amphifoxb and amphineurogenin , at the same developmental stages 12,35 . interestingly , the dorsal expression of both islet and amphicoe only occurs beyond the level of somite 5 , where the dorsal cells expressing them are displaced anteriorly to the accompanying expressing cells in the ventrolateral floor of the nerve cord 41 . however , though serially arranged , amphielav - expressing cells do not clearly match somite boundaries and are located on both the dorsal and ventral sides of the nerve cord , at all levels posterior to the cerebral vesicle 44 . it would seem that the only genes iteratively expressed beyond the level of somite 5 up to the latest stages of development analysed are amphicoe , amphielav and amphineurogenin , but always excluding the most posterior part of the neural tube 12,43,44 . the combinatorial iterative expression of all these genes , at different times and in distinct subsets of cells , suggests that the amphioxus nerve cord is internally organised in a variety of neurons , segmentally arranged in a dorso - ventral and an antero - posterior order , which might affect their relative connectivity and consequently their particular functions . although it is difficult to correlate the internal morphology of the embryonic and adult nerve cord , the differentiation events associated with the expression of the above - mentioned genes might only reflect the preliminary organisation of the anterior part of the adult nerve cord , which according to wicht and lacalli spans up to the level of myomere number 11 3 . however , this possibility is still to be explored , since the metamorphic and post - metamorphic larvae of amphioxus have rarely been used for gene expression studies . in recent years , the epidermis of amphioxus has received particular attention 50 , as a number of genes are specifically expressed by distinct epidermal cell populations during amphioxus embryogenesis , which indicates that distinct genetic programmes are switched on in particular cells and territories of epidermal tissue , previously thought to be uniform . while amphicoe and amphielav are also expressed in the central nervous system , by the time they do in epidermal cells , amphitrk expression is solely confined to the non - neural ectoderm during neurulation , yet is restrictively expressed in the central nervous system in adult amphioxus . the number of cells expressing amphielav is apparently higher than the number of cells expressing amphicoe or amphitrk , which suggests that the embryonic epidermis might contain sub - populations of neuronal cells , which might depend on the combinatorial or differential expression of certain genes , such as amphicoe or amphitrk , to abandon their epidermal condition . thus , the scattered epidermal expression of genes involved in neural specification or differentiation suggests a neurogenic potential for the embryonic epidermis of amphioxus , which would be able to generate neuronal cells from ordinary ectodermal cells , giving rise , among other cells perhaps , to the primary sensory neurons observed by scanning electron microscopy ( sem ) in the epidermis of late neurulae 43 . in this respect , the rostral expression of amphimsx and amphitrk at early larval stages has been related to the development of the corpuscles of de quatrefages , a multi - cellular organ composed of supporting cells and peripheral neurons that remain enclosed within a subepidermal capsule of connective tissue in the rostrum 21,51 . at least some of the peripheral neurons of the corpuscles of de quatrefages are primary neurons that send axonal processes to the central nervous system via the rostral nerves , but they are morphologically different from those primary neurons observed by sem at late neurula stages . the overlapping expression area of both amphineurogenin and amphipax6 in the ectoderm has been homologised with the vertebrate olfactory epithelia , which expresses the respective orthologous genes 12,20 . since amphioxus is devoid of placodes , it is very tempting to correlate these gene expression data with the presence of a putative rudimentary placode - like region , which could be defined as a relatively broad ectodermal area of the rostrum able to give rise to distinct sensorial organs and neuronal subpopulations , and located only in the anterior part of the embryonic ectoderm . furthermore , amphievxb is a highly derived gene , fruit of a specific cephalochordate duplication and a general , cephalochordate - specific role , in epidermal functions has been suggested for this gene 58 . if the embryonic epidermis of amphioxus is endowed with the neurogenic potential of generating assorted neuronal cell types , amphidll and ap-2 could participate in the epidermal versus non - epidermal sorting of the different cells in the embryonic skin of amphioxus . the neural crest is a vertebrate - specific embryonic cell population defined by its origins at the neural plate border , migratory capacity , pluripotency and characteristic gene expression profile . nevertheless , as noted in the previous section , amphioxus gene expression data demonstrate that particular epidermal cells have a distinctive gene expression profile , which would match the distinctive behaviour of these cells that migrate in a ventro - dorsal direction during neurulation . the absence of epidermal mitosis is consistent with the ciliation of most cells in the embryonic skin of amphioxus neurula . as in vertebrates , amphidral may be involved in the cell division shutdown , since its downregulation in the epidermis only happens when proliferation is resumed in this tissue , at advanced larval stages 13,61 . these are followed by the much smaller subpopulations expressing islet and amphierr , which , unlike those expressing amphicoe , amphielav and amphitrk , appear only in the trunk region 4 , 49 . as a common denominator , all these genes are involved in the specification of neuronal precursors or in neuronal differentiation , thereby strengthening the local neurogenic potential of the amphioxus embryonic epidermis . in tandem with this , one of the most striking features of amphitrk and amphielav expression is the dorsalisation of the signal as neurulation proceeds , which follows the same direction at the same stages of development of those migrating individual cells observed by dii tracing 51 . on taking this independent evidence together with the expression of the vertebrate trk and hu / elav orthologue genes in the neural crest and/or its derivatives , it is very tempting to establish parallelism between these cells and the vertebrate neural crest . the particular migration of vertebrate neural crest cells is also characterised by the precise definition of their individual routes , which are crucially dependent on an antero - posterior hox code interestingly , nested hox expression in scattered epidermal cells has been observed in the embryonic epidermis of amphioxus 4 by the time that migration begins and the earliest neural gene expression is already occurring . these results suggest that the embryonic skin of amphioxus is probably pre - patterned following an antero - posterior hox code , which in conjunction with other genes would divide the epidermis into distinct territories with particular gene expression profiles . collation of all the data set out here suggests that the individually migrating cells in amphioxus epidermis may well match cells with a particular gene expression profile that are responsible for the final differentiation into distinct types of peripheral sensory neurons , though these have much less pluripotency than in vertebrates . in summary , based on : i ) the presence of distinct genetic programs in individual embryonic epidermal cells ; ii ) the ability to migrate as individual cells ; iii ) the ability to detach , presumably before migration ; iv ) correlation with the antero - posterior boundaries of hox expression domains ; and v ) the partial pluripotency of these cells , i suggest that amphioxus possesses an embryonic cell population that exhibits most of the properties of vertebrate neural crest cells . thus , it is tempting to speculate that they might represent an intermediate evolutionary step that ended up in the deployment of vertebrate neural crest cells . however these two caveats can be reconciled if , in an evolutionary sense , the amphioxus nerve neural system represents an intermediate state , from a diffuse neural epidermal net all around the body , like that of hemichordates 64 , to full concentration on the dorsal side of the body , where the nerve tube cohabits in vertebrates with the embryonic germ of the neural crest and placodes . accumulating data on gene expression during amphioxus embryogenesis challenges the classic morphological simplicity of the amphioxus nervous system . spottily-expressed genes , neural and non - neural ectodermal specificity , cell migration in the ectoderm and cryptic complexity are all providing us with new insights into the neural genetic programs of this , our old beast , half - way along the path of construction of complex vertebrate nervous systems . in the near post - genomic future , more and more data will be arriving and , hopefully , experimental embryology ( by means of genetic manipulation of living embryos ) will help clarify the numerous question marks on the evolution of the nervous system , for which the friendly little amphioxus may have some answers . the migration and terminal differentiation of those neuronal precursors , in particular locations , could depend on the pre - patterning of the neurula epidermis . as in the neural tube , hox genes are expressed in a nested manner and , together with the expression of amphipax6 , amphien and amphicdx , divide the entire epidermis into meridians of differential expression .

at the beginning of the xx century , the cephalochordate amphioxus occupied the most prominent role in illuminating , at the morphological level , such a transition ( see 2 for an historical perspective ) . the nerve cord and the notochord cover together the entire length of the animal , except in the anterior region , where only the notochord reaches the rostral tip of the body ( fig . this exclusive rostral expansion of the notochord is a hallmark of the taxon cephalochordata ( corda for corda dorsalis , original name of the notochord - in the head ) . apart from the serially repeated exit points of the dorsal nerves , the nerve cord of amphioxus is devoid of rhombomeres or other external morphological indications of segmentation . the outgoing dorsal nerves , unsupplied with ganglia , contribute to the peripheral nervous system by innervating most of the organs of the body , shaping an irregular nerve net , where a number of peripheral neurons are also accommodated 3 in contrast with the anatomically non - regionalised central nervous system and the apparently disorganised peripheral nervous system , the discrete expression patterns of distinct genes and the specific distribution of certain proteins outline a completely different chronicle for the nervous system of amphioxus . most of the neural genes characterised to date reveal gapped expression domains within the developing central nervous system , and restricted expression to certain cell subpopulations within the embryonic epidermis . whatever its actual position , as the sister group of vertebrates , as the earliest chordate , or as ( less likely ) the earliest deuterostome 5 , its privileged phylogenetic position sets amphioxus in the appropriate place to cast light on some of the main transitions that occurred during animal evolution : the origin of deuterostomes , the origin of chordates , or the origin of vertebrates , all of them correlated with novel evolutionary features , and some of them linked to the elaboration of the most complex nervous systems . the present communion between modern morphology and the use of molecular biology tools is revealing an unexpected variety of cell types , territories and boundaries along the antero - posterior axis of the nerve cord and on the epidermis of amphioxus . here i attempt to summarize distinct patterns of gene expression , giving an overall view of the formation of the central and peripheral nervous systems of amphioxus and suggesting the possible origin of two characteristic neural features in vertebrates : the placodes and the neural crest . in amphioxus , the embryonic anterior - posterior axis is determined early ( by the time of gastrulation ) , as a consequence of the animal - vegetal polarity of the oocyte , induced by the sperm entry point during fertilisation . whether the notch and wnt signalling systems are involved in the specification of the dorso - ventral axis however , it has been suggested that wnt signals may be involved in restricting expression of genes such as amphisox1/2/3 , amphidral and amphidll to the ectoderm 7 . the expression of both largely overlaps at the early gastrula , on the outer dorsal epiblast ( ectoderm ) , thus indicating the existence of a dorso - ventral axis already at this stage . throughout gastrulation , some of the genes involved in neural patterning are expressed in the dorsal blastoporal lip , which has been regarded as the homolog of the amphibian spemann 's organiser 14 , crucially involved in neural induction . in vertebrates , the early nodal expression is involved in the establishment of the spemann 's organiser and induces the expression of organiser - specific genes such as goosecoid . therefore , amphinodal might well induce the expression of goosecoid in the dorsal blastoporal lip , where it overlaps with other organiser - related genes such as amhnf3 , ambra and amphifoxd , but not amphiotx 11 , 16 - 19 . thus , it is unclear to what extent the dorsal blastoporal lip functions , if it does , as a dorsal - ventral organiser , though it may have some properties related to the spemann 's organiser , such as influencing the formation of the neural plate and the underlying chordamesoderm . as gastrulation proceeds , some genes begin to be restrictively expressed in certain regions of the neuroectoderm , which may be an anticipation of the cryptic regionalisation of the neural tube and the epidermis observed in succeeding stages . as the gastrula elongates to reach the earliest neurula stage , the expression of amphibrn1/2/4 , initially located in the dorsal epiblast , gradually spans from the posterior to the anterior neural plate 25 . the initial expression of amphipax3/7 in the dorsal presumptive axial and paraxial mesoderm is additionally extended to the dorsal ectoderm , where it also appears at the lateral edges of the presumptive neural plate 26 . furthermore , the apposed ectodermal - mesodermal expression also observed for the amphioxus snail gene , which is detected in the dorsal presumptive somitic mesoderm and the overlying edges of the forming neural plate , and several other genes ( e.g. as the non - neural ectoderm covers the neural plate , the lateral edges of the latter curl up dorsally and fuse in the dorsal midline , underneath the non - neural ectoderm , to form the neural tube . the only regional difference lies at the anterior end , only regional difference lies at the anterior end only regional difference lies at the anterior end where the neural canal is slightly dilated into a cerebral vesicle , anterior to the level of somite 1 and up to the anterior part of somite 2 . this organ is the source of the reissner 's fiber , which fills the central neural canal , and is probably the homologue of the subcommisural organ of the vertebrate diencephalon 16 . amphid1 is also expressed in the cerebral vesicle at the level of the presumptive infundibular organ , while amphith is expressed at the level of the presumptive lamellar organ , which in turn is covered by the caudal expression of amphiotx 33,34 . however , amphioxus orthologues of other vertebrate midbrain and mhb ( midbrain - hindbrain boundary ) markers , such as amphievx , amphien , amphipax2/5/8 and amphiwnt1 , have failed to provide evidence for a true midbrain and mhb homolog in amphioxus 29,35 . together with the expression of amphiotx , the orthologues of which are markers of the vertebrate telencephalon , diencephalon and midbrain , and the territorial domain occupied by amphisim , combined gene expression suggests that the amphioxus cerebral vesicle is largely homologous to the vertebrate forebrain ( telencephalon plus diencephalon ) , although certain homology with the midbrain can not be completely ruled out . in the hypothetical absence of a midbrain homolog in amphioxus , the territory posterior to the cerebral vesicle , caudal to amphiotx expression at the level of somite 2 , has been widely seen as a homolog of the craniate hindbrain . spatially , the first hox gene expressed in the anterior hindbrain is amphihox1 ( somites 2 - 4 ) , followed by amphihox3 ( somites 4 - 8 ) and amphihox4 ( somites 6 - 8 ) 36 . amphifoxb is expressed segmentally as well , concomitantly with hox genes , though it shows a prominent gap of expression at the level of somite 5 , where the pigment spot will form and , intriguingly , where the expression of other segmental genes is also disrupted ( e.g. notwithstanding , amphinetrin expression in the floor plate lasts longer than amphisox1/2/3 expression does and always overlies its own expression in the dorsal notochord , even below the cerebral vesicle ( from which it is excluded ) , until the larval stages , when amphinetrin is lost at both anterior and posterior ends of the notochord . the long - lasting expression of amphinetrin in the floor plate suggests a role of the amphioxus floor plate in the control of axon path finding , possibly guiding newborn neurons within the nerve cord 37 . amphif - spondin expression by the end of neurulation largely overlaps the expression of amphinetrin , though covering broader areas within the neural tube , including the cerebral vesicle , where it shows a gap of expression just on the ventral side 38 . as mentioned previously , in general the expression of the different amphihairy genes in the neural tube is accompanied by a striped expression in the somitic mesoderm , but in the case of amphihairyc and amphihairyd , it is further escorted by their expression in the notochord 27 . though the expression of these three genes is undetectable in the ventral cerebral vesicle , it is clearly seen throughout the rest of the neural tube and along the whole length of the notochord , including the portion underlying the cerebral vesicle . although in amphioxus this structure is permanent , based on comparable gene expression patterns , it may well have properties similar to those of the vertebrate notochord , yet with a longer period of action that could extend beyond metamorphosis . in this regard , however , the role of the anterior notochord is obscured by the fact that genes such as amphinetrin , amhnf3 or amphihh seem to be unable to induce floor plate fates in the ventral cerebral vesicle , which suggests that the cells lying ventrally in the cerebral vesicle may not be competent to respond to their signals 37 . the amphicoe - expressing cells within the nerve cord show a periodicity matching the adjacent somites , in a pattern similar to those of amphifoxb and amphineurogenin , at the same developmental stages 12,35 . unlike amphikrox , shox and amphimnx expression , the amphicoe transcripts are not solely confined to the ventral part of the nerve cord , since they are also present in dorsal cells lying in comparable positions to those expressing islet . interestingly , the dorsal expression of both islet and amphicoe only occurs beyond the level of somite 5 , where the dorsal cells expressing them are displaced anteriorly to the accompanying expressing cells in the ventrolateral floor of the nerve cord 41 . it would seem that the only genes iteratively expressed beyond the level of somite 5 up to the latest stages of development analysed are amphicoe , amphielav and amphineurogenin , but always excluding the most posterior part of the neural tube 12,43,44 . the combinatorial iterative expression of all these genes , at different times and in distinct subsets of cells , suggests that the amphioxus nerve cord is internally organised in a variety of neurons , segmentally arranged in a dorso - ventral and an antero - posterior order , which might affect their relative connectivity and consequently their particular functions . although it is difficult to correlate the internal morphology of the embryonic and adult nerve cord , the differentiation events associated with the expression of the above - mentioned genes might only reflect the preliminary organisation of the anterior part of the adult nerve cord , which according to wicht and lacalli spans up to the level of myomere number 11 3 . in recent years , the epidermis of amphioxus has received particular attention 50 , as a number of genes are specifically expressed by distinct epidermal cell populations during amphioxus embryogenesis , which indicates that distinct genetic programmes are switched on in particular cells and territories of epidermal tissue , previously thought to be uniform . while amphicoe and amphielav are also expressed in the central nervous system , by the time they do in epidermal cells , amphitrk expression is solely confined to the non - neural ectoderm during neurulation , yet is restrictively expressed in the central nervous system in adult amphioxus . the number of cells expressing amphielav is apparently higher than the number of cells expressing amphicoe or amphitrk , which suggests that the embryonic epidermis might contain sub - populations of neuronal cells , which might depend on the combinatorial or differential expression of certain genes , such as amphicoe or amphitrk , to abandon their epidermal condition . thus , the scattered epidermal expression of genes involved in neural specification or differentiation suggests a neurogenic potential for the embryonic epidermis of amphioxus , which would be able to generate neuronal cells from ordinary ectodermal cells , giving rise , among other cells perhaps , to the primary sensory neurons observed by scanning electron microscopy ( sem ) in the epidermis of late neurulae 43 . in particular , the anterior epidermis appears to concentrate much of the epidermal gene expression studied to date : amphineurogenin , amphipax6 , islet , amphitrk , amphimsx , amphiotx , amphibmp2/4 , amphiwnt11 , amphifoxq2 and amphitob are all expressed in this region , though in a partially overlapping manner 12,16,20,21,41,51,52 - 55 . in this respect , the rostral expression of amphimsx and amphitrk at early larval stages has been related to the development of the corpuscles of de quatrefages , a multi - cellular organ composed of supporting cells and peripheral neurons that remain enclosed within a subepidermal capsule of connective tissue in the rostrum 21,51 . at least some of the peripheral neurons of the corpuscles of de quatrefages are primary neurons that send axonal processes to the central nervous system via the rostral nerves , but they are morphologically different from those primary neurons observed by sem at late neurula stages . the overlapping expression area of both amphineurogenin and amphipax6 in the ectoderm has been homologised with the vertebrate olfactory epithelia , which expresses the respective orthologous genes 12,20 . since amphioxus is devoid of placodes , it is very tempting to correlate these gene expression data with the presence of a putative rudimentary placode - like region , which could be defined as a relatively broad ectodermal area of the rostrum able to give rise to distinct sensorial organs and neuronal subpopulations , and located only in the anterior part of the embryonic ectoderm . if the embryonic epidermis of amphioxus is endowed with the neurogenic potential of generating assorted neuronal cell types , amphidll and ap-2 could participate in the epidermal versus non - epidermal sorting of the different cells in the embryonic skin of amphioxus . given that both genes are homogeneously expressed in this tissue , the decision between epidermal and neural would not rely on the expression of these genes , but on their expression together with other genes such as amphielav or other uncharacterised proneural genes . although the genetic machinery of neural crest appears to be at least partially conserved in both urochordates and cephalochordates 60 , there is no compelling evidence , to date , for any invertebrate embryonic cell population that has all the properties of the vertebrate neural crest . nevertheless , as noted in the previous section , amphioxus gene expression data demonstrate that particular epidermal cells have a distinctive gene expression profile , which would match the distinctive behaviour of these cells that migrate in a ventro - dorsal direction during neurulation . these results , together with the absence of apoptosis at the same stages and in tissue 63 , indicate that the migrated epidermal cells in late neurulae did not relocate by movements caused by the elimination or addition of nearby cells . in tandem with this , one of the most striking features of amphitrk and amphielav expression is the dorsalisation of the signal as neurulation proceeds , which follows the same direction at the same stages of development of those migrating individual cells observed by dii tracing 51 . on taking this independent evidence together with the expression of the vertebrate trk and hu / elav orthologue genes in the neural crest and/or its derivatives , it is very tempting to establish parallelism between these cells and the vertebrate neural crest . therefore , if these cells ( amphitrk- and amphielav - positive cells ) individually detached from the epidermal layer and entered the sub - epidermal plexus , until reaching the definitive ( and unknown ) location at which they would finally differentiate into particular neuronal types , they would conform some of the properties of vertebrate neural crest cells . the particular migration of vertebrate neural crest cells is also characterised by the precise definition of their individual routes , which are crucially dependent on an antero - posterior hox code interestingly , nested hox expression in scattered epidermal cells has been observed in the embryonic epidermis of amphioxus 4 by the time that migration begins and the earliest neural gene expression is already occurring . furthermore , under retinoic acid treatment the location of those individual epidermal cells that express hox genes is altered , as is that of the amphicoe , islet and amphierr - positive cells and , importantly , that of the primary neurons observed at late neurula stages 4 . these results suggest that the embryonic skin of amphioxus is probably pre - patterned following an antero - posterior hox code , which in conjunction with other genes would divide the epidermis into distinct territories with particular gene expression profiles . collation of all the data set out here suggests that the individually migrating cells in amphioxus epidermis may well match cells with a particular gene expression profile that are responsible for the final differentiation into distinct types of peripheral sensory neurons , though these have much less pluripotency than in vertebrates . in summary , based on : i ) the presence of distinct genetic programs in individual embryonic epidermal cells ; ii ) the ability to migrate as individual cells ; iii ) the ability to detach , presumably before migration ; iv ) correlation with the antero - posterior boundaries of hox expression domains ; and v ) the partial pluripotency of these cells , i suggest that amphioxus possesses an embryonic cell population that exhibits most of the properties of vertebrate neural crest cells . however these two caveats can be reconciled if , in an evolutionary sense , the amphioxus nerve neural system represents an intermediate state , from a diffuse neural epidermal net all around the body , like that of hemichordates 64 , to full concentration on the dorsal side of the body , where the nerve tube cohabits in vertebrates with the embryonic germ of the neural crest and placodes . spottily-expressed genes , neural and non - neural ectodermal specificity , cell migration in the ectoderm and cryptic complexity are all providing us with new insights into the neural genetic programs of this , our old beast , half - way along the path of construction of complex vertebrate nervous systems . in the near post - genomic future , more and more data will be arriving and , hopefully , experimental embryology ( by means of genetic manipulation of living embryos ) will help clarify the numerous question marks on the evolution of the nervous system , for which the friendly little amphioxus may have some answers . as in the neural tube , hox genes are expressed in a nested manner and , together with the expression of amphipax6 , amphien and amphicdx , divide the entire epidermis into meridians of differential expression .

carbon nanotubes ( cnts ) belong to the nanomaterials family . due to their unique specific properties ( e.g. , size , strength , and electrical conductivity ) , their use is planned in many industrial areas , including electronics , the medical and pharmaceutical industries , and aeronautics . cnts make up a complex family , comprising single - walled and multiwalled carbon nanotubes ( swcnts and mwcnts ) composed of single or multiple graphene sheets rolled into cylinders . cnts can also be functionalized for industrial purposes through modification of the nanotube surface with specific chemical groups . the biodurability and high length - to - width aspect ratio of cnts have raised questions related to their toxicity and effects on human health . their fibrous nature has led to particular concern surrounding the cnts , and parallels have been made with asbestos fibres and their effects on humans [ 2 , 3 ] . to date , occupational exposure to cnts remains poorly understood , but exposure can occur during their manufacture as well as during their industrial use , for example , in the machining or sanding of carbon parts . during the last decade , many toxicological studies have been published on the potential health effects of cnts , but the results have been sometimes conflicting . the discrepancy is mainly a result of differences in the type of cnt used ( shape , diameter , and being single - walled or multiwalled ) , the concentrations used , or the dispersion methods employed . moreover , few studies have analysed sw- and mwcnts in the same experimental model [ 511 ] . to illustrate this complexity , cnts have been shown to induce in vivo an inflammatory response after intratracheal instillation [ 1217 ] or intraperitoneal injection with fibrosis and granuloma [ 2 , 13 ] , but the effects were less clear after inhalation [ 14 , 18 ] . in vitro , genotoxic events have also been observed in vitro with the micronucleus assay [ 9 , 2224 ] and the comet assay [ 22 , 24 , 25 ] . in contrast , asakura et al . observed no induction of micronuclei or hgprt mutations in chl / iu lung cells , which raises concerns about the relevance of the choice of the cellular type according to nanomaterial and toxicological endpoints . the oxidative stress , induced after treatment with fibers and particles , can explain in part the biological effects observed . for carbon nanotubes , several works have shown that they were able to induce and increase ros production [ 2730 ] . the main objective of the present study was to determine the toxicological effects of cnts according to their physicochemical characteristics . however , as the majority of previous studies were conducted on immortalized cell lines and as syrian hamster embryo cells ( she ) are normal and easily implemented , we also compare the toxicological effects of cnts on she cells and on immortalized chinese hamster lung fibroblast v79 cells . this comparison will enable us to determine whether a normal cell model is more suitable than an immortalized cell line for evaluating the toxic effects of cnts . for this purpose , five commercially available cnts ( one swcnt , two dwcnts , and two mwcnts ) , which can potentially be found in the workplace , were tested in v79 and she cells for their in vitro genotoxicity ( comet and micronucleus assays ) , cytotoxicity , and oxidative stress induction ( dcfh - da fluorescent probe ) . three other laboratory - synthesized cnts ( one dwcnt and two mwcnts ) were tested for comparison . the single- and double - walled samples analysed in this study includeda purified single - walled carbon nanotube ( swcnt 1100 , nanocyl , belgium);a purified double - walled carbon nanotube ( dwcnt 2100 , nanocyl , belgium);a short , purified double - walled carbon nanotube ( dwcnt 2150 , nanocyl , belgium ) derived from grinding dwcnt 2100;a purified double - walled carbon nanotube ( dwef ) , donated by e. flahaut of cirimat / umr cnrs 5085 , toulouse , france . a purified single - walled carbon nanotube ( swcnt 1100 , nanocyl , belgium ) ; a purified double - walled carbon nanotube ( dwcnt 2100 , nanocyl , belgium ) ; a short , purified double - walled carbon nanotube ( dwcnt 2150 , nanocyl , belgium ) derived from grinding dwcnt 2100 ; a purified double - walled carbon nanotube ( dwef ) , donated by e. flahaut of cirimat / umr cnrs 5085 , toulouse , france . two multiwalled carbon nanotubes were also tested:(v)a purified multiwalled carbon nanotube ( mwcnt 3100 , nanocyl , belgium);(vi)a short , purified multiwalled carbon nanotube ( mwcnt 3150 , nanocyl , belgium ) , derived from grinding of mwcnt 3100 ; a purified multiwalled carbon nanotube ( mwcnt 3100 , nanocyl , belgium ) ; a short , purified multiwalled carbon nanotube ( mwcnt 3150 , nanocyl , belgium ) , derived from grinding of mwcnt 3100 ; two other mwcnt samples were provided by dr . d. begin ( lmspc - umr 7515-strasbourg ) , synthesized according to gulino et al . : ( vii)a raw multiwalled carbon nanotube ( mwcnt sbb);(viii)a purified multiwalled carbon nanotube ( mwcnt sbp).several criteria guided our choice of cnt samples . first , five of the samples are commercially available ( samples 1100 , 2100 , 2150 , 3100 , and 3150 ) and can therefore be encountered in the workplace . the other three samples ( dwef , sbb , and sbp ) were synthesized in research laboratories . second , each of the large cnt families is represented ( single- , double- , and multiwalled cnts ) . third , both short and long cnts were obtained in order to determine the biological effect of cnt length ( 2100 versus 2150 ; 3100 versus 3150 ) . finally , both raw and purified samples were chosen in order to determine the impact of the presence of chemical products other than carbon on cellular toxicity ( 2100 versus dwef ; sbb versus sbp ) . a raw multiwalled carbon nanotube ( mwcnt sbb ) ; a purified multiwalled carbon nanotube ( mwcnt sbp ) . the chemical contents of cnt samples were analysed by inductively coupled plasma mass spectrometry ( icp - ms ) ( spectro ciros ccd , germany ) . nanotube diameters and the number of walls present were measured by transmission electron microscopy ( tem ) ( philips cm20 , the netherlands ) . specific surface area was determined using the bet technique with a gas sorption analyzer ( asap 2020 micromeritics , france ) . cnt lengths were determined by the supplier . in order to obtain a homogeneous suspension ( estimated visually ) , the samples were placed in complete medium at the highest concentration used in in vitro assays and sonicated for 2.5 min . with a vibracell ( 50 w , 20 khz , bioblock scientific , france ) at 40% power . dls ( dynamic light scattering ) analysis was done to determine the agglomeration status of suspensions using a zetasizer nano zs apparatus ( malvern , france ) , but as mentioned before by tavares et al . and as recently commented on by the oecd , such technique ( designed for analysis of spherical particles ) did not give correct results ( data not shown ) . the alternative method , electronic microscopy , involves methods of sample preparation which induce changes in agglomeration status and thus is not perfectly adapted either . in the absence of adequate technique , the agglomeration status was unknown . the she and v79 cells were treated with cnts at concentrations ranging from 0.27 to 2.1 g / cm of cell culture dish ( free radical generation ) or from 0.23 to 3.75 g / cm ( other assays ) . these concentrations are in the same range as those previously used in our laboratory for studies of asbestos fibres . in a preliminary experiment , these concentrations induced no more than 5055% cytotoxicity as measured by the wst assay ( see below ) . v79 cells ( lung fibroblast from chinese hamster , atcc , usa , reference ccl-93 ) were selected for this study as they are one of the cell models recommended in ocde guideline number 487 for use in the in vitro micronucleus assay . cells were grown in dulbecco 's mem ( dmem ; invitrogen , france ) , supplemented with 10% fetal calf serum ( dutscher , france ) and 0.5% penicillin / streptomycin ( 5000 u-5000 g / ml , invitrogen , france ) . cells were incubated at 37c with 10% co2 , as recommended by the supplier for optimal culture with our medium . syrian hamster embryo ( she ) cell cultures were used as they are normal diploid cells , nongenetically modified , metabolically competent , and p53 effective and there is no known difference with those constituting the organism where they come from . they have been demonstrated to be suitable for genotoxicity assays [ 37 , 38 ] . cells were established from individual 13-day gestation foetuses ( inbred colony , inrs , france ) . the culture medium used was dulbecco 's mem ( dmem ; invitrogen , france ) , supplemented with 17% fetal calf serum ( dutscher , france ) and 0.5% penicillin / streptomycin ( 5000 u-5000 g / ml , invitrogen , france ) . cells were incubated at 37c and 10% co2 . 1 10 cells / ml ( v79 ) or 1.5 10 cells / ml ( she ) were seeded in 48 wells on a 96-well plate for 24 h. the cell cultures were then treated for 24 h with culture medium ( control ) or with sample suspensions in final concentrations between 0.23 and 3.75 g / cm of cell culture surface . the remaining 48 wells on the well plate received the same suspensions ( medium or cnt samples ) to ensure the absence of interference between cnts and wst-1 reagent . after treatment , 1/10 ( v / v ) wst-1 reagent ( roche diagnostics , france ) was added to each well for 3 h. the plates were then centrifuged at 4500 rpm for 5 min to eliminate the majority of cnts and therefore to avoid interference between the soluble formazan dye formed and the cnts at the time of the reading . the supernatant was transferred to new 96-well plates and optical density ( od ) was recorded at 450 nm and 690 nm with a microtiter plate reader ( synergy ht , biotek , france , ) . the delta od ( od450 nm od 690 nm ) was then calculated . data were expressed as % of control sem for each treatment concentration and compared using an anova - lsd test ( fisher 's least significant difference ) ( statgraphics centurion , statpoint technologies , usa ) . cell counting for the comet assay was performed with a coulter z1 ( beckman coulter , france ) ( data not shown ) . 5 10 v79 or she cells were treated with 0.27 to 2.1 g / cm of cnts for 24 h. thirty minutes before the end of treatment , 25 m of 2,7-dichlorodihydrofluorescin diacetate ( h2dcf - da , invitrogen , france ) was added to the cultures . cells were trypsinized and then centrifuged and then placed in hbss ( hank 's buffer saline solution ) with 50 g / ml of propidium iodide . a sample of nanometric anatase tio2 was used for the positive control at 9.2 g / cm . statistical analysis was performed using an anova - lsd test ( fisher 's least significant difference ) ( statgraphics centurion , statpoint technologies , usa ) . potential interference between cnts and dcf was tested by acellular assays , mixing h2dcf ( obtained by naoh treatment of h2dcf - da ) or dcf fluorescent probe ( sigma - aldrich , france ) and cnts at different concentrations ( from 1 to 250 g / ml , equivalent to 0.23 to 58 g / cm of cell culture dish ) . no interference was shown for up to 25 g / ml ( 5.8 g / cm ) of cnts in she cells ( data not shown ) . the fpg enzyme , a glycosylase , recognizes and specifically cuts modified bases such as 8-oxoguanine from dna , producing apurinic sites that are converted into strand breaks by the associated ap - endonuclease activity . therefore , dna strand breaks detected by the fpg modified comet assay provide a measure of oxidative dna damage . , with minor modifications . in brief , two duplicate comet slides were made for each treatment : one slide was treated with fpg and the other with the fpg buffer only . the she ( 2 10 ) or v79 ( 1 10 ) cells were treated for 24 hours either with cnts at concentrations ranging from 0.23 to 3.75 g / cm or with positive control methyl methanesulfonate ( mms , sigma - aldrich , france ) at 0.125 mm or with medium alone . approximately 20,000 cells were mixed in 600 l of 1% low melting agarose ( lma , sigma - aldrich , france ) and the mixture was transferred onto a slide precoated with normal melting agarose ( nma 1% , sigma - aldrich , france ) . slides were then immersed in lysis solution ( 2.5 m nacl , 100 mm na2edta , and 10 mm tris with 1% triton x-100 and 10% dmso added fresh ) and kept in the dark for 1 h at 4c . the slides were drained and incubated in the dark for 30 min at 37c , either in enzyme buffer alone or in fpg ( 5 u / ml ) in enzyme buffer ( 40 mm hepes , 0.1 m kcl , and 0.5 mm na2edta ; ph 8) . the slides were immersed in cold alkaline solution ( 300 mm naoh , 1 mm na2edta ; ph 13 ) for 20 min and electrophoresis was then performed in the same buffer at 0.7 v / cm for 40 min to allow the fragments of damaged dna to migrate towards the anode . the slides were then washed with 0.4 m tris - hcl for 15 min and stained with propidium iodide ( 2.5 g / ml ) . images of 100 randomly selected comets were acquired and analyzed for each sample ( comet assay iv , perceptive instruments , uk ) in order to evaluate the % tail dna used as a measure of dna damage . statistical analyses were performed on means using the anova - lsd test ( statgraphics centurion , statpoint technologies , usa ) . the concentration / tail dna relationship was determined by linear regression ( mixed model ) after logarithmic transformation of tail dna and concentration values ( stata 12.1 , college station , texas , usa ) . approximately 2.5 10 v79 cells and 5 10 she cells were seeded in labtek slides ( nunc a / s , denmark ) with 1 ml of culture medium . after 24 h , the cells were treated either with cnts at concentrations ranging from 0.23 to 3.75 g / cm or with positive control methyl methanesulfonate ( mms , sigma - aldrich , france ) at 0.25 mm or with medium alone for 24 h ( v79 cell doubling time : 1418 hours ; she cell doubling time : 1820 hours ) . at the end of treatment , cells were washed with pbs ( phosphate buffer saline , invitrogen , france ) and fixed in methanol for 15 min . slides were washed in pbs and drained and received one drop of pro long gold antifade reagent with dapi ( molecular probe , invitrogen , france ) . about 1000 cells were analysed at each concentration for the presence of micronuclei ( mn ) . cell proliferation / division was assessed through analysis of the mitotic index ( % of mitotic cells ) . statistical analysis of mn induction was performed on the pooled data of the three independent experiments using the chi - square test . the single - walled 1100 cnt sample contained 3.15 wt . % silica and 1.44 wt . % cobalt . the double - walled 2100 and 2150 cnt samples contained 2.69 and 2.48 wt . the multiwalled 3100 , 3150 , and sbp samples contained few impurities , but the mwcnt sbb contained 7.22 wt . % aluminium and 4.15 wt . % iron . the tem analyses revealed that most of the metal catalysts were located inside the carbon nanotubes . specific surface areas were higher for single- ( 1128 m / g ) and double - walled cnt ( 611 to 985 m / g ) than for the multiwalled cnt ( between 150 and 330 m / g ) . due to the association of carbon nanotubes in bundles , it was not possible to accurately measure their lengths . the external diameters of the carbon nanotube samples ranked from small to large as follows : 1100 ( 1.54 nm ) < dwef ( 1.63.4 nm ) < 21002150 ( 37 nm ) < 31003150 ( 1119 nm ) < sbb - sbp ( 977 nm ) . after dispersion in complete medium , optical microscopy observations showed that the mwcnts were better dispersed than both the double - walled and single - walled cnt ( 1100 ) , even though bundles were present in all samples . the production of reactive oxygen species ( ros ) is often associated with toxicological effects of particles or fibres . in order to address this issue , we performed ros detection in cells after treatment with cnts , using the cell - permeable dcfh - da fluorogenic probe . as shown in figures 1(a ) and 1(b ) , no increase in fluorescence intensity was induced by exposure of either cell type to the 1100 single - walled carbon nanotubes or by the 2100 double - walled carbon nanotubes . the 2150 and dwef samples induced significant increases in fluorescence with concentration in v79 cells ( figure 1(a ) ) but not in she cells ( figure 1(b ) ) . for the mwcnts in v79 cells , only the sbp sample did not induce significant increase in fluorescence . the 3150 induced significant increase at the highest dose , the 3100 at the two highest concentrations ( 1.05 and 2.1 g / cm ) , and sbb at 0.53 , 1.05 , and 2.1 g / cm . in she cells , all mwcnt samples were negative except sample sbb , which induced a significant increase at the highest concentration ( 2.1 g / cm ) ( figures 1(c ) and 1(d ) ) . cell viability was assessed after 24 h treatment with the carbon nanotube samples ( figure 2 ) . the data are reported as the percentage of control relative to the concentration . 24-hour exposure to the swcnt 1100 sample caused no modification of cell viability in either cell type , regardless of the concentration tested ( 0.23 to 3.75 g / cm ) . the 2100 dwcnt induced a significant reduction in cell viability at 3.75 g / cm in she cells but not in v79 cells . all the other sw- and dw - carbon nanotubes induced a concentration - dependent decrease in cell viability , which became significant at 3.75 g / cm in v79 cells and at 1.87 g / cm in she cells for the 2150 sample and at 1.88 and 3.75 g / cm in both cell types for the dwef sample . all the mwcnts induced significant decreases in cell viability at the two ( 3100 in v79 and she ; 3150 in she ) or three ( 3150 , sbb , and sbp ) highest concentrations . the effect on cell viability of the samples at 3.75 g / cm ranked in the following order : in v79 cells : 11002100 ( 100102% of control ) < 2150 ( 77% ) < dwef ( 74% ) < 3100-sbb ( 66% ) < 3150 ( 64% ) < sbp ( 59% ) ; in she cells : 1100 ( 106% ) < 2100 ( 87% ) < dwef ( 74% ) < 31003150 ( 67% ) < 2150 ( 63% ) < sbp ( 50% ) < sbb ( 47% ) . in v79 cells : 11002100 ( 100102% of control ) < 2150 ( 77% ) < dwef ( 74% ) < 3100-sbb ( 66% ) < 3150 ( 64% ) < sbp ( 59% ) ; in she cells : 1100 ( 106% ) < 2100 ( 87% ) < dwef ( 74% ) < 31003150 ( 67% ) < 2150 ( 63% ) < sbp ( 50% ) < sbb ( 47% ) . in conclusion , the mwcnts were found to be more cytotoxic than both the sw- or dw - nanotubes . two types of assay were used to evaluate the genotoxicity of carbon nanotubes in v79 and she cells : the comet assay and the micronucleus assay . results obtained following 24-hour treatment with 1100 , 2100 , 2150 , and dwef samples are presented in figure 3 . the positive control ( mms ) induced significant dna damage in both v79 and she cells , both with and without the fpg enzyme treatment . for the negative control ( medium alone ) , an increase in the number of dna breaks was observed after treatment of the slides with the fpg enzyme . with the exception of sample 2150 , treatment of the cells with the sw- or dwcnt samples induced no effect in either v79 or she cells , with or without fpg treatment . sample 2150 induced a significant increase in the number of dna breaks at 1.87 and 3.75 g / cm in the absence of fpg in she cells ( figure 3(f ) ) . negative results were also obtained in v79 cells with mwcnts ( figure 4 ) , regardless of the concentration tested and both with and without fpg treatment . in she cells , only the sbb and sbp samples showed a significant concentration - damage relationship , with a significant increase in dna breaks observed at the two highest concentrations ( figures 4(f ) and 4(h ) ) . we also observed a concentration - related increase in damage after fpg treatment , with a significant response at the highest concentration for the sbp sample ( figure 4(h ) ) . in v79 cells , sample 1100 induced a significant increase in micronucleated cells at concentrations of 0.94 and 1.87 g / cm but not at 3.75 g / cm . the results obtained from exposure to the double - walled cnts ( 2100 , 2150 , and dwef ) also showed one ( 2150 : 0.23 g / cm ) or two ( 2100 : 0.23 , 0.94 g / cm ; dwef : 0.47 , 0.94 g / cm ) significant concentrations . in she cells in contrast , samples 1100 , 2100 , and 2150 had no effect , and dwef only induced a significant increase in the number of micronucleated cells at 0.23 g / cm . the 3100 mwcnt induced a significant increase in the number of micronucleated cells at concentrations of 0.23 , 0.47 , and 1.87 g / cm in v79 cells and at 0.47 g / cm in she cells . the 3150 mwcnt exhibited similar genotoxic potential in that three ( 0.23 , 0.94 , and 1.87 g / cm ) and four ( 0.23 , 0.94 , 1.87 , and 3.75 g / cm ) of the concentrations tested induced significant increases in the number of micronucleated cells in she and v79 cell cultures , respectively . micronucleus formation was significant in v79 cells at all concentrations for sbb and sbp samples , with a concentration relationship observed for the sbb sample . the sbb and sbp samples were also positive in she cells but only at three concentrations ( 0.23 , 0.47 , and 0.94 g / cm ) for the sbp sample and four concentrations ( 0.23 , 0.47 , 0.94 , and 1.87 g / cm ) for the sbb sample . the v79 mitotic index shows that all cnts with the exception of the 1100 and dwef samples induce a decrease in the number of cells in mitosis . this effect was more pronounced for the mwcnts than for the single- or double - walled cnts and correlates with cell viability if sample dwef is excluded ( figure 2 ) . cells , only sbb and sbp , and to a lesser extent 2150 , induced a decrease in the mitotic index . the specific physicochemical properties of carbon nanotubes , associated with their high aspect ratios , have led many laboratories to initiate and conduct in vitro and in vivo toxicological studies . however , results are sometimes conflicting and despite these efforts it is difficult to draw any overall conclusions . in this work , eight cnts representative of each of the commonly encountered classes ( single- ( sw- ) , double- ( dw- ) , and multiwalled ( mw ) cnts , purified and raw ) were tested for their cytotoxicity and genotoxicity in she and v79 cells . v79 cells , which are recommended for the micronucleus assay ( oecd guideline number 487 ) , have also been used for comet assays in several studies . she cells were used as they are primary cells and are suitable for analyzing the genotoxic properties of chemicals and in particular the effects of fibres or particles [ 3638 ] . we have shown that , in our experimental conditions , mwcnts were more cytotoxic than their single- or double - walled equivalents in both cell types . she cells and v79 cells do not present any great differences in terms of sensitivity . because the sb samples induced 5055% cytotoxicity at concentrations of 3.75 g / cm , higher concentrations would not have been compatible with the other assays for evaluating the genotoxic potential of cnts . even though comparison with other studies is complex and risky because of differences between the materials and methods used , we note that our results differ from those obtained after 24-hour treatment in both macrophage nr8383 and human aortic endothelial cells in which the same toxicity was observed for sw- and mwcnts at concentrations of 31.2 g / cm and 1.4 g / cm , respectively . several hypotheses can be put forward to explain the discrepancy between these results . first , the different methods used for sample dispersion may have caused some discrepancy in biological assays as the cnts may have been dispersed to different extent . second , some cnts may interfere with the culture medium , leading to cytotoxicity through nutrient depletion [ 42 , 43 ] . we tested this hypothesis in a preliminary experiment by incubating the culture medium with cnts . after cnt elimination , no she cell cytotoxicity or cytostasis was induced by the medium ( data not shown ) . however , we observed only slight differences between the sbb and sbp samples . moreover , electronic microscopy showed that the metal particles are located inside the cnts and therefore do appear to be in contact with the surrounding medium . interestingly , as in our study , chen et al . observed that the 3150 sample induced cytotoxicity in a549 human lung epithelial cells ( 34% ) and in raw 264.7 murine macrophage cells ( 27% ) but at much higher concentrations ( 25 g / ml , approximately 15 g / cm ) than we observed . cell number decrease , as measured by the wst viability test , and the decrease in cell mitosis for the majority of samples in both cell types , as measured by the mitotic index , suggest that cnts can act on the cell cycle and block cell division , as was observed in c6 rat glioma cells with mwcnts one possible explanation for this could be that the action of cnts mainly takes place at the level of mitotic spindle as was demonstrated in the studies of sargent et al . [ 4547 ] . the action of cnts on the cell cycle should therefore be investigated in a future study , by analysis of the cell cycle , the dna repair system , dna synthesis , and the spindle apparatus . mwcnts , on the whole , also had greater effects than sw- and dwcnts in the genotoxicity assays . however , unlike in the cytotoxicity assays , some differences were observed in the responses of the two cell types . in the comet assay , none of the cnts induced a significant increase in dna damage in v79 cells , whereas sbb and sbp ( mwcnts ) and 2150 ( dwcnt ) induced significant increases in the number of dna breaks at the two highest concentrations in she cells . treatment with the fpg enzyme increased the level of dna breakage in both cell types ( see control assays with and without fpg in figures 3 and 4 ) , indicating that there was a background level of dna base modification in cells . however , no significant difference was observed between control fpg and treated fpg cells in v79 , and in she cells , only sbp triggered a significant increase in damage at 3.75 g / cm compared to the fpg treated control . similar results were obtained with the fpg enzyme by cavallo et al . in their investigation of mwcnt genotoxicity in a549 cells . an increase in the number of dna breaks induced by the fpg enzyme oxidative stress and production of reactive oxygen species are described as cytotoxic and genotoxic effectors which can lead to the production of oxidized bases . this was demonstrated in different cellular types with the cell - permeable dcfh - da fluorogenic probe after treatment with swcnts or mwcnts [ 30 , 49 , 50 ] . in our case , even though we were able to observe significant ros production with the dcfh - da probe for some cnts , no clear relationship could be identified between ros production and dna damage . the effects of cnt - induced ros production should be investigated in more detail by examining the levels of superoxide dismutase and glutathione and by using ros scavengers . cnt samples were also shown in our study to be capable of inducing micronucleated cells in both cell types , and the effect was seen to be more pronounced with mwcnts . the most genotoxic cnts were the 3150 mwcnt , whose length is described as short by the supplier , the raw and the purified sb samples . the decrease in micronucleated cell frequency at the highest concentrations may be explained by a cell cycle arrest , as was also suggested by the decrease in the mitotic index ( see the previous section ) . our results from the micronucleus assay corroborate those obtained by others with both sw- and mwcnts [ 9 , 20 ] . for example , migliore et al . , who demonstrated that sw- and mwcnts induce the formation of micronuclei in raw 264 cells , also showed that these cnts can induce dna damage . our results show that some cnts , and mainly the mwcnts , can induce cytotoxicity and genotoxicity in she and v79 cells . furthermore , because the cnts induced more micronucleated cells than dna damage and as cnt exposure provoked a cell cycle arrest as revealed by the evaluation of the mitotic index , we can hypothesize that cnts may act on the apparatus spindle during cell division . when looking at the in vivo studies for a comparison and even if such exercise is limited in terms of conclusions , the mwcnts seem to be more genotoxic than swcnts as we have shown in the present study . but , in vivo , data are limited and results obtained for the swcnts present some discrepancies . genotoxic effects have been seen in mouse or rat with swcnts by some authors [ 5254 ] but not by others [ 5558 ] . for mwcnts , results are less confusing with a majority of studies showing genotoxic effects [ 5961 ] . one study has compared sw- and mwcnts in the same model with the same methodology but in this work both sw- and mwcnts were unable to induce genotoxic effects [ 57 , 62 ] . a number of comments can be made regarding the responses of the two cellular types , taking into account that cnts are present in both cellular types as early as 3 h of treatment ( electronic microscopy analysis , data not shown ) : ( i ) cnt cytotoxicity is at almost the same level in both cell types ; ( ii ) more ros were generated in v79 cells than in she cells exposed to cnts ; ( iii ) more micronucleated cells were observed after cnt treatment in v79 cells , but no dna damage was revealed by the comet assay ; the opposite of that was observed in she cells for sbb and sbp . v79 cells are immortalized cells . as they can undergo an infinite number of cell divisions and even though no genotyping or metabolism data were available for this clone , the enzymatic content and gene expression profile for v79 cells are most probably modified at the level of cell cycle checkpoints and dna repair pathways . these differences can explain both the higher level of ros production compared to she cells and the higher background of dna breaks observed in control v79 compared to normal she cells . however , the p53 protein , which mediates the cellular response to dna damage and is involved in cell cycle regulation , apoptosis , and dna repair , does not appear to be able to explain these differences . indeed , v79 cells have already been described as defective for the functional p53 protein . as shown by chaung et al . , the v79 p53 sequence contains two mutation points that result in a nonfunctional protein . conversely , she cells are normal diploid cells with no alterations in the cell cycle pathway [ 65 , 66 ] and she cells also contain a normal p53 protein [ 67 , 68 ] . however , even if v79 cells do have a mutated p53 gene , we showed in the present study that cnts induced the same cytotoxicity and induced micronucleated cell formation in both cell types . , who found no difference in sensitivity to micronucleus induction and cytotoxicity in p53-wild and p53-null human lymphoblastoid cells . furthermore , the mitotic index suggests that a cell cycle arrest occurs in both cell types following exposure to cnts . thus , this blockage does not appear to be influenced by the presence or absence of a mutated p53 gene . to examine this further , as we suggested earlier , additional experiments should be conducted to investigate the cell cycle , spindle apparatus , and effectiveness of the dna repair system . an analysis , at the mrna and protein levels , of p53 and mdm2 ( e3 ubiquitin ligase that inactivates p53 by binding directly ) in she cells , could be also beneficial to better understanding of the response of these cells . as mentioned before , our results show that , for a given cnt , the ros generation can be different according to the cell type . in acellular assay , we have shown that all cnts were able to induce dcf fluorescence in phosphate buffer up to 25 mg / ml ( corresponding to 5.8 g / cm ) ( data not shown ) . as the basal level of ros was the same in terms of fluorescence intensity in both cell types and as cnts were able to induce ros in acellular assay , the level of ros cell generation seems to be specific to a combination between cnt and cellular type . these are preliminary results and ros production should be investigated in more detail by examining the levels of enzyme content of each cellular type , the response to ros scavengers , and so on . however , our results nevertheless suggest that no large differences exist between the v79 cell line and the she normal cells after cnt treatment . the two cellular types are thus complementary and a benefit can certainly be gained in using she cells as they are normal cells that are appropriate for the evaluation of nanomaterial cytotoxicity and genotoxicity . regarding the physicochemical properties and biological effects of cnts , the most pronounced cytotoxic and genotoxic effects were obtained with the multiwalled sbb and sbp samples , and the least toxic cnts in our experiments were the sw- and dwcnts . our data also demonstrate that , in our experimental conditions , there is no relationship between the toxicological effects of cnts and their metal contaminants . indeed , sbb , which contains 7.22% aluminium and 4.15% iron , presented near - identical toxicological effects to sbp , which contains only 0.86% iron . concerning surface area , our results suggest that increased toxicity is not correlated with a higher specific surface area . however , it is important to note that the bet method uses a gas to determine the surface area , and therefore the value obtained does not reflect the real surface area in contact with a liquid or biomolecules . furthermore , the agglomeration status of the suspension used , which we were unable to determine in this study , could directly influence the biological response . the biological impact of cnt length is also unclear from our experiments . even though the shorter 2150 sample was found to be more cytotoxic and induced more ros than the longer the long 3100 and short 3150 samples also presented no differences . in vivo , muller et al . found that ground cnts were less toxic than unground cnts but concluded that the agglomeration state of the cnts rather than their length was likely to be responsible for these differences . the same conclusion was reached by sato et al . in their in vitro and in vivo studies . ( 2012 ) observed a more toxic effect from short cnts than from long cnts , concluding that cnt length was indeed responsible for the observed difference in toxicity in c6 rat glioma cells . in our study , the only physical parameters that we were able to partially link to toxic effects were the number of walls and the outer diameters of the cnts . certainly , the thickest cnt samples ( sbb and sbp ) produced the most toxic effects ( in terms of both cytotoxicity and genotoxicity ) . the importance of cnt diameter as a parameter to be considered in toxicology assessment has previously been suggested in the work of fenoglio et al . . using two mwcnts of the same length range but with very different diameters , they showed that the thickest cnt was the least toxic in a murine macrophage cell line ( mh - s ) . in conclusion , this in vitro study demonstrates that exposure to some but not all cnts induces cytotoxic and genotoxic effects , to different extent depending on the cell type used . our results also suggest that some cnts may act on the cell cycle and on cellular division without having any genotoxic effect . because of their different physicochemical properties , cnts have different toxicological profiles . this suggests that it is not possible to draw any general conclusions regarding the toxicity of these nanomaterials .

carbon nanotubes ( cnts ) belong to a specific class of nanomaterials with unique properties . because of their anticipated use in a wide range of industrial applications , their toxicity is of increasing concern . in order to determine whether specific physicochemical characteristics of cnts are responsible for their toxicological effects , we investigated the cytotoxic and genotoxic effects of eight cnts representative of each of the commonly encountered classes : single- sw- , double- dw- , and multiwalled ( mw ) cnts , purified and raw . in addition , because most previous studies of cnt toxicity were conducted on immortalized cell lines , we decided to compare results obtained from v79 cells , an established cell line , with results from she ( syrian hamster embryo ) cells , an easy - to - handle normal cell model . after 24 hours of treatment , mwcnts were generally found to be more cytotoxic than sw- or dwcnts . mwcnts also provoked more genotoxic effects . no correlation could be found between cnt genotoxicity and metal impurities , length , surface area , or induction of cellular oxidative stress , but genotoxicity was seen to increase with cnt width . the toxicity observed for some cnts leads us to suggest that they might also act by interfering with the cell cycle , but no significant differences were observed between normal and immortalized cells .

1. Introduction 2. Material and Methods 3. Results 4. Discussion

carbon nanotubes ( cnts ) belong to the nanomaterials family . , size , strength , and electrical conductivity ) , their use is planned in many industrial areas , including electronics , the medical and pharmaceutical industries , and aeronautics . cnts make up a complex family , comprising single - walled and multiwalled carbon nanotubes ( swcnts and mwcnts ) composed of single or multiple graphene sheets rolled into cylinders . the biodurability and high length - to - width aspect ratio of cnts have raised questions related to their toxicity and effects on human health . their fibrous nature has led to particular concern surrounding the cnts , and parallels have been made with asbestos fibres and their effects on humans [ 2 , 3 ] . during the last decade , many toxicological studies have been published on the potential health effects of cnts , but the results have been sometimes conflicting . the discrepancy is mainly a result of differences in the type of cnt used ( shape , diameter , and being single - walled or multiwalled ) , the concentrations used , or the dispersion methods employed . observed no induction of micronuclei or hgprt mutations in chl / iu lung cells , which raises concerns about the relevance of the choice of the cellular type according to nanomaterial and toxicological endpoints . the oxidative stress , induced after treatment with fibers and particles , can explain in part the biological effects observed . for carbon nanotubes , several works have shown that they were able to induce and increase ros production [ 2730 ] . the main objective of the present study was to determine the toxicological effects of cnts according to their physicochemical characteristics . however , as the majority of previous studies were conducted on immortalized cell lines and as syrian hamster embryo cells ( she ) are normal and easily implemented , we also compare the toxicological effects of cnts on she cells and on immortalized chinese hamster lung fibroblast v79 cells . this comparison will enable us to determine whether a normal cell model is more suitable than an immortalized cell line for evaluating the toxic effects of cnts . for this purpose , five commercially available cnts ( one swcnt , two dwcnts , and two mwcnts ) , which can potentially be found in the workplace , were tested in v79 and she cells for their in vitro genotoxicity ( comet and micronucleus assays ) , cytotoxicity , and oxidative stress induction ( dcfh - da fluorescent probe ) . two multiwalled carbon nanotubes were also tested:(v)a purified multiwalled carbon nanotube ( mwcnt 3100 , nanocyl , belgium);(vi)a short , purified multiwalled carbon nanotube ( mwcnt 3150 , nanocyl , belgium ) , derived from grinding of mwcnt 3100 ; a purified multiwalled carbon nanotube ( mwcnt 3100 , nanocyl , belgium ) ; a short , purified multiwalled carbon nanotube ( mwcnt 3150 , nanocyl , belgium ) , derived from grinding of mwcnt 3100 ; two other mwcnt samples were provided by dr . first , five of the samples are commercially available ( samples 1100 , 2100 , 2150 , 3100 , and 3150 ) and can therefore be encountered in the workplace . second , each of the large cnt families is represented ( single- , double- , and multiwalled cnts ) . third , both short and long cnts were obtained in order to determine the biological effect of cnt length ( 2100 versus 2150 ; 3100 versus 3150 ) . finally , both raw and purified samples were chosen in order to determine the impact of the presence of chemical products other than carbon on cellular toxicity ( 2100 versus dwef ; sbb versus sbp ) . dls ( dynamic light scattering ) analysis was done to determine the agglomeration status of suspensions using a zetasizer nano zs apparatus ( malvern , france ) , but as mentioned before by tavares et al . v79 cells ( lung fibroblast from chinese hamster , atcc , usa , reference ccl-93 ) were selected for this study as they are one of the cell models recommended in ocde guideline number 487 for use in the in vitro micronucleus assay . syrian hamster embryo ( she ) cell cultures were used as they are normal diploid cells , nongenetically modified , metabolically competent , and p53 effective and there is no known difference with those constituting the organism where they come from . after treatment , 1/10 ( v / v ) wst-1 reagent ( roche diagnostics , france ) was added to each well for 3 h. the plates were then centrifuged at 4500 rpm for 5 min to eliminate the majority of cnts and therefore to avoid interference between the soluble formazan dye formed and the cnts at the time of the reading . 5 10 v79 or she cells were treated with 0.27 to 2.1 g / cm of cnts for 24 h. thirty minutes before the end of treatment , 25 m of 2,7-dichlorodihydrofluorescin diacetate ( h2dcf - da , invitrogen , france ) was added to the cultures . the she ( 2 10 ) or v79 ( 1 10 ) cells were treated for 24 hours either with cnts at concentrations ranging from 0.23 to 3.75 g / cm or with positive control methyl methanesulfonate ( mms , sigma - aldrich , france ) at 0.125 mm or with medium alone . at the end of treatment , cells were washed with pbs ( phosphate buffer saline , invitrogen , france ) and fixed in methanol for 15 min . the multiwalled 3100 , 3150 , and sbp samples contained few impurities , but the mwcnt sbb contained 7.22 wt . in order to address this issue , we performed ros detection in cells after treatment with cnts , using the cell - permeable dcfh - da fluorogenic probe . cell viability was assessed after 24 h treatment with the carbon nanotube samples ( figure 2 ) . all the other sw- and dw - carbon nanotubes induced a concentration - dependent decrease in cell viability , which became significant at 3.75 g / cm in v79 cells and at 1.87 g / cm in she cells for the 2150 sample and at 1.88 and 3.75 g / cm in both cell types for the dwef sample . the effect on cell viability of the samples at 3.75 g / cm ranked in the following order : in v79 cells : 11002100 ( 100102% of control ) < 2150 ( 77% ) < dwef ( 74% ) < 3100-sbb ( 66% ) < 3150 ( 64% ) < sbp ( 59% ) ; in she cells : 1100 ( 106% ) < 2100 ( 87% ) < dwef ( 74% ) < 31003150 ( 67% ) < 2150 ( 63% ) < sbp ( 50% ) < sbb ( 47% ) . in conclusion , the mwcnts were found to be more cytotoxic than both the sw- or dw - nanotubes . for the negative control ( medium alone ) , an increase in the number of dna breaks was observed after treatment of the slides with the fpg enzyme . with the exception of sample 2150 , treatment of the cells with the sw- or dwcnt samples induced no effect in either v79 or she cells , with or without fpg treatment . negative results were also obtained in v79 cells with mwcnts ( figure 4 ) , regardless of the concentration tested and both with and without fpg treatment . in she cells , only the sbb and sbp samples showed a significant concentration - damage relationship , with a significant increase in dna breaks observed at the two highest concentrations ( figures 4(f ) and 4(h ) ) . we also observed a concentration - related increase in damage after fpg treatment , with a significant response at the highest concentration for the sbp sample ( figure 4(h ) ) . the results obtained from exposure to the double - walled cnts ( 2100 , 2150 , and dwef ) also showed one ( 2150 : 0.23 g / cm ) or two ( 2100 : 0.23 , 0.94 g / cm ; dwef : 0.47 , 0.94 g / cm ) significant concentrations . the 3100 mwcnt induced a significant increase in the number of micronucleated cells at concentrations of 0.23 , 0.47 , and 1.87 g / cm in v79 cells and at 0.47 g / cm in she cells . the 3150 mwcnt exhibited similar genotoxic potential in that three ( 0.23 , 0.94 , and 1.87 g / cm ) and four ( 0.23 , 0.94 , 1.87 , and 3.75 g / cm ) of the concentrations tested induced significant increases in the number of micronucleated cells in she and v79 cell cultures , respectively . micronucleus formation was significant in v79 cells at all concentrations for sbb and sbp samples , with a concentration relationship observed for the sbb sample . the v79 mitotic index shows that all cnts with the exception of the 1100 and dwef samples induce a decrease in the number of cells in mitosis . cells , only sbb and sbp , and to a lesser extent 2150 , induced a decrease in the mitotic index . the specific physicochemical properties of carbon nanotubes , associated with their high aspect ratios , have led many laboratories to initiate and conduct in vitro and in vivo toxicological studies . in this work , eight cnts representative of each of the commonly encountered classes ( single- ( sw- ) , double- ( dw- ) , and multiwalled ( mw ) cnts , purified and raw ) were tested for their cytotoxicity and genotoxicity in she and v79 cells . v79 cells , which are recommended for the micronucleus assay ( oecd guideline number 487 ) , have also been used for comet assays in several studies . we have shown that , in our experimental conditions , mwcnts were more cytotoxic than their single- or double - walled equivalents in both cell types . because the sb samples induced 5055% cytotoxicity at concentrations of 3.75 g / cm , higher concentrations would not have been compatible with the other assays for evaluating the genotoxic potential of cnts . even though comparison with other studies is complex and risky because of differences between the materials and methods used , we note that our results differ from those obtained after 24-hour treatment in both macrophage nr8383 and human aortic endothelial cells in which the same toxicity was observed for sw- and mwcnts at concentrations of 31.2 g / cm and 1.4 g / cm , respectively . second , some cnts may interfere with the culture medium , leading to cytotoxicity through nutrient depletion [ 42 , 43 ] . moreover , electronic microscopy showed that the metal particles are located inside the cnts and therefore do appear to be in contact with the surrounding medium . cell number decrease , as measured by the wst viability test , and the decrease in cell mitosis for the majority of samples in both cell types , as measured by the mitotic index , suggest that cnts can act on the cell cycle and block cell division , as was observed in c6 rat glioma cells with mwcnts one possible explanation for this could be that the action of cnts mainly takes place at the level of mitotic spindle as was demonstrated in the studies of sargent et al . the action of cnts on the cell cycle should therefore be investigated in a future study , by analysis of the cell cycle , the dna repair system , dna synthesis , and the spindle apparatus . however , unlike in the cytotoxicity assays , some differences were observed in the responses of the two cell types . in the comet assay , none of the cnts induced a significant increase in dna damage in v79 cells , whereas sbb and sbp ( mwcnts ) and 2150 ( dwcnt ) induced significant increases in the number of dna breaks at the two highest concentrations in she cells . however , no significant difference was observed between control fpg and treated fpg cells in v79 , and in she cells , only sbp triggered a significant increase in damage at 3.75 g / cm compared to the fpg treated control . an increase in the number of dna breaks induced by the fpg enzyme oxidative stress and production of reactive oxygen species are described as cytotoxic and genotoxic effectors which can lead to the production of oxidized bases . this was demonstrated in different cellular types with the cell - permeable dcfh - da fluorogenic probe after treatment with swcnts or mwcnts [ 30 , 49 , 50 ] . in our case , even though we were able to observe significant ros production with the dcfh - da probe for some cnts , no clear relationship could be identified between ros production and dna damage . the effects of cnt - induced ros production should be investigated in more detail by examining the levels of superoxide dismutase and glutathione and by using ros scavengers . cnt samples were also shown in our study to be capable of inducing micronucleated cells in both cell types , and the effect was seen to be more pronounced with mwcnts . our results show that some cnts , and mainly the mwcnts , can induce cytotoxicity and genotoxicity in she and v79 cells . furthermore , because the cnts induced more micronucleated cells than dna damage and as cnt exposure provoked a cell cycle arrest as revealed by the evaluation of the mitotic index , we can hypothesize that cnts may act on the apparatus spindle during cell division . when looking at the in vivo studies for a comparison and even if such exercise is limited in terms of conclusions , the mwcnts seem to be more genotoxic than swcnts as we have shown in the present study . one study has compared sw- and mwcnts in the same model with the same methodology but in this work both sw- and mwcnts were unable to induce genotoxic effects [ 57 , 62 ] . a number of comments can be made regarding the responses of the two cellular types , taking into account that cnts are present in both cellular types as early as 3 h of treatment ( electronic microscopy analysis , data not shown ) : ( i ) cnt cytotoxicity is at almost the same level in both cell types ; ( ii ) more ros were generated in v79 cells than in she cells exposed to cnts ; ( iii ) more micronucleated cells were observed after cnt treatment in v79 cells , but no dna damage was revealed by the comet assay ; the opposite of that was observed in she cells for sbb and sbp . v79 cells are immortalized cells . as they can undergo an infinite number of cell divisions and even though no genotyping or metabolism data were available for this clone , the enzymatic content and gene expression profile for v79 cells are most probably modified at the level of cell cycle checkpoints and dna repair pathways . however , the p53 protein , which mediates the cellular response to dna damage and is involved in cell cycle regulation , apoptosis , and dna repair , does not appear to be able to explain these differences . conversely , she cells are normal diploid cells with no alterations in the cell cycle pathway [ 65 , 66 ] and she cells also contain a normal p53 protein [ 67 , 68 ] . to examine this further , as we suggested earlier , additional experiments should be conducted to investigate the cell cycle , spindle apparatus , and effectiveness of the dna repair system . an analysis , at the mrna and protein levels , of p53 and mdm2 ( e3 ubiquitin ligase that inactivates p53 by binding directly ) in she cells , could be also beneficial to better understanding of the response of these cells . as the basal level of ros was the same in terms of fluorescence intensity in both cell types and as cnts were able to induce ros in acellular assay , the level of ros cell generation seems to be specific to a combination between cnt and cellular type . these are preliminary results and ros production should be investigated in more detail by examining the levels of enzyme content of each cellular type , the response to ros scavengers , and so on . however , our results nevertheless suggest that no large differences exist between the v79 cell line and the she normal cells after cnt treatment . regarding the physicochemical properties and biological effects of cnts , the most pronounced cytotoxic and genotoxic effects were obtained with the multiwalled sbb and sbp samples , and the least toxic cnts in our experiments were the sw- and dwcnts . our data also demonstrate that , in our experimental conditions , there is no relationship between the toxicological effects of cnts and their metal contaminants . concerning surface area , our results suggest that increased toxicity is not correlated with a higher specific surface area . however , it is important to note that the bet method uses a gas to determine the surface area , and therefore the value obtained does not reflect the real surface area in contact with a liquid or biomolecules . furthermore , the agglomeration status of the suspension used , which we were unable to determine in this study , could directly influence the biological response . even though the shorter 2150 sample was found to be more cytotoxic and induced more ros than the longer the long 3100 and short 3150 samples also presented no differences . found that ground cnts were less toxic than unground cnts but concluded that the agglomeration state of the cnts rather than their length was likely to be responsible for these differences . ( 2012 ) observed a more toxic effect from short cnts than from long cnts , concluding that cnt length was indeed responsible for the observed difference in toxicity in c6 rat glioma cells . the importance of cnt diameter as a parameter to be considered in toxicology assessment has previously been suggested in the work of fenoglio et al . using two mwcnts of the same length range but with very different diameters , they showed that the thickest cnt was the least toxic in a murine macrophage cell line ( mh - s ) . in conclusion , this in vitro study demonstrates that exposure to some but not all cnts induces cytotoxic and genotoxic effects , to different extent depending on the cell type used . our results also suggest that some cnts may act on the cell cycle and on cellular division without having any genotoxic effect . because of their different physicochemical properties , cnts have different toxicological profiles .

the discrepancy is mainly a result of differences in the type of cnt used ( shape , diameter , and being single - walled or multiwalled ) , the concentrations used , or the dispersion methods employed . to illustrate this complexity , cnts have been shown to induce in vivo an inflammatory response after intratracheal instillation [ 1217 ] or intraperitoneal injection with fibrosis and granuloma [ 2 , 13 ] , but the effects were less clear after inhalation [ 14 , 18 ] . in vitro , genotoxic events have also been observed in vitro with the micronucleus assay [ 9 , 2224 ] and the comet assay [ 22 , 24 , 25 ] . observed no induction of micronuclei or hgprt mutations in chl / iu lung cells , which raises concerns about the relevance of the choice of the cellular type according to nanomaterial and toxicological endpoints . the main objective of the present study was to determine the toxicological effects of cnts according to their physicochemical characteristics . however , as the majority of previous studies were conducted on immortalized cell lines and as syrian hamster embryo cells ( she ) are normal and easily implemented , we also compare the toxicological effects of cnts on she cells and on immortalized chinese hamster lung fibroblast v79 cells . for this purpose , five commercially available cnts ( one swcnt , two dwcnts , and two mwcnts ) , which can potentially be found in the workplace , were tested in v79 and she cells for their in vitro genotoxicity ( comet and micronucleus assays ) , cytotoxicity , and oxidative stress induction ( dcfh - da fluorescent probe ) . the single- and double - walled samples analysed in this study includeda purified single - walled carbon nanotube ( swcnt 1100 , nanocyl , belgium);a purified double - walled carbon nanotube ( dwcnt 2100 , nanocyl , belgium);a short , purified double - walled carbon nanotube ( dwcnt 2150 , nanocyl , belgium ) derived from grinding dwcnt 2100;a purified double - walled carbon nanotube ( dwef ) , donated by e. flahaut of cirimat / umr cnrs 5085 , toulouse , france . a purified single - walled carbon nanotube ( swcnt 1100 , nanocyl , belgium ) ; a purified double - walled carbon nanotube ( dwcnt 2100 , nanocyl , belgium ) ; a short , purified double - walled carbon nanotube ( dwcnt 2150 , nanocyl , belgium ) derived from grinding dwcnt 2100 ; a purified double - walled carbon nanotube ( dwef ) , donated by e. flahaut of cirimat / umr cnrs 5085 , toulouse , france . two multiwalled carbon nanotubes were also tested:(v)a purified multiwalled carbon nanotube ( mwcnt 3100 , nanocyl , belgium);(vi)a short , purified multiwalled carbon nanotube ( mwcnt 3150 , nanocyl , belgium ) , derived from grinding of mwcnt 3100 ; a purified multiwalled carbon nanotube ( mwcnt 3100 , nanocyl , belgium ) ; a short , purified multiwalled carbon nanotube ( mwcnt 3150 , nanocyl , belgium ) , derived from grinding of mwcnt 3100 ; two other mwcnt samples were provided by dr . finally , both raw and purified samples were chosen in order to determine the impact of the presence of chemical products other than carbon on cellular toxicity ( 2100 versus dwef ; sbb versus sbp ) . v79 cells ( lung fibroblast from chinese hamster , atcc , usa , reference ccl-93 ) were selected for this study as they are one of the cell models recommended in ocde guideline number 487 for use in the in vitro micronucleus assay . 5 10 v79 or she cells were treated with 0.27 to 2.1 g / cm of cnts for 24 h. thirty minutes before the end of treatment , 25 m of 2,7-dichlorodihydrofluorescin diacetate ( h2dcf - da , invitrogen , france ) was added to the cultures . potential interference between cnts and dcf was tested by acellular assays , mixing h2dcf ( obtained by naoh treatment of h2dcf - da ) or dcf fluorescent probe ( sigma - aldrich , france ) and cnts at different concentrations ( from 1 to 250 g / ml , equivalent to 0.23 to 58 g / cm of cell culture dish ) . after 24 h , the cells were treated either with cnts at concentrations ranging from 0.23 to 3.75 g / cm or with positive control methyl methanesulfonate ( mms , sigma - aldrich , france ) at 0.25 mm or with medium alone for 24 h ( v79 cell doubling time : 1418 hours ; she cell doubling time : 1820 hours ) . specific surface areas were higher for single- ( 1128 m / g ) and double - walled cnt ( 611 to 985 m / g ) than for the multiwalled cnt ( between 150 and 330 m / g ) . the external diameters of the carbon nanotube samples ranked from small to large as follows : 1100 ( 1.54 nm ) < dwef ( 1.63.4 nm ) < 21002150 ( 37 nm ) < 31003150 ( 1119 nm ) < sbb - sbp ( 977 nm ) . after dispersion in complete medium , optical microscopy observations showed that the mwcnts were better dispersed than both the double - walled and single - walled cnt ( 1100 ) , even though bundles were present in all samples . in order to address this issue , we performed ros detection in cells after treatment with cnts , using the cell - permeable dcfh - da fluorogenic probe . as shown in figures 1(a ) and 1(b ) , no increase in fluorescence intensity was induced by exposure of either cell type to the 1100 single - walled carbon nanotubes or by the 2100 double - walled carbon nanotubes . the 2150 and dwef samples induced significant increases in fluorescence with concentration in v79 cells ( figure 1(a ) ) but not in she cells ( figure 1(b ) ) . the 3150 induced significant increase at the highest dose , the 3100 at the two highest concentrations ( 1.05 and 2.1 g / cm ) , and sbb at 0.53 , 1.05 , and 2.1 g / cm . in she cells , all mwcnt samples were negative except sample sbb , which induced a significant increase at the highest concentration ( 2.1 g / cm ) ( figures 1(c ) and 1(d ) ) . 24-hour exposure to the swcnt 1100 sample caused no modification of cell viability in either cell type , regardless of the concentration tested ( 0.23 to 3.75 g / cm ) . all the other sw- and dw - carbon nanotubes induced a concentration - dependent decrease in cell viability , which became significant at 3.75 g / cm in v79 cells and at 1.87 g / cm in she cells for the 2150 sample and at 1.88 and 3.75 g / cm in both cell types for the dwef sample . all the mwcnts induced significant decreases in cell viability at the two ( 3100 in v79 and she ; 3150 in she ) or three ( 3150 , sbb , and sbp ) highest concentrations . the effect on cell viability of the samples at 3.75 g / cm ranked in the following order : in v79 cells : 11002100 ( 100102% of control ) < 2150 ( 77% ) < dwef ( 74% ) < 3100-sbb ( 66% ) < 3150 ( 64% ) < sbp ( 59% ) ; in she cells : 1100 ( 106% ) < 2100 ( 87% ) < dwef ( 74% ) < 31003150 ( 67% ) < 2150 ( 63% ) < sbp ( 50% ) < sbb ( 47% ) . in v79 cells : 11002100 ( 100102% of control ) < 2150 ( 77% ) < dwef ( 74% ) < 3100-sbb ( 66% ) < 3150 ( 64% ) < sbp ( 59% ) ; in she cells : 1100 ( 106% ) < 2100 ( 87% ) < dwef ( 74% ) < 31003150 ( 67% ) < 2150 ( 63% ) < sbp ( 50% ) < sbb ( 47% ) . two types of assay were used to evaluate the genotoxicity of carbon nanotubes in v79 and she cells : the comet assay and the micronucleus assay . with the exception of sample 2150 , treatment of the cells with the sw- or dwcnt samples induced no effect in either v79 or she cells , with or without fpg treatment . sample 2150 induced a significant increase in the number of dna breaks at 1.87 and 3.75 g / cm in the absence of fpg in she cells ( figure 3(f ) ) . in she cells , only the sbb and sbp samples showed a significant concentration - damage relationship , with a significant increase in dna breaks observed at the two highest concentrations ( figures 4(f ) and 4(h ) ) . we also observed a concentration - related increase in damage after fpg treatment , with a significant response at the highest concentration for the sbp sample ( figure 4(h ) ) . the results obtained from exposure to the double - walled cnts ( 2100 , 2150 , and dwef ) also showed one ( 2150 : 0.23 g / cm ) or two ( 2100 : 0.23 , 0.94 g / cm ; dwef : 0.47 , 0.94 g / cm ) significant concentrations . in she cells in contrast , samples 1100 , 2100 , and 2150 had no effect , and dwef only induced a significant increase in the number of micronucleated cells at 0.23 g / cm . the 3100 mwcnt induced a significant increase in the number of micronucleated cells at concentrations of 0.23 , 0.47 , and 1.87 g / cm in v79 cells and at 0.47 g / cm in she cells . the 3150 mwcnt exhibited similar genotoxic potential in that three ( 0.23 , 0.94 , and 1.87 g / cm ) and four ( 0.23 , 0.94 , 1.87 , and 3.75 g / cm ) of the concentrations tested induced significant increases in the number of micronucleated cells in she and v79 cell cultures , respectively . the sbb and sbp samples were also positive in she cells but only at three concentrations ( 0.23 , 0.47 , and 0.94 g / cm ) for the sbp sample and four concentrations ( 0.23 , 0.47 , 0.94 , and 1.87 g / cm ) for the sbb sample . the v79 mitotic index shows that all cnts with the exception of the 1100 and dwef samples induce a decrease in the number of cells in mitosis . the specific physicochemical properties of carbon nanotubes , associated with their high aspect ratios , have led many laboratories to initiate and conduct in vitro and in vivo toxicological studies . in this work , eight cnts representative of each of the commonly encountered classes ( single- ( sw- ) , double- ( dw- ) , and multiwalled ( mw ) cnts , purified and raw ) were tested for their cytotoxicity and genotoxicity in she and v79 cells . she cells were used as they are primary cells and are suitable for analyzing the genotoxic properties of chemicals and in particular the effects of fibres or particles [ 3638 ] . because the sb samples induced 5055% cytotoxicity at concentrations of 3.75 g / cm , higher concentrations would not have been compatible with the other assays for evaluating the genotoxic potential of cnts . even though comparison with other studies is complex and risky because of differences between the materials and methods used , we note that our results differ from those obtained after 24-hour treatment in both macrophage nr8383 and human aortic endothelial cells in which the same toxicity was observed for sw- and mwcnts at concentrations of 31.2 g / cm and 1.4 g / cm , respectively . observed that the 3150 sample induced cytotoxicity in a549 human lung epithelial cells ( 34% ) and in raw 264.7 murine macrophage cells ( 27% ) but at much higher concentrations ( 25 g / ml , approximately 15 g / cm ) than we observed . cell number decrease , as measured by the wst viability test , and the decrease in cell mitosis for the majority of samples in both cell types , as measured by the mitotic index , suggest that cnts can act on the cell cycle and block cell division , as was observed in c6 rat glioma cells with mwcnts one possible explanation for this could be that the action of cnts mainly takes place at the level of mitotic spindle as was demonstrated in the studies of sargent et al . the action of cnts on the cell cycle should therefore be investigated in a future study , by analysis of the cell cycle , the dna repair system , dna synthesis , and the spindle apparatus . in the comet assay , none of the cnts induced a significant increase in dna damage in v79 cells , whereas sbb and sbp ( mwcnts ) and 2150 ( dwcnt ) induced significant increases in the number of dna breaks at the two highest concentrations in she cells . treatment with the fpg enzyme increased the level of dna breakage in both cell types ( see control assays with and without fpg in figures 3 and 4 ) , indicating that there was a background level of dna base modification in cells . however , no significant difference was observed between control fpg and treated fpg cells in v79 , and in she cells , only sbp triggered a significant increase in damage at 3.75 g / cm compared to the fpg treated control . an increase in the number of dna breaks induced by the fpg enzyme oxidative stress and production of reactive oxygen species are described as cytotoxic and genotoxic effectors which can lead to the production of oxidized bases . in our case , even though we were able to observe significant ros production with the dcfh - da probe for some cnts , no clear relationship could be identified between ros production and dna damage . the effects of cnt - induced ros production should be investigated in more detail by examining the levels of superoxide dismutase and glutathione and by using ros scavengers . furthermore , because the cnts induced more micronucleated cells than dna damage and as cnt exposure provoked a cell cycle arrest as revealed by the evaluation of the mitotic index , we can hypothesize that cnts may act on the apparatus spindle during cell division . when looking at the in vivo studies for a comparison and even if such exercise is limited in terms of conclusions , the mwcnts seem to be more genotoxic than swcnts as we have shown in the present study . one study has compared sw- and mwcnts in the same model with the same methodology but in this work both sw- and mwcnts were unable to induce genotoxic effects [ 57 , 62 ] . a number of comments can be made regarding the responses of the two cellular types , taking into account that cnts are present in both cellular types as early as 3 h of treatment ( electronic microscopy analysis , data not shown ) : ( i ) cnt cytotoxicity is at almost the same level in both cell types ; ( ii ) more ros were generated in v79 cells than in she cells exposed to cnts ; ( iii ) more micronucleated cells were observed after cnt treatment in v79 cells , but no dna damage was revealed by the comet assay ; the opposite of that was observed in she cells for sbb and sbp . as they can undergo an infinite number of cell divisions and even though no genotyping or metabolism data were available for this clone , the enzymatic content and gene expression profile for v79 cells are most probably modified at the level of cell cycle checkpoints and dna repair pathways . these differences can explain both the higher level of ros production compared to she cells and the higher background of dna breaks observed in control v79 compared to normal she cells . however , the p53 protein , which mediates the cellular response to dna damage and is involved in cell cycle regulation , apoptosis , and dna repair , does not appear to be able to explain these differences . however , even if v79 cells do have a mutated p53 gene , we showed in the present study that cnts induced the same cytotoxicity and induced micronucleated cell formation in both cell types . to examine this further , as we suggested earlier , additional experiments should be conducted to investigate the cell cycle , spindle apparatus , and effectiveness of the dna repair system . an analysis , at the mrna and protein levels , of p53 and mdm2 ( e3 ubiquitin ligase that inactivates p53 by binding directly ) in she cells , could be also beneficial to better understanding of the response of these cells . as the basal level of ros was the same in terms of fluorescence intensity in both cell types and as cnts were able to induce ros in acellular assay , the level of ros cell generation seems to be specific to a combination between cnt and cellular type . regarding the physicochemical properties and biological effects of cnts , the most pronounced cytotoxic and genotoxic effects were obtained with the multiwalled sbb and sbp samples , and the least toxic cnts in our experiments were the sw- and dwcnts . however , it is important to note that the bet method uses a gas to determine the surface area , and therefore the value obtained does not reflect the real surface area in contact with a liquid or biomolecules . in our study , the only physical parameters that we were able to partially link to toxic effects were the number of walls and the outer diameters of the cnts . in conclusion , this in vitro study demonstrates that exposure to some but not all cnts induces cytotoxic and genotoxic effects , to different extent depending on the cell type used .

systemic local anesthetic toxicity is a rare but potentially fatal complication that is intractable to conventional cardiopulmonary resuscitation . systemic local anesthetic toxicity also has a risk of progressing to " recurrent systemic local anesthetic toxicity after successful resuscitation " . intravenous infusion of les reverses intractable cardiac toxicity in an animal model , and les are effective for treating local anesthetic - induced cardiac toxicity . therefore , various studies associated with les have been actively conducted to understand the mechanism of lipid rescue and improve treatment regimens . intralipid 20% , which contains only 100% long - chain triglycerides , is commonly used to treat local anesthetic - induced systemic toxicity , whereas lipofundin mct / lct 20% which is composed of 50% long - chain triglycerides and 50% mediumchain triglycerides , is occasionally used to treat local anestheticinduced systemic toxicity . however , both types of le affect hemodynamics in an ex vivo model but the associated cellular mechanism remains unknown . therefore , we investigated the inotropic effect of two les ( intralipid or lipofundin mct / lct ) on a hanging heart model mounted in a langendorff perfusion system to elucidate the mechanism associated with changes in intracellular calcium level in myocardial cells . male sprague dawley rats ( koatech , pyeongtaek , korea ; weight , 250350 g ) were used for this study . all animals were maintained in accordance with the guidelines for the care and use of laboratory animals published by the us national institutes of health in 1996 . briefly , the animals received general anesthesia with an intramuscular injection of 15 mg / kg tiletamine / zolazepam ( zoletil50 ; virbac lab . , carros , france ) and 9 mg / kg of xylazine ( rompun ; bayer , seoul , korea ) . if the tail moved ( i.e. a sign of awakening ) during the operation , an additional 5 mg / kg tiletamine / zolazepam and 3 mg / kg xylazine were injected to maintain anesthesia . heparin ( 1000 iu / kg ) was administered through the femoral vein after anesthesia was induced . a tracheostomy was performed , and the animals were mechanically ventilated with room air via a 16 g catheter . the heart was mounted quickly on a langendorff perfusion system and perfused with modified krebs - henseleit solution ( 118 mm nacl , 4.7 mm kcl , 1.2 mm mgso4 , 1.2 mm kh2po4 , 2.4 mm cacl2 , 25 mm nahco3 , 11 mm glucose , and 0.03 mm edta ) , equilibrated to ph 7.4 with a mixture of 5% co2/95% o2 . the perfusion solution was maintained at 37 0.2 using thermostatically controlled water circulating system ( water bath ntt-2200 ; tokyo rikakikai co. ltd . , perfusion pressure was maintained at 70 mmhg with a 95 cm high fluid column and an overflow pump . the left atrium was removed and a 2f millar catheter ( model spr-407 ; millar instruments inc . , houston , tx , usa ) was inserted into the left ventricle . a pressure transducer ( model ml 118 ; adinstruments pty . sydney , australia ) was connected to a digital analysis system to measure left ventricular hemodynamic function . left ventricular systolic pressure ( lvsp ) , maximum rate of intraventricular pressure increase ( + dp / dtmax ) , maximum rate of intraventricular pressure decrease ( dp / dtmax ) , and heart rate ( hr ) were measured using a computer analysis program ( charttm5 pro ; adinstruments ) as described previously . the hanging hearts were perfused with modified krebs - henseleit solution for 10 min to stabilize prior to recording hemodynamic function ( n = 10 ) . hearts were excluded if the average values for the last 5 min of the stabilization period failed to meet the following criteria ; lvsp , 80130 mmhg ; and hr , 200350 beats / min . each experimental group was injected with the drug using an infusion pump ( auto syringe as50 infusion pump ; baxter , singapore ) through a three - way stopcock on the fluid column . the hanging hearts were assigned randomly to three groups as follows : ( 1 ) control group ( hearts in which 1 ml / kg modified krebs - henseleit solution was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; ( 2 ) il1 group ( hearts in which intralipid 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; and ( 3 ) mct1 group ( hearts in which lipofundin mct / lct 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) . , kaysville , ut , usa ; alpha = 0.05 , power = 92% ) . changes in intracellular calcium were examined by measuring increased fluorescence of fluo-3/acetoxymethyl ( am ) ( molecular probes , eugene , or , usa)-loaded cells with a confocal microscope ( fv1000 ; olympus , tokyo , japan ) . rat embryonic ventricular myocardial h9c2 cells were purchased from the american type culture collection ( manassas , va , usa ) and kept in complete growth medium ( dulbecco 's modified eagle 's medium ; invitrogen , carlsbad , ca , usa ) supplemented with 100% fetal bovine serum ( invitrogen ) at 37 in a humidified atmosphere of 5% co2 . basal and intracellular changes in calcium were examined by measuring increased fluorescence of fluo-3/am - loaded cells in the cytosol of h9c2 cells with a confocal microscope . h9c2 cells were attached to cover slips for 24 h at 37 before loading with 5 m fluo-3/am and incubating at 37 for 60 min . cover slips were inserted into a chamber mounted on the stage of the confocal microscope and rinsed with a warm normal bath solution ( 140 mm nacl , 5.0 mm kcl , 1.0 mm cacl2 , 1.0 mm mgcl2 , and10 mm hepes [ ph 7.4 ] ) . baseline measurements were taken on quiescent cells for 34 min prior to the application of le . fluorescent images were collected every 0.6 sec for fast signals and analyzed by fluoview image processing software ver . twenty signal curves were taken from one view for each and averaged after each le treatment ( n = 4 ) . the le concentration used was 0.01% , which induced a maximal increase in intracellular calcium in the preliminary study . all analyses were performed using spss statistical software ver . 18.0 for windows ( spss inc . , experimental outcomes were analyzed using one - way analysis of variance and the bonferroni post - hoc test . male sprague dawley rats ( koatech , pyeongtaek , korea ; weight , 250350 g ) were used for this study . all animals were maintained in accordance with the guidelines for the care and use of laboratory animals published by the us national institutes of health in 1996 . briefly , the animals received general anesthesia with an intramuscular injection of 15 mg / kg tiletamine / zolazepam ( zoletil50 ; virbac lab . , carros , france ) and 9 mg / kg of xylazine ( rompun ; bayer , seoul , korea ) . if the tail moved ( i.e. a sign of awakening ) during the operation , an additional 5 mg / kg tiletamine / zolazepam and 3 mg / kg xylazine were injected to maintain anesthesia . heparin ( 1000 iu / kg ) was administered through the femoral vein after anesthesia was induced . a tracheostomy was performed , and the animals were mechanically ventilated with room air via a 16 g catheter . the heart was mounted quickly on a langendorff perfusion system and perfused with modified krebs - henseleit solution ( 118 mm nacl , 4.7 mm kcl , 1.2 mm mgso4 , 1.2 mm kh2po4 , 2.4 mm cacl2 , 25 mm nahco3 , 11 mm glucose , and 0.03 mm edta ) , equilibrated to ph 7.4 with a mixture of 5% co2/95% o2 . the perfusion solution was maintained at 37 0.2 using thermostatically controlled water circulating system ( water bath ntt-2200 ; tokyo rikakikai co. ltd . , perfusion pressure was maintained at 70 mmhg with a 95 cm high fluid column and an overflow pump . the left atrium was removed and a 2f millar catheter ( model spr-407 ; millar instruments inc . , houston , tx , usa ) was inserted into the left ventricle . a pressure transducer ( model ml 118 ; adinstruments pty . sydney , australia ) was connected to a digital analysis system to measure left ventricular hemodynamic function . left ventricular systolic pressure ( lvsp ) , maximum rate of intraventricular pressure increase ( + dp / dtmax ) , maximum rate of intraventricular pressure decrease ( dp / dtmax ) , and heart rate ( hr ) were measured using a computer analysis program ( charttm5 pro ; adinstruments ) as described previously . the hanging hearts were perfused with modified krebs - henseleit solution for 10 min to stabilize prior to recording hemodynamic function ( n = 10 ) . hearts were excluded if the average values for the last 5 min of the stabilization period failed to meet the following criteria ; lvsp , 80130 mmhg ; and hr , 200350 beats / min . each experimental group was injected with the drug using an infusion pump ( auto syringe as50 infusion pump ; baxter , singapore ) through a three - way stopcock on the fluid column . the hanging hearts were assigned randomly to three groups as follows : ( 1 ) control group ( hearts in which 1 ml / kg modified krebs - henseleit solution was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; ( 2 ) il1 group ( hearts in which intralipid 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; and ( 3 ) mct1 group ( hearts in which lipofundin mct / lct 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) . , kaysville , ut , usa ; alpha = 0.05 , power = 92% ) . changes in intracellular calcium were examined by measuring increased fluorescence of fluo-3/acetoxymethyl ( am ) ( molecular probes , eugene , or , usa)-loaded cells with a confocal microscope ( fv1000 ; olympus , tokyo , japan ) . rat embryonic ventricular myocardial h9c2 cells were purchased from the american type culture collection ( manassas , va , usa ) and kept in complete growth medium ( dulbecco 's modified eagle 's medium ; invitrogen , carlsbad , ca , usa ) supplemented with 100% fetal bovine serum ( invitrogen ) at 37 in a humidified atmosphere of 5% co2 . basal and intracellular changes in calcium were examined by measuring increased fluorescence of fluo-3/am - loaded cells in the cytosol of h9c2 cells with a confocal microscope . h9c2 cells were attached to cover slips for 24 h at 37 before loading with 5 m fluo-3/am and incubating at 37 for 60 min . cover slips were inserted into a chamber mounted on the stage of the confocal microscope and rinsed with a warm normal bath solution ( 140 mm nacl , 5.0 mm kcl , 1.0 mm cacl2 , 1.0 mm mgcl2 , and10 mm hepes [ ph 7.4 ] ) . baseline measurements were taken on quiescent cells for 34 min prior to the application of le . fluorescent images were collected every 0.6 sec for fast signals and analyzed by fluoview image processing software ver . twenty signal curves were taken from one view for each and averaged after each le treatment ( n = 4 ) . the le concentration used was 0.01% , which induced a maximal increase in intracellular calcium in the preliminary study . male sprague dawley rats ( koatech , pyeongtaek , korea ; weight , 250350 g ) were used for this study . all animals were maintained in accordance with the guidelines for the care and use of laboratory animals published by the us national institutes of health in 1996 . briefly , the animals received general anesthesia with an intramuscular injection of 15 mg / kg tiletamine / zolazepam ( zoletil50 ; virbac lab . , carros , france ) and 9 mg / kg of xylazine ( rompun ; bayer , seoul , korea ) . if the tail moved ( i.e. a sign of awakening ) during the operation , an additional 5 mg / kg tiletamine / zolazepam and 3 mg / kg xylazine were injected to maintain anesthesia . heparin ( 1000 iu / kg ) was administered through the femoral vein after anesthesia was induced . a tracheostomy was performed , and the animals were mechanically ventilated with room air via a 16 g catheter . the heart was mounted quickly on a langendorff perfusion system and perfused with modified krebs - henseleit solution ( 118 mm nacl , 4.7 mm kcl , 1.2 mm mgso4 , 1.2 mm kh2po4 , 2.4 mm cacl2 , 25 mm nahco3 , 11 mm glucose , and 0.03 mm edta ) , equilibrated to ph 7.4 with a mixture of 5% co2/95% o2 . the perfusion solution was maintained at 37 0.2 using thermostatically controlled water circulating system ( water bath ntt-2200 ; tokyo rikakikai co. ltd . , perfusion pressure was maintained at 70 mmhg with a 95 cm high fluid column and an overflow pump . the left atrium was removed and a 2f millar catheter ( model spr-407 ; millar instruments inc . , houston , tx , usa ) was inserted into the left ventricle . a pressure transducer ( model ml 118 ; adinstruments pty . sydney , australia ) was connected to a digital analysis system to measure left ventricular hemodynamic function . left ventricular systolic pressure ( lvsp ) , maximum rate of intraventricular pressure increase ( + dp / dtmax ) , maximum rate of intraventricular pressure decrease ( dp / dtmax ) , and heart rate ( hr ) were measured using a computer analysis program ( charttm5 pro ; adinstruments ) as described previously . the hanging hearts were perfused with modified krebs - henseleit solution for 10 min to stabilize prior to recording hemodynamic function ( n = 10 ) . hearts were excluded if the average values for the last 5 min of the stabilization period failed to meet the following criteria ; lvsp , 80130 mmhg ; and hr , 200350 beats / min . each experimental group was injected with the drug using an infusion pump ( auto syringe as50 infusion pump ; baxter , singapore ) through a three - way stopcock on the fluid column . the hanging hearts were assigned randomly to three groups as follows : ( 1 ) control group ( hearts in which 1 ml / kg modified krebs - henseleit solution was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; ( 2 ) il1 group ( hearts in which intralipid 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; and ( 3 ) mct1 group ( hearts in which lipofundin mct / lct 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) . , kaysville , ut , usa ; alpha = 0.05 , power = 92% ) . changes in intracellular calcium were examined by measuring increased fluorescence of fluo-3/acetoxymethyl ( am ) ( molecular probes , eugene , or , usa)-loaded cells with a confocal microscope ( fv1000 ; olympus , tokyo , japan ) . rat embryonic ventricular myocardial h9c2 cells were purchased from the american type culture collection ( manassas , va , usa ) and kept in complete growth medium ( dulbecco 's modified eagle 's medium ; invitrogen , carlsbad , ca , usa ) supplemented with 100% fetal bovine serum ( invitrogen ) at 37 in a humidified atmosphere of 5% co2 . basal and intracellular changes in calcium were examined by measuring increased fluorescence of fluo-3/am - loaded cells in the cytosol of h9c2 cells with a confocal microscope . h9c2 cells were attached to cover slips for 24 h at 37 before loading with 5 m fluo-3/am and incubating at 37 for 60 min . cover slips were inserted into a chamber mounted on the stage of the confocal microscope and rinsed with a warm normal bath solution ( 140 mm nacl , 5.0 mm kcl , 1.0 mm cacl2 , 1.0 mm mgcl2 , and10 mm hepes [ ph 7.4 ] ) . baseline measurements were taken on quiescent cells for 34 min prior to the application of le . fluorescent images were collected every 0.6 sec for fast signals and analyzed by fluoview image processing software ver . twenty signal curves were taken from one view for each and averaged after each le treatment ( n = 4 ) . the le concentration used was 0.01% , which induced a maximal increase in intracellular calcium in the preliminary study . all analyses were performed using spss statistical software ver . 18.0 for windows ( spss inc . , chicago , il , usa ) . experimental outcomes were analyzed using one - way analysis of variance and the bonferroni post - hoc test . the ratio of lvsp in the mct1 group ( 110.6% 6.4% ) increased significantly compared to that in the control group ( 103.2% 2.1% , p = 0.003 , 95% confidence interval [ ci ] , 2.412.5 ) and the il1 group ( 104.1% 3.7% , p = 0.009 , 95% ci , 1.411.6 ) ( fig . the ratio of + dp / dtmax ( control , 98.5% 6.9% ; il1 , 103.2% 7.2% ; mct1 , 108.7% 15.7% ) , dp / dtmax ( control , 97.6% 10.0% ; il1 , 103.9% 6.0% ; mct1 , 102.1% 9.6% ) , and hr ( control , 93.2% 7.8% ; il1 , 96.6% 1.5% ; mct1 , 94.0% 5.0% ) were not significant between the groups . the change of intracellular calcium level was estimated by comparing the cellular fluorescence ratio with the basal fluorescence ratio using the fluo-3/am total calcium imaging technique . lipofundin mct / lct ( 0.01% ) increased intracellular calcium levels more than that of intralipid ( 0.01% ) ( il group : 3.03 0.53 ; mct group : 3.92 0.60 ; p < 0.05 ; 95% ci , 0.01.9 ) . the ratio of lvsp in the mct1 group ( 110.6% 6.4% ) increased significantly compared to that in the control group ( 103.2% 2.1% , p = 0.003 , 95% confidence interval [ ci ] , 2.412.5 ) and the il1 group ( 104.1% 3.7% , p = 0.009 , 95% ci , 1.411.6 ) ( fig . the ratio of + dp / dtmax ( control , 98.5% 6.9% ; il1 , 103.2% 7.2% ; mct1 , 108.7% 15.7% ) , dp / dtmax ( control , 97.6% 10.0% ; il1 , 103.9% 6.0% ; mct1 , 102.1% 9.6% ) , and hr ( control , 93.2% 7.8% ; il1 , 96.6% 1.5% ; mct1 , 94.0% 5.0% ) were not significant between the groups . the change of intracellular calcium level was estimated by comparing the cellular fluorescence ratio with the basal fluorescence ratio using the fluo-3/am total calcium imaging technique . lipofundin mct / lct ( 0.01% ) increased intracellular calcium levels more than that of intralipid ( 0.01% ) ( il group : 3.03 0.53 ; mct group : 3.92 0.60 ; p < 0.05 ; 95% ci , 0.01.9 ) . the major findings of our study are : 1 ) the lipofundin mct / lct infusion significantly increased lvsp in hanging hearts , 2 ) lipofundin mct / lct has had more of a positive inotropic effect than that of intralipid , and 3 ) the le treatments increased the intracellular calcium levels in h9c2 cells . lipofundin mct / lct increased intracellular calcium levels more than that of intralipid. although there are some reports indicate that long - chain triglycerides has have virtually no effect on hemodynamics , it is a generally accepted that le has a positive hemodynamic effect . however , a mixture of medium - chain triglycerides , long - chain triglycerides , and omega-3 polyunsaturated fatty acid emulsions increase mean aortic blood pressure . thus , we investigated the effect of commercially available les ( intralipid 20% and lipofundin mct / lct 20% ) on hemodynamics in an ex vivo heart model . as results , lipofundin mct / lct had a more positive inotropic effect than that of intralipid. therefore , careful observations of hemodynamic changes are needed during bolus infusion of les . in particular , lipofundin mct / lct should be used with caution as a total parenteral nutrition component in critically ill patients with compromised cardiovascular function . the second aim of this study was to elucidate the mechanism responsible for the le - induced increase in lvsp and intracellular calcium levels in myocardial cells . long chain fatty acids directly activate calcium channels at some lipid sites near the channels or on the channel protein itself , as assessed by the standard whole cell voltage clamp technique in ventricular myocytes . therefore , the positive inotropic effect of lipofundin mct / lct shown on the hearts connected to the langendorff perfusion system may be associated with cardiac myocyte calcium concentration . however , one study showed that 10% intralipid exerts a modest positive inotropic effect , although the intracellular calcium remains unchanged by intralipid . thus , we measured the change in calcium current after adding les to rat cardiac myoblastic h9c2 cells to clarify their effect on calcium current . most of the signal transduction pathways that stimulate inotropy ultimately involve calcium either by increasing calcium influx , by increasing release of calcium from the sarcoplasmic reticulum , or by sensitizing troponin - c to calcium . in this study , we found that les increased intracellular calcium level more than three times in h9c2 cells , and that lipofundin mct / lct increased intracellular calcium level more than that of intralipid , suggesting that lipofundin mct / lct has a more positive inotropic effect than that of intralipid. les are used for various clinical purposes and have become an integral part of parenteral nutrition , effective treatment for local anesthetic - induced cardiovascular collapse , and treatment for lipophilic drug toxicity . the mechanism of action of les for treating local anesthetic - induced cardiac toxicity is not completely understood . however , local anesthetic - induced systemic toxicity is caused by inhibiting inotropic and metabotropic cell signal systems and possibly mitochondrial metabolism . the " lipid sink theory " ( reduced tissue binding by re - establishing equilibrium in the plasma lipid phase ) and the " energetic - metabolic effect " ( reversal of inhibited mitochondrial fatty acid transport ) are the most widely known theories for the mechanism of action of les for treating local anesthetic - induced systemic toxicity . in addition , a " positive inotropic effect " is proposed as another mechanism . in this regard , lipofundin mct / lct seems to be a more effective le for treating this condition than intralipid. our study had some limitations . however , we did not verify the source of the increase or the channel involved . thus , further research regarding the effect of calcium channel blockers on le - induced increase in calcium is needed to elucidate the calcium source . third , we showed previously that 3 ml / kg intralipid increases lvsp in an in vivo rat model . however , as this large volume of le could change the hemodynamics in an ex vivo heart model , we chose 1 ml / kg in this study . we assume that this may have affected the limited increase in lvsp after the intralipid treatment despite the increased intracellular calcium level . in conclusion particularly , lipofundin mct / lct had more of a positive inotropic effect than that of intralipid. the inotropic effect of les may be related to increased intracellular calcium levels in the heart .

backgroundlipid emulsions have been used to treat various drug toxicities and for total parenteral nutrition therapy . their usefulness has also been confirmed in patients with local anesthetic - induced cardiac toxicity . the purpose of this study was to measure the hemodynamic and composition effects of lipid emulsions and to elucidate the mechanism associated with changes in intracellular calcium levels in myocardiocytes.methodswe measured hemodynamic effects using a digital analysis system after intralipid and lipofundin mct / lct were infused into hearts hanging in a langendorff perfusion system . we measured the effects of the lipid emulsions on intracellular calcium levels in h9c2 cells by confocal microscopy.resultsinfusion of lipofundin mct / lct 20% ( 1 ml / kg ) resulted in a significant increase in left ventricular systolic pressure compared to that after infusing modified krebs - henseleit solution ( 1 ml / kg ) ( p = 0.003 , 95% confidence interval [ ci ] , 2.412.5 ) . lipofundin mct / lct 20% had a more positive inotropic effect than that of intralipid 20% ( p = 0.009 , 95% ci , 1.411.6 ) . both lipid emulsion treatments increased intracellular calcium levels . lipofundin mct / lct ( 0.01% ) increased intracellular calcium level more than that of 0.01% intralipid ( p < 0.05 , 95% ci , 0.01.9).conclusionsthese two lipid emulsions had different inotropic effects depending on their triglyceride component . the inotropic effect of lipid emulsions could be related with intracellular calcium level .

Introduction Materials and Methods Experimental protocols Experiment 1 Experiment 2 Statistical analysis Results Effect of LE on hemodynamic functions of hanging hearts in a Langendorff perfusion system Effects of the lipid emulsions on intracellular calcium in H9c2 cells Discussion

intravenous infusion of les reverses intractable cardiac toxicity in an animal model , and les are effective for treating local anesthetic - induced cardiac toxicity . therefore , various studies associated with les have been actively conducted to understand the mechanism of lipid rescue and improve treatment regimens . intralipid 20% , which contains only 100% long - chain triglycerides , is commonly used to treat local anesthetic - induced systemic toxicity , whereas lipofundin mct / lct 20% which is composed of 50% long - chain triglycerides and 50% mediumchain triglycerides , is occasionally used to treat local anestheticinduced systemic toxicity . therefore , we investigated the inotropic effect of two les ( intralipid or lipofundin mct / lct ) on a hanging heart model mounted in a langendorff perfusion system to elucidate the mechanism associated with changes in intracellular calcium level in myocardial cells . male sprague dawley rats ( koatech , pyeongtaek , korea ; weight , 250350 g ) were used for this study . briefly , the animals received general anesthesia with an intramuscular injection of 15 mg / kg tiletamine / zolazepam ( zoletil50 ; virbac lab . a sign of awakening ) during the operation , an additional 5 mg / kg tiletamine / zolazepam and 3 mg / kg xylazine were injected to maintain anesthesia . heparin ( 1000 iu / kg ) was administered through the femoral vein after anesthesia was induced . the heart was mounted quickly on a langendorff perfusion system and perfused with modified krebs - henseleit solution ( 118 mm nacl , 4.7 mm kcl , 1.2 mm mgso4 , 1.2 mm kh2po4 , 2.4 mm cacl2 , 25 mm nahco3 , 11 mm glucose , and 0.03 mm edta ) , equilibrated to ph 7.4 with a mixture of 5% co2/95% o2 . sydney , australia ) was connected to a digital analysis system to measure left ventricular hemodynamic function . left ventricular systolic pressure ( lvsp ) , maximum rate of intraventricular pressure increase ( + dp / dtmax ) , maximum rate of intraventricular pressure decrease ( dp / dtmax ) , and heart rate ( hr ) were measured using a computer analysis program ( charttm5 pro ; adinstruments ) as described previously . the hanging hearts were perfused with modified krebs - henseleit solution for 10 min to stabilize prior to recording hemodynamic function ( n = 10 ) . hearts were excluded if the average values for the last 5 min of the stabilization period failed to meet the following criteria ; lvsp , 80130 mmhg ; and hr , 200350 beats / min . the hanging hearts were assigned randomly to three groups as follows : ( 1 ) control group ( hearts in which 1 ml / kg modified krebs - henseleit solution was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; ( 2 ) il1 group ( hearts in which intralipid 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; and ( 3 ) mct1 group ( hearts in which lipofundin mct / lct 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) . , kaysville , ut , usa ; alpha = 0.05 , power = 92% ) . changes in intracellular calcium were examined by measuring increased fluorescence of fluo-3/acetoxymethyl ( am ) ( molecular probes , eugene , or , usa)-loaded cells with a confocal microscope ( fv1000 ; olympus , tokyo , japan ) . rat embryonic ventricular myocardial h9c2 cells were purchased from the american type culture collection ( manassas , va , usa ) and kept in complete growth medium ( dulbecco 's modified eagle 's medium ; invitrogen , carlsbad , ca , usa ) supplemented with 100% fetal bovine serum ( invitrogen ) at 37 in a humidified atmosphere of 5% co2 . basal and intracellular changes in calcium were examined by measuring increased fluorescence of fluo-3/am - loaded cells in the cytosol of h9c2 cells with a confocal microscope . h9c2 cells were attached to cover slips for 24 h at 37 before loading with 5 m fluo-3/am and incubating at 37 for 60 min . cover slips were inserted into a chamber mounted on the stage of the confocal microscope and rinsed with a warm normal bath solution ( 140 mm nacl , 5.0 mm kcl , 1.0 mm cacl2 , 1.0 mm mgcl2 , and10 mm hepes [ ph 7.4 ] ) . the le concentration used was 0.01% , which induced a maximal increase in intracellular calcium in the preliminary study . briefly , the animals received general anesthesia with an intramuscular injection of 15 mg / kg tiletamine / zolazepam ( zoletil50 ; virbac lab . , carros , france ) and 9 mg / kg of xylazine ( rompun ; bayer , seoul , korea ) . a sign of awakening ) during the operation , an additional 5 mg / kg tiletamine / zolazepam and 3 mg / kg xylazine were injected to maintain anesthesia . heparin ( 1000 iu / kg ) was administered through the femoral vein after anesthesia was induced . the heart was mounted quickly on a langendorff perfusion system and perfused with modified krebs - henseleit solution ( 118 mm nacl , 4.7 mm kcl , 1.2 mm mgso4 , 1.2 mm kh2po4 , 2.4 mm cacl2 , 25 mm nahco3 , 11 mm glucose , and 0.03 mm edta ) , equilibrated to ph 7.4 with a mixture of 5% co2/95% o2 . sydney , australia ) was connected to a digital analysis system to measure left ventricular hemodynamic function . left ventricular systolic pressure ( lvsp ) , maximum rate of intraventricular pressure increase ( + dp / dtmax ) , maximum rate of intraventricular pressure decrease ( dp / dtmax ) , and heart rate ( hr ) were measured using a computer analysis program ( charttm5 pro ; adinstruments ) as described previously . the hanging hearts were perfused with modified krebs - henseleit solution for 10 min to stabilize prior to recording hemodynamic function ( n = 10 ) . hearts were excluded if the average values for the last 5 min of the stabilization period failed to meet the following criteria ; lvsp , 80130 mmhg ; and hr , 200350 beats / min . the hanging hearts were assigned randomly to three groups as follows : ( 1 ) control group ( hearts in which 1 ml / kg modified krebs - henseleit solution was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; ( 2 ) il1 group ( hearts in which intralipid 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; and ( 3 ) mct1 group ( hearts in which lipofundin mct / lct 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) . , kaysville , ut , usa ; alpha = 0.05 , power = 92% ) . changes in intracellular calcium were examined by measuring increased fluorescence of fluo-3/acetoxymethyl ( am ) ( molecular probes , eugene , or , usa)-loaded cells with a confocal microscope ( fv1000 ; olympus , tokyo , japan ) . rat embryonic ventricular myocardial h9c2 cells were purchased from the american type culture collection ( manassas , va , usa ) and kept in complete growth medium ( dulbecco 's modified eagle 's medium ; invitrogen , carlsbad , ca , usa ) supplemented with 100% fetal bovine serum ( invitrogen ) at 37 in a humidified atmosphere of 5% co2 . basal and intracellular changes in calcium were examined by measuring increased fluorescence of fluo-3/am - loaded cells in the cytosol of h9c2 cells with a confocal microscope . h9c2 cells were attached to cover slips for 24 h at 37 before loading with 5 m fluo-3/am and incubating at 37 for 60 min . cover slips were inserted into a chamber mounted on the stage of the confocal microscope and rinsed with a warm normal bath solution ( 140 mm nacl , 5.0 mm kcl , 1.0 mm cacl2 , 1.0 mm mgcl2 , and10 mm hepes [ ph 7.4 ] ) . the le concentration used was 0.01% , which induced a maximal increase in intracellular calcium in the preliminary study . briefly , the animals received general anesthesia with an intramuscular injection of 15 mg / kg tiletamine / zolazepam ( zoletil50 ; virbac lab . , carros , france ) and 9 mg / kg of xylazine ( rompun ; bayer , seoul , korea ) . heparin ( 1000 iu / kg ) was administered through the femoral vein after anesthesia was induced . the heart was mounted quickly on a langendorff perfusion system and perfused with modified krebs - henseleit solution ( 118 mm nacl , 4.7 mm kcl , 1.2 mm mgso4 , 1.2 mm kh2po4 , 2.4 mm cacl2 , 25 mm nahco3 , 11 mm glucose , and 0.03 mm edta ) , equilibrated to ph 7.4 with a mixture of 5% co2/95% o2 . sydney , australia ) was connected to a digital analysis system to measure left ventricular hemodynamic function . left ventricular systolic pressure ( lvsp ) , maximum rate of intraventricular pressure increase ( + dp / dtmax ) , maximum rate of intraventricular pressure decrease ( dp / dtmax ) , and heart rate ( hr ) were measured using a computer analysis program ( charttm5 pro ; adinstruments ) as described previously . the hanging hearts were perfused with modified krebs - henseleit solution for 10 min to stabilize prior to recording hemodynamic function ( n = 10 ) . the hanging hearts were assigned randomly to three groups as follows : ( 1 ) control group ( hearts in which 1 ml / kg modified krebs - henseleit solution was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; ( 2 ) il1 group ( hearts in which intralipid 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; and ( 3 ) mct1 group ( hearts in which lipofundin mct / lct 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) . , kaysville , ut , usa ; alpha = 0.05 , power = 92% ) . changes in intracellular calcium were examined by measuring increased fluorescence of fluo-3/acetoxymethyl ( am ) ( molecular probes , eugene , or , usa)-loaded cells with a confocal microscope ( fv1000 ; olympus , tokyo , japan ) . rat embryonic ventricular myocardial h9c2 cells were purchased from the american type culture collection ( manassas , va , usa ) and kept in complete growth medium ( dulbecco 's modified eagle 's medium ; invitrogen , carlsbad , ca , usa ) supplemented with 100% fetal bovine serum ( invitrogen ) at 37 in a humidified atmosphere of 5% co2 . basal and intracellular changes in calcium were examined by measuring increased fluorescence of fluo-3/am - loaded cells in the cytosol of h9c2 cells with a confocal microscope . h9c2 cells were attached to cover slips for 24 h at 37 before loading with 5 m fluo-3/am and incubating at 37 for 60 min . cover slips were inserted into a chamber mounted on the stage of the confocal microscope and rinsed with a warm normal bath solution ( 140 mm nacl , 5.0 mm kcl , 1.0 mm cacl2 , 1.0 mm mgcl2 , and10 mm hepes [ ph 7.4 ] ) . the le concentration used was 0.01% , which induced a maximal increase in intracellular calcium in the preliminary study . the ratio of lvsp in the mct1 group ( 110.6% 6.4% ) increased significantly compared to that in the control group ( 103.2% 2.1% , p = 0.003 , 95% confidence interval [ ci ] , 2.412.5 ) and the il1 group ( 104.1% 3.7% , p = 0.009 , 95% ci , 1.411.6 ) ( fig . the change of intracellular calcium level was estimated by comparing the cellular fluorescence ratio with the basal fluorescence ratio using the fluo-3/am total calcium imaging technique . lipofundin mct / lct ( 0.01% ) increased intracellular calcium levels more than that of intralipid ( 0.01% ) ( il group : 3.03 0.53 ; mct group : 3.92 0.60 ; p < 0.05 ; 95% ci , 0.01.9 ) . the ratio of lvsp in the mct1 group ( 110.6% 6.4% ) increased significantly compared to that in the control group ( 103.2% 2.1% , p = 0.003 , 95% confidence interval [ ci ] , 2.412.5 ) and the il1 group ( 104.1% 3.7% , p = 0.009 , 95% ci , 1.411.6 ) ( fig . the change of intracellular calcium level was estimated by comparing the cellular fluorescence ratio with the basal fluorescence ratio using the fluo-3/am total calcium imaging technique . lipofundin mct / lct ( 0.01% ) increased intracellular calcium levels more than that of intralipid ( 0.01% ) ( il group : 3.03 0.53 ; mct group : 3.92 0.60 ; p < 0.05 ; 95% ci , 0.01.9 ) . the major findings of our study are : 1 ) the lipofundin mct / lct infusion significantly increased lvsp in hanging hearts , 2 ) lipofundin mct / lct has had more of a positive inotropic effect than that of intralipid , and 3 ) the le treatments increased the intracellular calcium levels in h9c2 cells . lipofundin mct / lct increased intracellular calcium levels more than that of intralipid. thus , we investigated the effect of commercially available les ( intralipid 20% and lipofundin mct / lct 20% ) on hemodynamics in an ex vivo heart model . as results , lipofundin mct / lct had a more positive inotropic effect than that of intralipid. in particular , lipofundin mct / lct should be used with caution as a total parenteral nutrition component in critically ill patients with compromised cardiovascular function . the second aim of this study was to elucidate the mechanism responsible for the le - induced increase in lvsp and intracellular calcium levels in myocardial cells . therefore , the positive inotropic effect of lipofundin mct / lct shown on the hearts connected to the langendorff perfusion system may be associated with cardiac myocyte calcium concentration . however , one study showed that 10% intralipid exerts a modest positive inotropic effect , although the intracellular calcium remains unchanged by intralipid . thus , we measured the change in calcium current after adding les to rat cardiac myoblastic h9c2 cells to clarify their effect on calcium current . most of the signal transduction pathways that stimulate inotropy ultimately involve calcium either by increasing calcium influx , by increasing release of calcium from the sarcoplasmic reticulum , or by sensitizing troponin - c to calcium . in this study , we found that les increased intracellular calcium level more than three times in h9c2 cells , and that lipofundin mct / lct increased intracellular calcium level more than that of intralipid , suggesting that lipofundin mct / lct has a more positive inotropic effect than that of intralipid. les are used for various clinical purposes and have become an integral part of parenteral nutrition , effective treatment for local anesthetic - induced cardiovascular collapse , and treatment for lipophilic drug toxicity . the mechanism of action of les for treating local anesthetic - induced cardiac toxicity is not completely understood . however , local anesthetic - induced systemic toxicity is caused by inhibiting inotropic and metabotropic cell signal systems and possibly mitochondrial metabolism . the " lipid sink theory " ( reduced tissue binding by re - establishing equilibrium in the plasma lipid phase ) and the " energetic - metabolic effect " ( reversal of inhibited mitochondrial fatty acid transport ) are the most widely known theories for the mechanism of action of les for treating local anesthetic - induced systemic toxicity . in addition , a " positive inotropic effect " is proposed as another mechanism . in this regard , lipofundin mct / lct seems to be a more effective le for treating this condition than intralipid. however , we did not verify the source of the increase or the channel involved . thus , further research regarding the effect of calcium channel blockers on le - induced increase in calcium is needed to elucidate the calcium source . third , we showed previously that 3 ml / kg intralipid increases lvsp in an in vivo rat model . however , as this large volume of le could change the hemodynamics in an ex vivo heart model , we chose 1 ml / kg in this study . we assume that this may have affected the limited increase in lvsp after the intralipid treatment despite the increased intracellular calcium level . in conclusion particularly , lipofundin mct / lct had more of a positive inotropic effect than that of intralipid. the inotropic effect of les may be related to increased intracellular calcium levels in the heart .

intravenous infusion of les reverses intractable cardiac toxicity in an animal model , and les are effective for treating local anesthetic - induced cardiac toxicity . therefore , various studies associated with les have been actively conducted to understand the mechanism of lipid rescue and improve treatment regimens . intralipid 20% , which contains only 100% long - chain triglycerides , is commonly used to treat local anesthetic - induced systemic toxicity , whereas lipofundin mct / lct 20% which is composed of 50% long - chain triglycerides and 50% mediumchain triglycerides , is occasionally used to treat local anestheticinduced systemic toxicity . however , both types of le affect hemodynamics in an ex vivo model but the associated cellular mechanism remains unknown . therefore , we investigated the inotropic effect of two les ( intralipid or lipofundin mct / lct ) on a hanging heart model mounted in a langendorff perfusion system to elucidate the mechanism associated with changes in intracellular calcium level in myocardial cells . all animals were maintained in accordance with the guidelines for the care and use of laboratory animals published by the us national institutes of health in 1996 . briefly , the animals received general anesthesia with an intramuscular injection of 15 mg / kg tiletamine / zolazepam ( zoletil50 ; virbac lab . , carros , france ) and 9 mg / kg of xylazine ( rompun ; bayer , seoul , korea ) . a sign of awakening ) during the operation , an additional 5 mg / kg tiletamine / zolazepam and 3 mg / kg xylazine were injected to maintain anesthesia . heparin ( 1000 iu / kg ) was administered through the femoral vein after anesthesia was induced . a tracheostomy was performed , and the animals were mechanically ventilated with room air via a 16 g catheter . the heart was mounted quickly on a langendorff perfusion system and perfused with modified krebs - henseleit solution ( 118 mm nacl , 4.7 mm kcl , 1.2 mm mgso4 , 1.2 mm kh2po4 , 2.4 mm cacl2 , 25 mm nahco3 , 11 mm glucose , and 0.03 mm edta ) , equilibrated to ph 7.4 with a mixture of 5% co2/95% o2 . left ventricular systolic pressure ( lvsp ) , maximum rate of intraventricular pressure increase ( + dp / dtmax ) , maximum rate of intraventricular pressure decrease ( dp / dtmax ) , and heart rate ( hr ) were measured using a computer analysis program ( charttm5 pro ; adinstruments ) as described previously . the hanging hearts were perfused with modified krebs - henseleit solution for 10 min to stabilize prior to recording hemodynamic function ( n = 10 ) . hearts were excluded if the average values for the last 5 min of the stabilization period failed to meet the following criteria ; lvsp , 80130 mmhg ; and hr , 200350 beats / min . each experimental group was injected with the drug using an infusion pump ( auto syringe as50 infusion pump ; baxter , singapore ) through a three - way stopcock on the fluid column . the hanging hearts were assigned randomly to three groups as follows : ( 1 ) control group ( hearts in which 1 ml / kg modified krebs - henseleit solution was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; ( 2 ) il1 group ( hearts in which intralipid 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; and ( 3 ) mct1 group ( hearts in which lipofundin mct / lct 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) . changes in intracellular calcium were examined by measuring increased fluorescence of fluo-3/acetoxymethyl ( am ) ( molecular probes , eugene , or , usa)-loaded cells with a confocal microscope ( fv1000 ; olympus , tokyo , japan ) . rat embryonic ventricular myocardial h9c2 cells were purchased from the american type culture collection ( manassas , va , usa ) and kept in complete growth medium ( dulbecco 's modified eagle 's medium ; invitrogen , carlsbad , ca , usa ) supplemented with 100% fetal bovine serum ( invitrogen ) at 37 in a humidified atmosphere of 5% co2 . basal and intracellular changes in calcium were examined by measuring increased fluorescence of fluo-3/am - loaded cells in the cytosol of h9c2 cells with a confocal microscope . cover slips were inserted into a chamber mounted on the stage of the confocal microscope and rinsed with a warm normal bath solution ( 140 mm nacl , 5.0 mm kcl , 1.0 mm cacl2 , 1.0 mm mgcl2 , and10 mm hepes [ ph 7.4 ] ) . the le concentration used was 0.01% , which induced a maximal increase in intracellular calcium in the preliminary study . , experimental outcomes were analyzed using one - way analysis of variance and the bonferroni post - hoc test . all animals were maintained in accordance with the guidelines for the care and use of laboratory animals published by the us national institutes of health in 1996 . briefly , the animals received general anesthesia with an intramuscular injection of 15 mg / kg tiletamine / zolazepam ( zoletil50 ; virbac lab . , carros , france ) and 9 mg / kg of xylazine ( rompun ; bayer , seoul , korea ) . a sign of awakening ) during the operation , an additional 5 mg / kg tiletamine / zolazepam and 3 mg / kg xylazine were injected to maintain anesthesia . heparin ( 1000 iu / kg ) was administered through the femoral vein after anesthesia was induced . a tracheostomy was performed , and the animals were mechanically ventilated with room air via a 16 g catheter . the heart was mounted quickly on a langendorff perfusion system and perfused with modified krebs - henseleit solution ( 118 mm nacl , 4.7 mm kcl , 1.2 mm mgso4 , 1.2 mm kh2po4 , 2.4 mm cacl2 , 25 mm nahco3 , 11 mm glucose , and 0.03 mm edta ) , equilibrated to ph 7.4 with a mixture of 5% co2/95% o2 . left ventricular systolic pressure ( lvsp ) , maximum rate of intraventricular pressure increase ( + dp / dtmax ) , maximum rate of intraventricular pressure decrease ( dp / dtmax ) , and heart rate ( hr ) were measured using a computer analysis program ( charttm5 pro ; adinstruments ) as described previously . hearts were excluded if the average values for the last 5 min of the stabilization period failed to meet the following criteria ; lvsp , 80130 mmhg ; and hr , 200350 beats / min . each experimental group was injected with the drug using an infusion pump ( auto syringe as50 infusion pump ; baxter , singapore ) through a three - way stopcock on the fluid column . the hanging hearts were assigned randomly to three groups as follows : ( 1 ) control group ( hearts in which 1 ml / kg modified krebs - henseleit solution was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; ( 2 ) il1 group ( hearts in which intralipid 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; and ( 3 ) mct1 group ( hearts in which lipofundin mct / lct 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) . changes in intracellular calcium were examined by measuring increased fluorescence of fluo-3/acetoxymethyl ( am ) ( molecular probes , eugene , or , usa)-loaded cells with a confocal microscope ( fv1000 ; olympus , tokyo , japan ) . rat embryonic ventricular myocardial h9c2 cells were purchased from the american type culture collection ( manassas , va , usa ) and kept in complete growth medium ( dulbecco 's modified eagle 's medium ; invitrogen , carlsbad , ca , usa ) supplemented with 100% fetal bovine serum ( invitrogen ) at 37 in a humidified atmosphere of 5% co2 . basal and intracellular changes in calcium were examined by measuring increased fluorescence of fluo-3/am - loaded cells in the cytosol of h9c2 cells with a confocal microscope . cover slips were inserted into a chamber mounted on the stage of the confocal microscope and rinsed with a warm normal bath solution ( 140 mm nacl , 5.0 mm kcl , 1.0 mm cacl2 , 1.0 mm mgcl2 , and10 mm hepes [ ph 7.4 ] ) . the le concentration used was 0.01% , which induced a maximal increase in intracellular calcium in the preliminary study . all animals were maintained in accordance with the guidelines for the care and use of laboratory animals published by the us national institutes of health in 1996 . briefly , the animals received general anesthesia with an intramuscular injection of 15 mg / kg tiletamine / zolazepam ( zoletil50 ; virbac lab . , carros , france ) and 9 mg / kg of xylazine ( rompun ; bayer , seoul , korea ) . a sign of awakening ) during the operation , an additional 5 mg / kg tiletamine / zolazepam and 3 mg / kg xylazine were injected to maintain anesthesia . heparin ( 1000 iu / kg ) was administered through the femoral vein after anesthesia was induced . the heart was mounted quickly on a langendorff perfusion system and perfused with modified krebs - henseleit solution ( 118 mm nacl , 4.7 mm kcl , 1.2 mm mgso4 , 1.2 mm kh2po4 , 2.4 mm cacl2 , 25 mm nahco3 , 11 mm glucose , and 0.03 mm edta ) , equilibrated to ph 7.4 with a mixture of 5% co2/95% o2 . left ventricular systolic pressure ( lvsp ) , maximum rate of intraventricular pressure increase ( + dp / dtmax ) , maximum rate of intraventricular pressure decrease ( dp / dtmax ) , and heart rate ( hr ) were measured using a computer analysis program ( charttm5 pro ; adinstruments ) as described previously . hearts were excluded if the average values for the last 5 min of the stabilization period failed to meet the following criteria ; lvsp , 80130 mmhg ; and hr , 200350 beats / min . the hanging hearts were assigned randomly to three groups as follows : ( 1 ) control group ( hearts in which 1 ml / kg modified krebs - henseleit solution was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; ( 2 ) il1 group ( hearts in which intralipid 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) ; and ( 3 ) mct1 group ( hearts in which lipofundin mct / lct 20% ( 1 ml / kg ) was infused through the three - way stopcock using an infusion pump ( n = 10 ) . changes in intracellular calcium were examined by measuring increased fluorescence of fluo-3/acetoxymethyl ( am ) ( molecular probes , eugene , or , usa)-loaded cells with a confocal microscope ( fv1000 ; olympus , tokyo , japan ) . rat embryonic ventricular myocardial h9c2 cells were purchased from the american type culture collection ( manassas , va , usa ) and kept in complete growth medium ( dulbecco 's modified eagle 's medium ; invitrogen , carlsbad , ca , usa ) supplemented with 100% fetal bovine serum ( invitrogen ) at 37 in a humidified atmosphere of 5% co2 . basal and intracellular changes in calcium were examined by measuring increased fluorescence of fluo-3/am - loaded cells in the cytosol of h9c2 cells with a confocal microscope . cover slips were inserted into a chamber mounted on the stage of the confocal microscope and rinsed with a warm normal bath solution ( 140 mm nacl , 5.0 mm kcl , 1.0 mm cacl2 , 1.0 mm mgcl2 , and10 mm hepes [ ph 7.4 ] ) . the le concentration used was 0.01% , which induced a maximal increase in intracellular calcium in the preliminary study . the ratio of lvsp in the mct1 group ( 110.6% 6.4% ) increased significantly compared to that in the control group ( 103.2% 2.1% , p = 0.003 , 95% confidence interval [ ci ] , 2.412.5 ) and the il1 group ( 104.1% 3.7% , p = 0.009 , 95% ci , 1.411.6 ) ( fig . the ratio of + dp / dtmax ( control , 98.5% 6.9% ; il1 , 103.2% 7.2% ; mct1 , 108.7% 15.7% ) , dp / dtmax ( control , 97.6% 10.0% ; il1 , 103.9% 6.0% ; mct1 , 102.1% 9.6% ) , and hr ( control , 93.2% 7.8% ; il1 , 96.6% 1.5% ; mct1 , 94.0% 5.0% ) were not significant between the groups . lipofundin mct / lct ( 0.01% ) increased intracellular calcium levels more than that of intralipid ( 0.01% ) ( il group : 3.03 0.53 ; mct group : 3.92 0.60 ; p < 0.05 ; 95% ci , 0.01.9 ) . the ratio of lvsp in the mct1 group ( 110.6% 6.4% ) increased significantly compared to that in the control group ( 103.2% 2.1% , p = 0.003 , 95% confidence interval [ ci ] , 2.412.5 ) and the il1 group ( 104.1% 3.7% , p = 0.009 , 95% ci , 1.411.6 ) ( fig . the ratio of + dp / dtmax ( control , 98.5% 6.9% ; il1 , 103.2% 7.2% ; mct1 , 108.7% 15.7% ) , dp / dtmax ( control , 97.6% 10.0% ; il1 , 103.9% 6.0% ; mct1 , 102.1% 9.6% ) , and hr ( control , 93.2% 7.8% ; il1 , 96.6% 1.5% ; mct1 , 94.0% 5.0% ) were not significant between the groups . lipofundin mct / lct ( 0.01% ) increased intracellular calcium levels more than that of intralipid ( 0.01% ) ( il group : 3.03 0.53 ; mct group : 3.92 0.60 ; p < 0.05 ; 95% ci , 0.01.9 ) . the major findings of our study are : 1 ) the lipofundin mct / lct infusion significantly increased lvsp in hanging hearts , 2 ) lipofundin mct / lct has had more of a positive inotropic effect than that of intralipid , and 3 ) the le treatments increased the intracellular calcium levels in h9c2 cells . however , a mixture of medium - chain triglycerides , long - chain triglycerides , and omega-3 polyunsaturated fatty acid emulsions increase mean aortic blood pressure . thus , we investigated the effect of commercially available les ( intralipid 20% and lipofundin mct / lct 20% ) on hemodynamics in an ex vivo heart model . the second aim of this study was to elucidate the mechanism responsible for the le - induced increase in lvsp and intracellular calcium levels in myocardial cells . long chain fatty acids directly activate calcium channels at some lipid sites near the channels or on the channel protein itself , as assessed by the standard whole cell voltage clamp technique in ventricular myocytes . therefore , the positive inotropic effect of lipofundin mct / lct shown on the hearts connected to the langendorff perfusion system may be associated with cardiac myocyte calcium concentration . thus , we measured the change in calcium current after adding les to rat cardiac myoblastic h9c2 cells to clarify their effect on calcium current . most of the signal transduction pathways that stimulate inotropy ultimately involve calcium either by increasing calcium influx , by increasing release of calcium from the sarcoplasmic reticulum , or by sensitizing troponin - c to calcium . in this study , we found that les increased intracellular calcium level more than three times in h9c2 cells , and that lipofundin mct / lct increased intracellular calcium level more than that of intralipid , suggesting that lipofundin mct / lct has a more positive inotropic effect than that of intralipid. les are used for various clinical purposes and have become an integral part of parenteral nutrition , effective treatment for local anesthetic - induced cardiovascular collapse , and treatment for lipophilic drug toxicity . the " lipid sink theory " ( reduced tissue binding by re - establishing equilibrium in the plasma lipid phase ) and the " energetic - metabolic effect " ( reversal of inhibited mitochondrial fatty acid transport ) are the most widely known theories for the mechanism of action of les for treating local anesthetic - induced systemic toxicity . thus , further research regarding the effect of calcium channel blockers on le - induced increase in calcium is needed to elucidate the calcium source . in conclusion particularly , lipofundin mct / lct had more of a positive inotropic effect than that of intralipid.

united nations estimated that , in 2006 , the number of elderly people in the world was 678,923,000 , which is expected to increase to 1,968,153,000 by 2050 . in iran , the population is moving toward senility . according to the latest statistics of population census in 2011 , lifestyle modification and paying attention to quality of life could significantly increase functionality and independence of the elderly and help them control multiple complications of aging and their different treatments . on the other hand , moving to a nursing home is considered to be one of the most stressful events of an individual 's life and is usually accompanied with depression , anxiety , insomnia , and attempted suicide . according to corbin and strauss ( 1988 ) , despite enhancement of living condition in the new residence , seniors who were forced to move to a nursing home would experience more cardiovascular diseases and strokes and would refer to hospitals more than those who did not move to a nursing home . depression and anxiety are the most common mental problems among the elderly and mostly remain undiagnosed or untreated , affecting the elderly 's quality of life and increasing their living expenses . different studies have mentioned that seniors living in nursing homes have poorer health condition than those living with their families . for example , in a study conducted by momeni et al . to compare the general health of residents of nursing homes and nonresidence elderlies , 254 seniors were evaluated using general health questionnaire-28 ( ghq-28 ) . results showed that the residents of nursing homes experienced more depression , physical , and social dysfunction symptoms than nonresidence seniors . health is a multidimensional matter in a way that individual 's physical problems affect their mind and their mental problems affect their body and both of these would affect the society and social disorders would affect other aspects of health . therefore , health improvement measures must consider all the aspects of personal ( physical , mental , and spiritual ) and general health of the society . health education has a key role in most of health improvement interventions by focusing on training people to participate in self - care activities , which provides a background for prevention . one of the most important methods for compatibility with old age and having a healthy and successful life is general and professional training for the elderly and their families regarding the needs and problems of old age using different educational tools . the trainer nurse , first , determines the special learning needs of the target population , and then prepares an appropriate philosophical view for providing those learning needs ; considers educational principles that could increase learning ; evaluates educational issues such as the target group 's special concerns , obstacles to learning , and appropriate technological strategies to facilitate the learning process ; designs and executes the educational program ; and eventually assesses the effect of the educational program on learning and behaviors . in a study conducted by badertscher et al . in 2012 in switzerland to evaluate the approaches , barriers , and facilitators of elderly health improvement in general practitioners opinion as a focused group study and through interviews results showed that two of the most important perceived barriers by this group concerning the elderly health improvement were lack of time and inadequate reimbursement for following preventive and health improvement recommendations . in their opinion , one of the interventions that could be performed to improve the health of the elderly is involving nurses in health improvement and consultation interventions , which was widely discussed by the case group . according to imhof et al . , self - care programs and ability to manage the disease , that must be executed for patients with special diseases such as diabetes or cardiovascular diseases , have desirable results in the community and health care centers . nowadays , authorities have realized that to reach an active and healthy old age all the aspects of physical , mental , social , economic , and spiritual heath must be considered , and since most of the diseases and problems of old age are due to unhealthy lifestyle , the basics of health in these aspects must be determined by using the right methods and improving the quality of life from the very first stages of life . considering all the abovementioned concerns , most of the studies in the field of elderly health have been evaluations and less attention has been paid to educational interventions during old age based on the needs assessment of the trainees . therefore , community health nursing , which is one of the most important pillars of the health team for community health improvement , must apply appropriate measures including educational interventions , which is one of the most important health improvement tools , to reach this goal . lack of information regarding elderly health education based on their needs assessment that has been conducted through interviewing them and their caregivers , elderly 's special educational needs about their health condition ( which is the same as general health meaning physical , mental and social health of the elderly ) , and also the need for paying more attention to residents of nursing homes due to their undesirable living conditions compared to nonresidence elderly , led us to conduct a study to evaluate the effect of need - oriented education on the general health of seniors living at nursing homes . this is a semi - experimental study that evaluated the effect of independent variable of need - oriented educational intervention on dependent variable of general health score in the case group . in both the case and control groups , this study was conducted at the sadeghieh nursing home of isfahan , after obtaining permission from the ethics committee of isfahan university of medical sciences and written informed consent form from the participants . it must be noted that this center was selected as the study environment due to its availability and large number of residents . the inclusion criteria were than 60-year - old individuals , being familiar with farsi language , having the intellectual ability to understand the contents and physical ability to participate in sessions , having motivation for participating in educational sessions , and being able to at least read and write . the exclusion criteria were not participating in sessions for any reason , participant 's death or sickness during the intervention , and missing more than 3 sessions . in the beginning , a pilot study was conducted to assess the educational needs . for this pilot study , after interviewing the senior manager of the center , 2 personnel ( care givers ) and 10 elderly who satisfied the inclusion criteria , educational need assessment was conducted . need assessment was conducted using agreement assessment technique , which is a standard method for need assessment that starts with an open answer question such as if you were supposed to be educated about your health issues , what were the subjects you would like to learn ? after interviewing center 's management , staff , and residents regarding health education for residents of the nursing home ( educational needs that are rooted in behavioral factors of the elderly , that could be said are the same as their health problems ) 5 educational needs that had the most repetition among problems and proposed needs were selected . at the second stage , for prioritizing the selected needs , each of the participants were asked to give a score from 1 to 5 to each of the selected needs . then , by summing up all the numbers , the needs were prioritized as follows : ( 1 ) musculoskeletal pains and physical inactivity , ( 2 ) chronic diseases ( high blood pressure , diabetes , and alzheimer ) , ( 3 ) undesirable mental health , ( 4 ) poor nutrition , and ( 5 ) inappropriate personal health . considering the pilot study , the educational program was designed to educate the elderly about their health needs which was assessed at the need assessment stage . for reaching the abovementioned educational objectives , eight 1-hour sessions were conducted for teaching the educational content according to the developed educational needs of the elderly . at each session , 15 minutes were allocated to physical exercises and 45 minutes were for lectures and discussions about the subject of the session . all the participants were obliged to do the physical exercise and take part in discussions . for better learning and more effectiveness of the training , the elderly were divided into four groups ( two groups of women and two groups of men ) . then , 70 seniors who satisfied the inclusion criteria ( with their written consent forms ) were selected through simple sampling . for data collection , a two - part questionnaire including demographic characteristics of the participants ( age , sex , marital status , educational level , duration of living at the nursing home , and underlying diseases ) and ghq-28 was used before and after the educational intervention . this questionnaire has 28 questions and evaluates four subscales of somatic symptoms , anxiety , social impairment and depression , pathological symptoms , and positive moods and wellbeing for an individual from 1 month prior to the intervention up to the time of the intervention . each choice has a score from 0 to 3 , and in total the least score could be 0 and the highest score could be 48 . this questionnaire evaluates four dimensions of physical health , anxiety , social functioning , and depression in the participants . according to the study of noorbala et al . , the cut - off point for the total score of this questionnaire is 23 , implying that the score of 23 and less indicates healthy individual and higher scores refer to possible disorders . ghq-28 is a standard tool that has been used by different researchers in many different studies , and its reliability has been reported to be 7895% . selected participants were assigned into two groups of case and control by random allocation . it must be noted that physical exercises were conducted by the researcher who was trained by a physical education expert and the moves were approved by a specialist . during the intervention , the control group received the usual care and a booklet containing the educational content that was discussed at the sessions for the case group . after all the sessions ended , ghq28 was completed by both the groups 1 week and 1 month after the intervention , and the mean scores of general health before and 1 week and 1 month after the intervention were compared to each other between both groups and for each group separately . regarding the 1-month interval between evaluations of general health , it must be noted that this interval was to evaluate the durability of the training . other studies that have used the same questionnaire also had a time interval between evaluations ; in some studies , the interval was 2 months , in some 1 month , and in some other 1.5 month . because in the present study we were dealing with some sort of behavioral change , the participants must have had enough time to exercise the training , and if they would be applied correctly , they could increase the level of general health . data was analyzed using the statistical package for the social sciences ( spss inc . , chicago , il , usa ) and through independent t - test , paired t - test , chi - square test and mann whitney test . at each session , 15 minutes were allocated to physical exercises and 45 minutes were for lectures and discussions about the subject of the session . all the participants were obliged to do the physical exercise and take part in discussions . for better learning and more effectiveness of the training , the elderly were divided into four groups ( two groups of women and two groups of men ) . then , 70 seniors who satisfied the inclusion criteria ( with their written consent forms ) were selected through simple sampling . for data collection , a two - part questionnaire including demographic characteristics of the participants ( age , sex , marital status , educational level , duration of living at the nursing home , and underlying diseases ) and ghq-28 was used before and after the educational intervention . this questionnaire has 28 questions and evaluates four subscales of somatic symptoms , anxiety , social impairment and depression , pathological symptoms , and positive moods and wellbeing for an individual from 1 month prior to the intervention up to the time of the intervention . each choice has a score from 0 to 3 , and in total the least score could be 0 and the highest score could be 48 . this questionnaire evaluates four dimensions of physical health , anxiety , social functioning , and depression in the participants . according to the study of noorbala et al . , the cut - off point for the total score of this questionnaire is 23 , implying that the score of 23 and less indicates healthy individual and higher scores refer to possible disorders . ghq-28 is a standard tool that has been used by different researchers in many different studies , and its reliability has been reported to be 7895% . selected participants were assigned into two groups of case and control by random allocation . it must be noted that physical exercises were conducted by the researcher who was trained by a physical education expert and the moves were approved by a specialist . during the intervention , the control group received the usual care and a booklet containing the educational content that was discussed at the sessions for the case group . after all the sessions ended , ghq28 was completed by both the groups 1 week and 1 month after the intervention , and the mean scores of general health before and 1 week and 1 month after the intervention were compared to each other between both groups and for each group separately . regarding the 1-month interval between evaluations of general health , it must be noted that this interval was to evaluate the durability of the training . other studies that have used the same questionnaire also had a time interval between evaluations ; in some studies , the interval was 2 months , in some 1 month , and in some other 1.5 month . because in the present study we were dealing with some sort of behavioral change , the participants must have had enough time to exercise the training , and if they would be applied correctly , they could increase the level of general health . data was analyzed using the statistical package for the social sciences ( spss inc . , chicago , il , usa ) and through independent t - test , paired t - test , chi - square test and mann whitney test . for this study among 320 seniors living at the sadeghieh nursing home , 70 who satisfied the inclusion criteria were selected through simple sampling and were randomly allocated to two groups of 35 participants in each group . lost samples until the end of the study were one from the control group and two from the case group ; hence finally , analysis was conducted among 33 participants of the case group and 34 participants of the control group . the mean age of the case and the control group was 74.2 and 76.2 years , respectively . in the case group , 16 were females ( 48.5% ) and 17 were males ( 51.8% ) , and in the control group , 15 were females ( 44.1% ) and 19 were males ( 55.9% ) . the mean duration of living at the nursing home was 2.2 years in the case group and 2.4 years in the control group . regarding marital status , 6 were single , 17 were married , 40 were widowed , and 4 were divorced . according to table 1 , independent t - test showed no significant difference between the mean of age and duration of living at the nursing home of both the groups . chi - square test showed no significant difference between the frequency of sex and marital status in both groups . chi - square test also showed no significant difference between the frequency of underlying diseases in both the groups . whitney test showed no significant difference between the educational level of both groups . comparing the mean ( standard deviation ) of age , duration of living at nursing house , and the frequency ( percent ) of sex and marital status in the case and the control group according to table 2 , variance analysis with repeated observations showed a significant difference between the mean score of general health at three different time intervals for the case group ( p < 0.001 ) , however , this difference was not significant for the control group . according to table 3 , independent t - test showed no significant difference between the mean score of general health of both groups before the intervention , however , 1 week and 1 month after the intervention , the difference between the mean score of general health of both groups showed a significant difference ( p < 0.001 ) . variance analysis with repeated observations showed no significant difference between the mean score of general health at three time intervals in the control group , however , this difference was significant in the case group ( p < 0.001 ) . in addition , independent t - test showed that the mean of changes in total score of general health of the case group 1 week and 1 month after the intervention compared to the score before the intervention was significantly more than the control group ( p < 0.001 ) . comparing the mean score of general health in different aspects between the case and the control group before and 1 week and 1 month after the intervention comparing the mean score of general health between the case and the control group before and 1 week and 1 month after the intervention results of this study confirmed the theory that need - oriented educational interventions have positive effects on general health of the elderly living at the sadeghieh nursing home of isfahan . in line with the results of the present study , gharamani et al . in 2009 , in their study titled improving the quality of life of the elderly men living at kahrizak nursing home based on educational intervention , showed that educational intervention had a positive and significant effect on improvement of quality of life in elderly men ( p < 0.05 ) . with regard to the methods and results of this study , educational needs of the elderly were achieved by analyzing the quality of life questionnaire before the intervention , however , in the present study , need assessment was conducted by interviewing the participants , the managers and staff of the center ; this method provides a more comprehensive view of educational needs as well as the problems of the residents of the nursing home . in addition , educational planning based on this type of assessment , because it is based on the needs of the target population and the subjects that they have declared they need , could have a more desirable effect on health improvement ; however , in the study by ghahramani et al . educational intervention had a positive and significant effect on the improvement of quality of life in the elderly men too . also ghahramani et al . only studied the elderly men but in the present study elderly women were included too and this could make the results more generalizable . however , in the present study , considering the differences between the needs of men and women , common needs that were confirmed by the elderly themselves were selected . in the study of mortazavi titled the effect of regular physical exercise on mental health of the elderly of shahrekord results showed a significant reduction in the score of general health after the intervention in the case group compared to their score before the intervention . on the other hand , mortazavi et al . in their study showed that , although physical exercise had a positive effect on the mental health , it was not effective on anxiety , depression , and stress . the present study was specifically conducted on the residents of the nursing home , and the educational intervention included both physical exercises and lectures and discussions regarding their pre - determined needs and health problems , and results showed that need - oriented educational intervention could affect all the aspects of general health toward improvement . as it was mentioned before , a trainer nurse , by evaluating the educational needs of the target population in different aspects of health including physical , mental , and social would prepare a special educational protocol for individuals considering their facilities and conditions , and therefore , it could be said that the odds of reaching desirable results are higher . moreover , it could be said that , in the present study , conducting physical exercises in groups and providing a place and time for the elderly to have more social interaction with each other could have been effective on their mental health . in the study by yumin et al . in 2011 titled the effect of functional mobility and balance on the health - related quality of life in the elderly living at home and living at nursing homes in turkey , results showed that balanced training in elderly care and rehabilitation programs could improve functional independence and increase health - related quality of life . according to the results of this study , it could also be said that regular physical exercise has a positive effect on enhancement of physical functioning , and consequently improvement health level in every aspect , i.e. , physical , mental , and social . as shown in the present study , educational intervention , of which physical exercise was an important part of it , could have improved the level of general health in the elderly in its all the four fields . on the other hand , these physical exercises were simple and practicable moves for the elderly that were chosen from the physical exercises recommended by ministry of health and mentioned in guidebooks for improving healthy lifestyle in old age , as well as by the instructions of a physical education specialist . furthermore , practicing physical exercises in groups , due to their entertaining atmosphere , would give the elderly more motivation for taking part in physical activities . clark et al . , in 2011 , conducted a clinical trial in america titled the effect of lifestyle interventions on the health improvement of seniors living independently . the results revealed that the case group showed more desirable changes than the control group regarding their scores of pain , vital signs , social functioning , mental health , satisfaction with life , and depression symptoms . therefore , it could be concluded from the results that , since this program is cost - effective and applicable on a large population , by performing such programs physical and mental health of the elderly could be improved . results of the present study showed that group interventions could have improved health level and quality of life in the elderly . as mentioned before , moving to a nursing home is considered as one of the most stressful events of an individual 's life . many studies have shown that living environment of the elderly is an important and effective factor in their health and longevity . many studies have revealed higher levels of mental health among the elderly living with their families compared to those living in nursing homes . momeni in their study reported that the mean score of general health of the elderly residents of nursing homes in all the 4 subscales was higher than those who were living among the community . in addition , ghasemi in their study revealed that the mean score of quality of life in the elderly living with their families was higher than those living at nursing homes . according to the results of the present study , it could be said that training based on need assessment from the target population , because it evaluated all the health aspects including physical , mental and social , and educational program has been designed based on that could have more effect than usual training . therefore , need - oriented education , which is one of the most important roles of community health nursing , could be one of the effective , safe , and inexpensive methods for improving the general health and preventing the complications of senescence and diseases of the old age among the residents of nursing homes .

introduction : improving the health of the elderly must be based on their special needs that can be achieved through self - needs assessments through education , which is one of the most important tools in the field of health improvement . the aim of the present study was to evaluate the effect of need - oriented educational interventions on the general health of the elderly living in nursing homes.materials and methods : the present study is a clinical trial conducted among the elderly living at the sadeghieh nursing home of isfahan , iran . seventy elderly people who satisfied the inclusion criteria were selected and divided into two groups of case and control . the case group went through 8 sessions of group therapy for educating participants who were previously investigated through needs assessment . the control group received the usual health care . the general health level of the participants was evaluated before and 1 week and 1 month after the intervention using goldberg 's general health questionnaire . data were analyzed using chi - square test , independent t - test , paired t - test , mann whitney test , and variance analysis.results:the score of general health had no significant difference between both the groups before the intervention , however , the difference in the score between both the groups 1 week and 1 month after the intervention was significant ( 1 week after the intervention , the scores were 15.6 and 30.3 , and 1 month after the intervention , the scores were 16 and 32.2 , respectively ) ( p < 0.001).conclusions : need - oriented educational intervention is an effective , safe , and inexpensive method for improving the general health of the elderly living in nursing homes .

Introduction Materials and Methods The syllabus of sessions Ethical considerations Results Discussion Conclusion Financial support and sponsorship Conflicts of interest

united nations estimated that , in 2006 , the number of elderly people in the world was 678,923,000 , which is expected to increase to 1,968,153,000 by 2050 . according to the latest statistics of population census in 2011 , lifestyle modification and paying attention to quality of life could significantly increase functionality and independence of the elderly and help them control multiple complications of aging and their different treatments . on the other hand , moving to a nursing home is considered to be one of the most stressful events of an individual 's life and is usually accompanied with depression , anxiety , insomnia , and attempted suicide . according to corbin and strauss ( 1988 ) , despite enhancement of living condition in the new residence , seniors who were forced to move to a nursing home would experience more cardiovascular diseases and strokes and would refer to hospitals more than those who did not move to a nursing home . depression and anxiety are the most common mental problems among the elderly and mostly remain undiagnosed or untreated , affecting the elderly 's quality of life and increasing their living expenses . different studies have mentioned that seniors living in nursing homes have poorer health condition than those living with their families . to compare the general health of residents of nursing homes and nonresidence elderlies , 254 seniors were evaluated using general health questionnaire-28 ( ghq-28 ) . results showed that the residents of nursing homes experienced more depression , physical , and social dysfunction symptoms than nonresidence seniors . health is a multidimensional matter in a way that individual 's physical problems affect their mind and their mental problems affect their body and both of these would affect the society and social disorders would affect other aspects of health . therefore , health improvement measures must consider all the aspects of personal ( physical , mental , and spiritual ) and general health of the society . health education has a key role in most of health improvement interventions by focusing on training people to participate in self - care activities , which provides a background for prevention . one of the most important methods for compatibility with old age and having a healthy and successful life is general and professional training for the elderly and their families regarding the needs and problems of old age using different educational tools . the trainer nurse , first , determines the special learning needs of the target population , and then prepares an appropriate philosophical view for providing those learning needs ; considers educational principles that could increase learning ; evaluates educational issues such as the target group 's special concerns , obstacles to learning , and appropriate technological strategies to facilitate the learning process ; designs and executes the educational program ; and eventually assesses the effect of the educational program on learning and behaviors . in 2012 in switzerland to evaluate the approaches , barriers , and facilitators of elderly health improvement in general practitioners opinion as a focused group study and through interviews results showed that two of the most important perceived barriers by this group concerning the elderly health improvement were lack of time and inadequate reimbursement for following preventive and health improvement recommendations . in their opinion , one of the interventions that could be performed to improve the health of the elderly is involving nurses in health improvement and consultation interventions , which was widely discussed by the case group . , self - care programs and ability to manage the disease , that must be executed for patients with special diseases such as diabetes or cardiovascular diseases , have desirable results in the community and health care centers . nowadays , authorities have realized that to reach an active and healthy old age all the aspects of physical , mental , social , economic , and spiritual heath must be considered , and since most of the diseases and problems of old age are due to unhealthy lifestyle , the basics of health in these aspects must be determined by using the right methods and improving the quality of life from the very first stages of life . considering all the abovementioned concerns , most of the studies in the field of elderly health have been evaluations and less attention has been paid to educational interventions during old age based on the needs assessment of the trainees . therefore , community health nursing , which is one of the most important pillars of the health team for community health improvement , must apply appropriate measures including educational interventions , which is one of the most important health improvement tools , to reach this goal . lack of information regarding elderly health education based on their needs assessment that has been conducted through interviewing them and their caregivers , elderly 's special educational needs about their health condition ( which is the same as general health meaning physical , mental and social health of the elderly ) , and also the need for paying more attention to residents of nursing homes due to their undesirable living conditions compared to nonresidence elderly , led us to conduct a study to evaluate the effect of need - oriented education on the general health of seniors living at nursing homes . this is a semi - experimental study that evaluated the effect of independent variable of need - oriented educational intervention on dependent variable of general health score in the case group . in both the case and control groups , this study was conducted at the sadeghieh nursing home of isfahan , after obtaining permission from the ethics committee of isfahan university of medical sciences and written informed consent form from the participants . the inclusion criteria were than 60-year - old individuals , being familiar with farsi language , having the intellectual ability to understand the contents and physical ability to participate in sessions , having motivation for participating in educational sessions , and being able to at least read and write . the exclusion criteria were not participating in sessions for any reason , participant 's death or sickness during the intervention , and missing more than 3 sessions . in the beginning , a pilot study was conducted to assess the educational needs . for this pilot study , after interviewing the senior manager of the center , 2 personnel ( care givers ) and 10 elderly who satisfied the inclusion criteria , educational need assessment was conducted . need assessment was conducted using agreement assessment technique , which is a standard method for need assessment that starts with an open answer question such as if you were supposed to be educated about your health issues , what were the subjects you would like to learn ? after interviewing center 's management , staff , and residents regarding health education for residents of the nursing home ( educational needs that are rooted in behavioral factors of the elderly , that could be said are the same as their health problems ) 5 educational needs that had the most repetition among problems and proposed needs were selected . at the second stage , for prioritizing the selected needs , each of the participants were asked to give a score from 1 to 5 to each of the selected needs . then , by summing up all the numbers , the needs were prioritized as follows : ( 1 ) musculoskeletal pains and physical inactivity , ( 2 ) chronic diseases ( high blood pressure , diabetes , and alzheimer ) , ( 3 ) undesirable mental health , ( 4 ) poor nutrition , and ( 5 ) inappropriate personal health . considering the pilot study , the educational program was designed to educate the elderly about their health needs which was assessed at the need assessment stage . for reaching the abovementioned educational objectives , eight 1-hour sessions were conducted for teaching the educational content according to the developed educational needs of the elderly . for better learning and more effectiveness of the training , the elderly were divided into four groups ( two groups of women and two groups of men ) . then , 70 seniors who satisfied the inclusion criteria ( with their written consent forms ) were selected through simple sampling . for data collection , a two - part questionnaire including demographic characteristics of the participants ( age , sex , marital status , educational level , duration of living at the nursing home , and underlying diseases ) and ghq-28 was used before and after the educational intervention . this questionnaire has 28 questions and evaluates four subscales of somatic symptoms , anxiety , social impairment and depression , pathological symptoms , and positive moods and wellbeing for an individual from 1 month prior to the intervention up to the time of the intervention . this questionnaire evaluates four dimensions of physical health , anxiety , social functioning , and depression in the participants . , the cut - off point for the total score of this questionnaire is 23 , implying that the score of 23 and less indicates healthy individual and higher scores refer to possible disorders . selected participants were assigned into two groups of case and control by random allocation . during the intervention , the control group received the usual care and a booklet containing the educational content that was discussed at the sessions for the case group . after all the sessions ended , ghq28 was completed by both the groups 1 week and 1 month after the intervention , and the mean scores of general health before and 1 week and 1 month after the intervention were compared to each other between both groups and for each group separately . regarding the 1-month interval between evaluations of general health , it must be noted that this interval was to evaluate the durability of the training . other studies that have used the same questionnaire also had a time interval between evaluations ; in some studies , the interval was 2 months , in some 1 month , and in some other 1.5 month . because in the present study we were dealing with some sort of behavioral change , the participants must have had enough time to exercise the training , and if they would be applied correctly , they could increase the level of general health . , chicago , il , usa ) and through independent t - test , paired t - test , chi - square test and mann whitney test . for better learning and more effectiveness of the training , the elderly were divided into four groups ( two groups of women and two groups of men ) . then , 70 seniors who satisfied the inclusion criteria ( with their written consent forms ) were selected through simple sampling . for data collection , a two - part questionnaire including demographic characteristics of the participants ( age , sex , marital status , educational level , duration of living at the nursing home , and underlying diseases ) and ghq-28 was used before and after the educational intervention . this questionnaire has 28 questions and evaluates four subscales of somatic symptoms , anxiety , social impairment and depression , pathological symptoms , and positive moods and wellbeing for an individual from 1 month prior to the intervention up to the time of the intervention . this questionnaire evaluates four dimensions of physical health , anxiety , social functioning , and depression in the participants . , the cut - off point for the total score of this questionnaire is 23 , implying that the score of 23 and less indicates healthy individual and higher scores refer to possible disorders . ghq-28 is a standard tool that has been used by different researchers in many different studies , and its reliability has been reported to be 7895% . selected participants were assigned into two groups of case and control by random allocation . during the intervention , the control group received the usual care and a booklet containing the educational content that was discussed at the sessions for the case group . after all the sessions ended , ghq28 was completed by both the groups 1 week and 1 month after the intervention , and the mean scores of general health before and 1 week and 1 month after the intervention were compared to each other between both groups and for each group separately . regarding the 1-month interval between evaluations of general health , it must be noted that this interval was to evaluate the durability of the training . other studies that have used the same questionnaire also had a time interval between evaluations ; in some studies , the interval was 2 months , in some 1 month , and in some other 1.5 month . because in the present study we were dealing with some sort of behavioral change , the participants must have had enough time to exercise the training , and if they would be applied correctly , they could increase the level of general health . , chicago , il , usa ) and through independent t - test , paired t - test , chi - square test and mann whitney test . for this study among 320 seniors living at the sadeghieh nursing home , 70 who satisfied the inclusion criteria were selected through simple sampling and were randomly allocated to two groups of 35 participants in each group . lost samples until the end of the study were one from the control group and two from the case group ; hence finally , analysis was conducted among 33 participants of the case group and 34 participants of the control group . the mean age of the case and the control group was 74.2 and 76.2 years , respectively . in the case group , 16 were females ( 48.5% ) and 17 were males ( 51.8% ) , and in the control group , 15 were females ( 44.1% ) and 19 were males ( 55.9% ) . the mean duration of living at the nursing home was 2.2 years in the case group and 2.4 years in the control group . according to table 1 , independent t - test showed no significant difference between the mean of age and duration of living at the nursing home of both the groups . chi - square test showed no significant difference between the frequency of sex and marital status in both groups . chi - square test also showed no significant difference between the frequency of underlying diseases in both the groups . whitney test showed no significant difference between the educational level of both groups . comparing the mean ( standard deviation ) of age , duration of living at nursing house , and the frequency ( percent ) of sex and marital status in the case and the control group according to table 2 , variance analysis with repeated observations showed a significant difference between the mean score of general health at three different time intervals for the case group ( p < 0.001 ) , however , this difference was not significant for the control group . according to table 3 , independent t - test showed no significant difference between the mean score of general health of both groups before the intervention , however , 1 week and 1 month after the intervention , the difference between the mean score of general health of both groups showed a significant difference ( p < 0.001 ) . variance analysis with repeated observations showed no significant difference between the mean score of general health at three time intervals in the control group , however , this difference was significant in the case group ( p < 0.001 ) . in addition , independent t - test showed that the mean of changes in total score of general health of the case group 1 week and 1 month after the intervention compared to the score before the intervention was significantly more than the control group ( p < 0.001 ) . comparing the mean score of general health in different aspects between the case and the control group before and 1 week and 1 month after the intervention comparing the mean score of general health between the case and the control group before and 1 week and 1 month after the intervention results of this study confirmed the theory that need - oriented educational interventions have positive effects on general health of the elderly living at the sadeghieh nursing home of isfahan . in line with the results of the present study , gharamani et al . in 2009 , in their study titled improving the quality of life of the elderly men living at kahrizak nursing home based on educational intervention , showed that educational intervention had a positive and significant effect on improvement of quality of life in elderly men ( p < 0.05 ) . with regard to the methods and results of this study , educational needs of the elderly were achieved by analyzing the quality of life questionnaire before the intervention , however , in the present study , need assessment was conducted by interviewing the participants , the managers and staff of the center ; this method provides a more comprehensive view of educational needs as well as the problems of the residents of the nursing home . in addition , educational planning based on this type of assessment , because it is based on the needs of the target population and the subjects that they have declared they need , could have a more desirable effect on health improvement ; however , in the study by ghahramani et al . educational intervention had a positive and significant effect on the improvement of quality of life in the elderly men too . only studied the elderly men but in the present study elderly women were included too and this could make the results more generalizable . however , in the present study , considering the differences between the needs of men and women , common needs that were confirmed by the elderly themselves were selected . in the study of mortazavi titled the effect of regular physical exercise on mental health of the elderly of shahrekord results showed a significant reduction in the score of general health after the intervention in the case group compared to their score before the intervention . in their study showed that , although physical exercise had a positive effect on the mental health , it was not effective on anxiety , depression , and stress . the present study was specifically conducted on the residents of the nursing home , and the educational intervention included both physical exercises and lectures and discussions regarding their pre - determined needs and health problems , and results showed that need - oriented educational intervention could affect all the aspects of general health toward improvement . as it was mentioned before , a trainer nurse , by evaluating the educational needs of the target population in different aspects of health including physical , mental , and social would prepare a special educational protocol for individuals considering their facilities and conditions , and therefore , it could be said that the odds of reaching desirable results are higher . moreover , it could be said that , in the present study , conducting physical exercises in groups and providing a place and time for the elderly to have more social interaction with each other could have been effective on their mental health . in 2011 titled the effect of functional mobility and balance on the health - related quality of life in the elderly living at home and living at nursing homes in turkey , results showed that balanced training in elderly care and rehabilitation programs could improve functional independence and increase health - related quality of life . according to the results of this study , it could also be said that regular physical exercise has a positive effect on enhancement of physical functioning , and consequently improvement health level in every aspect , i.e. as shown in the present study , educational intervention , of which physical exercise was an important part of it , could have improved the level of general health in the elderly in its all the four fields . on the other hand , these physical exercises were simple and practicable moves for the elderly that were chosen from the physical exercises recommended by ministry of health and mentioned in guidebooks for improving healthy lifestyle in old age , as well as by the instructions of a physical education specialist . , in 2011 , conducted a clinical trial in america titled the effect of lifestyle interventions on the health improvement of seniors living independently . the results revealed that the case group showed more desirable changes than the control group regarding their scores of pain , vital signs , social functioning , mental health , satisfaction with life , and depression symptoms . therefore , it could be concluded from the results that , since this program is cost - effective and applicable on a large population , by performing such programs physical and mental health of the elderly could be improved . results of the present study showed that group interventions could have improved health level and quality of life in the elderly . as mentioned before , moving to a nursing home is considered as one of the most stressful events of an individual 's life . many studies have shown that living environment of the elderly is an important and effective factor in their health and longevity . many studies have revealed higher levels of mental health among the elderly living with their families compared to those living in nursing homes . momeni in their study reported that the mean score of general health of the elderly residents of nursing homes in all the 4 subscales was higher than those who were living among the community . in addition , ghasemi in their study revealed that the mean score of quality of life in the elderly living with their families was higher than those living at nursing homes . according to the results of the present study , it could be said that training based on need assessment from the target population , because it evaluated all the health aspects including physical , mental and social , and educational program has been designed based on that could have more effect than usual training . therefore , need - oriented education , which is one of the most important roles of community health nursing , could be one of the effective , safe , and inexpensive methods for improving the general health and preventing the complications of senescence and diseases of the old age among the residents of nursing homes .

united nations estimated that , in 2006 , the number of elderly people in the world was 678,923,000 , which is expected to increase to 1,968,153,000 by 2050 . according to the latest statistics of population census in 2011 , lifestyle modification and paying attention to quality of life could significantly increase functionality and independence of the elderly and help them control multiple complications of aging and their different treatments . on the other hand , moving to a nursing home is considered to be one of the most stressful events of an individual 's life and is usually accompanied with depression , anxiety , insomnia , and attempted suicide . according to corbin and strauss ( 1988 ) , despite enhancement of living condition in the new residence , seniors who were forced to move to a nursing home would experience more cardiovascular diseases and strokes and would refer to hospitals more than those who did not move to a nursing home . depression and anxiety are the most common mental problems among the elderly and mostly remain undiagnosed or untreated , affecting the elderly 's quality of life and increasing their living expenses . different studies have mentioned that seniors living in nursing homes have poorer health condition than those living with their families . to compare the general health of residents of nursing homes and nonresidence elderlies , 254 seniors were evaluated using general health questionnaire-28 ( ghq-28 ) . results showed that the residents of nursing homes experienced more depression , physical , and social dysfunction symptoms than nonresidence seniors . health is a multidimensional matter in a way that individual 's physical problems affect their mind and their mental problems affect their body and both of these would affect the society and social disorders would affect other aspects of health . therefore , health improvement measures must consider all the aspects of personal ( physical , mental , and spiritual ) and general health of the society . health education has a key role in most of health improvement interventions by focusing on training people to participate in self - care activities , which provides a background for prevention . one of the most important methods for compatibility with old age and having a healthy and successful life is general and professional training for the elderly and their families regarding the needs and problems of old age using different educational tools . the trainer nurse , first , determines the special learning needs of the target population , and then prepares an appropriate philosophical view for providing those learning needs ; considers educational principles that could increase learning ; evaluates educational issues such as the target group 's special concerns , obstacles to learning , and appropriate technological strategies to facilitate the learning process ; designs and executes the educational program ; and eventually assesses the effect of the educational program on learning and behaviors . in 2012 in switzerland to evaluate the approaches , barriers , and facilitators of elderly health improvement in general practitioners opinion as a focused group study and through interviews results showed that two of the most important perceived barriers by this group concerning the elderly health improvement were lack of time and inadequate reimbursement for following preventive and health improvement recommendations . in their opinion , one of the interventions that could be performed to improve the health of the elderly is involving nurses in health improvement and consultation interventions , which was widely discussed by the case group . , self - care programs and ability to manage the disease , that must be executed for patients with special diseases such as diabetes or cardiovascular diseases , have desirable results in the community and health care centers . nowadays , authorities have realized that to reach an active and healthy old age all the aspects of physical , mental , social , economic , and spiritual heath must be considered , and since most of the diseases and problems of old age are due to unhealthy lifestyle , the basics of health in these aspects must be determined by using the right methods and improving the quality of life from the very first stages of life . considering all the abovementioned concerns , most of the studies in the field of elderly health have been evaluations and less attention has been paid to educational interventions during old age based on the needs assessment of the trainees . therefore , community health nursing , which is one of the most important pillars of the health team for community health improvement , must apply appropriate measures including educational interventions , which is one of the most important health improvement tools , to reach this goal . lack of information regarding elderly health education based on their needs assessment that has been conducted through interviewing them and their caregivers , elderly 's special educational needs about their health condition ( which is the same as general health meaning physical , mental and social health of the elderly ) , and also the need for paying more attention to residents of nursing homes due to their undesirable living conditions compared to nonresidence elderly , led us to conduct a study to evaluate the effect of need - oriented education on the general health of seniors living at nursing homes . this is a semi - experimental study that evaluated the effect of independent variable of need - oriented educational intervention on dependent variable of general health score in the case group . in both the case and control groups , this study was conducted at the sadeghieh nursing home of isfahan , after obtaining permission from the ethics committee of isfahan university of medical sciences and written informed consent form from the participants . the inclusion criteria were than 60-year - old individuals , being familiar with farsi language , having the intellectual ability to understand the contents and physical ability to participate in sessions , having motivation for participating in educational sessions , and being able to at least read and write . in the beginning , a pilot study was conducted to assess the educational needs . for this pilot study , after interviewing the senior manager of the center , 2 personnel ( care givers ) and 10 elderly who satisfied the inclusion criteria , educational need assessment was conducted . need assessment was conducted using agreement assessment technique , which is a standard method for need assessment that starts with an open answer question such as if you were supposed to be educated about your health issues , what were the subjects you would like to learn ? after interviewing center 's management , staff , and residents regarding health education for residents of the nursing home ( educational needs that are rooted in behavioral factors of the elderly , that could be said are the same as their health problems ) 5 educational needs that had the most repetition among problems and proposed needs were selected . at the second stage , for prioritizing the selected needs , each of the participants were asked to give a score from 1 to 5 to each of the selected needs . then , by summing up all the numbers , the needs were prioritized as follows : ( 1 ) musculoskeletal pains and physical inactivity , ( 2 ) chronic diseases ( high blood pressure , diabetes , and alzheimer ) , ( 3 ) undesirable mental health , ( 4 ) poor nutrition , and ( 5 ) inappropriate personal health . considering the pilot study , the educational program was designed to educate the elderly about their health needs which was assessed at the need assessment stage . for reaching the abovementioned educational objectives , eight 1-hour sessions were conducted for teaching the educational content according to the developed educational needs of the elderly . at each session , 15 minutes were allocated to physical exercises and 45 minutes were for lectures and discussions about the subject of the session . all the participants were obliged to do the physical exercise and take part in discussions . for better learning and more effectiveness of the training , the elderly were divided into four groups ( two groups of women and two groups of men ) . for data collection , a two - part questionnaire including demographic characteristics of the participants ( age , sex , marital status , educational level , duration of living at the nursing home , and underlying diseases ) and ghq-28 was used before and after the educational intervention . this questionnaire has 28 questions and evaluates four subscales of somatic symptoms , anxiety , social impairment and depression , pathological symptoms , and positive moods and wellbeing for an individual from 1 month prior to the intervention up to the time of the intervention . each choice has a score from 0 to 3 , and in total the least score could be 0 and the highest score could be 48 . this questionnaire evaluates four dimensions of physical health , anxiety , social functioning , and depression in the participants . , the cut - off point for the total score of this questionnaire is 23 , implying that the score of 23 and less indicates healthy individual and higher scores refer to possible disorders . ghq-28 is a standard tool that has been used by different researchers in many different studies , and its reliability has been reported to be 7895% . during the intervention , the control group received the usual care and a booklet containing the educational content that was discussed at the sessions for the case group . after all the sessions ended , ghq28 was completed by both the groups 1 week and 1 month after the intervention , and the mean scores of general health before and 1 week and 1 month after the intervention were compared to each other between both groups and for each group separately . regarding the 1-month interval between evaluations of general health , it must be noted that this interval was to evaluate the durability of the training . other studies that have used the same questionnaire also had a time interval between evaluations ; in some studies , the interval was 2 months , in some 1 month , and in some other 1.5 month . because in the present study we were dealing with some sort of behavioral change , the participants must have had enough time to exercise the training , and if they would be applied correctly , they could increase the level of general health . at each session , 15 minutes were allocated to physical exercises and 45 minutes were for lectures and discussions about the subject of the session . all the participants were obliged to do the physical exercise and take part in discussions . for better learning and more effectiveness of the training , the elderly were divided into four groups ( two groups of women and two groups of men ) . for data collection , a two - part questionnaire including demographic characteristics of the participants ( age , sex , marital status , educational level , duration of living at the nursing home , and underlying diseases ) and ghq-28 was used before and after the educational intervention . this questionnaire has 28 questions and evaluates four subscales of somatic symptoms , anxiety , social impairment and depression , pathological symptoms , and positive moods and wellbeing for an individual from 1 month prior to the intervention up to the time of the intervention . each choice has a score from 0 to 3 , and in total the least score could be 0 and the highest score could be 48 . this questionnaire evaluates four dimensions of physical health , anxiety , social functioning , and depression in the participants . , the cut - off point for the total score of this questionnaire is 23 , implying that the score of 23 and less indicates healthy individual and higher scores refer to possible disorders . ghq-28 is a standard tool that has been used by different researchers in many different studies , and its reliability has been reported to be 7895% . during the intervention , the control group received the usual care and a booklet containing the educational content that was discussed at the sessions for the case group . after all the sessions ended , ghq28 was completed by both the groups 1 week and 1 month after the intervention , and the mean scores of general health before and 1 week and 1 month after the intervention were compared to each other between both groups and for each group separately . regarding the 1-month interval between evaluations of general health , it must be noted that this interval was to evaluate the durability of the training . other studies that have used the same questionnaire also had a time interval between evaluations ; in some studies , the interval was 2 months , in some 1 month , and in some other 1.5 month . because in the present study we were dealing with some sort of behavioral change , the participants must have had enough time to exercise the training , and if they would be applied correctly , they could increase the level of general health . for this study among 320 seniors living at the sadeghieh nursing home , 70 who satisfied the inclusion criteria were selected through simple sampling and were randomly allocated to two groups of 35 participants in each group . lost samples until the end of the study were one from the control group and two from the case group ; hence finally , analysis was conducted among 33 participants of the case group and 34 participants of the control group . the mean age of the case and the control group was 74.2 and 76.2 years , respectively . in the case group , 16 were females ( 48.5% ) and 17 were males ( 51.8% ) , and in the control group , 15 were females ( 44.1% ) and 19 were males ( 55.9% ) . the mean duration of living at the nursing home was 2.2 years in the case group and 2.4 years in the control group . according to table 1 , independent t - test showed no significant difference between the mean of age and duration of living at the nursing home of both the groups . chi - square test showed no significant difference between the frequency of sex and marital status in both groups . chi - square test also showed no significant difference between the frequency of underlying diseases in both the groups . comparing the mean ( standard deviation ) of age , duration of living at nursing house , and the frequency ( percent ) of sex and marital status in the case and the control group according to table 2 , variance analysis with repeated observations showed a significant difference between the mean score of general health at three different time intervals for the case group ( p < 0.001 ) , however , this difference was not significant for the control group . according to table 3 , independent t - test showed no significant difference between the mean score of general health of both groups before the intervention , however , 1 week and 1 month after the intervention , the difference between the mean score of general health of both groups showed a significant difference ( p < 0.001 ) . variance analysis with repeated observations showed no significant difference between the mean score of general health at three time intervals in the control group , however , this difference was significant in the case group ( p < 0.001 ) . in addition , independent t - test showed that the mean of changes in total score of general health of the case group 1 week and 1 month after the intervention compared to the score before the intervention was significantly more than the control group ( p < 0.001 ) . comparing the mean score of general health in different aspects between the case and the control group before and 1 week and 1 month after the intervention comparing the mean score of general health between the case and the control group before and 1 week and 1 month after the intervention results of this study confirmed the theory that need - oriented educational interventions have positive effects on general health of the elderly living at the sadeghieh nursing home of isfahan . in line with the results of the present study , gharamani et al . in 2009 , in their study titled improving the quality of life of the elderly men living at kahrizak nursing home based on educational intervention , showed that educational intervention had a positive and significant effect on improvement of quality of life in elderly men ( p < 0.05 ) . with regard to the methods and results of this study , educational needs of the elderly were achieved by analyzing the quality of life questionnaire before the intervention , however , in the present study , need assessment was conducted by interviewing the participants , the managers and staff of the center ; this method provides a more comprehensive view of educational needs as well as the problems of the residents of the nursing home . in addition , educational planning based on this type of assessment , because it is based on the needs of the target population and the subjects that they have declared they need , could have a more desirable effect on health improvement ; however , in the study by ghahramani et al . educational intervention had a positive and significant effect on the improvement of quality of life in the elderly men too . only studied the elderly men but in the present study elderly women were included too and this could make the results more generalizable . however , in the present study , considering the differences between the needs of men and women , common needs that were confirmed by the elderly themselves were selected . in the study of mortazavi titled the effect of regular physical exercise on mental health of the elderly of shahrekord results showed a significant reduction in the score of general health after the intervention in the case group compared to their score before the intervention . in their study showed that , although physical exercise had a positive effect on the mental health , it was not effective on anxiety , depression , and stress . the present study was specifically conducted on the residents of the nursing home , and the educational intervention included both physical exercises and lectures and discussions regarding their pre - determined needs and health problems , and results showed that need - oriented educational intervention could affect all the aspects of general health toward improvement . as it was mentioned before , a trainer nurse , by evaluating the educational needs of the target population in different aspects of health including physical , mental , and social would prepare a special educational protocol for individuals considering their facilities and conditions , and therefore , it could be said that the odds of reaching desirable results are higher . moreover , it could be said that , in the present study , conducting physical exercises in groups and providing a place and time for the elderly to have more social interaction with each other could have been effective on their mental health . in 2011 titled the effect of functional mobility and balance on the health - related quality of life in the elderly living at home and living at nursing homes in turkey , results showed that balanced training in elderly care and rehabilitation programs could improve functional independence and increase health - related quality of life . according to the results of this study , it could also be said that regular physical exercise has a positive effect on enhancement of physical functioning , and consequently improvement health level in every aspect , i.e. as shown in the present study , educational intervention , of which physical exercise was an important part of it , could have improved the level of general health in the elderly in its all the four fields . on the other hand , these physical exercises were simple and practicable moves for the elderly that were chosen from the physical exercises recommended by ministry of health and mentioned in guidebooks for improving healthy lifestyle in old age , as well as by the instructions of a physical education specialist . furthermore , practicing physical exercises in groups , due to their entertaining atmosphere , would give the elderly more motivation for taking part in physical activities . , in 2011 , conducted a clinical trial in america titled the effect of lifestyle interventions on the health improvement of seniors living independently . the results revealed that the case group showed more desirable changes than the control group regarding their scores of pain , vital signs , social functioning , mental health , satisfaction with life , and depression symptoms . therefore , it could be concluded from the results that , since this program is cost - effective and applicable on a large population , by performing such programs physical and mental health of the elderly could be improved . results of the present study showed that group interventions could have improved health level and quality of life in the elderly . as mentioned before , moving to a nursing home is considered as one of the most stressful events of an individual 's life . many studies have shown that living environment of the elderly is an important and effective factor in their health and longevity . many studies have revealed higher levels of mental health among the elderly living with their families compared to those living in nursing homes . momeni in their study reported that the mean score of general health of the elderly residents of nursing homes in all the 4 subscales was higher than those who were living among the community . in addition , ghasemi in their study revealed that the mean score of quality of life in the elderly living with their families was higher than those living at nursing homes . according to the results of the present study , it could be said that training based on need assessment from the target population , because it evaluated all the health aspects including physical , mental and social , and educational program has been designed based on that could have more effect than usual training . therefore , need - oriented education , which is one of the most important roles of community health nursing , could be one of the effective , safe , and inexpensive methods for improving the general health and preventing the complications of senescence and diseases of the old age among the residents of nursing homes .

cancer is an aberrant net accumulation of atypical cells , which can arise from an excess of proliferation , an insufficiency of apoptosis , or a combination of the two . the frequency of apoptosis could contribute to cell loss in tumours and promote tumour regression . thus , in cancer therapy , the focus is on strategies that suppress tumour growth by activating the apoptotic program in the cell . evidence accumulated to this date has established that many agents of cancer chemotherapy affect tumour cell killing through launching the mechanisms of apoptosis . manifestations of apoptosis are easily discernible by the appearance of cell shrinkage , membrane blebbing , chromatin condensation , dna cleavage , and finally , fragmentation of the cell into membrane - bound apoptotic bodies . expressed as inactive proenzymes , caspases are members of a family of cysteine proteases that play a central role in the apoptotic pathway . two major mechanisms exist that initiate the caspase cascade : the extrinsic , involving caspase-8 ; and the intrinsic pathway , involving caspase-9 as the apical caspase . observations from several studies have suggested that a caspase-8 pathway can be up - regulated after drug treatment , and these include the drugs cisplatin , etoposide , doxorubicin and methothrexate . once activated , caspase-8 is thought to activate the downstream caspases by proteolytic cleavage of their zymogen forms , thus amplifying the caspase signal . the other initiator caspase , caspase-9 , controls the apoptotic response to lethal cellular insults such as ionizing radiation or certain chemotherapeutic drugs . in many systems , release of cytochrome c from the mitochondria to cytosol has been demonstrated to be a crucial step in the activation of apoptosis [ 12 - 14 ] . once released from mitochondria , cytochrome c acts as a co - factor and interacts with apaf-1 and procaspase-9 , which in turn activates caspase-9 . the role of active caspase-8 and -9 is to generate the active forms of downstream executioner caspases , including caspase-3 and -7 , by limited proteolysis , and thereby transmit the apoptotic signal to the execution phase . activation of these executioner caspases during apoptosis results in the cleavage of critical cellular substrates , thus disabling critical homeostatic and repair enzymes as well as key structural components that culminate in cell death . styrylpyrone derivative ( spd ) is a pharmacologically active compound extracted from the plant goniothalamus sp . of the annonaceae family . among the species of goniothalamus previous studies on spd suggest this bioactive compound as an antiproliferative and selective cytotoxic agent . in vitro , spd was found to selectively inhibit the proliferation of several cancer cell lines without being significantly cytotoxic towards non - malignant cells [ 19 - 21 ] . on in vivo models , spd is reported to be capable of tumoricidal and tumoristatic effects on experimental rats with mammary tumours . recent work done to elucidate spd 's mechanism of action found evidence that spd modulates the gene expression of bcl-2 and bax in ovarian carcinoma . in breast cancer cells , spd induces an increase of the proapoptotic bax protein , culminating in cell death by apoptosis . in this study we show that procaspase-8 was not activated in mcf-7 cells but caspase-9 activation was detected in response to spd treatment , with the release of cytochrome c into the cytosol . this was followed by the activation of the executioner caspase-7 . to further examine the involvement of this executioner caspase , we found that caspase-7 activity decreased and apoptosis was abrogated when spd - treated cells were preincubated with the caspase-7 inhibitor , ac - devd - cho , suggesting a caspase-7-dependent apoptotic pathway induced by spd . dna condensation and fragmentation characteristic of apoptotic cells were examined and quantitated by the tunel assay and nuclear fluorochrome hoechst 33258 . as reported previously , spd induced apoptosis in mcf-7 cells at 10 m in a time - dependent manner ( figure 1 ) . spd treatment ( 10 m ) significantly increased the level of apoptosis in mcf-7 cells when compared to untreated controls , as judged by apoptotic morphology by nuclear staining and dna fragmentation by tunel assay described in the experimental procedures . results were presented as the means sd of 6 independent experiments . during apoptosis , initiator caspases are activated in response to proapoptotic signals . by sds - page and subsequent western blot analysis with a caspase-8 specific antibody , it was found that spd treatment did not lead to the activation of the initiator caspase-8 . procaspase-8 , expressed in two functionally active isoforms , caspase-8a and caspase-8b was not processed . from immunoblotting , the two bands observed were the 55/50-kda procaspase-8 isoforms ( figure 2 ) , similarly reported by sun et al . , and dirsch et al . , and the active p18 subunit could not be detected . as processing of this caspase did not occur , it is possible that the other initiator caspase , caspase-9 may be involved in spd - induced apoptosis . proteins from mcf-7 cells treated with 10 m spd for the indicated times were resolved on 12% sds - page and submitted to western blotting with an anti - procaspase-8 antibody . two bands were observed , corresponding to the uncleaved 55/50-kda procaspase-8 isoforms . the active p18 subunit was not detected . samples were also detected for procaspase-9 with an anti - procaspase-9 antibody ( clone b40 ) . the anti - caspase-9 antibodies recognized the proenzyme ; and the decrease of this band indicated activation of caspase-9 . when proteins from cytosolic fractions of mcf-7 cells treated with 10 m spd were resolved on 15% sds - page and submitted to immunoblotting with the cytochrome c antibody ( clone 7h8.2c12 ) , increasing amounts of cytochrome c all blots were then washed and reprobed with -actin to confirm equal loading . from immunoblot analysis , untreated mcf-7 cells exhibited the ~48-kda proform of caspase-9 . when mcf-7 cells were treated with 10 m spd , the observed zymogen of caspase-9 slowly diminished in the course of the experiment ( figure 2 ) . the disappearance of the procaspase-9 band reflects the processing of the zymogen to generate the active form of caspase-9 , as has been interpreted in previous reports . for activation of caspase-9 , a multimeric structure termed the apoptosome is involved , consisting of cytochrome c , the apoptotic protease activating factor-1 ( apaf-1 ) , and atp or datp . cytochrome c seems to be a major trigger for the assembly of this complex , and various studies have found that cytochrome c is released from the mitochondria into the cytosol during cell death [ 29 - 31 ] . when cytochrome c levels in the cytosol were examined , we detected increasing levels in the spd - treated mcf-7 cells ( figure 2 ) . untreated control cells did not exhibit similar high levels of cytochrome c , indicating that the release of cytochrome c from the mitochondria into the cytosol was an effect of spd treatment . the role of the initiator caspase-9 is to generate the active forms of executioner caspase-3 and -7 by limited proteolysis , and thereby transmit the apoptotic signal to the execution phase . as with previous reports , caspase-3 activity was not detected ( data not shown ) . immunoblot analyses of lysates obtained from mcf-7 cells treated with spd at 10 m found that caspase-7 was cleaved to the 17-kda fragment required for its activation ( figure 3 ) . when the activity of caspase-7 was assayed , spd - treated cells showed increase in activity compared to untreated controls ( figure 4 ) . to confirm that the spd - induced apoptotic cell death was due to the involvement of caspase-7 , cells were also treated with spd in the presence of the specific inhibitor of caspase-7 , ac - devd - cho . spd - treated cells preincubated with the inhibitor exhibited repressed caspase-7 devdase activity . also , preincubation of mcf-7 cells with this inhibitor at 50 m to 100 m inhibited apoptosis and brought apoptotic levels down to the level similar to controls ( figure 5 ) , thus purporting an apoptotic pathway dependent on caspase-7 . proteins from mcf-7 cells treated with 10 m spd for the indicated times were resolved on a 15% page and submitted to western blotting using a monoclonal antibody against caspase-7 ( b94 - 1 ) , which recognizes the pro- ( 35-kda ) and the active ( 17-kda ) forms of caspase-7 . following spd treatment , the caspase-7 proenzyme was cleaved , generating the catalytically - active 17-kda fragment . the activity of caspase-7 was measured with a colorimetric assay kit ( chemicon ) that recognizes cleavage of the sequence devd by active caspase-7 . caspase-7 activity increased when cells were treated with spd , indicating the catalytic activation of this executioner caspase . when mcf-7 cells were incubated with the caspase-7 inhibitor , devd - cho , prior to spd treatment , caspase-7 devdase activity diminished . when mcf-7 cells were incubated with the caspase-7 inhibitor , devd - cho at ( a ) 50 m and ( b ) 100 m , prior to spd treatment , apoptosis levels decreased to control untreated levels as detected by nuclear staining , suggesting the important role played by this executioner caspase in spd - induced apoptosis . ( c ) results were presented as the means sd of 3 independent experiments . dna condensation and fragmentation characteristic of apoptotic cells were examined and quantitated by the tunel assay and nuclear fluorochrome hoechst 33258 . as reported previously , spd induced apoptosis in mcf-7 cells at 10 m in a time - dependent manner ( figure 1 ) . spd treatment ( 10 m ) significantly increased the level of apoptosis in mcf-7 cells when compared to untreated controls , as judged by apoptotic morphology by nuclear staining and dna fragmentation by tunel assay described in the experimental procedures . during apoptosis , initiator caspases are activated in response to proapoptotic signals . by sds - page and subsequent western blot analysis with a caspase-8 specific antibody , it was found that spd treatment did not lead to the activation of the initiator caspase-8 . procaspase-8 , expressed in two functionally active isoforms , caspase-8a and caspase-8b was not processed . from immunoblotting , the two bands observed were the 55/50-kda procaspase-8 isoforms ( figure 2 ) , similarly reported by sun et al . , and dirsch et al . , and the active p18 subunit could not be detected . as processing of this caspase did not occur , it is possible that the other initiator caspase , caspase-9 may be involved in spd - induced apoptosis . proteins from mcf-7 cells treated with 10 m spd for the indicated times were resolved on 12% sds - page and submitted to western blotting with an anti - procaspase-8 antibody . two bands were observed , corresponding to the uncleaved 55/50-kda procaspase-8 isoforms . the active p18 subunit was not detected . samples were also detected for procaspase-9 with an anti - procaspase-9 antibody ( clone b40 ) . the anti - caspase-9 antibodies recognized the proenzyme ; and the decrease of this band indicated activation of caspase-9 . when proteins from cytosolic fractions of mcf-7 cells treated with 10 m spd were resolved on 15% sds - page and submitted to immunoblotting with the cytochrome c antibody ( clone 7h8.2c12 ) , increasing amounts of cytochrome c from immunoblot analysis , untreated mcf-7 cells exhibited the ~48-kda proform of caspase-9 . when mcf-7 cells were treated with 10 m spd , the observed zymogen of caspase-9 slowly diminished in the course of the experiment ( figure 2 ) . the disappearance of the procaspase-9 band reflects the processing of the zymogen to generate the active form of caspase-9 , as has been interpreted in previous reports . for activation of caspase-9 , a multimeric structure termed the apoptosome is involved , consisting of cytochrome c , the apoptotic protease activating factor-1 ( apaf-1 ) , and atp or datp . cytochrome c seems to be a major trigger for the assembly of this complex , and various studies have found that cytochrome c is released from the mitochondria into the cytosol during cell death [ 29 - 31 ] . when cytochrome c levels in the cytosol were examined , we detected increasing levels in the spd - treated mcf-7 cells ( figure 2 ) . untreated control cells did not exhibit similar high levels of cytochrome c , indicating that the release of cytochrome c from the mitochondria into the cytosol was an effect of spd treatment . the role of the initiator caspase-9 is to generate the active forms of executioner caspase-3 and -7 by limited proteolysis , and thereby transmit the apoptotic signal to the execution phase . here as with previous reports , caspase-3 activity was not detected ( data not shown ) . immunoblot analyses of lysates obtained from mcf-7 cells treated with spd at 10 m found that caspase-7 was cleaved to the 17-kda fragment required for its activation ( figure 3 ) . when the activity of caspase-7 was assayed , spd - treated cells showed increase in activity compared to untreated controls ( figure 4 ) . to confirm that the spd - induced apoptotic cell death was due to the involvement of caspase-7 , cells were also treated with spd in the presence of the specific inhibitor of caspase-7 , ac - devd - cho . spd - treated cells preincubated with the inhibitor exhibited repressed caspase-7 devdase activity . also , preincubation of mcf-7 cells with this inhibitor at 50 m to 100 m inhibited apoptosis and brought apoptotic levels down to the level similar to controls ( figure 5 ) , thus purporting an apoptotic pathway dependent on caspase-7 . proteins from mcf-7 cells treated with 10 m spd for the indicated times were resolved on a 15% page and submitted to western blotting using a monoclonal antibody against caspase-7 ( b94 - 1 ) , which recognizes the pro- ( 35-kda ) and the active ( 17-kda ) forms of caspase-7 . following spd treatment , the caspase-7 proenzyme was cleaved , generating the catalytically - active 17-kda fragment . the activity of caspase-7 was measured with a colorimetric assay kit ( chemicon ) that recognizes cleavage of the sequence devd by active caspase-7 . caspase-7 activity increased when cells were treated with spd , indicating the catalytic activation of this executioner caspase . when mcf-7 cells were incubated with the caspase-7 inhibitor , devd - cho , prior to spd treatment , caspase-7 devdase activity diminished . when mcf-7 cells were incubated with the caspase-7 inhibitor , devd - cho at ( a ) 50 m and ( b ) 100 m , prior to spd treatment , apoptosis levels decreased to control untreated levels as detected by nuclear staining , suggesting the important role played by this executioner caspase in spd - induced apoptosis . ( c ) results were presented as the means sd of 3 independent experiments . there is an increasing realization that chemotherapeutic agents act primarily by inducing cancer cell death through the mechanisms of apoptosis . however , there are many cancers that are intrinsically resistant to apoptosis , making it vital to develop novel drugs for combination chemotherapy . in the present study , we provide evidence that a compound of plant - origin , spd , may be a promising new anticancer agent for human breast cancers . previously , we have shown that mcf-7 cells treated with spd displayed elevated levels of apoptosis and a marked increase in the expression of the proapoptotic bax protein . in addition to the loss of viability , bax expression produces other typical manifestations leading to apoptosis , namely caspase activation . here , we found that caspase-9 was activated , together with accumulation of cytochrome c in the cytosol . caspase-9 can activate downstream executioner caspases including caspase-7 , which is termed caspase-3-like due to its similarity in specificity with caspase-3 . caspase-3 deficiency in mcf-7 is due to a deletion mutation in exon 3 of the gene . previous studies on mcf-7 with exogenously - expressed caspase-3 indicates that caspase-3 plays an important role in apoptotic pathways . studies using etoposide and doxorubicin , active chemotherapeutic agents and key adjuvant drugs for breast cancer treatment , concluded that mcf-7 cells were sensitized to apoptosis only when these cells were reconstituted with caspase-3 . chemoresistance is often caused by aberrant apoptosis that in some instances has been related to defects in caspase activation . given the importance of caspase-3 in apoptotic execution , it is then postulated that caspase-3 deficiency might significantly contribute to chemotherapeutic resistance . in our studies , we observed manifestations of apoptosis in spd - treated mcf-7 cells . previous reports by hishikawa and colleagues and heerdt et al . , have also demonstrated similar apoptotic hallmarks in mcf-7 cells when induced with connective tissue growth factor ( ctgf ) and tributyrin , respectively . these suggest that the mechanism for induction of apoptosis is present and functional in mcf-7 cells , but is dependent on the external stimuli . caspase-7 is highly related to caspase-3 and shows the same synthetic substrate specificity in vitro suggesting that caspase-3 and -7 have possibly overlapping roles in apoptosis . without caspase-3 , spd - treated mcf-7 cells may utilize an alternate caspase pathway to affect cell death . here , we demonstrated that caspase-7 was activated in spd - induced apoptosis . in mcf-7 cells treated with spd , overexpression of full - length caspase-7 in the mcf-7 does not induce apoptosis , whereas the activated 17-kda subunit induces apoptotic cell death . activation of executioner caspases , caspase-3 or -7 results in the cleavage of critical cellular substrates and homeostatic enzymes , bringing about the manifestations of apoptosis . experimental inhibition of apoptosis by peptide caspase inhibitors presents the opportunity to investigate the importance of this protease family . when mcf-7 cells were preincubated with the caspase-7 inhibitor ac - devd - cho before treatment with spd , apoptosis levels decreased to a level similar to controls . cell death was thus inhibited and treated cells had morphology similar to controls , further supporting the involvement of caspase-7 in spd - induced apoptosis . previous reports have found that mcf-7 cells are relatively insensitive to many chemotherapeutic agents due to the absence of caspase-3 . our studies here have shown that the mechanism for apoptosis is functional in mcf-7 and spd is able to induce an alternate caspase pathway , possibly via caspase-7 . tumours accumulate mutations that increase their resistance to apoptotic inducers ; e.g. abrogation of caspase-3 has been associated with acquired multidrug resistance . therefore , finding new therapeutic agents that induce tumour cell apoptosis in a manner independent of caspase-3 may have important clinical implications . by not requiring caspase-3 , spd may evoke an apoptotic pathway different from clinical oncology drugs such as doxorubucin and etoposide , thus making it a promising agent for combination chemotherapy that merits further study . mcf-7 human mammary carcinoma cells were obtained from the american type culture collection ( atcc ) and maintained in dmem supplemented with 10% fetal bovine serum and 2 mm glutamine . styrylpyrone derivative ( spd ) was isolated from the bark of goniothalamus umbrosus as described previously . briefly , cells were treated with spd at 10 m for various incubation times . for inhibitor studies , cells were incubated with the caspase-7 inhibitor , ac - devd - cho ( n - acetyl - asp - glu - val - asp - al ) ( sigma - aldrich ) 1 h prior to spd treatment . after treatment periods , floating and trypsinized adherent cells were collected and washed with phosphate - buffered saline ( pbs ) . after washing , cells were incubated in the nuclear fluorochrome hoechst 33258 ( sigma ) at a final concentration of 30 g / ml at room temperature for 30 min . nuclear morphology was then examined with a zeiss fluorescent microscope and apoptotic cells were counted . dna fragmentation characteristic of apoptotic cells was quantified by tdt - mediated dutp nick end labeling ( tunel ) with the apoptosis detection kit , fluorescein ( promega ) according to the manufacturer 's instructions . to calculate the percentage of tunel - positive cells , four random microscopic fields at 100 and 400 , treated cells were harvested by centrifugation and washed with ice - cold phosphate - buffered saline and resuspended in 5 volumes of extraction buffer containing 250 mm sucrose . cells were homogenized and the homogenates were centrifuged twice at 750 g for 10 min at 4c . the supernatant was then centrifuged at 10,000 g for 15 min at 4c , and the resulting mitochondrial pellets were discarded . as previously described , cells were scraped with a rubber policeman after incubation in extraction buffer and put on ice . cells were then submitted to 3 freeze - thaw cycles and centrifuged at 10,000 rpm for 20 min at -4c . supernatant was collected and added with 1:1 sample buffer and boiled at 95c for 5 min . after protein concentration was determined by standard procedures , protein aliquots of 20 g were applied to 12% or 15% sds - polyacrylamide gels for separation . after electrophoresis , proteins were blotted onto polivynyl - difluoride membranes ( polyscreen , nen life science ) . membranes were dried , preblocked with 5% non - fat milk in phosphate - buffered saline and 0.1% tween-20 , then incubated with a primary antibody for caspase-8 , caspase-9 ( clone b40 ) , caspase-7 ( clone b94 - 1 ) or cytochrome c ( clone 7h8.2c12 ) ( all from pharmingen ) , and detected with horseradish peroxidase - labeled antibodies to rabbit or mouse igg . following exposure on a kodak biomax x - ray film , densitometry analysis was done with a gs 670 imaging densitometer with the software molecular analyst ( bio rad ) . blots were stripped with re - blot plus ( chemicon ) before reprobing with -actin antibody to determine equal loading . caspase-7 activity in the caspase-3-deficient mcf-7 cells was assayed with the colorimetric assay kit ( chemicon international , usa ) that provides a means to assay the activity of caspases that recognize the sequence devd . briefly , mcf-7 cells were treated with spd at 10 m for 24 and 48 h. after the treatment period , cells were counted and then pelleted at 1,500 rpm for 10 min . cells were then resuspended in chilled cell lysis buffer and incubated on ice before centrifugation at 10,000 g for 5 min . the supernatant ( cytosolic extract ) was then transferred to a fresh microcentrifuge tube and put on ice . the protein concentration for each sample set assay mixture was prepared in a 96-well plate and mixed with assay buffer , dh2o and the caspase substrate , ac - devd - pna , and incubated at 37c for 1.5 h. for inhibitor studies , the sample was pre - incubated with the caspase-7 inhibitor , ac - devd - cho , for 10 min at room temperature before adding the substrate solution . after incubation , samples were read with a dynex mrx microtiter plate reader at 405 nm . increase in caspase-7 activity was then determined by comparing the od reading from the spd - treated samples with the level of the untreated control . mcf-7 human mammary carcinoma cells were obtained from the american type culture collection ( atcc ) and maintained in dmem supplemented with 10% fetal bovine serum and 2 mm glutamine . styrylpyrone derivative ( spd ) was isolated from the bark of goniothalamus umbrosus as described previously . briefly , cells were treated with spd at 10 m for various incubation times . for inhibitor studies , cells were incubated with the caspase-7 inhibitor , ac - devd - cho ( n - acetyl - asp - glu - val - asp - al ) ( sigma - aldrich ) 1 h prior to spd treatment . after treatment periods , floating and trypsinized adherent cells were collected and washed with phosphate - buffered saline ( pbs ) . after washing , cells were incubated in the nuclear fluorochrome hoechst 33258 ( sigma ) at a final concentration of 30 g / ml at room temperature for 30 min . nuclear morphology was then examined with a zeiss fluorescent microscope and apoptotic cells were counted . dna fragmentation characteristic of apoptotic cells was quantified by tdt - mediated dutp nick end labeling ( tunel ) with the apoptosis detection kit , fluorescein ( promega ) according to the manufacturer 's instructions . to calculate the percentage of tunel - positive cells , four random microscopic fields at 100 and 400 briefly , treated cells were harvested by centrifugation and washed with ice - cold phosphate - buffered saline and resuspended in 5 volumes of extraction buffer containing 250 mm sucrose . cells were homogenized and the homogenates were centrifuged twice at 750 g for 10 min at 4c . the supernatant was then centrifuged at 10,000 g for 15 min at 4c , and the resulting mitochondrial pellets were discarded . as previously described , cells were scraped with a rubber policeman after incubation in extraction buffer and put on ice . cells were then submitted to 3 freeze - thaw cycles and centrifuged at 10,000 rpm for 20 min at -4c . supernatant was collected and added with 1:1 sample buffer and boiled at 95c for 5 min . after protein concentration was determined by standard procedures , protein aliquots of 20 g were applied to 12% or 15% sds - polyacrylamide gels for separation . after electrophoresis , proteins were blotted onto polivynyl - difluoride membranes ( polyscreen , nen life science ) . membranes were dried , preblocked with 5% non - fat milk in phosphate - buffered saline and 0.1% tween-20 , then incubated with a primary antibody for caspase-8 , caspase-9 ( clone b40 ) , caspase-7 ( clone b94 - 1 ) or cytochrome c ( clone 7h8.2c12 ) ( all from pharmingen ) , and detected with horseradish peroxidase - labeled antibodies to rabbit or mouse igg . following exposure on a kodak biomax x - ray film , densitometry analysis was done with a gs 670 imaging densitometer with the software molecular analyst ( bio rad ) . blots were stripped with re - blot plus ( chemicon ) before reprobing with -actin antibody to determine equal loading . caspase-7 activity in the caspase-3-deficient mcf-7 cells was assayed with the colorimetric assay kit ( chemicon international , usa ) that provides a means to assay the activity of caspases that recognize the sequence devd . briefly , mcf-7 cells were treated with spd at 10 m for 24 and 48 h. after the treatment period , cells were counted and then pelleted at 1,500 rpm for 10 min . cells were then resuspended in chilled cell lysis buffer and incubated on ice before centrifugation at 10,000 g for 5 min . the supernatant ( cytosolic extract ) was then transferred to a fresh microcentrifuge tube and put on ice . the protein concentration for each sample set assay mixture was prepared in a 96-well plate and mixed with assay buffer , dh2o and the caspase substrate , ac - devd - pna , and incubated at 37c for 1.5 h. for inhibitor studies , the sample was pre - incubated with the caspase-7 inhibitor , ac - devd - cho , for 10 min at room temperature before adding the substrate solution . after incubation , samples were read with a dynex mrx microtiter plate reader at 405 nm . increase in caspase-7 activity was then determined by comparing the od reading from the spd - treated samples with the level of the untreated control .

backgroundstyrylpyrone derivative ( spd ) is a plant - derived pharmacologically active compound extracted from goniothalamus sp . previously , we have reported that spd inhibited the proliferation of mcf-7 human breast cancer cells by inducing apoptotic cell death , while having minimal effects on non - malignant cells . here , we attempt to further elucidate the mode of action of spd.resultswe found that the intrinsic apoptotic pathway was invoked , with the accumulation of cytosolic cytochrome c and processing of the initiator caspase-9 . cleaved products of procaspase-8 were not detected . next , the executioner caspase-7 was cleaved and activated in response to spd treatment . to confirm that apoptosis was induced following caspase-7 activation , the caspase inhibitor ac - devd - cho was used . pre - incubation of cells with this inhibitor reversed apoptosis levels and caspase-7 activity in spd - treated cells to untreated levels.conclusionstaken together , these results suggest spd as a potent antiproliferative agent on mcf-7 cells by inducing apoptosis in a caspase-7-dependent manner .

Background Results SPD induced apoptotic cell death Procaspase-8 is not processed in SPD-treated cells Involvement of caspase-9 and cytochrome c The "executioner", caspase-7, is activated in SPD-induced apoptosis Discussion Methods Cell culture Apoptotic index and nuclear morphology Isolation of cytosolic fractions Protein extraction for caspases Western Blotting Caspase-7 activity assay

cancer is an aberrant net accumulation of atypical cells , which can arise from an excess of proliferation , an insufficiency of apoptosis , or a combination of the two . manifestations of apoptosis are easily discernible by the appearance of cell shrinkage , membrane blebbing , chromatin condensation , dna cleavage , and finally , fragmentation of the cell into membrane - bound apoptotic bodies . two major mechanisms exist that initiate the caspase cascade : the extrinsic , involving caspase-8 ; and the intrinsic pathway , involving caspase-9 as the apical caspase . in many systems , release of cytochrome c from the mitochondria to cytosol has been demonstrated to be a crucial step in the activation of apoptosis [ 12 - 14 ] . once released from mitochondria , cytochrome c acts as a co - factor and interacts with apaf-1 and procaspase-9 , which in turn activates caspase-9 . styrylpyrone derivative ( spd ) is a pharmacologically active compound extracted from the plant goniothalamus sp . of the annonaceae family . in vitro , spd was found to selectively inhibit the proliferation of several cancer cell lines without being significantly cytotoxic towards non - malignant cells [ 19 - 21 ] . recent work done to elucidate spd 's mechanism of action found evidence that spd modulates the gene expression of bcl-2 and bax in ovarian carcinoma . in breast cancer cells , spd induces an increase of the proapoptotic bax protein , culminating in cell death by apoptosis . in this study we show that procaspase-8 was not activated in mcf-7 cells but caspase-9 activation was detected in response to spd treatment , with the release of cytochrome c into the cytosol . this was followed by the activation of the executioner caspase-7 . to further examine the involvement of this executioner caspase , we found that caspase-7 activity decreased and apoptosis was abrogated when spd - treated cells were preincubated with the caspase-7 inhibitor , ac - devd - cho , suggesting a caspase-7-dependent apoptotic pathway induced by spd . as reported previously , spd induced apoptosis in mcf-7 cells at 10 m in a time - dependent manner ( figure 1 ) . spd treatment ( 10 m ) significantly increased the level of apoptosis in mcf-7 cells when compared to untreated controls , as judged by apoptotic morphology by nuclear staining and dna fragmentation by tunel assay described in the experimental procedures . during apoptosis , initiator caspases are activated in response to proapoptotic signals . by sds - page and subsequent western blot analysis with a caspase-8 specific antibody , it was found that spd treatment did not lead to the activation of the initiator caspase-8 . from immunoblotting , the two bands observed were the 55/50-kda procaspase-8 isoforms ( figure 2 ) , similarly reported by sun et al . as processing of this caspase did not occur , it is possible that the other initiator caspase , caspase-9 may be involved in spd - induced apoptosis . the active p18 subunit was not detected . when proteins from cytosolic fractions of mcf-7 cells treated with 10 m spd were resolved on 15% sds - page and submitted to immunoblotting with the cytochrome c antibody ( clone 7h8.2c12 ) , increasing amounts of cytochrome c all blots were then washed and reprobed with -actin to confirm equal loading . when mcf-7 cells were treated with 10 m spd , the observed zymogen of caspase-9 slowly diminished in the course of the experiment ( figure 2 ) . the disappearance of the procaspase-9 band reflects the processing of the zymogen to generate the active form of caspase-9 , as has been interpreted in previous reports . for activation of caspase-9 , a multimeric structure termed the apoptosome is involved , consisting of cytochrome c , the apoptotic protease activating factor-1 ( apaf-1 ) , and atp or datp . cytochrome c seems to be a major trigger for the assembly of this complex , and various studies have found that cytochrome c is released from the mitochondria into the cytosol during cell death [ 29 - 31 ] . when cytochrome c levels in the cytosol were examined , we detected increasing levels in the spd - treated mcf-7 cells ( figure 2 ) . untreated control cells did not exhibit similar high levels of cytochrome c , indicating that the release of cytochrome c from the mitochondria into the cytosol was an effect of spd treatment . the role of the initiator caspase-9 is to generate the active forms of executioner caspase-3 and -7 by limited proteolysis , and thereby transmit the apoptotic signal to the execution phase . as with previous reports , caspase-3 activity was not detected ( data not shown ) . immunoblot analyses of lysates obtained from mcf-7 cells treated with spd at 10 m found that caspase-7 was cleaved to the 17-kda fragment required for its activation ( figure 3 ) . when the activity of caspase-7 was assayed , spd - treated cells showed increase in activity compared to untreated controls ( figure 4 ) . to confirm that the spd - induced apoptotic cell death was due to the involvement of caspase-7 , cells were also treated with spd in the presence of the specific inhibitor of caspase-7 , ac - devd - cho . spd - treated cells preincubated with the inhibitor exhibited repressed caspase-7 devdase activity . also , preincubation of mcf-7 cells with this inhibitor at 50 m to 100 m inhibited apoptosis and brought apoptotic levels down to the level similar to controls ( figure 5 ) , thus purporting an apoptotic pathway dependent on caspase-7 . proteins from mcf-7 cells treated with 10 m spd for the indicated times were resolved on a 15% page and submitted to western blotting using a monoclonal antibody against caspase-7 ( b94 - 1 ) , which recognizes the pro- ( 35-kda ) and the active ( 17-kda ) forms of caspase-7 . following spd treatment , the caspase-7 proenzyme was cleaved , generating the catalytically - active 17-kda fragment . the activity of caspase-7 was measured with a colorimetric assay kit ( chemicon ) that recognizes cleavage of the sequence devd by active caspase-7 . when mcf-7 cells were incubated with the caspase-7 inhibitor , devd - cho , prior to spd treatment , caspase-7 devdase activity diminished . when mcf-7 cells were incubated with the caspase-7 inhibitor , devd - cho at ( a ) 50 m and ( b ) 100 m , prior to spd treatment , apoptosis levels decreased to control untreated levels as detected by nuclear staining , suggesting the important role played by this executioner caspase in spd - induced apoptosis . as reported previously , spd induced apoptosis in mcf-7 cells at 10 m in a time - dependent manner ( figure 1 ) . spd treatment ( 10 m ) significantly increased the level of apoptosis in mcf-7 cells when compared to untreated controls , as judged by apoptotic morphology by nuclear staining and dna fragmentation by tunel assay described in the experimental procedures . during apoptosis , initiator caspases are activated in response to proapoptotic signals . by sds - page and subsequent western blot analysis with a caspase-8 specific antibody , it was found that spd treatment did not lead to the activation of the initiator caspase-8 . as processing of this caspase did not occur , it is possible that the other initiator caspase , caspase-9 may be involved in spd - induced apoptosis . when proteins from cytosolic fractions of mcf-7 cells treated with 10 m spd were resolved on 15% sds - page and submitted to immunoblotting with the cytochrome c antibody ( clone 7h8.2c12 ) , increasing amounts of cytochrome c from immunoblot analysis , untreated mcf-7 cells exhibited the ~48-kda proform of caspase-9 . when mcf-7 cells were treated with 10 m spd , the observed zymogen of caspase-9 slowly diminished in the course of the experiment ( figure 2 ) . the disappearance of the procaspase-9 band reflects the processing of the zymogen to generate the active form of caspase-9 , as has been interpreted in previous reports . for activation of caspase-9 , a multimeric structure termed the apoptosome is involved , consisting of cytochrome c , the apoptotic protease activating factor-1 ( apaf-1 ) , and atp or datp . cytochrome c seems to be a major trigger for the assembly of this complex , and various studies have found that cytochrome c is released from the mitochondria into the cytosol during cell death [ 29 - 31 ] . when cytochrome c levels in the cytosol were examined , we detected increasing levels in the spd - treated mcf-7 cells ( figure 2 ) . untreated control cells did not exhibit similar high levels of cytochrome c , indicating that the release of cytochrome c from the mitochondria into the cytosol was an effect of spd treatment . the role of the initiator caspase-9 is to generate the active forms of executioner caspase-3 and -7 by limited proteolysis , and thereby transmit the apoptotic signal to the execution phase . immunoblot analyses of lysates obtained from mcf-7 cells treated with spd at 10 m found that caspase-7 was cleaved to the 17-kda fragment required for its activation ( figure 3 ) . when the activity of caspase-7 was assayed , spd - treated cells showed increase in activity compared to untreated controls ( figure 4 ) . to confirm that the spd - induced apoptotic cell death was due to the involvement of caspase-7 , cells were also treated with spd in the presence of the specific inhibitor of caspase-7 , ac - devd - cho . spd - treated cells preincubated with the inhibitor exhibited repressed caspase-7 devdase activity . also , preincubation of mcf-7 cells with this inhibitor at 50 m to 100 m inhibited apoptosis and brought apoptotic levels down to the level similar to controls ( figure 5 ) , thus purporting an apoptotic pathway dependent on caspase-7 . proteins from mcf-7 cells treated with 10 m spd for the indicated times were resolved on a 15% page and submitted to western blotting using a monoclonal antibody against caspase-7 ( b94 - 1 ) , which recognizes the pro- ( 35-kda ) and the active ( 17-kda ) forms of caspase-7 . following spd treatment , the caspase-7 proenzyme was cleaved , generating the catalytically - active 17-kda fragment . the activity of caspase-7 was measured with a colorimetric assay kit ( chemicon ) that recognizes cleavage of the sequence devd by active caspase-7 . caspase-7 activity increased when cells were treated with spd , indicating the catalytic activation of this executioner caspase . when mcf-7 cells were incubated with the caspase-7 inhibitor , devd - cho , prior to spd treatment , caspase-7 devdase activity diminished . when mcf-7 cells were incubated with the caspase-7 inhibitor , devd - cho at ( a ) 50 m and ( b ) 100 m , prior to spd treatment , apoptosis levels decreased to control untreated levels as detected by nuclear staining , suggesting the important role played by this executioner caspase in spd - induced apoptosis . there is an increasing realization that chemotherapeutic agents act primarily by inducing cancer cell death through the mechanisms of apoptosis . in the present study , we provide evidence that a compound of plant - origin , spd , may be a promising new anticancer agent for human breast cancers . previously , we have shown that mcf-7 cells treated with spd displayed elevated levels of apoptosis and a marked increase in the expression of the proapoptotic bax protein . here , we found that caspase-9 was activated , together with accumulation of cytochrome c in the cytosol . caspase-3 deficiency in mcf-7 is due to a deletion mutation in exon 3 of the gene . studies using etoposide and doxorubicin , active chemotherapeutic agents and key adjuvant drugs for breast cancer treatment , concluded that mcf-7 cells were sensitized to apoptosis only when these cells were reconstituted with caspase-3 . in our studies , we observed manifestations of apoptosis in spd - treated mcf-7 cells . these suggest that the mechanism for induction of apoptosis is present and functional in mcf-7 cells , but is dependent on the external stimuli . without caspase-3 , spd - treated mcf-7 cells may utilize an alternate caspase pathway to affect cell death . here , we demonstrated that caspase-7 was activated in spd - induced apoptosis . in mcf-7 cells treated with spd , overexpression of full - length caspase-7 in the mcf-7 does not induce apoptosis , whereas the activated 17-kda subunit induces apoptotic cell death . when mcf-7 cells were preincubated with the caspase-7 inhibitor ac - devd - cho before treatment with spd , apoptosis levels decreased to a level similar to controls . cell death was thus inhibited and treated cells had morphology similar to controls , further supporting the involvement of caspase-7 in spd - induced apoptosis . previous reports have found that mcf-7 cells are relatively insensitive to many chemotherapeutic agents due to the absence of caspase-3 . therefore , finding new therapeutic agents that induce tumour cell apoptosis in a manner independent of caspase-3 may have important clinical implications . by not requiring caspase-3 , spd may evoke an apoptotic pathway different from clinical oncology drugs such as doxorubucin and etoposide , thus making it a promising agent for combination chemotherapy that merits further study . styrylpyrone derivative ( spd ) was isolated from the bark of goniothalamus umbrosus as described previously . for inhibitor studies , cells were incubated with the caspase-7 inhibitor , ac - devd - cho ( n - acetyl - asp - glu - val - asp - al ) ( sigma - aldrich ) 1 h prior to spd treatment . membranes were dried , preblocked with 5% non - fat milk in phosphate - buffered saline and 0.1% tween-20 , then incubated with a primary antibody for caspase-8 , caspase-9 ( clone b40 ) , caspase-7 ( clone b94 - 1 ) or cytochrome c ( clone 7h8.2c12 ) ( all from pharmingen ) , and detected with horseradish peroxidase - labeled antibodies to rabbit or mouse igg . following exposure on a kodak biomax x - ray film , densitometry analysis was done with a gs 670 imaging densitometer with the software molecular analyst ( bio rad ) . caspase-7 activity in the caspase-3-deficient mcf-7 cells was assayed with the colorimetric assay kit ( chemicon international , usa ) that provides a means to assay the activity of caspases that recognize the sequence devd . the protein concentration for each sample set assay mixture was prepared in a 96-well plate and mixed with assay buffer , dh2o and the caspase substrate , ac - devd - pna , and incubated at 37c for 1.5 h. for inhibitor studies , the sample was pre - incubated with the caspase-7 inhibitor , ac - devd - cho , for 10 min at room temperature before adding the substrate solution . increase in caspase-7 activity was then determined by comparing the od reading from the spd - treated samples with the level of the untreated control . mcf-7 human mammary carcinoma cells were obtained from the american type culture collection ( atcc ) and maintained in dmem supplemented with 10% fetal bovine serum and 2 mm glutamine . styrylpyrone derivative ( spd ) was isolated from the bark of goniothalamus umbrosus as described previously . for inhibitor studies , cells were incubated with the caspase-7 inhibitor , ac - devd - cho ( n - acetyl - asp - glu - val - asp - al ) ( sigma - aldrich ) 1 h prior to spd treatment . dna fragmentation characteristic of apoptotic cells was quantified by tdt - mediated dutp nick end labeling ( tunel ) with the apoptosis detection kit , fluorescein ( promega ) according to the manufacturer 's instructions . membranes were dried , preblocked with 5% non - fat milk in phosphate - buffered saline and 0.1% tween-20 , then incubated with a primary antibody for caspase-8 , caspase-9 ( clone b40 ) , caspase-7 ( clone b94 - 1 ) or cytochrome c ( clone 7h8.2c12 ) ( all from pharmingen ) , and detected with horseradish peroxidase - labeled antibodies to rabbit or mouse igg . caspase-7 activity in the caspase-3-deficient mcf-7 cells was assayed with the colorimetric assay kit ( chemicon international , usa ) that provides a means to assay the activity of caspases that recognize the sequence devd . the protein concentration for each sample set assay mixture was prepared in a 96-well plate and mixed with assay buffer , dh2o and the caspase substrate , ac - devd - pna , and incubated at 37c for 1.5 h. for inhibitor studies , the sample was pre - incubated with the caspase-7 inhibitor , ac - devd - cho , for 10 min at room temperature before adding the substrate solution . increase in caspase-7 activity was then determined by comparing the od reading from the spd - treated samples with the level of the untreated control .

cancer is an aberrant net accumulation of atypical cells , which can arise from an excess of proliferation , an insufficiency of apoptosis , or a combination of the two . thus , in cancer therapy , the focus is on strategies that suppress tumour growth by activating the apoptotic program in the cell . evidence accumulated to this date has established that many agents of cancer chemotherapy affect tumour cell killing through launching the mechanisms of apoptosis . manifestations of apoptosis are easily discernible by the appearance of cell shrinkage , membrane blebbing , chromatin condensation , dna cleavage , and finally , fragmentation of the cell into membrane - bound apoptotic bodies . expressed as inactive proenzymes , caspases are members of a family of cysteine proteases that play a central role in the apoptotic pathway . two major mechanisms exist that initiate the caspase cascade : the extrinsic , involving caspase-8 ; and the intrinsic pathway , involving caspase-9 as the apical caspase . observations from several studies have suggested that a caspase-8 pathway can be up - regulated after drug treatment , and these include the drugs cisplatin , etoposide , doxorubicin and methothrexate . in many systems , release of cytochrome c from the mitochondria to cytosol has been demonstrated to be a crucial step in the activation of apoptosis [ 12 - 14 ] . the role of active caspase-8 and -9 is to generate the active forms of downstream executioner caspases , including caspase-3 and -7 , by limited proteolysis , and thereby transmit the apoptotic signal to the execution phase . activation of these executioner caspases during apoptosis results in the cleavage of critical cellular substrates , thus disabling critical homeostatic and repair enzymes as well as key structural components that culminate in cell death . styrylpyrone derivative ( spd ) is a pharmacologically active compound extracted from the plant goniothalamus sp . among the species of goniothalamus previous studies on spd suggest this bioactive compound as an antiproliferative and selective cytotoxic agent . in vitro , spd was found to selectively inhibit the proliferation of several cancer cell lines without being significantly cytotoxic towards non - malignant cells [ 19 - 21 ] . on in vivo models , spd is reported to be capable of tumoricidal and tumoristatic effects on experimental rats with mammary tumours . recent work done to elucidate spd 's mechanism of action found evidence that spd modulates the gene expression of bcl-2 and bax in ovarian carcinoma . in breast cancer cells , spd induces an increase of the proapoptotic bax protein , culminating in cell death by apoptosis . in this study we show that procaspase-8 was not activated in mcf-7 cells but caspase-9 activation was detected in response to spd treatment , with the release of cytochrome c into the cytosol . to further examine the involvement of this executioner caspase , we found that caspase-7 activity decreased and apoptosis was abrogated when spd - treated cells were preincubated with the caspase-7 inhibitor , ac - devd - cho , suggesting a caspase-7-dependent apoptotic pathway induced by spd . dna condensation and fragmentation characteristic of apoptotic cells were examined and quantitated by the tunel assay and nuclear fluorochrome hoechst 33258 . as reported previously , spd induced apoptosis in mcf-7 cells at 10 m in a time - dependent manner ( figure 1 ) . spd treatment ( 10 m ) significantly increased the level of apoptosis in mcf-7 cells when compared to untreated controls , as judged by apoptotic morphology by nuclear staining and dna fragmentation by tunel assay described in the experimental procedures . by sds - page and subsequent western blot analysis with a caspase-8 specific antibody , it was found that spd treatment did not lead to the activation of the initiator caspase-8 . as processing of this caspase did not occur , it is possible that the other initiator caspase , caspase-9 may be involved in spd - induced apoptosis . proteins from mcf-7 cells treated with 10 m spd for the indicated times were resolved on 12% sds - page and submitted to western blotting with an anti - procaspase-8 antibody . the anti - caspase-9 antibodies recognized the proenzyme ; and the decrease of this band indicated activation of caspase-9 . when proteins from cytosolic fractions of mcf-7 cells treated with 10 m spd were resolved on 15% sds - page and submitted to immunoblotting with the cytochrome c antibody ( clone 7h8.2c12 ) , increasing amounts of cytochrome c all blots were then washed and reprobed with -actin to confirm equal loading . from immunoblot analysis , untreated mcf-7 cells exhibited the ~48-kda proform of caspase-9 . when mcf-7 cells were treated with 10 m spd , the observed zymogen of caspase-9 slowly diminished in the course of the experiment ( figure 2 ) . the disappearance of the procaspase-9 band reflects the processing of the zymogen to generate the active form of caspase-9 , as has been interpreted in previous reports . for activation of caspase-9 , a multimeric structure termed the apoptosome is involved , consisting of cytochrome c , the apoptotic protease activating factor-1 ( apaf-1 ) , and atp or datp . cytochrome c seems to be a major trigger for the assembly of this complex , and various studies have found that cytochrome c is released from the mitochondria into the cytosol during cell death [ 29 - 31 ] . when cytochrome c levels in the cytosol were examined , we detected increasing levels in the spd - treated mcf-7 cells ( figure 2 ) . untreated control cells did not exhibit similar high levels of cytochrome c , indicating that the release of cytochrome c from the mitochondria into the cytosol was an effect of spd treatment . the role of the initiator caspase-9 is to generate the active forms of executioner caspase-3 and -7 by limited proteolysis , and thereby transmit the apoptotic signal to the execution phase . immunoblot analyses of lysates obtained from mcf-7 cells treated with spd at 10 m found that caspase-7 was cleaved to the 17-kda fragment required for its activation ( figure 3 ) . when the activity of caspase-7 was assayed , spd - treated cells showed increase in activity compared to untreated controls ( figure 4 ) . to confirm that the spd - induced apoptotic cell death was due to the involvement of caspase-7 , cells were also treated with spd in the presence of the specific inhibitor of caspase-7 , ac - devd - cho . spd - treated cells preincubated with the inhibitor exhibited repressed caspase-7 devdase activity . also , preincubation of mcf-7 cells with this inhibitor at 50 m to 100 m inhibited apoptosis and brought apoptotic levels down to the level similar to controls ( figure 5 ) , thus purporting an apoptotic pathway dependent on caspase-7 . proteins from mcf-7 cells treated with 10 m spd for the indicated times were resolved on a 15% page and submitted to western blotting using a monoclonal antibody against caspase-7 ( b94 - 1 ) , which recognizes the pro- ( 35-kda ) and the active ( 17-kda ) forms of caspase-7 . following spd treatment , the caspase-7 proenzyme was cleaved , generating the catalytically - active 17-kda fragment . caspase-7 activity increased when cells were treated with spd , indicating the catalytic activation of this executioner caspase . when mcf-7 cells were incubated with the caspase-7 inhibitor , devd - cho at ( a ) 50 m and ( b ) 100 m , prior to spd treatment , apoptosis levels decreased to control untreated levels as detected by nuclear staining , suggesting the important role played by this executioner caspase in spd - induced apoptosis . dna condensation and fragmentation characteristic of apoptotic cells were examined and quantitated by the tunel assay and nuclear fluorochrome hoechst 33258 . as reported previously , spd induced apoptosis in mcf-7 cells at 10 m in a time - dependent manner ( figure 1 ) . spd treatment ( 10 m ) significantly increased the level of apoptosis in mcf-7 cells when compared to untreated controls , as judged by apoptotic morphology by nuclear staining and dna fragmentation by tunel assay described in the experimental procedures . by sds - page and subsequent western blot analysis with a caspase-8 specific antibody , it was found that spd treatment did not lead to the activation of the initiator caspase-8 . as processing of this caspase did not occur , it is possible that the other initiator caspase , caspase-9 may be involved in spd - induced apoptosis . proteins from mcf-7 cells treated with 10 m spd for the indicated times were resolved on 12% sds - page and submitted to western blotting with an anti - procaspase-8 antibody . the anti - caspase-9 antibodies recognized the proenzyme ; and the decrease of this band indicated activation of caspase-9 . when proteins from cytosolic fractions of mcf-7 cells treated with 10 m spd were resolved on 15% sds - page and submitted to immunoblotting with the cytochrome c antibody ( clone 7h8.2c12 ) , increasing amounts of cytochrome c from immunoblot analysis , untreated mcf-7 cells exhibited the ~48-kda proform of caspase-9 . when mcf-7 cells were treated with 10 m spd , the observed zymogen of caspase-9 slowly diminished in the course of the experiment ( figure 2 ) . the disappearance of the procaspase-9 band reflects the processing of the zymogen to generate the active form of caspase-9 , as has been interpreted in previous reports . for activation of caspase-9 , a multimeric structure termed the apoptosome is involved , consisting of cytochrome c , the apoptotic protease activating factor-1 ( apaf-1 ) , and atp or datp . cytochrome c seems to be a major trigger for the assembly of this complex , and various studies have found that cytochrome c is released from the mitochondria into the cytosol during cell death [ 29 - 31 ] . when cytochrome c levels in the cytosol were examined , we detected increasing levels in the spd - treated mcf-7 cells ( figure 2 ) . untreated control cells did not exhibit similar high levels of cytochrome c , indicating that the release of cytochrome c from the mitochondria into the cytosol was an effect of spd treatment . the role of the initiator caspase-9 is to generate the active forms of executioner caspase-3 and -7 by limited proteolysis , and thereby transmit the apoptotic signal to the execution phase . when the activity of caspase-7 was assayed , spd - treated cells showed increase in activity compared to untreated controls ( figure 4 ) . to confirm that the spd - induced apoptotic cell death was due to the involvement of caspase-7 , cells were also treated with spd in the presence of the specific inhibitor of caspase-7 , ac - devd - cho . spd - treated cells preincubated with the inhibitor exhibited repressed caspase-7 devdase activity . also , preincubation of mcf-7 cells with this inhibitor at 50 m to 100 m inhibited apoptosis and brought apoptotic levels down to the level similar to controls ( figure 5 ) , thus purporting an apoptotic pathway dependent on caspase-7 . proteins from mcf-7 cells treated with 10 m spd for the indicated times were resolved on a 15% page and submitted to western blotting using a monoclonal antibody against caspase-7 ( b94 - 1 ) , which recognizes the pro- ( 35-kda ) and the active ( 17-kda ) forms of caspase-7 . caspase-7 activity increased when cells were treated with spd , indicating the catalytic activation of this executioner caspase . when mcf-7 cells were incubated with the caspase-7 inhibitor , devd - cho at ( a ) 50 m and ( b ) 100 m , prior to spd treatment , apoptosis levels decreased to control untreated levels as detected by nuclear staining , suggesting the important role played by this executioner caspase in spd - induced apoptosis . in the present study , we provide evidence that a compound of plant - origin , spd , may be a promising new anticancer agent for human breast cancers . previously , we have shown that mcf-7 cells treated with spd displayed elevated levels of apoptosis and a marked increase in the expression of the proapoptotic bax protein . here , we found that caspase-9 was activated , together with accumulation of cytochrome c in the cytosol . studies using etoposide and doxorubicin , active chemotherapeutic agents and key adjuvant drugs for breast cancer treatment , concluded that mcf-7 cells were sensitized to apoptosis only when these cells were reconstituted with caspase-3 . in our studies , we observed manifestations of apoptosis in spd - treated mcf-7 cells . , have also demonstrated similar apoptotic hallmarks in mcf-7 cells when induced with connective tissue growth factor ( ctgf ) and tributyrin , respectively . these suggest that the mechanism for induction of apoptosis is present and functional in mcf-7 cells , but is dependent on the external stimuli . caspase-7 is highly related to caspase-3 and shows the same synthetic substrate specificity in vitro suggesting that caspase-3 and -7 have possibly overlapping roles in apoptosis . here , we demonstrated that caspase-7 was activated in spd - induced apoptosis . in mcf-7 cells treated with spd , overexpression of full - length caspase-7 in the mcf-7 does not induce apoptosis , whereas the activated 17-kda subunit induces apoptotic cell death . activation of executioner caspases , caspase-3 or -7 results in the cleavage of critical cellular substrates and homeostatic enzymes , bringing about the manifestations of apoptosis . experimental inhibition of apoptosis by peptide caspase inhibitors presents the opportunity to investigate the importance of this protease family . when mcf-7 cells were preincubated with the caspase-7 inhibitor ac - devd - cho before treatment with spd , apoptosis levels decreased to a level similar to controls . cell death was thus inhibited and treated cells had morphology similar to controls , further supporting the involvement of caspase-7 in spd - induced apoptosis . our studies here have shown that the mechanism for apoptosis is functional in mcf-7 and spd is able to induce an alternate caspase pathway , possibly via caspase-7 . therefore , finding new therapeutic agents that induce tumour cell apoptosis in a manner independent of caspase-3 may have important clinical implications . for inhibitor studies , cells were incubated with the caspase-7 inhibitor , ac - devd - cho ( n - acetyl - asp - glu - val - asp - al ) ( sigma - aldrich ) 1 h prior to spd treatment . dna fragmentation characteristic of apoptotic cells was quantified by tdt - mediated dutp nick end labeling ( tunel ) with the apoptosis detection kit , fluorescein ( promega ) according to the manufacturer 's instructions . membranes were dried , preblocked with 5% non - fat milk in phosphate - buffered saline and 0.1% tween-20 , then incubated with a primary antibody for caspase-8 , caspase-9 ( clone b40 ) , caspase-7 ( clone b94 - 1 ) or cytochrome c ( clone 7h8.2c12 ) ( all from pharmingen ) , and detected with horseradish peroxidase - labeled antibodies to rabbit or mouse igg . caspase-7 activity in the caspase-3-deficient mcf-7 cells was assayed with the colorimetric assay kit ( chemicon international , usa ) that provides a means to assay the activity of caspases that recognize the sequence devd . the protein concentration for each sample set assay mixture was prepared in a 96-well plate and mixed with assay buffer , dh2o and the caspase substrate , ac - devd - pna , and incubated at 37c for 1.5 h. for inhibitor studies , the sample was pre - incubated with the caspase-7 inhibitor , ac - devd - cho , for 10 min at room temperature before adding the substrate solution . increase in caspase-7 activity was then determined by comparing the od reading from the spd - treated samples with the level of the untreated control . for inhibitor studies , cells were incubated with the caspase-7 inhibitor , ac - devd - cho ( n - acetyl - asp - glu - val - asp - al ) ( sigma - aldrich ) 1 h prior to spd treatment . dna fragmentation characteristic of apoptotic cells was quantified by tdt - mediated dutp nick end labeling ( tunel ) with the apoptosis detection kit , fluorescein ( promega ) according to the manufacturer 's instructions . membranes were dried , preblocked with 5% non - fat milk in phosphate - buffered saline and 0.1% tween-20 , then incubated with a primary antibody for caspase-8 , caspase-9 ( clone b40 ) , caspase-7 ( clone b94 - 1 ) or cytochrome c ( clone 7h8.2c12 ) ( all from pharmingen ) , and detected with horseradish peroxidase - labeled antibodies to rabbit or mouse igg . the protein concentration for each sample set assay mixture was prepared in a 96-well plate and mixed with assay buffer , dh2o and the caspase substrate , ac - devd - pna , and incubated at 37c for 1.5 h. for inhibitor studies , the sample was pre - incubated with the caspase-7 inhibitor , ac - devd - cho , for 10 min at room temperature before adding the substrate solution .

enantiomerically pure alcohols and amines constitute important synthetic building blocks and key targets in the manufacturing of a wide range of chemical products , such as agrochemicals , food additives , fragrances , and pharmaceuticals . consequently , significant efforts have been dedicated to the enantioselective synthesis of these compounds , including catalytic protocols for carbon - heteroatom bond formation , hydrogenations of ketones / imines , nucleophilic addition to carbonyl compounds , and kinetic resolution ( kr ) . of these methods , enzymatic kr of racemic mixtures is the most common way to access enantiomerically pure alcohols and amines on an industrial scale , owing to its high performance in terms of activity and selectivity . for the kr of these compounds , the process can be made either ( r)- or ( s)-selective depending on whether a lipase or a serine protease is chosen as the enzymatic resolving agent . in most of the reported kr protocols of alcohols and amines , the enzyme resolves the racemic substrate through selective acylation of one of its enantiomers , which allows for the isolation of the enantiopure alcohol or amine using conventional purification techniques . the acyl group being transferred to substrate by the enzyme comes from a so - called acyl donor , which is added to the reaction in at least equimolar amounts in regard to the substrate . since this transesterification process is fully reversible , highly activated esters or enol esters are commonly employed as acyl donors to push the reactions toward the formation of the acylated product . unfortunately , enzymatic kr , as all other resolution methods , suffers from the limitation that the maximum theoretical yield is only 50% . an efficient way to overcome this drawback and achieve a theoretical yield of 100% is to combine the resolution process with in situ racemization in a so - called dynamic kinetic resolution ( dkr ) ( scheme 1 ) . to date , a variety of protocols for the racemization of alcohols and amines have been developed , and these involve for example acid / base catalysts , transition - metal complexes , metal nanoparticles , or enzymes . however , the design of a successful dkr system is far from simple , given that the following requirements must be fulfilled : ( i ) the kr must display a sufficient enantioselectivity ( e value = kfast / kslow 20 ) ; ( ii ) the enzyme and the racemization catalyst must be compatible with one another ; ( iii ) the rate of racemization ( krac ) must be at least 10 times faster than the enzyme - catalyzed reaction of the slow reacting enantiomer ( kslow ) ; and ( iv ) the racemization catalyst must not react with the product formed from the resolution . among these requirements , the compatibility between the enzyme and racemization catalyst is generally the critical issue , since these catalysts often operate optimally under very different condition . it is also common that the racemization catalyst interferes with the enzymatic resolution or that the enzyme and its accompanying additives ( e.g. , surfactants and stabilizers ) have an inhibitory effect on the racemization catalyst . as a result of this compatibility issue , the identification of reaction conditions that enable both high enantioselectivity of the kr and efficient racemization has been a reoccurring challenge within the field of dkr . this perspective summarizes the key features of the extensive research that has been dedicated to the chemoenzymatic dkr of alcohols and amines during the past two decades . the aim is to cover both the biological and chemical aspects of the dkr , by discussing topics ranging from the design of enzyme - compatible racemization catalysts to enzyme engineering . further , we wish to point out the current state - of - the - art dkr protocols and their respective limitations , in an attempt to highlight novel avenues for future research . in 1996 , williams reported the dkr of an allylic acetate derivative by the combinative use of pseudomonas fluorescens lipase and pdcl2(mecn)2 to give the corresponding allylic alcohol in 81% yield and 96% ee after 19 days ( scheme 2a ) . in this reaction , the acetylated alcohol was deracemized by the coupling of a lipase - catalyzed ester hydrolysis to a racemization proceeding via palladium(ii)-mediated 1,3-acetate shift . although , the reaction proceeded at an impractical rate , this seminal study was an important first step that demonstrated the possibility of combining transition metal catalysis with enzyme catalysis for achieving dkr . in a subsequent study , williams and co - workers developed a method for the dkr of secondary alcohols lacking adjacent c = c double bonds by utilizing racemization catalysts operating through a reversible hydrogen - transfer mechanism . among the studied catalysts , rh2(oac)4 gave the best results and was combined with lipase - catalyzed transesterification , affording ( r)-1-phenylethanol with 60% conversion and 98% ee ( scheme 2b ) . unfortunately , this method suffers from several critical drawbacks , such as a low conversion of the overall dkr and the necessity of both o - phenanthroline and acetophenone as additives for efficient racemization . however , despite the disadvantages of this particular dkr system , this study was important since it demonstrated the potential of using metal catalysts operating via transfer hydrogenation mechanisms as a general method for racemizing alcohols . this work became a great source of inspiration for subsequent catalyst design , triggering the development of a great number of racemization catalysts based on different transition metals , which all functioned through different transfer hydrogenation mechanisms ( vide infra ) . unfortunately , it is beyond the scope and purpose of this perspective to provide an in - depth mechanistic discussion for these transfer hydrogenative racemization processes , and thus we kindly refer interested readers to some recent reviews that cover this topic . following the pioneering work of williams , the group of bckvall developed the first practical system for the dkr of secondary alcohols , which involved candida antarctica lipase b ( calb ) immobilized on acrylic resin ( also known under the trade name novozyme-435 ) and shvo s dimeric ruthenium complex 1 ( scheme 3 ) . this protocol was found to be compatible with a wide range of aliphatic and benzylic alcohols , providing the corresponding ( r)-acetates in high yields and ee s . a drawback of this dkr system is that the shvo complex 1 requires 70 c to efficiently split into the two monomeric species 1a and 1b , which mediates racemization through an outer sphere redox mechanism . because of this heat activation , the shvo complex 1 can only be combined with thermostable lipases , which limits the number of enzymes that can be used in the dkr . for example , sensitive serine proteases that exhibit ( s)-selectivity according to kazlauskas rule can not be used together with 1 . another issue of the shvo complex 1 was that it had to be used together with activated aryl esters such as p - chlorophenyl acetate , since simpler alkenyl acetates as acyl donors were found to interfere with the racemization and lead to substantial formation of ketone side products . despite these limitations , complex 1 has been successfully combined with several lipases for the dkr of -azido alcohols , benzoins , -halo alcohols , heteroaryl ethanols , hydroxylalkanephosphonates , -hydroxy amides , hydroxyl acid esters , hydroxyl aldehydes , -hydroxy alkyl sulfones , and -nitrile alcohols . in addition , complex 1 has been applied as racemization catalyst in the dkr of -substituted primary alcohols , where racemization occurs through enolization of the intermediate aldehydes . subsequent research aimed at discovering more active catalysts that could efficiently racemize alcohols under milder reaction conditions , enabling the use of a wider range of enzymes for dkr . the group of park reported on ( -indenyl)rucl(pph3)2 , 2 , as an efficient racemization catalyst for alcohols at room temperature , which unlike 1 only produced negligible amounts of ketone byproduct . however , a severe drawback of complex 2 is that it requires koh to display catalytic activity , which is detrimental for applications in dkr as the base can hydrolyze the product acetates and also cause enzyme deactivation . it was later found that complex 2 could instead be activated by o2 and et3n , but unfortunately a higher reaction temperature ( 60 c ) was needed for efficient racemization in this case . in the latter study , complex 2 was successfully combined with pseudomonas cepacia lipase ( ps - c ) for the dkr of a small scope of simple secondary alcohols ( scheme 4 ) . the initial success achieved by combining enzymes with ruthenium - based complexes strongly influenced the subsequent research within the field of dkr , which resulted in the development of a number of protocols utilizing different transfer hydrogenation - type ruthenium complexes as the racemization catalyst . however , the first main breakthrough came in 2002 when the group of park prepared the monomeric ruthenium aminocyclopentadienyl complex 3 and demonstrated that it could efficiently racemize secondary alcohols at room temperature . unlike complex 1 , this racemization catalyst did not require any heat activation but was activated by kotbu . in contrast to koh , kotbu shows a higher compatibility with most dkr systems commonly used . complex 3 was successfully paired with novozyme-435 for the synthesis of a variety of functionalized aliphatic and benzylic ( r)-acetates in high yields and ee s at room temperature ( scheme 5 ) . an advantage of this dkr protocol was that the cheap and readily available isopropenyl acetate could be used as the acyl donor instead of activated esters , such as p - chlorophenyl acetate . unfortunately , the dkr reactions were found to progress slowly , requiring reaction times of up to 7 days , which is in sharp contrast to the separate racemization and kr reactions , which were generally complete within a few hours . this significant difference in efficiency between the dkr and the separate reactions suggests that complex 3 and calb are not fully compatible with one another , leading to partial deactivation of both catalysts . because of its good racemization activity at room temperature , complex 3 could also be combined with the more sensitive protease subtilisin carlsberg , which opened up for ( s)-selective dkr protocols of secondary alcohols . in addition , the group of kim and park has in a recent study demonstrated that complex 3 can be used together with ionic surfactant - stabilized burkholderia cepacia lipase for the dkr of allylic secondary alcohols at room temperature in excellent yields and ee s . shortly after the development of 3 , the group of bckvall prepared a related monomeric ruthenium pentaarylcyclopentadiene complex 4 , which proved to be a highly efficient catalyst that managed to fully racemize enantiomerically pure 1-phenylethanol within 10 min , even at catalyst loadings as low as 0.5 mol % . this racemization catalyst displayed many similarities to complex 3 , both in terms of structure and activation method ; however , the absence of an amino - functionality in the cyclopentadienyl ligand resulted in an improved compatibility with enzymes . thus , combination of complex 4 with calb ( novozym-435 ) afforded a fast dkr of secondary alcohols , e.g. , 1-phenylethanol was transformed to its acetate in high yield and > 99% ee in 3 h. complex 4 paved the way for a new generation of dkr and dynamic asymmetric transformation ( dykat ) protocols involving several different enzymes , many of which had not been possible to incorporate previously . dkr protocols utilizing complex 4 have been applied to the deracemization of a wide range of functionalized secondary alcohols in excellent yields and ee s , including aliphatic alcohols , allylic alcohols , chlorohydrins , diols , homoallylic alcohols , and n - heterocyclic 1,2-amino alcohols ( figure 1 ) . in the case of the dkr of 1-phenylethanol , a large scale reaction with only 0.05 mol % of complex 4 was carried out on a 1 mol - scale to furnish 159 g ( 97% yield ) of the corresponding ( r)-acetate in 99.8% ee . dkr and dykat systems involving complex 4 have also been employed in the synthesis of several biologically relevant molecules and pharmaceuticals . as with complex 3 , the racemization activity of 4 at room temperature allowed it to be combined with subtilisin carlsberg for ( s)-selective dkr protocols . for some chlorohydrins and for alcohols containing a distant olefin group , such as homoallylic alcohols and 5-hexen-2-ol , racemization occurs significantly slower with ruthenium - based catalysts such as 4 , which calls for increased reaction temperatures in the dkrs . for these substrates , further research into more efficient racemization protocols one promising way to achieve a more efficient racemization of chlorohydrins could perhaps be to match the electronic properties of catalyst and substrate as recently reported by the group of bckvall . in this study , it was found that a highly electron - deficient analogue of complex 4 gave a 1030 times faster racemization of chlorohydrins than the standard catalyst . the authors ascribed the improved racemization rate to the higher efficiency of the electron - deficient catalyst in abstracting the hydride from this electron - deficient class of substrates . in recent years , several analogous enzyme - compatible racemization catalysts based on the cyclopentadienyl ruthenium core have been synthesized ( figure 2 ) . particularly , the group of kim and park has made several key contributions to the field of alcohol dkr by developing ruthenium - based racemization catalysts exhibiting improved stability and broader scope . one of these catalysts is the benzyloxy derivative 5 that has been used as a racemization catalyst in the dkr of a number of aliphatic and benzylic secondary alcohols under air atmosphere . the possibility to run the reactions open to air constitutes a significant practical improvement compared to the previous systems involving catalysts 3 and 4 , which both require the use of dry and inert conditions to prevent catalyst deactivation . another advantage of the benzyloxy motif was that it could be exploited as a handle for linking 5 onto polystyrene to create a heterogeneous version . interestingly , the dkrs involving the polymer - bound catalyst 6 gave comparable results in terms of yield and ee to those employing homogeneous 5 , demonstrating that heterogenization of the catalyst had a negligible effect on the racemization activity . moreover , catalyst 6 exhibited good recyclability that allowed it to be reused three times in the dkr of 1-phenylethanol , where the ( r)-acetate could be obtained in 95% yield and 99% ee over all cycles . in a subsequent study , catalyst 6 was employed in the key step of the synthesis of the enantiomerically pure pharmaceutical ( )-rivastigmine . kim , park , and co - workers have also developed another air - stable analogue of complex 4 by replacing one of its carbon monoxide ligands with pph3 . the resulting catalyst 7 could be activated at room temperature by ag2o and used together with novozyme-435 for the dkr of a small set of aliphatic and benzylic alcohols in excellent ee s . recently , the group of kim and park reported on an interesting ruthenium complex 8 containing an acyl substituted cyclopentadienyl ligand . this catalyst allowed for a significant extension of the scope of enantiomerically pure secondary alcohols that can be accessed through chemoenzymatic dkr . by combining catalyst 8 with ionic surfactant - coated burkholderia cepacia lipase , the dkr of a variety of secondary alcohols was accomplished at 2560 c , including -arylpropargyl alcohols , b(pin)-substituted benzylic alcohols , -chloro alcohols , and tms - propargyl alcohols . the groups of leino and kanerva reported on the preparation of the related pentabenzylcyclopentadienyl ruthenium complex 9 , which displayed comparable activity and scope of utility to that of complex 4 . however , an advantage of 9 is that its benzyl - substituted ligand can be conveniently synthesized on a large scale from the simple and cheap starting materials cyclopentadiene and benzyl alcohol . this can be compared to the syntheses of complexes 1 and 3 - 8 , which require the significantly more expensive precursor tetraphenylcyclopentadienone . another useful racemization catalyst , which does not require the use of strong alkoxide bases , was very recently reported by nolan and co - workers . the cationic ruthenium indenyl complex 10 is efficiently activated by the mild base , k2co3 , and was successfully combined with novozyme-435 for the dkr of a variety of secondary alcohols in high yields and ee s at room temperature . recently , the group of martn - matute showed that a ruthenium catalyst , formed in situ from the readily available complex [ ru(p - cymene)cl2]2 and the ligand 1,4-bis(diphenylphosphino)butane , could be employed in combination with lipase tl from pseudomonas stutzeri for the efficient dkr of -hydroxy ketones at room temperature . the dkr of these substrates provides straightforward access to a variety of functionalized molecules , such as enantiomerically pure amino alcohol and diol derivatives . so far this perspective has mainly described protocols employing ruthenium - based racemization catalysts , but it is important to also highlight the work on secondary alcohol dkr that involves other metals . for instance , feringa , de vries , and co - workers have developed a procedure for synthesizing enantiomerically pure epoxides in one step from the corresponding chlorohydrins , by utilizing the cationic iridacycle 11 together with a doubly mutated haloalcohol dehalogenase ( hhec ) in a biphasic system comprising toluene and 50 mm hepes buffer ( scheme 6 ) . as with the monomeric ruthenium - based racemization catalysts 3 - 9 , iridacycle 11 was activated by kotbu , enabling efficient dkr at room temperature . iridacycle 11 displayed an intriguing complementary reactivity to the ruthenium systems by exhibiting a significantly higher racemization activity and selectivity toward chlorohydrins compared to conventional benzylic secondary alcohols . another iridium - catalyzed protocol for the base - free dkr of nonfunctionalized aliphatic and benzylic secondary alcohols was disclosed by marr et al . this dkr utilized a series of piano - stool-type iridium nhc complexes together with calb . a general topic of concern regarding ruthenium- and iridium - based catalytic systems for racemization is the relatively high cost and low natural availability of these metals . therefore , efforts have been made to develop more cost - effective and readily accessible metal catalysts . an example addressing this requirement is the alme3/binol / calb system designed by berkessel et al . , which was used for the dkr of both aliphatic and benzylic alcohols at room temperature . in addition , a number of vanadium - based catalytic protocols for the dkr of alcohols have been developed during the past decade . akai and co - workers demonstrated that the oxyvanadium(v ) complex [ vo(osiph3)3 ) ] could racemize secondary allylic alcohols through 1,3-transposition of the hydroxyl group under mild reaction conditions . accordingly , this catalyst was found to be compatible with several lipases , such as burkholderia cepacia lipase , novozyme-435 and pseudomonas fluorescens lipase , which allowed for dkr of a wide range of linear and cyclic allylic secondary alcohols . moreover , the developed methodology could be used to transform a stereoisomeric mixture of dienols into a single dienyl acetate product in excellent yield and ee ( scheme 7 ) . the authors also prepared heterogeneous analogues of this oxyvanadium(v ) catalyst , which were immobilized on both a polymer and a mesoporous silica . the latter heterogeneous catalyst proved to be recyclable over six cycles without any loss in activity , and furthermore , it was capable of racemizing benzylic , heteroaromatic and propargylic alcohols . the ability of this catalyst to mediate the racemization of substrates lacking the allylic alcohol motif indicates that it can also operate through a more general dehydrative mechanism proceeding via a carbocation intermediate . another heterogeneous protocol for the dkr of secondary alcohols involving vanadium catalysis was reported by wuyts et al . in this system , voso4 was combined with novozyme-435 to achieve deracemization of several benzylic alcohols in octane at 80 c . there are also a number of reports on the use of heterogeneous acids and zeolites as racemization catalysts together with lipases for dkr of secondary alcohols . however , the major limitation of most of these protocols is that they can only racemize alcohols through a dehydration mechanism , which limits their scope to substrates that can form stable carbocations . furthermore , many of these systems suffer from reduced yields of the desired dkr products due to substantial formation of elimination side products . in sharp contrast to secondary alcohols , tertiary alcohols are a significantly more cumbersome class of substrates for which there exist no practical dkr protocols . although , there are a few enzymes that can resolve tertiary alcohols , it has proven difficult to couple these kr processes to in situ racemization . since the quaternary stereocenter of tertiary alcohols lacks a hydride substituent , it is not possible to utilize any of the transfer hydrogenation - type racemization catalysts . thus , the list of available racemization catalysts for tertiary alcohols is primarily limited to those operating through dehydrative mechanisms ( e.g. , lewis acids or vanadium catalysts ) proceeding via the formation of a tertiary carbocation . the latter carbocation intermediate is formed much more readily than the corresponding secondary one , which should facilitate the racemization of tertiary alcohols . the development of a general and practical dkr protocol for tertiary alcohols would be considered as an important milestone within the field of asymmetric synthesis , given the high prevalence of this structural motif in natural products and pharmaceuticals . as with alcohols , there are a variety of efficient methods for obtaining enantiomerically pure amines by the use of enzymatic kr . however , the available dkr protocols are drastically fewer in number due to the lack of efficient amine racemization catalysts . the main reason for the difficulty of racemizing amines is that they can act as strong coordinating ligands , which may lead to inhibition or even complete deactivation of the metal catalysts . thus , high temperatures are generally required to disrupt this undesired coordination and promote the racemization reaction . as previously discussed , the use of elevated reaction temperatures is undesirable from a dkr perspective , since it restricts the set of enzymes that can be employed . an additional challenge associated with the racemization of amines is that the generated imine intermediate is highly reactive and can thus take part in several side reactions , which reduces the yield of the desired dkr product . for example , the imine is prone to undergo hydrolysis into the corresponding ketone in the presence of water . the imine intermediate can also be subject to nucleophilic attack by another amine molecule to produce an aminal , which upon elimination of an ammonium ion forms a secondary imine that can be further reduced to a secondary amine byproduct . it has been found that both of these side reactions are usually favored by an elevated temperature , which further highlights the importance of efficient and mild amine racemization protocols . the first dkr of an amine was reported by reetz and schimossek in 1996 , where resolution of 1-phenylethylamine was accomplished by coupling calb - catalyzed amine acylation to pd / c - catalyzed racemization . unfortunately , the dkr reaction , which was performed in triethylamine at 5055 c using ethyl acetate as the acyl donor , was found to proceed slowly , and despite a reaction time of 8 days , it only gave a moderate conversion of 60% . following this work , the group of bckvall demonstrated that the shvo dimer 1 can be used as an efficient racemization catalyst for primary amines at 110 c . as a result of the high temperature , the enzymatic resolution was run separately at a lower temperature , and therefore the racemization and resolution had to be done stepwise . however , this problem was later circumvented by changing to the methoxy - substituted shvo analogue 12 , which enabled efficient racemization at 90 c . by using complex 12 together with novozyme-435 , the one - pot dkr of several aliphatic and benzylic primary amines was achieved in high yields and excellent ee s ( scheme 8) . this protocol enabled the dkr of 1-phenylethylamine to be performed on a multigram scale , with a low catalytic loading ( 1.25 mol % ) and with a substrate concentration of up to 0.9 m , affording the corresponding ( r)-amide in good isolated yield and 98% ee . a noteworthy feature of the dkr protocol involving 12 was that isopropyl acetate could be used as the acyl donor . although , this acyl donor may seem as the ideal choice given its low price and high availability , the use of carboxylic esters as acylating agents is generally undesired in amine dkr as they give an amide product that requires harsh reaction conditions to be reconverted back to the amine . commonly , strong acids and elevated temperatures are required to cleave the stable amide bond , which may be detrimental for substrates containing sensitive functional groups . to address this issue , bckvall and co - workers developed an improved procedure for the dkr of both aliphatic and benzylic primary amines involving complex 12 and calb that worked efficiently with dibenzyl carbonate as the acyl donor . in contrast to the amide functionality , the installed benzyloxy carbonyl group can be easily removed under mild reaction conditions through pd - catalyzed hydrogenolysis . together , the broad substrate scope involving both aliphatic and benzylic primary amines , the possibility of using carbonate - based acyl donors and the scalability make this catalytic protocol one of the most practical methods for the dkr of primary amines available to date . the group of bckvall has also demonstrated that complex 12 can be used in combination with the related enzyme candida antarctica lipase a ( cala ) immobilized on siliceous mesocellular foam ( mcf ) for the dkr of -amino esters . inspired by the seminal findings of reetz and schimossek , several research groups continued to study heterogeneous racemization protocols based on palladium for application in amine dkr . the first steps toward a practical dkr method for amines using this strategy were taken by jacobs and co - workers with their investigation on how alkaline earth supports affected the racemization activity of immobilized pd particles . among the tested catalysts , pd on baso4 was found to exhibit the highest activity and selectivity . the dkr with this racemization catalyst was performed at 70 c under 0.1 bar of h2 , using novozyme-435 as the resolving agent and either ethyl acetate or isopropyl acetate as the acyl donor . under these reaction conditions , a range of benzylic primary amines were converted into the corresponding ( r)-amides in high yields and ee s within 2472 h ( scheme 10 ) . andrade et al . later demonstrated that this protocol can also be applied for the dkr of selenium - containing benzylic primary amines with good results . however , a significant drawback of this dkr system is that it is limited mainly to benzylic amines , while aliphatic primary amines generally react too slowly . the only aliphatic amine that was tolerated by this system was 1-methyl-3-phenylpropylamine , which contained a distant aryl group that was most likely the reason for the success . despite , the presence of an aromatic moiety in the structure , this aliphatic amine was found to racemize significantly more slowly than the benzylic substrates and thus the corresponding dkr required both elevated temperatures and longer reaction times to give satisfactory results . in a subsequent study , the group of de vos demonstrated that the activity and selectivity of the pd / baso4 and pd / caco3 catalysts in the racemization of primary amines could be improved by using microwave irradiation as an alternative heating method . the reason for this phenomenon is that metal clusters are capable of efficiently absorbing microwave irradiation , which results in the generation of so - called hot - spots that can reach a temperature that exceeds that of the surrounding reaction media . the use of microwave irradiation was also found to lead to faster dkrs , although the ee s of these reactions were generally lower than those performed with conventional heating in an oil bath , because of a more facile background chemical acylation under the employed microwave conditions . a useful dkr method involving heterogeneous palladium has been reported by kim , park and co - workers ( scheme 9 ) . in this protocol , nanoparticulate pd immobilized on alo(oh ) is employed as the racemization catalyst together with novozyme-435 . this catalyst combination proved effective in the dkr of a range of benzylic primary amines , enabling the preparation of the corresponding ( r)-amide products in high yields and excellent ee s . however , in line with the protocol developed by jacobs and co - workers , this catalytic system required significantly harsher reaction conditions for the dkr of aliphatic substrates ( 12 mol % pd , 100 c and 1 atm h2 ) . interestingly , both the pd nanocatalyst and the enzyme could be recycled eight times in the dkr of 1-methyl-3-phenylpropylamine without any observable decrease in either conversion or ee . in a subsequent study , the group of kim and park extended the scope of this method to also include -amino amides . in addition , bckvall and co - workers have applied the pd / alo(oh ) catalyst in combination with cala - mcf for the dkr of -amino esters . xu et al . reported on the preparation of a heterogeneous racemization catalyst based on pd immobilized on a layered double - hydroxide - dodecyl sulfate anion support and demonstrated that it could be used with novozyme-435 for the dkr of benzylic primary amines at 55 c . unfortunately this protocol suffered from several drawbacks , such as high catalyst loadings , dilute substrate concentrations , and the need of the activated ester 4-chlorophenyl valerate as acyl donor . recently , the group of bckvall also developed a palladium - based heterogeneous racemization catalyst , consisting of 1.53.0 nm - sized pd nanoparticles immobilized on aminopropyl - functionalized mcf ( amp - mcf ) . this pd nanocatalyst ( pd - amp - mcf ) exhibited high activity in the racemization of 1-phenylethylamine , and moreover it displayed good enzyme - compatibility that allowed it to be used in dkr . the pd - amp - mcf catalyst was combined with novozyme-435 for the dkr of a range of primary benzylic amines at 70 c , producing the corresponding ( r)-amides in high yields and excellent ee s ( scheme 10 ) . furthermore , by increasing the catalytic amount of palladium from 1.25 to 5.0 mol % it was possible to maintain an efficient racemization even at 50 c , which allowed the catalyst to be used in a dkr of 1-phenylethylamine with the sensitive enzyme amano lipase ps - c1 ( burkholderia cepacia lipase immobilized on ceramic beads ) . remarkably , this is the first time that amano lipase ps - c1 has been successfully utilized in a dkr of an amine . it is also important to highlight the fact that the amount of pd nanocatalyst used in these dkr reactions with reasonably short reaction times is lower than previously reported for primary amines and that the reactions are run at a substrate concentration of 0.4 m , which is significantly higher than that used in previously reported systems . other practical advantages of the pd - amp - mcf were that it displayed high stability and low leaching , which allowed it to be recycled up to four times in the dkr of 1-phenylethylamine without any observable decrease in performance . as with other palladium - based systems , this dkr system does not work well for aliphatic amines . the group of li has also studied this system for the dkr of primary amines ; however , they used a slightly different version of the pd - amp - mcf catalyst that was impregnated with k2co3 and contained a lower palladium loading than the one used by bckvall and co - workers ( 2.0 versus 8.0 wt % pd ) . even though this alternative protocol allowed for an efficient and selective dkr of several amines , it is difficult to compare its performance to the system published by the group of bckvall , as it was studied under very different reaction conditions involving increased enzyme loadings and significantly lower substrate concentrations . the mcf material that was employed by the group of bckvall and li to support the pd nanoparticles has also been used to immobilize cala . with this versatility of the mcf in mind , the group of bckvall explored the possibility of co - immobilizing pd nanoparticles and an enzyme into the cavities of this support . this was done by first preparing the pd - amp - mcf catalyst with a moderate loading of palladium to leave a number of free aminopropyl groups for the enzyme , then functionalizing the free aminopropyl groups of the support with glutaraldehyde , and finally exploiting the aldehyde groups as linkers for the anchoring of calb . by this co - immobilization strategy , a metalloenzyme - resembling bifunctional catalyst was obtained that can perform both racemization and kr ( figure 3 ) . this hybrid catalyst was evaluated in the dkr of 1-phenylethylamine using ethyl methoxy acetate as the acyl donor under 1 atm . of h2 at 70 c . under these conditions the desired ( r)-amide product was obtained in 99% yield and 99% ee within 16 h. interestingly , the reaction involving the hybrid catalyst was found to proceed faster than that of separately supported pd(0)-amp - mcf and calb - mcf , highlighting that the close proximity of the two catalysts increases the rate of the dkr . the hybrid catalyst could be recycled , but unfortunately it was found to exhibit diminished activity from the third cycle as a result of partial enzyme denaturation caused by the hydrophilic silica support surface . dkr of an amine with a bifunctional biomimetic catalyst in which pd nanoparticles and a lipase ( calb ) are co - immobilized in mcf . it is not only palladium- and ruthenium - based racemization catalysts that have been employed in amine dkr systems . the group of de vos showed that both raney ni and raney co catalysts could be combined with novozyme-435 for the dkr of primary amines . unfortunately , these dkrs were found to proceed slowly , and even though they were performed at 7080 c for 25 days , the conversions and ee s were generally low . on the other hand , this catalytic system displayed an interesting preference for aliphatic primary amines , allowing for a faster dkr of these substrates . surprisingly , by following the ee of the starting material throughout the reaction , it was established that the long reaction time was due to a rate - limiting kr process . normally , the enzymatic kr of primary amines should proceed fast at these high temperatures . these results therefore suggest that the raney metal catalysts have an inhibitory effect on the enzyme . it was found that enzyme poisoning was caused by leaching of cobalt and nickel ions and that this problem could be circumvented by performing the kr and racemization in separate pots in a successive manner . however , this greatly diminished the practical utility of this dkr protocol . another interesting dkr system based on homogeneous [ ircp*i2]2 and candida rugosa lipase was reported by page and co - workers and was used for the deracemization of a secondary amine on a multigram scale . the dkr of secondary amines is significantly more challenging , because the extra substituent on the nitrogen brings additional steric bulk that prevents the substrate from being accepted by the enzyme . despite the fact that several secondary amines could be efficiently racemized by the iridium catalyst , the authors only managed to construct a working dkr for one isoquinoline derivative ( scheme 11 ) . in nature , the stereoinversion of some amino acids is carried out by a family of racemases that utilize pyridoxal phosphate as the catalytically active prosthetic group . these enzymes operate through a so - called schiff base - type mechanism , where the catalytically active pyridoxal phosphate group reacts with an amino acid to produce an imine intermediate , from which racemization occurs via a base - mediated enamine - imine interconversion . inspired by this class of natural racemases , felten et al . developed a synthetic active - site analogue by complexating zn(otf)2 to picolinaldehyde this racemization catalyst was successfully combined with the enzyme alacase ( another name for subtilisin carlsberg ) for the dkr of a small series of -branched amino acids in high enantioselectivity at room temperature . recently , several reports on metal - free methods for the dkr of amines have appeared . the group of gil and bertrand has shown that racemization of amines can be achieved by the use of in situ generated sulfanyl radicals and that it is possible to couple this process to enzymatic acylation . although this method has so far only been applied to the dkr of simple primary amines containing no or little functionality , it is promising since the racemization occurs under mild reaction conditions , which enables the use of sensitive proteases . another well - established method to racemize amino acid derivatives containing acidic -protons is to employ a base that is sufficiently strong to deprotonate these substrates . for example , tessaro and co - workers have successfully utilized the organic base 1,8-diazabicycloundec-7-ene ( dbu ) as a racemization catalyst in the mild dkr of several amino acid derivatives by combining it with subtilisin - catalyzed thioester hydrolysis . so far , this perspective has focused almost entirely on the design of efficient racemization catalysts as a means to broaden the scope of the dkr methodology . however , it is important not to overlook the considerable amount of work that has been done to improve enzymes as biocatalysts . the properties of an enzyme can be improved by immobilization , cross - linking , surfactant - stabilization , or enzyme engineering / directed evolution . the latter topic has been greatly propelled by advances in the field of molecular biology and genetic engineering , which has led to the development of new recombinant technologies that makes it possible to incorporate tailor - made dna fragments into organisms , such as escherichia coli and pichia pastoris , and use them as hosts for the expression of mutant enzymes with novel properties . unfortunately , it is beyond the scope of this perspective to summarize all key contributions to this vast research area , and therefore we kindly refer interested readers to a number of excellent recent reviews that cover this topic thoroughly . instead , our aim is to describe some selected techniques that we believe have the potential of making an impact on the field of alcohol and amine dkr . a classical way to improve the thermostability of enzymes and thus make them available for a wider range of dkr protocols is to immobilize them on a heterogeneous support . in fact , the majority of the commercially available enzymes that are used for dkr today are already supported on various types of carriers , including ceramic beads , diatomaceous earth , ionic liquids , resins , and silicas . in addition to improving the thermostability , the immobilization of enzymes also lead to several practical advantages , such as easier handling , simpler separation , and possibilities of recycling . another intriguing method to improve the general performance of enzymes is to polymerize them into so - called cross - linked enzyme aggregates ( cleas ) by the use of a bifunctional cross - linking agent . typically , glutaraldehyde is used for this purpose as it can react with free lysine residues on the surface of two neighboring enzyme molecules and covalently link them together through stable schiff - base - type bonds . this cross - linking leads to the formation and precipitation of large insoluble enzyme aggregates , which can be easily separated by either centrifugation or filtration . remarkably , these cleas often display comparable catalytic activity to that of the free enzyme , suggesting that the enzyme is locked in its active conformation in the aggregate and that diffusion of substrate into the enzyme s active site is not significantly hindered . furthermore , this aggregation strategy has been shown to lead to dramatic improvements of the stability of the enzyme toward elevated temperatures , hostile solvents , and autoproteolysis . these improvements are a direct consequence of the decrease in flexibility , which suppresses deactivation through denaturation . an additional advantage of the clea methodology is that it combines the processes of enzyme purification and immobilization into a single operation . consequently , it is possible to apply this method directly on crude extracts instead of pure enzyme solutions . today , a substantial number of cleas based on lipases and proteases have been developed and successfully used for the preparation of enantiomerically pure alcohols and amines . despite these achievements , there is to the best of our knowledge only one group , who has so far studied clea for applications in dkr . tessaro and co - workers studied the advantages of the clea methodology in dkrs of amino acid derivatives involving subtilisin carlsberg and dbu . here , subtilisin carlsberg was found to exhibit a higher tolerance to dbu when it was turned into a clea , which enabled a more efficient dkr . the promising results from this work suggest that the clea methodology could find applications in other dkr protocols as well . for example , in the case of amine dkr , where the racemization catalysts require elevated reaction temperatures , there is a need for thermostable enzymes and here the use of cleas could be advantageous . an alternative approach to prepare heterogeneous enzyme composites reminiscent of the clea methodology was recently reported by the group of zare . in this method , flower - shaped protein - inorganic hybrid nanostructures could be generated upon addition of cu(ii ) ions to enzymes , such as cala , carbonic anhydrase , laccase , and -lactalbumin . as in the case with the cleas , the nanoflowers were shown to exhibit a significantly higher thermostability than the free enzymes . interestingly , the nanoflowers also exhibited significantly enhanced catalytic activities , which were ascribed to the high surface area and the confinement of the enzymes in the nanoflowers . synthesized a similar protein - inorganic hybrid by mixing calb with pd(oac)2 in aqueous media . in this reaction , the calb acts as a reducing agent for the pd(ii ) ions , which leads to the formation of small pd nanoparticles within the emerging polymeric enzyme composite . the pd / calb composite was shown to display both acylation and racemization activity , which allowed it to be employed as a bifunctional catalyst in the dkr of 1-phenylethylamine in excellent yield and ee ( scheme 12 ) . for most dkr applications , it is crucial that the enzyme operates efficiently in organic solvents since most substrates and racemization catalysts are not soluble in aqueous media . one way to improve the activity and stability of a protease in organic solvents is to coat it with a lipid or surfactant before lyophilization . this treatment generates a reversed micelle around the enzyme with concomitant solubilization of small amounts of water , which provides the protease with a stable aqueous microenvironment that is maintained even when it is suspended in an organic solvent . several examples on the successful use of surfactants to stabilize enzymes and enable dkrs have already been presented in this perspective . alternative ways of obtaining enzyme mutants with improved properties involve rational enzyme design and directed evolution . in the rational design approach , amino acid residues that are anticipated to play a key role for the function of the enzymes are first identified by the means of for example x - ray crystallography , homology studies , or computational models . these amino acids are then selectively replaced by other amino acid residues that are expected to yield a mutant variant displaying the desired properties . unfortunately , the structural information on the enzyme that is needed to guide such efforts is in most cases limited , which imposes a severe restriction on when this methodology can be utilized . in fact , even with this knowledge in hand , it is often difficult to predict what structural modifications that should be incorporated in order to improve the performance of the enzyme . this is because our understanding of the chemical principles that govern the function and stability of enzymes is still very limited . despite this issue , rational design has been successfully used at multiple occasions for improving the performance of lipases . for example , hult and co - workers used a rational design approach to create a calb mutant that exhibited reverse enantioselectivity ( s ) as well as an improved substrate tolerance toward bulky secondary alcohols . interestingly , this dramatic alteration of the catalytic properties was achieved by exchanging a single amino acid residue in the so - called stereospecificity pocket of the enzyme . in the wild - type calb , the fast - reacting enantiomer places its medium - sized group in the stereoselectivity pocket and its large group toward the entrance of the active site . the access of the large group to the stereoselectivity pocket is effectively prevented by three sterically demanding amino acid residues : thr42 , ser47 and trp104 . the authors identified that a mutant with fundamentally different substrate preference could be generated by changing the sterically demanding trp104 to a smaller alanine residue . subsequently , bckvall and co - workers used this enzyme variant , denoted as calb w104a , together with complex 4 for the ( s)-selective dkr of a series of bulky 1-phenylalkanols in high yields and ee s ( scheme 13 ) . following this study , the group of bckvall and hult explored calb w104 as the resolving enzyme for diarylmethanols ; however , satisfactory e values were only obtained for substrates where the two aryl substituents differed significantly in size . as a result , the development of a dkr protocol for the latter substrate class was never pursued . the group of kim and park recently solved the dkr of this substrate class by using ruthenium complex 8 together with activated lipoprotein lipase . recently , ema , sakai , and co - workers redesigned burkholderia cepacia lipase by introducing two alterations , i287f and i290a , into the catalytically active site using a rational design approach . this double mutant removed a substantial part of the steric congestion in the active site , which enabled this enzyme variant to accept a wide range of extremely bulky secondary alcohols . furthermore , it was found that the phenylalanine residue introduced at position 287 could participate in an additional c h/-interaction with the substrate alcohol , which helped to stabilize the transition state of the acylation reaction and led to an improved ( r)-selectivity of the enzyme . although , the authors only evaluated this burkholderia cepacia lipase variant for kr purposes , it is reasonable to envision that a mild dkr protocol could be constructed by combining this enzyme with any of the available ruthenium - based racemization catalysts . in comparison to rational design , generation of large enzyme libraries with subsequent screening and selection ( e.g. , directed evolution ) is a more useful method for accessing enzyme mutants with improved properties . in this directed evolution approach , natural evolution is artificially mimicked under laboratory settings to create a darwinian - type selection process that will favor emergence of a desired mutant . in practice , this is done by performing iterative cycles of : ( i ) generation of gene libraries from the parent wild - type enzyme by the use of various mutagenesis techniques ; ( ii ) expression of the corresponding enzymes from the gene libraries ; ( iii ) screening of the enzyme mutants for a desired property using various high - throughput methods ; and ( iv ) selecting an improved mutant as a template for the next round of mutagenesis / expression / screening ( figure 4 ) . to date , the directed evolution methodology has been successfully used to modulate several properties of enzymes , including solvent tolerance , thermostability , and higher enantioselectivity for a broader scope of substrates . schematic representation of the directed evolution methodology , where an iterative number of mutagenesis , expression , screening and selection cycles is conducted until an enzyme mutant with desired properties has been obtained . the field of alcohol dkr has certainly advanced significantly during the past two decades and reached a high level of maturity . today , a wide range of functionalized primary and secondary alcohols can be efficiently resolved by the use of chemoenzymatic dkr . the key to this progress has been the successful design of several racemization catalysts , particularly those based on ruthenium , that can racemize alcohols under mild reaction conditions , thereby enabling the use of an increased number of enzymes . ultimately , it is the enzyme component of the dkr that determines what types of substrates that can be resolved and which enantiomer of the product that is favored . therefore , it is essential to have access to dkr protocols that involve a variety of enzymes . so far , lipases have been the enzymes of choice for dkrs , owing to their high activity and selectivity . moreover , these enzymes are associated with a number of practical advantages , including high commercial availability , high thermostability , and good tolerance toward organic reaction media . however , as mentioned previously most naturally occurring lipases preferentially give ( r)-selective resolution of secondary alcohols , and this imposes a limitation for the dkr , since the ( s)-product can not be accessed directly . it is therefore important to have access to ( s)-selective enzymes so that the ( s)-product can be prepared directly by dkr . examples of ( s)-selective enzymes in kr of alcohols are serine proteases , but they are unfortunately not very thermostable . for most secondary alcohols , racemization can be accomplished in reasonable times at room temperature thanks to the most recently developed ruthenium catalysts , which enable dkr systems involving serine proteases . however , for certain challenging substrate classes , such as chlorohydrins and alcohols containing distant olefin groups , the performance of the available ruthenium catalysts is not sufficient to allow for mild dkr s , and here there exists an opportunity for new catalyst design . another important topic of research within the field of alcohol dkr is to develop efficient racemization protocols for tertiary alcohols , which are compatible with the currently available enzymatic kr processes . in contrast to alcohols , the available dkr systems for amines are significantly fewer in number due to challenges associated with the racemization of these substrates . despite the considerable amount of research that has been dedicated to amine dkr , most of the reported protocols still involve racemization catalysts that require high reaction temperatures to function efficiently , which greatly restrict the set of enzymes that can be employed . moreover , the majority of these dkr protocols have only been successful with substrates that are readily racemized , such as -amino acid derivatives and benzylic amines . when it comes to aliphatic amines , the available dkr protocols are significantly fewer in number and generally involve harsh reaction conditions . here , the recently developed metal - free methods to racemize amines by the use of sulfanyl radicals show great promise and might hold the key to mild dkr of both aliphatic and benzylic amines . however , a major concern regarding the racemization by sulfanyl radicals is that it has so far only been combined with a limited number of enzymes , and it is still unclear how widely applicable this method is for dkr . research efforts dedicated toward improving the enzyme component will also play an important role in advancing the field of alcohol and amine dkr . with available molecular biological techniques , chemists now have access to methods for improving and expanding the portfolio of enzymes provided by nature . in particular , evolution of enzymes via generation of large libraries with subsequent screening and selection is a highly useful method for obtaining new enzyme variants with improved properties . until very recently , all screening studies on lipase libraries for increased enantioselectivity had dealt with the hydrolysis of esters in an aqueous medium . however , most dkrs of alcohols and amines are carried out as transacylations in an organic solvent . recently , a method was reported that enables evolution of a lipase for transacylation of secondary alcohols in organic solvent , and it was demonstrated that cala gave a double mutant ( calay93l / l367i ) with a significantly improved e value , 100 vs 3 , in the transacylation of 1-phenylethanol in isooctane . this method is promising and may provide new improved enzymes for the dkr of alcohols and amines . another promising technique to improve the thermostability of enzymes is the clea methodology , where enzymes are converted into heterogeneous aggregates through treatment with a bifunctional cross - linking agent . it is expected that the clea methodology will find future applications in dkr of alcohols and amines .

chemoenzymatic dynamic kinetic resolution ( dkr ) constitutes a convenient and efficient method to access enantiomerically pure alcohol and amine derivatives . this perspective highlights the work carried out within this field during the past two decades and pinpoints important avenues for future research . first , the perspective will summarize the more developed area of alcohol dkr , by delineating the way from the earliest proof - of - concept protocols to the current state - of - the - art systems that allows for the highly efficient and selective preparation of a wide range of enantiomerically pure alcohol derivatives . thereafter , the perspective will focus on the more challenging dkr of amines , by presenting the currently available homogeneous and heterogeneous methods and their respective limitations . in these two parts , significant attention will be dedicated to the design of efficient racemization methods as an important means of developing milder dkr protocols . in the final part of the perspective , a brief overview of the research that has been devoted toward improving enzymes as biocatalysts is presented .

Introduction Early Combinations of Metal Catalysts and Enzymes for the DKR of Secondary Alcohols Development of More Practical Protocols for the DKR of More Functionalized Alcohols The more Challenging DKR of Amines Methods for Improving Enzymes As Biocatalysts Summary and Outlook

enantiomerically pure alcohols and amines constitute important synthetic building blocks and key targets in the manufacturing of a wide range of chemical products , such as agrochemicals , food additives , fragrances , and pharmaceuticals . consequently , significant efforts have been dedicated to the enantioselective synthesis of these compounds , including catalytic protocols for carbon - heteroatom bond formation , hydrogenations of ketones / imines , nucleophilic addition to carbonyl compounds , and kinetic resolution ( kr ) . of these methods , enzymatic kr of racemic mixtures is the most common way to access enantiomerically pure alcohols and amines on an industrial scale , owing to its high performance in terms of activity and selectivity . in most of the reported kr protocols of alcohols and amines , the enzyme resolves the racemic substrate through selective acylation of one of its enantiomers , which allows for the isolation of the enantiopure alcohol or amine using conventional purification techniques . an efficient way to overcome this drawback and achieve a theoretical yield of 100% is to combine the resolution process with in situ racemization in a so - called dynamic kinetic resolution ( dkr ) ( scheme 1 ) . however , the design of a successful dkr system is far from simple , given that the following requirements must be fulfilled : ( i ) the kr must display a sufficient enantioselectivity ( e value = kfast / kslow 20 ) ; ( ii ) the enzyme and the racemization catalyst must be compatible with one another ; ( iii ) the rate of racemization ( krac ) must be at least 10 times faster than the enzyme - catalyzed reaction of the slow reacting enantiomer ( kslow ) ; and ( iv ) the racemization catalyst must not react with the product formed from the resolution . as a result of this compatibility issue , the identification of reaction conditions that enable both high enantioselectivity of the kr and efficient racemization has been a reoccurring challenge within the field of dkr . this perspective summarizes the key features of the extensive research that has been dedicated to the chemoenzymatic dkr of alcohols and amines during the past two decades . the aim is to cover both the biological and chemical aspects of the dkr , by discussing topics ranging from the design of enzyme - compatible racemization catalysts to enzyme engineering . further , we wish to point out the current state - of - the - art dkr protocols and their respective limitations , in an attempt to highlight novel avenues for future research . following the pioneering work of williams , the group of bckvall developed the first practical system for the dkr of secondary alcohols , which involved candida antarctica lipase b ( calb ) immobilized on acrylic resin ( also known under the trade name novozyme-435 ) and shvo s dimeric ruthenium complex 1 ( scheme 3 ) . this protocol was found to be compatible with a wide range of aliphatic and benzylic alcohols , providing the corresponding ( r)-acetates in high yields and ee s . despite these limitations , complex 1 has been successfully combined with several lipases for the dkr of -azido alcohols , benzoins , -halo alcohols , heteroaryl ethanols , hydroxylalkanephosphonates , -hydroxy amides , hydroxyl acid esters , hydroxyl aldehydes , -hydroxy alkyl sulfones , and -nitrile alcohols . in addition , complex 1 has been applied as racemization catalyst in the dkr of -substituted primary alcohols , where racemization occurs through enolization of the intermediate aldehydes . in the latter study , complex 2 was successfully combined with pseudomonas cepacia lipase ( ps - c ) for the dkr of a small scope of simple secondary alcohols ( scheme 4 ) . the initial success achieved by combining enzymes with ruthenium - based complexes strongly influenced the subsequent research within the field of dkr , which resulted in the development of a number of protocols utilizing different transfer hydrogenation - type ruthenium complexes as the racemization catalyst . in addition , the group of kim and park has in a recent study demonstrated that complex 3 can be used together with ionic surfactant - stabilized burkholderia cepacia lipase for the dkr of allylic secondary alcohols at room temperature in excellent yields and ee s . shortly after the development of 3 , the group of bckvall prepared a related monomeric ruthenium pentaarylcyclopentadiene complex 4 , which proved to be a highly efficient catalyst that managed to fully racemize enantiomerically pure 1-phenylethanol within 10 min , even at catalyst loadings as low as 0.5 mol % . dkr protocols utilizing complex 4 have been applied to the deracemization of a wide range of functionalized secondary alcohols in excellent yields and ee s , including aliphatic alcohols , allylic alcohols , chlorohydrins , diols , homoallylic alcohols , and n - heterocyclic 1,2-amino alcohols ( figure 1 ) . in the case of the dkr of 1-phenylethanol , a large scale reaction with only 0.05 mol % of complex 4 was carried out on a 1 mol - scale to furnish 159 g ( 97% yield ) of the corresponding ( r)-acetate in 99.8% ee . particularly , the group of kim and park has made several key contributions to the field of alcohol dkr by developing ruthenium - based racemization catalysts exhibiting improved stability and broader scope . one of these catalysts is the benzyloxy derivative 5 that has been used as a racemization catalyst in the dkr of a number of aliphatic and benzylic secondary alcohols under air atmosphere . interestingly , the dkrs involving the polymer - bound catalyst 6 gave comparable results in terms of yield and ee to those employing homogeneous 5 , demonstrating that heterogenization of the catalyst had a negligible effect on the racemization activity . in a subsequent study , catalyst 6 was employed in the key step of the synthesis of the enantiomerically pure pharmaceutical ( )-rivastigmine . the resulting catalyst 7 could be activated at room temperature by ag2o and used together with novozyme-435 for the dkr of a small set of aliphatic and benzylic alcohols in excellent ee s . this catalyst allowed for a significant extension of the scope of enantiomerically pure secondary alcohols that can be accessed through chemoenzymatic dkr . by combining catalyst 8 with ionic surfactant - coated burkholderia cepacia lipase , the dkr of a variety of secondary alcohols was accomplished at 2560 c , including -arylpropargyl alcohols , b(pin)-substituted benzylic alcohols , -chloro alcohols , and tms - propargyl alcohols . the groups of leino and kanerva reported on the preparation of the related pentabenzylcyclopentadienyl ruthenium complex 9 , which displayed comparable activity and scope of utility to that of complex 4 . the cationic ruthenium indenyl complex 10 is efficiently activated by the mild base , k2co3 , and was successfully combined with novozyme-435 for the dkr of a variety of secondary alcohols in high yields and ee s at room temperature . recently , the group of martn - matute showed that a ruthenium catalyst , formed in situ from the readily available complex [ ru(p - cymene)cl2]2 and the ligand 1,4-bis(diphenylphosphino)butane , could be employed in combination with lipase tl from pseudomonas stutzeri for the efficient dkr of -hydroxy ketones at room temperature . the dkr of these substrates provides straightforward access to a variety of functionalized molecules , such as enantiomerically pure amino alcohol and diol derivatives . so far this perspective has mainly described protocols employing ruthenium - based racemization catalysts , but it is important to also highlight the work on secondary alcohol dkr that involves other metals . for instance , feringa , de vries , and co - workers have developed a procedure for synthesizing enantiomerically pure epoxides in one step from the corresponding chlorohydrins , by utilizing the cationic iridacycle 11 together with a doubly mutated haloalcohol dehalogenase ( hhec ) in a biphasic system comprising toluene and 50 mm hepes buffer ( scheme 6 ) . in addition , a number of vanadium - based catalytic protocols for the dkr of alcohols have been developed during the past decade . accordingly , this catalyst was found to be compatible with several lipases , such as burkholderia cepacia lipase , novozyme-435 and pseudomonas fluorescens lipase , which allowed for dkr of a wide range of linear and cyclic allylic secondary alcohols . the development of a general and practical dkr protocol for tertiary alcohols would be considered as an important milestone within the field of asymmetric synthesis , given the high prevalence of this structural motif in natural products and pharmaceuticals . however , the available dkr protocols are drastically fewer in number due to the lack of efficient amine racemization catalysts . it has been found that both of these side reactions are usually favored by an elevated temperature , which further highlights the importance of efficient and mild amine racemization protocols . following this work , the group of bckvall demonstrated that the shvo dimer 1 can be used as an efficient racemization catalyst for primary amines at 110 c . together , the broad substrate scope involving both aliphatic and benzylic primary amines , the possibility of using carbonate - based acyl donors and the scalability make this catalytic protocol one of the most practical methods for the dkr of primary amines available to date . in a subsequent study , the group of de vos demonstrated that the activity and selectivity of the pd / baso4 and pd / caco3 catalysts in the racemization of primary amines could be improved by using microwave irradiation as an alternative heating method . this catalyst combination proved effective in the dkr of a range of benzylic primary amines , enabling the preparation of the corresponding ( r)-amide products in high yields and excellent ee s . reported on the preparation of a heterogeneous racemization catalyst based on pd immobilized on a layered double - hydroxide - dodecyl sulfate anion support and demonstrated that it could be used with novozyme-435 for the dkr of benzylic primary amines at 55 c . the pd - amp - mcf catalyst was combined with novozyme-435 for the dkr of a range of primary benzylic amines at 70 c , producing the corresponding ( r)-amides in high yields and excellent ee s ( scheme 10 ) . furthermore , by increasing the catalytic amount of palladium from 1.25 to 5.0 mol % it was possible to maintain an efficient racemization even at 50 c , which allowed the catalyst to be used in a dkr of 1-phenylethylamine with the sensitive enzyme amano lipase ps - c1 ( burkholderia cepacia lipase immobilized on ceramic beads ) . other practical advantages of the pd - amp - mcf were that it displayed high stability and low leaching , which allowed it to be recycled up to four times in the dkr of 1-phenylethylamine without any observable decrease in performance . the group of li has also studied this system for the dkr of primary amines ; however , they used a slightly different version of the pd - amp - mcf catalyst that was impregnated with k2co3 and contained a lower palladium loading than the one used by bckvall and co - workers ( 2.0 versus 8.0 wt % pd ) . even though this alternative protocol allowed for an efficient and selective dkr of several amines , it is difficult to compare its performance to the system published by the group of bckvall , as it was studied under very different reaction conditions involving increased enzyme loadings and significantly lower substrate concentrations . on the other hand , this catalytic system displayed an interesting preference for aliphatic primary amines , allowing for a faster dkr of these substrates . the dkr of secondary amines is significantly more challenging , because the extra substituent on the nitrogen brings additional steric bulk that prevents the substrate from being accepted by the enzyme . developed a synthetic active - site analogue by complexating zn(otf)2 to picolinaldehyde this racemization catalyst was successfully combined with the enzyme alacase ( another name for subtilisin carlsberg ) for the dkr of a small series of -branched amino acids in high enantioselectivity at room temperature . recently , several reports on metal - free methods for the dkr of amines have appeared . so far , this perspective has focused almost entirely on the design of efficient racemization catalysts as a means to broaden the scope of the dkr methodology . however , it is important not to overlook the considerable amount of work that has been done to improve enzymes as biocatalysts . the latter topic has been greatly propelled by advances in the field of molecular biology and genetic engineering , which has led to the development of new recombinant technologies that makes it possible to incorporate tailor - made dna fragments into organisms , such as escherichia coli and pichia pastoris , and use them as hosts for the expression of mutant enzymes with novel properties . instead , our aim is to describe some selected techniques that we believe have the potential of making an impact on the field of alcohol and amine dkr . today , a substantial number of cleas based on lipases and proteases have been developed and successfully used for the preparation of enantiomerically pure alcohols and amines . interestingly , the nanoflowers also exhibited significantly enhanced catalytic activities , which were ascribed to the high surface area and the confinement of the enzymes in the nanoflowers . in this reaction , the calb acts as a reducing agent for the pd(ii ) ions , which leads to the formation of small pd nanoparticles within the emerging polymeric enzyme composite . in the rational design approach , amino acid residues that are anticipated to play a key role for the function of the enzymes are first identified by the means of for example x - ray crystallography , homology studies , or computational models . in the wild - type calb , the fast - reacting enantiomer places its medium - sized group in the stereoselectivity pocket and its large group toward the entrance of the active site . subsequently , bckvall and co - workers used this enzyme variant , denoted as calb w104a , together with complex 4 for the ( s)-selective dkr of a series of bulky 1-phenylalkanols in high yields and ee s ( scheme 13 ) . as a result , the development of a dkr protocol for the latter substrate class was never pursued . this double mutant removed a substantial part of the steric congestion in the active site , which enabled this enzyme variant to accept a wide range of extremely bulky secondary alcohols . in practice , this is done by performing iterative cycles of : ( i ) generation of gene libraries from the parent wild - type enzyme by the use of various mutagenesis techniques ; ( ii ) expression of the corresponding enzymes from the gene libraries ; ( iii ) screening of the enzyme mutants for a desired property using various high - throughput methods ; and ( iv ) selecting an improved mutant as a template for the next round of mutagenesis / expression / screening ( figure 4 ) . the field of alcohol dkr has certainly advanced significantly during the past two decades and reached a high level of maturity . today , a wide range of functionalized primary and secondary alcohols can be efficiently resolved by the use of chemoenzymatic dkr . another important topic of research within the field of alcohol dkr is to develop efficient racemization protocols for tertiary alcohols , which are compatible with the currently available enzymatic kr processes . despite the considerable amount of research that has been dedicated to amine dkr , most of the reported protocols still involve racemization catalysts that require high reaction temperatures to function efficiently , which greatly restrict the set of enzymes that can be employed . when it comes to aliphatic amines , the available dkr protocols are significantly fewer in number and generally involve harsh reaction conditions . research efforts dedicated toward improving the enzyme component will also play an important role in advancing the field of alcohol and amine dkr . recently , a method was reported that enables evolution of a lipase for transacylation of secondary alcohols in organic solvent , and it was demonstrated that cala gave a double mutant ( calay93l / l367i ) with a significantly improved e value , 100 vs 3 , in the transacylation of 1-phenylethanol in isooctane .

enantiomerically pure alcohols and amines constitute important synthetic building blocks and key targets in the manufacturing of a wide range of chemical products , such as agrochemicals , food additives , fragrances , and pharmaceuticals . consequently , significant efforts have been dedicated to the enantioselective synthesis of these compounds , including catalytic protocols for carbon - heteroatom bond formation , hydrogenations of ketones / imines , nucleophilic addition to carbonyl compounds , and kinetic resolution ( kr ) . of these methods , enzymatic kr of racemic mixtures is the most common way to access enantiomerically pure alcohols and amines on an industrial scale , owing to its high performance in terms of activity and selectivity . in most of the reported kr protocols of alcohols and amines , the enzyme resolves the racemic substrate through selective acylation of one of its enantiomers , which allows for the isolation of the enantiopure alcohol or amine using conventional purification techniques . to date , a variety of protocols for the racemization of alcohols and amines have been developed , and these involve for example acid / base catalysts , transition - metal complexes , metal nanoparticles , or enzymes . however , the design of a successful dkr system is far from simple , given that the following requirements must be fulfilled : ( i ) the kr must display a sufficient enantioselectivity ( e value = kfast / kslow 20 ) ; ( ii ) the enzyme and the racemization catalyst must be compatible with one another ; ( iii ) the rate of racemization ( krac ) must be at least 10 times faster than the enzyme - catalyzed reaction of the slow reacting enantiomer ( kslow ) ; and ( iv ) the racemization catalyst must not react with the product formed from the resolution . as a result of this compatibility issue , the identification of reaction conditions that enable both high enantioselectivity of the kr and efficient racemization has been a reoccurring challenge within the field of dkr . in 1996 , williams reported the dkr of an allylic acetate derivative by the combinative use of pseudomonas fluorescens lipase and pdcl2(mecn)2 to give the corresponding allylic alcohol in 81% yield and 96% ee after 19 days ( scheme 2a ) . among the studied catalysts , rh2(oac)4 gave the best results and was combined with lipase - catalyzed transesterification , affording ( r)-1-phenylethanol with 60% conversion and 98% ee ( scheme 2b ) . this work became a great source of inspiration for subsequent catalyst design , triggering the development of a great number of racemization catalysts based on different transition metals , which all functioned through different transfer hydrogenation mechanisms ( vide infra ) . unfortunately , it is beyond the scope and purpose of this perspective to provide an in - depth mechanistic discussion for these transfer hydrogenative racemization processes , and thus we kindly refer interested readers to some recent reviews that cover this topic . following the pioneering work of williams , the group of bckvall developed the first practical system for the dkr of secondary alcohols , which involved candida antarctica lipase b ( calb ) immobilized on acrylic resin ( also known under the trade name novozyme-435 ) and shvo s dimeric ruthenium complex 1 ( scheme 3 ) . another issue of the shvo complex 1 was that it had to be used together with activated aryl esters such as p - chlorophenyl acetate , since simpler alkenyl acetates as acyl donors were found to interfere with the racemization and lead to substantial formation of ketone side products . despite these limitations , complex 1 has been successfully combined with several lipases for the dkr of -azido alcohols , benzoins , -halo alcohols , heteroaryl ethanols , hydroxylalkanephosphonates , -hydroxy amides , hydroxyl acid esters , hydroxyl aldehydes , -hydroxy alkyl sulfones , and -nitrile alcohols . the initial success achieved by combining enzymes with ruthenium - based complexes strongly influenced the subsequent research within the field of dkr , which resulted in the development of a number of protocols utilizing different transfer hydrogenation - type ruthenium complexes as the racemization catalyst . however , the first main breakthrough came in 2002 when the group of park prepared the monomeric ruthenium aminocyclopentadienyl complex 3 and demonstrated that it could efficiently racemize secondary alcohols at room temperature . in addition , the group of kim and park has in a recent study demonstrated that complex 3 can be used together with ionic surfactant - stabilized burkholderia cepacia lipase for the dkr of allylic secondary alcohols at room temperature in excellent yields and ee s . shortly after the development of 3 , the group of bckvall prepared a related monomeric ruthenium pentaarylcyclopentadiene complex 4 , which proved to be a highly efficient catalyst that managed to fully racemize enantiomerically pure 1-phenylethanol within 10 min , even at catalyst loadings as low as 0.5 mol % . this racemization catalyst displayed many similarities to complex 3 , both in terms of structure and activation method ; however , the absence of an amino - functionality in the cyclopentadienyl ligand resulted in an improved compatibility with enzymes . , 1-phenylethanol was transformed to its acetate in high yield and > 99% ee in 3 h. complex 4 paved the way for a new generation of dkr and dynamic asymmetric transformation ( dykat ) protocols involving several different enzymes , many of which had not been possible to incorporate previously . dkr protocols utilizing complex 4 have been applied to the deracemization of a wide range of functionalized secondary alcohols in excellent yields and ee s , including aliphatic alcohols , allylic alcohols , chlorohydrins , diols , homoallylic alcohols , and n - heterocyclic 1,2-amino alcohols ( figure 1 ) . in the case of the dkr of 1-phenylethanol , a large scale reaction with only 0.05 mol % of complex 4 was carried out on a 1 mol - scale to furnish 159 g ( 97% yield ) of the corresponding ( r)-acetate in 99.8% ee . interestingly , the dkrs involving the polymer - bound catalyst 6 gave comparable results in terms of yield and ee to those employing homogeneous 5 , demonstrating that heterogenization of the catalyst had a negligible effect on the racemization activity . by combining catalyst 8 with ionic surfactant - coated burkholderia cepacia lipase , the dkr of a variety of secondary alcohols was accomplished at 2560 c , including -arylpropargyl alcohols , b(pin)-substituted benzylic alcohols , -chloro alcohols , and tms - propargyl alcohols . recently , the group of martn - matute showed that a ruthenium catalyst , formed in situ from the readily available complex [ ru(p - cymene)cl2]2 and the ligand 1,4-bis(diphenylphosphino)butane , could be employed in combination with lipase tl from pseudomonas stutzeri for the efficient dkr of -hydroxy ketones at room temperature . for instance , feringa , de vries , and co - workers have developed a procedure for synthesizing enantiomerically pure epoxides in one step from the corresponding chlorohydrins , by utilizing the cationic iridacycle 11 together with a doubly mutated haloalcohol dehalogenase ( hhec ) in a biphasic system comprising toluene and 50 mm hepes buffer ( scheme 6 ) . this protocol enabled the dkr of 1-phenylethylamine to be performed on a multigram scale , with a low catalytic loading ( 1.25 mol % ) and with a substrate concentration of up to 0.9 m , affording the corresponding ( r)-amide in good isolated yield and 98% ee . to address this issue , bckvall and co - workers developed an improved procedure for the dkr of both aliphatic and benzylic primary amines involving complex 12 and calb that worked efficiently with dibenzyl carbonate as the acyl donor . together , the broad substrate scope involving both aliphatic and benzylic primary amines , the possibility of using carbonate - based acyl donors and the scalability make this catalytic protocol one of the most practical methods for the dkr of primary amines available to date . the group of bckvall has also demonstrated that complex 12 can be used in combination with the related enzyme candida antarctica lipase a ( cala ) immobilized on siliceous mesocellular foam ( mcf ) for the dkr of -amino esters . despite , the presence of an aromatic moiety in the structure , this aliphatic amine was found to racemize significantly more slowly than the benzylic substrates and thus the corresponding dkr required both elevated temperatures and longer reaction times to give satisfactory results . in a subsequent study , the group of de vos demonstrated that the activity and selectivity of the pd / baso4 and pd / caco3 catalysts in the racemization of primary amines could be improved by using microwave irradiation as an alternative heating method . the use of microwave irradiation was also found to lead to faster dkrs , although the ee s of these reactions were generally lower than those performed with conventional heating in an oil bath , because of a more facile background chemical acylation under the employed microwave conditions . however , in line with the protocol developed by jacobs and co - workers , this catalytic system required significantly harsher reaction conditions for the dkr of aliphatic substrates ( 12 mol % pd , 100 c and 1 atm h2 ) . reported on the preparation of a heterogeneous racemization catalyst based on pd immobilized on a layered double - hydroxide - dodecyl sulfate anion support and demonstrated that it could be used with novozyme-435 for the dkr of benzylic primary amines at 55 c . recently , the group of bckvall also developed a palladium - based heterogeneous racemization catalyst , consisting of 1.53.0 nm - sized pd nanoparticles immobilized on aminopropyl - functionalized mcf ( amp - mcf ) . this pd nanocatalyst ( pd - amp - mcf ) exhibited high activity in the racemization of 1-phenylethylamine , and moreover it displayed good enzyme - compatibility that allowed it to be used in dkr . the pd - amp - mcf catalyst was combined with novozyme-435 for the dkr of a range of primary benzylic amines at 70 c , producing the corresponding ( r)-amides in high yields and excellent ee s ( scheme 10 ) . furthermore , by increasing the catalytic amount of palladium from 1.25 to 5.0 mol % it was possible to maintain an efficient racemization even at 50 c , which allowed the catalyst to be used in a dkr of 1-phenylethylamine with the sensitive enzyme amano lipase ps - c1 ( burkholderia cepacia lipase immobilized on ceramic beads ) . it is also important to highlight the fact that the amount of pd nanocatalyst used in these dkr reactions with reasonably short reaction times is lower than previously reported for primary amines and that the reactions are run at a substrate concentration of 0.4 m , which is significantly higher than that used in previously reported systems . other practical advantages of the pd - amp - mcf were that it displayed high stability and low leaching , which allowed it to be recycled up to four times in the dkr of 1-phenylethylamine without any observable decrease in performance . the group of li has also studied this system for the dkr of primary amines ; however , they used a slightly different version of the pd - amp - mcf catalyst that was impregnated with k2co3 and contained a lower palladium loading than the one used by bckvall and co - workers ( 2.0 versus 8.0 wt % pd ) . even though this alternative protocol allowed for an efficient and selective dkr of several amines , it is difficult to compare its performance to the system published by the group of bckvall , as it was studied under very different reaction conditions involving increased enzyme loadings and significantly lower substrate concentrations . this was done by first preparing the pd - amp - mcf catalyst with a moderate loading of palladium to leave a number of free aminopropyl groups for the enzyme , then functionalizing the free aminopropyl groups of the support with glutaraldehyde , and finally exploiting the aldehyde groups as linkers for the anchoring of calb . under these conditions the desired ( r)-amide product was obtained in 99% yield and 99% ee within 16 h. interestingly , the reaction involving the hybrid catalyst was found to proceed faster than that of separately supported pd(0)-amp - mcf and calb - mcf , highlighting that the close proximity of the two catalysts increases the rate of the dkr . these enzymes operate through a so - called schiff base - type mechanism , where the catalytically active pyridoxal phosphate group reacts with an amino acid to produce an imine intermediate , from which racemization occurs via a base - mediated enamine - imine interconversion . developed a synthetic active - site analogue by complexating zn(otf)2 to picolinaldehyde this racemization catalyst was successfully combined with the enzyme alacase ( another name for subtilisin carlsberg ) for the dkr of a small series of -branched amino acids in high enantioselectivity at room temperature . for example , tessaro and co - workers have successfully utilized the organic base 1,8-diazabicycloundec-7-ene ( dbu ) as a racemization catalyst in the mild dkr of several amino acid derivatives by combining it with subtilisin - catalyzed thioester hydrolysis . the latter topic has been greatly propelled by advances in the field of molecular biology and genetic engineering , which has led to the development of new recombinant technologies that makes it possible to incorporate tailor - made dna fragments into organisms , such as escherichia coli and pichia pastoris , and use them as hosts for the expression of mutant enzymes with novel properties . remarkably , these cleas often display comparable catalytic activity to that of the free enzyme , suggesting that the enzyme is locked in its active conformation in the aggregate and that diffusion of substrate into the enzyme s active site is not significantly hindered . in this method , flower - shaped protein - inorganic hybrid nanostructures could be generated upon addition of cu(ii ) ions to enzymes , such as cala , carbonic anhydrase , laccase , and -lactalbumin . the pd / calb composite was shown to display both acylation and racemization activity , which allowed it to be employed as a bifunctional catalyst in the dkr of 1-phenylethylamine in excellent yield and ee ( scheme 12 ) . in the rational design approach , amino acid residues that are anticipated to play a key role for the function of the enzymes are first identified by the means of for example x - ray crystallography , homology studies , or computational models . following this study , the group of bckvall and hult explored calb w104 as the resolving enzyme for diarylmethanols ; however , satisfactory e values were only obtained for substrates where the two aryl substituents differed significantly in size . in practice , this is done by performing iterative cycles of : ( i ) generation of gene libraries from the parent wild - type enzyme by the use of various mutagenesis techniques ; ( ii ) expression of the corresponding enzymes from the gene libraries ; ( iii ) screening of the enzyme mutants for a desired property using various high - throughput methods ; and ( iv ) selecting an improved mutant as a template for the next round of mutagenesis / expression / screening ( figure 4 ) . despite the considerable amount of research that has been dedicated to amine dkr , most of the reported protocols still involve racemization catalysts that require high reaction temperatures to function efficiently , which greatly restrict the set of enzymes that can be employed . here , the recently developed metal - free methods to racemize amines by the use of sulfanyl radicals show great promise and might hold the key to mild dkr of both aliphatic and benzylic amines . recently , a method was reported that enables evolution of a lipase for transacylation of secondary alcohols in organic solvent , and it was demonstrated that cala gave a double mutant ( calay93l / l367i ) with a significantly improved e value , 100 vs 3 , in the transacylation of 1-phenylethanol in isooctane .

over - the - counter ( otc ) and prescription drug misuse are the nonmedical use of medication , which is used without a prescription or for the feeling caused by the drug . otc and prescription drugs are fast becoming the drugs of choice for adolescents , with the national center on addiction and substance abuse reporting a 212% increase in prescription drug misuse among adolescents between the 1992 and 2003 period . the national survey on drug use and health and the drug abuse warning network have also documented an increase in otc drug misuse since 1992 . compton and volkow have reported that pain medication is second only to marijuana in being the most popular drug of choice for adolescents . given that parent and peer influence have been highlighted as major predictors of substance use in adolescents and that pain medications are the most widely abused prescription drugs , we hypothesize that parent and peer influence will affect pain medication misuse , specifically misuse to get high ( recreational use ) , in the same way these influences affect marijuana use . very little is known about what is responsible for the increase in prevalence and incidence rates of prescription drug misuse . it has been suggested that adolescents ' and young adults ' otc and prescription drug misuse could be explained by their perception that such drugs are safer than illicit drugs , by the ease of access to drugs and by lower societal stigma [ 1117 ] . additionally , because otc drugs are easily purchased , they can be used as an alternative when a prescription drug misuser is unable to access their preferred drug as well as mixed with alcohol to achieve a some researchers have shown that there is a strong relationship between illicit drug use and prescription drug misuse [ 13 , 1924 ] . additionally , ford highlighted the importance of family and school bonds as protective factors against misuse . ford also suggested that adolescents whose parents and peers disapprove of substance use and whose peers use drugs are more likely to engage in prescription drug misuse . marijuana use , on the other hand , has been thoroughly studied , and clear correlates of marijuana use in adolescents have been identified across the literature . specifically , sensation - seeking , risk perception of use , parent and peer attitudes towards use , adolescent and peers ' delinquent behavior , peer use , adolescent tobacco and alcohol use , and demographic variables including gender and ethnicity [ 2732 ] have all been shown to be related to adolescent marijuana use . to better understand the relationship between illicit drug use and pain medication recreational use , we explored the extent to which three known predictors of marijuana use were similarly predictive of otc and prescription pain medication recreational use . specifically , we explored the extent to which risk perception , parent disapproval , and peer disapproval were predictive of recreational use and whether risk perception mediated the effects of parent and peer disapproval . parent and peer disapproval have been strongly linked to risk perception and use in the marijuana literature [ 9 , 28 , 31 , 33 ] , hence their inclusion in the model ( see figure 1 ) . additionally , risk perception has been indicated in the health risk behaviors literature and alluded to in the prescription drug literature as a contributing variable in adolescents ' decisions to engage in these behaviors [ 12 , 14 , 27 , 34 ] . we examined the extent to which these relationships were gender dependent as the marijuana use literature has suggested that different variables are predictive of males ' and females ' use . additionally , because illicit drug use is correlated with otc and prescription drugs misuse [ 13 , 20 , 21 ] , we propose that the relationship between risk perception , parent and peer disapproval , and otc and prescription pain medication recreational use will differ for marijuana users and nonusers . this study differs from ford , in that our measures of parents ' and peers ' disapproval of misuse ( specifically recreational use ) are specific to otc and prescription drug recreational use independent of other substance use . this is especially important since previous research has shown that perceptions of prescription drug misuse often differ from perceptions of illicit drug use [ 15 , 17 ] , and these differing perceptions will subsequently affect peers ' and possibly parents ' level of disapproval . additionally , the inclusion of risk perception as a predictor variable provides some clinical utility to the study , in that it investigates the role of an easily targetable variable for intervention to prevent or decrease misuse . the inclusion of separate path analyses for otc pain medication recreational use , prescription pain medication recreational use , and marijuana use also provides for a comparison of predictors of use of the different drugs . the sample consisted of 465 college students between the ages of 18 and 24 years ( mage = 18.57 , sd = 0.86 ) . the majority of participants ( 91% ) were 18 or 19 years of age ; the remaining 9% were ages 2024 years . nineteen percent of participants ( n = 88 ) reported some ( nonzero ) lifetime otc or prescription drug recreational use with 13% ( n = 60 ) and 11% ( n = 50 ) reporting otc and prescription pain medication recreational use , respectively . twenty - nine percent of participants ( n = 134 ) reported nonzero lifetime marijuana use . risk perception / perceived susceptibility was measured by perceived personal risk , which was the extent to which participants felt they would be at risk of getting sick or hurt if they recreationally used otc pain medication , prescription pain medications , or marijuana , respectively . risk perception was measured on a 7-point evaluation scale ranging from no risk at all ( 1 ) to very much at risk ( 7 ) . to assess for parents ' and peers ' disapproval , participants were asked to report on how they felt their parents and peers would feel about them engaging in the recreational use of otc pain medication , prescription pain medication , or marijuana on a 5-point likert scale ranging from strongly approve ( 1 ) to strongly disapprove ( 5 ) . recreational use or misuse of otc and prescription pain medication was defined as use of medications to get high . participants were asked about their lifetime misuse of otc pain medication , prescription pain medication , and marijuana use on a 5-point frequency scale ( see table 1 ) . responses included never ( 1 ) , 15 times ( 2 ) , 619 times ( 3 ) , 2040 times ( 4 ) , and more than 40 times ( 5 ) . otc pain medication recreational use included the misuse of otc tylenol , motrin , advil , aleve , ibuprofen , and aspirin . prescription pain medication recreational use included the misuse of vicodin , codeine , oxycontin , and percocet . participants also reported on their recreational use of other classifications of otc and prescription drugs including tranquilizers , stimulants , sedatives , steroids , and cough and cold syrup , although these items were not included in the analyses . data was collected as part of a larger project , media influences on health risk behaviors and prescription drug use in young adults . data was collected using a survey developed by the authors specifically for the project . participants were invited to complete an online survey at computer workstations separated by desk partitions . the survey was administered via http://www.surveymonkey.com/. the online survey took approximately 3550 minutes to complete , and participants received research credit for their class upon completion of the survey . to ensure participant privacy , descriptive statistics are shown in table 2 ( for otc pain medication and prescription pain medication variables ) and table 3 ( for marijuana variables ) . path models were estimated separately for each set of variables for one of the three drugs : otc pain medication , prescription pain medication , and marijuana . parent and peer disapproval of misuse of a drug were used to predict risk perception for that drug . both disapproval variables and risk perception misuse of the drug was entered using two variables , one representing whether a respondent reported zero misuse of the drug versus nonzero misuse and the other representing amount of use for respondents who reported a nonzero amount . this model allowed for the testing of whether risk perception mediated the effects of the disapproval variables on the misuse variables , which was done using the joint significance test . for each drug , the path model was first estimated using data from all participants . it was then of interest to test whether any of the associations among the variables differed as a function of gender or as a function of use or nonuse of the other drugs . each path model was reestimated first with gender as a grouping variable and second with misuse of marijuana ( for otc and prescription pain medication models ) or otc and prescription pain medication misuse ( for the marijuana model ) . in order to be used as grouping variables , misuse variables were dichotomized into zero use versus nonzero use . because otc and prescription pain medication misuse were combined into a single grouping variable , a zero represented no misuse of either type and respondents reporting any use of either type , were scored as nonzero . the misuse - dependent variables were each entered using two variables in the model by way of the twopart feature of mplus , which scores all participants on a dichotomous use variable depending on whether they reported zero or nonzero misuse and also score amount of misuse as a separate continuous variable . amount of misuse is scored as missing for respondents who have zero use , but data from these participants was still able to be included in the models because mplus can estimate models using full information maximum likelihood , which can include partially complete data . as the amount of misuse was skewed , the mlr estimator , a variation of maximum likelihood that is more robust to nonnormal data than the more typical maximum likelihood , was used to estimate the models . regarding descriptive statistics , parent disapproval was positively correlated with peer disapproval and risk perception for marijuana and otc pain and prescription pain medication misuse variables . parent disapproval for otc pain medication was also negatively correlated with marijuana use and lifetime use . among marijuana use variables , parent disapproval was negatively correlated with lifetime use ; however , this was only true for participants who did not report any pain medication misuse . peer disapproval was positively correlated with risk perception and negatively correlated with lifetime use for marijuana and otc pain and prescription pain medication misuse variables . peer disapproval of otc pain and prescription pain medication misuse was also negatively correlated with marijuana use . risk perception was positively correlated with gender and negatively correlated with lifetime use and marijuana use for both types of pain medication variables . for marijuana variables , risk perception however , risk perception was only positively correlated with gender among participants who reported no pain medication misuse . lifetime use was positively correlated with marijuana use and negatively correlated with gender for the prescription pain variables . because mplus uses numerical integration to estimate models with dichotomous - dependent variables , it does not report the usual fit statistics or fit indices for such models . fit of the models was not an issue , though , as they included all possible associations among the variables ( note that no association between the two misuse variables could be included because the continuous variable only took on nonmissing values for respondents scoring one on the dichotomous variable ) and so were effectively saturated . note that because the data is cross - sectional , the term predictor is used for statistical purposes rather than to indicate causality . results for the path models are shown in table 4 . for otc pain medication misuse , peer disapproval ( = .516 , p < .05 ) significantly predicted risk perception , with higher levels of peer disapproval predicting higher levels of risk perception . both peer disapproval ( = .401 , p < .05 ) and risk perception ( = .221 , p < .05 ) significantly predicted dichotomous misuse , with higher levels of peer disapproval and risk perception predicting lower likelihood of nonzero misuse . because peer disapproval predicted risk perception and risk perception predicted dichotomous misuse , risk perception was a significant mediator of the effect of peer disapproval on dichotomous misuse . there were no significant predictors of amount of misuse for respondents who reported a nonzero amount . multiple groups analyses revealed no significant differences in path coefficients as a function of either gender groups ( (8 ) = 3.44 , p > .05 ) or groups defined by zero or nonzero marijuana misuse ( (8 ) = 7.60 , p > .05 ) . for prescription pain medication misuse , both parent disapproval ( = .183 , p < .05 ) and peer disapproval ( = .425 , p < .05 ) , significantly predicted risk perception , with higher levels of parent and peer disapproval predicting higher levels of risk perception . both peer disapproval ( = .424 , p < .05 ) and risk perception ( = .188 , p < .05 ) significantly predicted dichotomous misuse , with higher levels of peer disapproval and risk perception predicting lower likelihood of nonzero misuse . because peer disapproval predicted risk perception and risk perception predicted dichotomous misuse , risk perception was a significant mediator of the effect of peer disapproval on dichotomous misuse . parent disapproval ( = .286 , p < .05 ) significantly predicted amount of misuse for respondents who reported a nonzero amount , but in an unexpected direction : higher levels of disapproval predicted greater amounts of misuse . multiple groups analyses revealed no significant differences in path coefficients as a function of either gender groups ( (8 ) = 12.35 , p > .05 ) or groups defined by zero or nonzero marijuana misuse ( (8 ) = 3.13 , p > .05 ) . for marijuana misuse , both parent disapproval ( = .162 , p < .05 ) and peer disapproval ( = .493 , p < .05 ) significantly predicted risk perceptions , with higher levels of parent and peer disapproval predicting higher levels of risk perception . peer disapproval ( = .356 , p < .05 ) and risk perception ( = .409 , p < .05 ) significantly predicted dichotomous misuse , with higher levels of peer disapproval and risk perception predicting lower likelihood of nonzero misuse . parent disapproval also significantly predicted dichotomous misuse , but this effect varied depending on whether respondents were in the zero misuse or nonzero misuse group for otc and prescription pain medications , so it is discussed below . both peer disapproval ( = .262 , p < .05 ) and risk perception ( = .454 , p < .05 ) were significant predictors of amount of misuse for respondents who reported a nonzero amount , with higher levels of disapproval and risk perception predicting smaller amounts of use . because both parent and peer disapproval predicted risk perception and risk perception predicted both dichotomous misuse and amount of misuse , risk perception was a significant mediator of the effect of both parent and peer disapproval on both dichotomous misuse and amount of misuse . multiple group analyses revealed no significant differences in path coefficients as a function of gender groups ( (8 ) = 10.02 , p > .05 ) . there was a significant difference in path coefficients as a function of groups defined by zero or nonzero use of otc or prescription pain medications ( (8 ) = 18.81 , p < .05 ) . tests of invariance of the individual paths revealed that the only path to differ significantly was for parent disapproval predicting dichotomous misuse . for respondents reporting zero otc or prescription pain medication misuse , this association was significant and negative ( = .131 , p < .05 ) , so higher levels of disapproval predicted lower likelihoods of nonzero misuse . for respondents reporting nonzero otc or prescription pain misuse , this association was significant and positive ( = .282 , p < .05 ) , so higher levels of disapproval predicted higher likelihoods of nonzero misuse . to add to the understanding of otc and prescription pain medication misuse and its relationship with other substance abuse in adolescents and young adults , we explored the relationship between parent and peer disapproval of recreational use , risk perception of recreational use , and reported recreational use . the results of this study indicate that similar to marijuana use , risk perception of otc and prescription pain medication recreational use are influenced by parent and peer disapproval of recreational use , and use itself is influenced by peer disapproval and risk perception of recreational use . noteworthy was the ability of peer disapproval of misuse to predict dichotomous misuse over and above the mediated effect through risk perception , for all three drugs : otc and prescription pain medication misuse and marijuana use . this is consistent with findings in the substance abuse literature [ 31 , 33 ] and suggests that peer subculture may either encourage or act as a protective variable against adolescents engaging in the recreational use of all substances including prescription drugs . this also highlights the role of development in adolescents ' decisions to engage in health risk behaviors ; that is , they are in a period in their lives where peers are their key counterplayers and they may follow the norms outlined by their subgroup ; hence , peer disapproval has more influence on their perceptions of harm and use . these results also suggest that in order to increase risk perception and reduce recreational use of otc and prescription pain medication , program planners should work towards reducing the acceptability of otc and prescription pain medication misuse among adolescents , since they influence each others ' perceptions . the office of national drug policy and partnership for a drug - free america launched a prescription drug abuse media campaign targeting parents and health and school professionals ; however , no ads were directly marketed toward adolescents and young adults . based on the significant influence of peer disapproval on risk perception and use , future campaigns should target adolescents and young adults . parent disapproval was predictive of risk perception of prescription pain medication recreational use and marijuana use . we speculate that adolescents may take their parents ' opinions into consideration when formulating their risk perceptions on both marijuana and prescription pain medication as opposed to otc pain medication because of the restrictive access to these substances ; however , this hypothesis should be explored in future studies . parent disapproval of misuse failed to directly predict dichotomous misuse of otc and prescription pain medications ( although it did have a mediated effect through risk perception for prescription pain medications ) . this highlights the need for parents and by extension other adults working with adolescents to focus less on preventing behavior via direct methods ( e.g. , just say no campaigns ) and focus more on educating adolescents on the risks associated with the behavior . the goal of redirecting adults ' focus on educating adolescents on the risks of otc and prescription pain misuse is to influence other correlates of use , such as peer disapproval and risk perception , in order to prevent or decrease use . parent disapproval predicting dichotomous misuse of marijuana is consistent with the marijuana use literature [ 39 , 40 ] . however , the finding of reverse relationships when the sample was divided into otc and prescription pain drugs zero and nonzero misusers provides some additional data about the relationship of use for the top two drugs of choice for adolescents . specifically , adolescents who engaged in otc and prescription pain misuse were more likely to be marijuana users the more their parents disapproved of marijuana use , whereas those who were zero otc and prescription pain misusers were less likely to engage in marijuana use if their parents disapproved of marijuana use . these results are suggestive of a deviant and high sensation - seeking subculture trend in adolescents who engage in both pain medication and marijuana use and this subculture is seen across the substance abuse literature [ 9 , 31 , 33 ] . it is possible that among this subculture , parent interventions may encourage adolescents to engage in substance use , providing further need for parents to focus less on preventing behavior and more on highlighting the risks associated with the behaviors . another explanation may be that of reverse causality , whereby the more the adolescents engage in use , the more their parents disapprove of their behavior . although risk perception was significantly predictive of use in the general models , the regression weights and significance values suggest that it was a weaker predictor for otc and prescription pain medication recreational use than for marijuana use . these findings suggest that although risk perception may play an important role in adolescents ' decision making regarding substance use and other health risk behaviors [ 28 , 34 , 41 , 42 ] , risk perception is less of an important variable in adolescents ' decision - making regarding recreational use of otc and prescription pain medication . additional variables that are uncommon to the substance abuse literature , such as lower societal stigma , and comparative risk perception to street drugs may be more important in adolescents ' decision making regarding prescription drug recreational use [ 12 , 14 ] . additionally , risk perception may interact with other variables not studied here to explain adolescents ' decision making regarding use ( e.g. , general attitudes toward prescription drug use , frequency of family and friends ' medical use of prescription drugs ) . future studies should explore how other correlates of recreational use of otc and prescription pain medication interact with risk perception to influence misuse . this study was restricted to recreational users of otc and prescription pain medication ; these users do not encompass all nonmedical use ; therefore , generalizations about misuse are limited . another limitation regarding generalization is the use of a convenience sample of college students ; future studies should test this model on a community sample . another limitation of this study is that the psychosocial characteristics of the sample were not considered . adolescents and young adults are highly influenced by developmental characteristics and their external environment and future studies should explore the extent to which developmental characteristics interact with these environmental variables to predict risk perception and use . additionally , future studies should retest these paths with a larger sample ( preferably including more participants who report nonzero use ) to check for consistency of the results across samples . future studies should also compare otc and prescription pain medication misuse to other illicit drug use and other health risk behaviors . finally , the present study was cross - sectional , so temporal precedence can not be established for the assumed causal relationships between variables . despite these limitations , this study provides evidence that suggests that predictors of otc and prescription pain medication recreational use are similar to predictors of marijuana use in adolescents in that peer disapproval is a significant predictor of risk perception and misuse . additionally , both peer and parent disapproval are significant predictors of risk perception of prescription pain medication misuse . unlike marijuana use , otc and prescription pain medication misuse in adolescents are relatively less explained by risk perception suggesting that other variables may be responsible for adolescents ' decision to engage in the misuse of otc and prescription pain medication .

parent and peer disapproval were examined as potential predictors of recreational use of over - the - counter ( otc ) and prescription pain medication . risk perception was studied as a potential mediator of the effects of parent and peer disapproval . four hundred and sixty - five college students ( mage = 18.57 , sd = 0.86 ) were recruited between september 2009 and september 2010 . participants completed an online survey about their recreational medication use , other substance use , and correlates of use . path analyses showed that predictors of otc and prescription pain medication recreational use are largely similar to predictors of marijuana use in college students such that risk perception mediated both the effect of parent and peer disapproval on dichotomous misuse , and peer disapproval had a significant direct effect on dichotomous misuse . prevention interventions for recreational use of pain medication should target risk perception and peer disapproval .

1. Introduction 2. Methods 3. Results 4. Comments/Discussion

over - the - counter ( otc ) and prescription drug misuse are the nonmedical use of medication , which is used without a prescription or for the feeling caused by the drug . otc and prescription drugs are fast becoming the drugs of choice for adolescents , with the national center on addiction and substance abuse reporting a 212% increase in prescription drug misuse among adolescents between the 1992 and 2003 period . compton and volkow have reported that pain medication is second only to marijuana in being the most popular drug of choice for adolescents . given that parent and peer influence have been highlighted as major predictors of substance use in adolescents and that pain medications are the most widely abused prescription drugs , we hypothesize that parent and peer influence will affect pain medication misuse , specifically misuse to get high ( recreational use ) , in the same way these influences affect marijuana use . it has been suggested that adolescents ' and young adults ' otc and prescription drug misuse could be explained by their perception that such drugs are safer than illicit drugs , by the ease of access to drugs and by lower societal stigma [ 1117 ] . additionally , because otc drugs are easily purchased , they can be used as an alternative when a prescription drug misuser is unable to access their preferred drug as well as mixed with alcohol to achieve a some researchers have shown that there is a strong relationship between illicit drug use and prescription drug misuse [ 13 , 1924 ] . ford also suggested that adolescents whose parents and peers disapprove of substance use and whose peers use drugs are more likely to engage in prescription drug misuse . marijuana use , on the other hand , has been thoroughly studied , and clear correlates of marijuana use in adolescents have been identified across the literature . specifically , sensation - seeking , risk perception of use , parent and peer attitudes towards use , adolescent and peers ' delinquent behavior , peer use , adolescent tobacco and alcohol use , and demographic variables including gender and ethnicity [ 2732 ] have all been shown to be related to adolescent marijuana use . to better understand the relationship between illicit drug use and pain medication recreational use , we explored the extent to which three known predictors of marijuana use were similarly predictive of otc and prescription pain medication recreational use . specifically , we explored the extent to which risk perception , parent disapproval , and peer disapproval were predictive of recreational use and whether risk perception mediated the effects of parent and peer disapproval . parent and peer disapproval have been strongly linked to risk perception and use in the marijuana literature [ 9 , 28 , 31 , 33 ] , hence their inclusion in the model ( see figure 1 ) . additionally , risk perception has been indicated in the health risk behaviors literature and alluded to in the prescription drug literature as a contributing variable in adolescents ' decisions to engage in these behaviors [ 12 , 14 , 27 , 34 ] . we examined the extent to which these relationships were gender dependent as the marijuana use literature has suggested that different variables are predictive of males ' and females ' use . additionally , because illicit drug use is correlated with otc and prescription drugs misuse [ 13 , 20 , 21 ] , we propose that the relationship between risk perception , parent and peer disapproval , and otc and prescription pain medication recreational use will differ for marijuana users and nonusers . this study differs from ford , in that our measures of parents ' and peers ' disapproval of misuse ( specifically recreational use ) are specific to otc and prescription drug recreational use independent of other substance use . this is especially important since previous research has shown that perceptions of prescription drug misuse often differ from perceptions of illicit drug use [ 15 , 17 ] , and these differing perceptions will subsequently affect peers ' and possibly parents ' level of disapproval . additionally , the inclusion of risk perception as a predictor variable provides some clinical utility to the study , in that it investigates the role of an easily targetable variable for intervention to prevent or decrease misuse . the inclusion of separate path analyses for otc pain medication recreational use , prescription pain medication recreational use , and marijuana use also provides for a comparison of predictors of use of the different drugs . the sample consisted of 465 college students between the ages of 18 and 24 years ( mage = 18.57 , sd = 0.86 ) . the majority of participants ( 91% ) were 18 or 19 years of age ; the remaining 9% were ages 2024 years . nineteen percent of participants ( n = 88 ) reported some ( nonzero ) lifetime otc or prescription drug recreational use with 13% ( n = 60 ) and 11% ( n = 50 ) reporting otc and prescription pain medication recreational use , respectively . twenty - nine percent of participants ( n = 134 ) reported nonzero lifetime marijuana use . risk perception / perceived susceptibility was measured by perceived personal risk , which was the extent to which participants felt they would be at risk of getting sick or hurt if they recreationally used otc pain medication , prescription pain medications , or marijuana , respectively . risk perception was measured on a 7-point evaluation scale ranging from no risk at all ( 1 ) to very much at risk ( 7 ) . to assess for parents ' and peers ' disapproval , participants were asked to report on how they felt their parents and peers would feel about them engaging in the recreational use of otc pain medication , prescription pain medication , or marijuana on a 5-point likert scale ranging from strongly approve ( 1 ) to strongly disapprove ( 5 ) . recreational use or misuse of otc and prescription pain medication was defined as use of medications to get high . participants were asked about their lifetime misuse of otc pain medication , prescription pain medication , and marijuana use on a 5-point frequency scale ( see table 1 ) . responses included never ( 1 ) , 15 times ( 2 ) , 619 times ( 3 ) , 2040 times ( 4 ) , and more than 40 times ( 5 ) . otc pain medication recreational use included the misuse of otc tylenol , motrin , advil , aleve , ibuprofen , and aspirin . prescription pain medication recreational use included the misuse of vicodin , codeine , oxycontin , and percocet . participants also reported on their recreational use of other classifications of otc and prescription drugs including tranquilizers , stimulants , sedatives , steroids , and cough and cold syrup , although these items were not included in the analyses . data was collected as part of a larger project , media influences on health risk behaviors and prescription drug use in young adults . participants were invited to complete an online survey at computer workstations separated by desk partitions . the online survey took approximately 3550 minutes to complete , and participants received research credit for their class upon completion of the survey . to ensure participant privacy , descriptive statistics are shown in table 2 ( for otc pain medication and prescription pain medication variables ) and table 3 ( for marijuana variables ) . path models were estimated separately for each set of variables for one of the three drugs : otc pain medication , prescription pain medication , and marijuana . parent and peer disapproval of misuse of a drug were used to predict risk perception for that drug . both disapproval variables and risk perception misuse of the drug was entered using two variables , one representing whether a respondent reported zero misuse of the drug versus nonzero misuse and the other representing amount of use for respondents who reported a nonzero amount . this model allowed for the testing of whether risk perception mediated the effects of the disapproval variables on the misuse variables , which was done using the joint significance test . it was then of interest to test whether any of the associations among the variables differed as a function of gender or as a function of use or nonuse of the other drugs . each path model was reestimated first with gender as a grouping variable and second with misuse of marijuana ( for otc and prescription pain medication models ) or otc and prescription pain medication misuse ( for the marijuana model ) . because otc and prescription pain medication misuse were combined into a single grouping variable , a zero represented no misuse of either type and respondents reporting any use of either type , were scored as nonzero . the misuse - dependent variables were each entered using two variables in the model by way of the twopart feature of mplus , which scores all participants on a dichotomous use variable depending on whether they reported zero or nonzero misuse and also score amount of misuse as a separate continuous variable . amount of misuse is scored as missing for respondents who have zero use , but data from these participants was still able to be included in the models because mplus can estimate models using full information maximum likelihood , which can include partially complete data . regarding descriptive statistics , parent disapproval was positively correlated with peer disapproval and risk perception for marijuana and otc pain and prescription pain medication misuse variables . parent disapproval for otc pain medication was also negatively correlated with marijuana use and lifetime use . among marijuana use variables , parent disapproval was negatively correlated with lifetime use ; however , this was only true for participants who did not report any pain medication misuse . peer disapproval was positively correlated with risk perception and negatively correlated with lifetime use for marijuana and otc pain and prescription pain medication misuse variables . peer disapproval of otc pain and prescription pain medication misuse was also negatively correlated with marijuana use . risk perception was positively correlated with gender and negatively correlated with lifetime use and marijuana use for both types of pain medication variables . for marijuana variables , risk perception however , risk perception was only positively correlated with gender among participants who reported no pain medication misuse . lifetime use was positively correlated with marijuana use and negatively correlated with gender for the prescription pain variables . fit of the models was not an issue , though , as they included all possible associations among the variables ( note that no association between the two misuse variables could be included because the continuous variable only took on nonmissing values for respondents scoring one on the dichotomous variable ) and so were effectively saturated . for otc pain medication misuse , peer disapproval ( = .516 , p < .05 ) significantly predicted risk perception , with higher levels of peer disapproval predicting higher levels of risk perception . both peer disapproval ( = .401 , p < .05 ) and risk perception ( = .221 , p < .05 ) significantly predicted dichotomous misuse , with higher levels of peer disapproval and risk perception predicting lower likelihood of nonzero misuse . because peer disapproval predicted risk perception and risk perception predicted dichotomous misuse , risk perception was a significant mediator of the effect of peer disapproval on dichotomous misuse . there were no significant predictors of amount of misuse for respondents who reported a nonzero amount . multiple groups analyses revealed no significant differences in path coefficients as a function of either gender groups ( (8 ) = 3.44 , p > .05 ) or groups defined by zero or nonzero marijuana misuse ( (8 ) = 7.60 , p > .05 ) . for prescription pain medication misuse , both parent disapproval ( = .183 , p < .05 ) and peer disapproval ( = .425 , p < .05 ) , significantly predicted risk perception , with higher levels of parent and peer disapproval predicting higher levels of risk perception . both peer disapproval ( = .424 , p < .05 ) and risk perception ( = .188 , p < .05 ) significantly predicted dichotomous misuse , with higher levels of peer disapproval and risk perception predicting lower likelihood of nonzero misuse . because peer disapproval predicted risk perception and risk perception predicted dichotomous misuse , risk perception was a significant mediator of the effect of peer disapproval on dichotomous misuse . multiple groups analyses revealed no significant differences in path coefficients as a function of either gender groups ( (8 ) = 12.35 , p > .05 ) or groups defined by zero or nonzero marijuana misuse ( (8 ) = 3.13 , p > .05 ) . for marijuana misuse , both parent disapproval ( = .162 , p < .05 ) and peer disapproval ( = .493 , p < .05 ) significantly predicted risk perceptions , with higher levels of parent and peer disapproval predicting higher levels of risk perception . peer disapproval ( = .356 , p < .05 ) and risk perception ( = .409 , p < .05 ) significantly predicted dichotomous misuse , with higher levels of peer disapproval and risk perception predicting lower likelihood of nonzero misuse . parent disapproval also significantly predicted dichotomous misuse , but this effect varied depending on whether respondents were in the zero misuse or nonzero misuse group for otc and prescription pain medications , so it is discussed below . both peer disapproval ( = .262 , p < .05 ) and risk perception ( = .454 , p < .05 ) were significant predictors of amount of misuse for respondents who reported a nonzero amount , with higher levels of disapproval and risk perception predicting smaller amounts of use . because both parent and peer disapproval predicted risk perception and risk perception predicted both dichotomous misuse and amount of misuse , risk perception was a significant mediator of the effect of both parent and peer disapproval on both dichotomous misuse and amount of misuse . multiple group analyses revealed no significant differences in path coefficients as a function of gender groups ( (8 ) = 10.02 , p > .05 ) . there was a significant difference in path coefficients as a function of groups defined by zero or nonzero use of otc or prescription pain medications ( (8 ) = 18.81 , p < .05 ) . tests of invariance of the individual paths revealed that the only path to differ significantly was for parent disapproval predicting dichotomous misuse . for respondents reporting zero otc or prescription pain medication misuse , this association was significant and negative ( = .131 , p < .05 ) , so higher levels of disapproval predicted lower likelihoods of nonzero misuse . for respondents reporting nonzero otc or prescription pain misuse , this association was significant and positive ( = .282 , p < .05 ) , so higher levels of disapproval predicted higher likelihoods of nonzero misuse . to add to the understanding of otc and prescription pain medication misuse and its relationship with other substance abuse in adolescents and young adults , we explored the relationship between parent and peer disapproval of recreational use , risk perception of recreational use , and reported recreational use . the results of this study indicate that similar to marijuana use , risk perception of otc and prescription pain medication recreational use are influenced by parent and peer disapproval of recreational use , and use itself is influenced by peer disapproval and risk perception of recreational use . noteworthy was the ability of peer disapproval of misuse to predict dichotomous misuse over and above the mediated effect through risk perception , for all three drugs : otc and prescription pain medication misuse and marijuana use . this is consistent with findings in the substance abuse literature [ 31 , 33 ] and suggests that peer subculture may either encourage or act as a protective variable against adolescents engaging in the recreational use of all substances including prescription drugs . this also highlights the role of development in adolescents ' decisions to engage in health risk behaviors ; that is , they are in a period in their lives where peers are their key counterplayers and they may follow the norms outlined by their subgroup ; hence , peer disapproval has more influence on their perceptions of harm and use . these results also suggest that in order to increase risk perception and reduce recreational use of otc and prescription pain medication , program planners should work towards reducing the acceptability of otc and prescription pain medication misuse among adolescents , since they influence each others ' perceptions . based on the significant influence of peer disapproval on risk perception and use , future campaigns should target adolescents and young adults . parent disapproval was predictive of risk perception of prescription pain medication recreational use and marijuana use . we speculate that adolescents may take their parents ' opinions into consideration when formulating their risk perceptions on both marijuana and prescription pain medication as opposed to otc pain medication because of the restrictive access to these substances ; however , this hypothesis should be explored in future studies . parent disapproval of misuse failed to directly predict dichotomous misuse of otc and prescription pain medications ( although it did have a mediated effect through risk perception for prescription pain medications ) . , just say no campaigns ) and focus more on educating adolescents on the risks associated with the behavior . the goal of redirecting adults ' focus on educating adolescents on the risks of otc and prescription pain misuse is to influence other correlates of use , such as peer disapproval and risk perception , in order to prevent or decrease use . parent disapproval predicting dichotomous misuse of marijuana is consistent with the marijuana use literature [ 39 , 40 ] . however , the finding of reverse relationships when the sample was divided into otc and prescription pain drugs zero and nonzero misusers provides some additional data about the relationship of use for the top two drugs of choice for adolescents . specifically , adolescents who engaged in otc and prescription pain misuse were more likely to be marijuana users the more their parents disapproved of marijuana use , whereas those who were zero otc and prescription pain misusers were less likely to engage in marijuana use if their parents disapproved of marijuana use . these results are suggestive of a deviant and high sensation - seeking subculture trend in adolescents who engage in both pain medication and marijuana use and this subculture is seen across the substance abuse literature [ 9 , 31 , 33 ] . it is possible that among this subculture , parent interventions may encourage adolescents to engage in substance use , providing further need for parents to focus less on preventing behavior and more on highlighting the risks associated with the behaviors . another explanation may be that of reverse causality , whereby the more the adolescents engage in use , the more their parents disapprove of their behavior . although risk perception was significantly predictive of use in the general models , the regression weights and significance values suggest that it was a weaker predictor for otc and prescription pain medication recreational use than for marijuana use . these findings suggest that although risk perception may play an important role in adolescents ' decision making regarding substance use and other health risk behaviors [ 28 , 34 , 41 , 42 ] , risk perception is less of an important variable in adolescents ' decision - making regarding recreational use of otc and prescription pain medication . additional variables that are uncommon to the substance abuse literature , such as lower societal stigma , and comparative risk perception to street drugs may be more important in adolescents ' decision making regarding prescription drug recreational use [ 12 , 14 ] . additionally , risk perception may interact with other variables not studied here to explain adolescents ' decision making regarding use ( e.g. , general attitudes toward prescription drug use , frequency of family and friends ' medical use of prescription drugs ) . future studies should explore how other correlates of recreational use of otc and prescription pain medication interact with risk perception to influence misuse . this study was restricted to recreational users of otc and prescription pain medication ; these users do not encompass all nonmedical use ; therefore , generalizations about misuse are limited . another limitation regarding generalization is the use of a convenience sample of college students ; future studies should test this model on a community sample . another limitation of this study is that the psychosocial characteristics of the sample were not considered . adolescents and young adults are highly influenced by developmental characteristics and their external environment and future studies should explore the extent to which developmental characteristics interact with these environmental variables to predict risk perception and use . additionally , future studies should retest these paths with a larger sample ( preferably including more participants who report nonzero use ) to check for consistency of the results across samples . future studies should also compare otc and prescription pain medication misuse to other illicit drug use and other health risk behaviors . despite these limitations , this study provides evidence that suggests that predictors of otc and prescription pain medication recreational use are similar to predictors of marijuana use in adolescents in that peer disapproval is a significant predictor of risk perception and misuse . additionally , both peer and parent disapproval are significant predictors of risk perception of prescription pain medication misuse . unlike marijuana use , otc and prescription pain medication misuse in adolescents are relatively less explained by risk perception suggesting that other variables may be responsible for adolescents ' decision to engage in the misuse of otc and prescription pain medication .

over - the - counter ( otc ) and prescription drug misuse are the nonmedical use of medication , which is used without a prescription or for the feeling caused by the drug . otc and prescription drugs are fast becoming the drugs of choice for adolescents , with the national center on addiction and substance abuse reporting a 212% increase in prescription drug misuse among adolescents between the 1992 and 2003 period . the national survey on drug use and health and the drug abuse warning network have also documented an increase in otc drug misuse since 1992 . compton and volkow have reported that pain medication is second only to marijuana in being the most popular drug of choice for adolescents . given that parent and peer influence have been highlighted as major predictors of substance use in adolescents and that pain medications are the most widely abused prescription drugs , we hypothesize that parent and peer influence will affect pain medication misuse , specifically misuse to get high ( recreational use ) , in the same way these influences affect marijuana use . very little is known about what is responsible for the increase in prevalence and incidence rates of prescription drug misuse . it has been suggested that adolescents ' and young adults ' otc and prescription drug misuse could be explained by their perception that such drugs are safer than illicit drugs , by the ease of access to drugs and by lower societal stigma [ 1117 ] . additionally , because otc drugs are easily purchased , they can be used as an alternative when a prescription drug misuser is unable to access their preferred drug as well as mixed with alcohol to achieve a some researchers have shown that there is a strong relationship between illicit drug use and prescription drug misuse [ 13 , 1924 ] . additionally , ford highlighted the importance of family and school bonds as protective factors against misuse . ford also suggested that adolescents whose parents and peers disapprove of substance use and whose peers use drugs are more likely to engage in prescription drug misuse . marijuana use , on the other hand , has been thoroughly studied , and clear correlates of marijuana use in adolescents have been identified across the literature . specifically , sensation - seeking , risk perception of use , parent and peer attitudes towards use , adolescent and peers ' delinquent behavior , peer use , adolescent tobacco and alcohol use , and demographic variables including gender and ethnicity [ 2732 ] have all been shown to be related to adolescent marijuana use . to better understand the relationship between illicit drug use and pain medication recreational use , we explored the extent to which three known predictors of marijuana use were similarly predictive of otc and prescription pain medication recreational use . specifically , we explored the extent to which risk perception , parent disapproval , and peer disapproval were predictive of recreational use and whether risk perception mediated the effects of parent and peer disapproval . parent and peer disapproval have been strongly linked to risk perception and use in the marijuana literature [ 9 , 28 , 31 , 33 ] , hence their inclusion in the model ( see figure 1 ) . additionally , risk perception has been indicated in the health risk behaviors literature and alluded to in the prescription drug literature as a contributing variable in adolescents ' decisions to engage in these behaviors [ 12 , 14 , 27 , 34 ] . we examined the extent to which these relationships were gender dependent as the marijuana use literature has suggested that different variables are predictive of males ' and females ' use . additionally , because illicit drug use is correlated with otc and prescription drugs misuse [ 13 , 20 , 21 ] , we propose that the relationship between risk perception , parent and peer disapproval , and otc and prescription pain medication recreational use will differ for marijuana users and nonusers . this study differs from ford , in that our measures of parents ' and peers ' disapproval of misuse ( specifically recreational use ) are specific to otc and prescription drug recreational use independent of other substance use . this is especially important since previous research has shown that perceptions of prescription drug misuse often differ from perceptions of illicit drug use [ 15 , 17 ] , and these differing perceptions will subsequently affect peers ' and possibly parents ' level of disapproval . additionally , the inclusion of risk perception as a predictor variable provides some clinical utility to the study , in that it investigates the role of an easily targetable variable for intervention to prevent or decrease misuse . the inclusion of separate path analyses for otc pain medication recreational use , prescription pain medication recreational use , and marijuana use also provides for a comparison of predictors of use of the different drugs . the sample consisted of 465 college students between the ages of 18 and 24 years ( mage = 18.57 , sd = 0.86 ) . the majority of participants ( 91% ) were 18 or 19 years of age ; the remaining 9% were ages 2024 years . nineteen percent of participants ( n = 88 ) reported some ( nonzero ) lifetime otc or prescription drug recreational use with 13% ( n = 60 ) and 11% ( n = 50 ) reporting otc and prescription pain medication recreational use , respectively . twenty - nine percent of participants ( n = 134 ) reported nonzero lifetime marijuana use . risk perception / perceived susceptibility was measured by perceived personal risk , which was the extent to which participants felt they would be at risk of getting sick or hurt if they recreationally used otc pain medication , prescription pain medications , or marijuana , respectively . risk perception was measured on a 7-point evaluation scale ranging from no risk at all ( 1 ) to very much at risk ( 7 ) . to assess for parents ' and peers ' disapproval , participants were asked to report on how they felt their parents and peers would feel about them engaging in the recreational use of otc pain medication , prescription pain medication , or marijuana on a 5-point likert scale ranging from strongly approve ( 1 ) to strongly disapprove ( 5 ) . recreational use or misuse of otc and prescription pain medication was defined as use of medications to get high . participants were asked about their lifetime misuse of otc pain medication , prescription pain medication , and marijuana use on a 5-point frequency scale ( see table 1 ) . responses included never ( 1 ) , 15 times ( 2 ) , 619 times ( 3 ) , 2040 times ( 4 ) , and more than 40 times ( 5 ) . otc pain medication recreational use included the misuse of otc tylenol , motrin , advil , aleve , ibuprofen , and aspirin . prescription pain medication recreational use included the misuse of vicodin , codeine , oxycontin , and percocet . participants also reported on their recreational use of other classifications of otc and prescription drugs including tranquilizers , stimulants , sedatives , steroids , and cough and cold syrup , although these items were not included in the analyses . data was collected as part of a larger project , media influences on health risk behaviors and prescription drug use in young adults . the online survey took approximately 3550 minutes to complete , and participants received research credit for their class upon completion of the survey . to ensure participant privacy , descriptive statistics are shown in table 2 ( for otc pain medication and prescription pain medication variables ) and table 3 ( for marijuana variables ) . path models were estimated separately for each set of variables for one of the three drugs : otc pain medication , prescription pain medication , and marijuana . parent and peer disapproval of misuse of a drug were used to predict risk perception for that drug . both disapproval variables and risk perception misuse of the drug was entered using two variables , one representing whether a respondent reported zero misuse of the drug versus nonzero misuse and the other representing amount of use for respondents who reported a nonzero amount . this model allowed for the testing of whether risk perception mediated the effects of the disapproval variables on the misuse variables , which was done using the joint significance test . it was then of interest to test whether any of the associations among the variables differed as a function of gender or as a function of use or nonuse of the other drugs . each path model was reestimated first with gender as a grouping variable and second with misuse of marijuana ( for otc and prescription pain medication models ) or otc and prescription pain medication misuse ( for the marijuana model ) . because otc and prescription pain medication misuse were combined into a single grouping variable , a zero represented no misuse of either type and respondents reporting any use of either type , were scored as nonzero . the misuse - dependent variables were each entered using two variables in the model by way of the twopart feature of mplus , which scores all participants on a dichotomous use variable depending on whether they reported zero or nonzero misuse and also score amount of misuse as a separate continuous variable . amount of misuse is scored as missing for respondents who have zero use , but data from these participants was still able to be included in the models because mplus can estimate models using full information maximum likelihood , which can include partially complete data . as the amount of misuse was skewed , the mlr estimator , a variation of maximum likelihood that is more robust to nonnormal data than the more typical maximum likelihood , was used to estimate the models . regarding descriptive statistics , parent disapproval was positively correlated with peer disapproval and risk perception for marijuana and otc pain and prescription pain medication misuse variables . parent disapproval for otc pain medication was also negatively correlated with marijuana use and lifetime use . among marijuana use variables , parent disapproval was negatively correlated with lifetime use ; however , this was only true for participants who did not report any pain medication misuse . peer disapproval was positively correlated with risk perception and negatively correlated with lifetime use for marijuana and otc pain and prescription pain medication misuse variables . peer disapproval of otc pain and prescription pain medication misuse was also negatively correlated with marijuana use . risk perception was positively correlated with gender and negatively correlated with lifetime use and marijuana use for both types of pain medication variables . for marijuana variables , risk perception however , risk perception was only positively correlated with gender among participants who reported no pain medication misuse . lifetime use was positively correlated with marijuana use and negatively correlated with gender for the prescription pain variables . fit of the models was not an issue , though , as they included all possible associations among the variables ( note that no association between the two misuse variables could be included because the continuous variable only took on nonmissing values for respondents scoring one on the dichotomous variable ) and so were effectively saturated . note that because the data is cross - sectional , the term predictor is used for statistical purposes rather than to indicate causality . for otc pain medication misuse , peer disapproval ( = .516 , p < .05 ) significantly predicted risk perception , with higher levels of peer disapproval predicting higher levels of risk perception . both peer disapproval ( = .401 , p < .05 ) and risk perception ( = .221 , p < .05 ) significantly predicted dichotomous misuse , with higher levels of peer disapproval and risk perception predicting lower likelihood of nonzero misuse . because peer disapproval predicted risk perception and risk perception predicted dichotomous misuse , risk perception was a significant mediator of the effect of peer disapproval on dichotomous misuse . there were no significant predictors of amount of misuse for respondents who reported a nonzero amount . multiple groups analyses revealed no significant differences in path coefficients as a function of either gender groups ( (8 ) = 3.44 , p > .05 ) or groups defined by zero or nonzero marijuana misuse ( (8 ) = 7.60 , p > .05 ) . for prescription pain medication misuse , both parent disapproval ( = .183 , p < .05 ) and peer disapproval ( = .425 , p < .05 ) , significantly predicted risk perception , with higher levels of parent and peer disapproval predicting higher levels of risk perception . both peer disapproval ( = .424 , p < .05 ) and risk perception ( = .188 , p < .05 ) significantly predicted dichotomous misuse , with higher levels of peer disapproval and risk perception predicting lower likelihood of nonzero misuse . because peer disapproval predicted risk perception and risk perception predicted dichotomous misuse , risk perception was a significant mediator of the effect of peer disapproval on dichotomous misuse . parent disapproval ( = .286 , p < .05 ) significantly predicted amount of misuse for respondents who reported a nonzero amount , but in an unexpected direction : higher levels of disapproval predicted greater amounts of misuse . multiple groups analyses revealed no significant differences in path coefficients as a function of either gender groups ( (8 ) = 12.35 , p > .05 ) or groups defined by zero or nonzero marijuana misuse ( (8 ) = 3.13 , p > .05 ) . for marijuana misuse , both parent disapproval ( = .162 , p < .05 ) and peer disapproval ( = .493 , p < .05 ) significantly predicted risk perceptions , with higher levels of parent and peer disapproval predicting higher levels of risk perception . peer disapproval ( = .356 , p < .05 ) and risk perception ( = .409 , p < .05 ) significantly predicted dichotomous misuse , with higher levels of peer disapproval and risk perception predicting lower likelihood of nonzero misuse . parent disapproval also significantly predicted dichotomous misuse , but this effect varied depending on whether respondents were in the zero misuse or nonzero misuse group for otc and prescription pain medications , so it is discussed below . both peer disapproval ( = .262 , p < .05 ) and risk perception ( = .454 , p < .05 ) were significant predictors of amount of misuse for respondents who reported a nonzero amount , with higher levels of disapproval and risk perception predicting smaller amounts of use . because both parent and peer disapproval predicted risk perception and risk perception predicted both dichotomous misuse and amount of misuse , risk perception was a significant mediator of the effect of both parent and peer disapproval on both dichotomous misuse and amount of misuse . multiple group analyses revealed no significant differences in path coefficients as a function of gender groups ( (8 ) = 10.02 , p > .05 ) . there was a significant difference in path coefficients as a function of groups defined by zero or nonzero use of otc or prescription pain medications ( (8 ) = 18.81 , p < .05 ) . tests of invariance of the individual paths revealed that the only path to differ significantly was for parent disapproval predicting dichotomous misuse . for respondents reporting zero otc or prescription pain medication misuse , this association was significant and negative ( = .131 , p < .05 ) , so higher levels of disapproval predicted lower likelihoods of nonzero misuse . for respondents reporting nonzero otc or prescription pain misuse , this association was significant and positive ( = .282 , p < .05 ) , so higher levels of disapproval predicted higher likelihoods of nonzero misuse . to add to the understanding of otc and prescription pain medication misuse and its relationship with other substance abuse in adolescents and young adults , we explored the relationship between parent and peer disapproval of recreational use , risk perception of recreational use , and reported recreational use . the results of this study indicate that similar to marijuana use , risk perception of otc and prescription pain medication recreational use are influenced by parent and peer disapproval of recreational use , and use itself is influenced by peer disapproval and risk perception of recreational use . noteworthy was the ability of peer disapproval of misuse to predict dichotomous misuse over and above the mediated effect through risk perception , for all three drugs : otc and prescription pain medication misuse and marijuana use . this is consistent with findings in the substance abuse literature [ 31 , 33 ] and suggests that peer subculture may either encourage or act as a protective variable against adolescents engaging in the recreational use of all substances including prescription drugs . this also highlights the role of development in adolescents ' decisions to engage in health risk behaviors ; that is , they are in a period in their lives where peers are their key counterplayers and they may follow the norms outlined by their subgroup ; hence , peer disapproval has more influence on their perceptions of harm and use . these results also suggest that in order to increase risk perception and reduce recreational use of otc and prescription pain medication , program planners should work towards reducing the acceptability of otc and prescription pain medication misuse among adolescents , since they influence each others ' perceptions . the office of national drug policy and partnership for a drug - free america launched a prescription drug abuse media campaign targeting parents and health and school professionals ; however , no ads were directly marketed toward adolescents and young adults . based on the significant influence of peer disapproval on risk perception and use , future campaigns should target adolescents and young adults . we speculate that adolescents may take their parents ' opinions into consideration when formulating their risk perceptions on both marijuana and prescription pain medication as opposed to otc pain medication because of the restrictive access to these substances ; however , this hypothesis should be explored in future studies . parent disapproval of misuse failed to directly predict dichotomous misuse of otc and prescription pain medications ( although it did have a mediated effect through risk perception for prescription pain medications ) . this highlights the need for parents and by extension other adults working with adolescents to focus less on preventing behavior via direct methods ( e.g. , just say no campaigns ) and focus more on educating adolescents on the risks associated with the behavior . the goal of redirecting adults ' focus on educating adolescents on the risks of otc and prescription pain misuse is to influence other correlates of use , such as peer disapproval and risk perception , in order to prevent or decrease use . however , the finding of reverse relationships when the sample was divided into otc and prescription pain drugs zero and nonzero misusers provides some additional data about the relationship of use for the top two drugs of choice for adolescents . specifically , adolescents who engaged in otc and prescription pain misuse were more likely to be marijuana users the more their parents disapproved of marijuana use , whereas those who were zero otc and prescription pain misusers were less likely to engage in marijuana use if their parents disapproved of marijuana use . these results are suggestive of a deviant and high sensation - seeking subculture trend in adolescents who engage in both pain medication and marijuana use and this subculture is seen across the substance abuse literature [ 9 , 31 , 33 ] . it is possible that among this subculture , parent interventions may encourage adolescents to engage in substance use , providing further need for parents to focus less on preventing behavior and more on highlighting the risks associated with the behaviors . another explanation may be that of reverse causality , whereby the more the adolescents engage in use , the more their parents disapprove of their behavior . although risk perception was significantly predictive of use in the general models , the regression weights and significance values suggest that it was a weaker predictor for otc and prescription pain medication recreational use than for marijuana use . these findings suggest that although risk perception may play an important role in adolescents ' decision making regarding substance use and other health risk behaviors [ 28 , 34 , 41 , 42 ] , risk perception is less of an important variable in adolescents ' decision - making regarding recreational use of otc and prescription pain medication . additional variables that are uncommon to the substance abuse literature , such as lower societal stigma , and comparative risk perception to street drugs may be more important in adolescents ' decision making regarding prescription drug recreational use [ 12 , 14 ] . future studies should explore how other correlates of recreational use of otc and prescription pain medication interact with risk perception to influence misuse . adolescents and young adults are highly influenced by developmental characteristics and their external environment and future studies should explore the extent to which developmental characteristics interact with these environmental variables to predict risk perception and use . finally , the present study was cross - sectional , so temporal precedence can not be established for the assumed causal relationships between variables . despite these limitations , this study provides evidence that suggests that predictors of otc and prescription pain medication recreational use are similar to predictors of marijuana use in adolescents in that peer disapproval is a significant predictor of risk perception and misuse . unlike marijuana use , otc and prescription pain medication misuse in adolescents are relatively less explained by risk perception suggesting that other variables may be responsible for adolescents ' decision to engage in the misuse of otc and prescription pain medication .

complementary and alternative medicine ( cam ) is an additional health care system , comprising mind / body practices and natural products that are not regarded as part of conventional medicine12 ) . conventional medicine is used together when complementary medicine is applied , and alternative medicine is employed instead of conventional medicine1 ) . most patients who try to take non - prevailing approaches use them together with conventional treatments1 ) . natural products include herbs , minerals , vitamins , and probiotics , and mind / body practices include acupuncture , massage therapy , meditation , movement therapies , relaxation techniques , spinal manipulation , and others1 ) . neurologic diseases in children often tend to have a chronic course and require long - term management . children with chronic health conditions use cam more frequently3 ) , but it was found that only around 30% of patients with epilepsy and 50% of children with chronic health conditions who use cam reported this behavior to their physicians456 ) . physicians should be aware of their patients ' use of cam and provide accurate information about cam to enable patients to make the right decisions . this review aims to investigate the characteristics of the use of cam in children with neurologic diseases such as epilepsy , migraine , cerebral palsy , and congenital malformations in order to improve management through analysis of several studies of cam use in children with chronic neurologic conditions2678910 ) . further , this review will discuss the differences in the use of cam between children and adults with neurologic diseases , and between patients with neurologic and nonneurologic conditions . barnes et al.9 ) reported that approximately 12% of children ( total sample size [ n]=9,417 ) used cam in the united states ( us ) , and mccann and newell3 ) reported that 12% of healthy children ( n=25 ) used cam in the united kingdom ( uk ) . they also reported that 40% ( n=75 ) of children with chronic health conditions used cam in the uk , and this proportion is much higher than that in healthy children3 ) . in the study by barnes et al.9 ) , the most common modalities used were natural products ( neither vitamin nor mineral ) and osteopathic or chiropractic manipulation . regarding significant factors , parental use of cam was associated with cam use in children compared to children whose parents did not use cam ( 24% vs. 5% ) . parental use of cam was also found to be a strongly associated factor in a study by birdee et al.11 ) . additionally , for both adults and children , worry about cost was found to be one of the factors related to the use of cam and delaying using conventional medicine , and patients were more likely to use cam when expenses were a concern than when expenses were not a problem for conventional medicine9 ) . other factors significantly associated with pediatric use of cam , in the study of birdee et al.11 ) , were adolescent age rather than that of toddler or infant , parent with a college education , more frequent use of prescription medication , stress or anxiety , musculoskeletal conditions , dermatologic conditions , and sinus infection . in a recent study on patterns of cam use in pediatric patients with common neurologic conditions ( headaches , migraines , seizures ) , pediatric patients with common neurological conditions were found to use cam significantly more compared to children without these conditions ( 24% [ n=586 ] vs.13% [ n=7,083])7 ) . pediatric patients with neurologic conditions used biological therapies with a similar frequency and mind / body practices with a significantly higher frequency compared to other pediatric patients using cam . among mind / body practices , deep breathing was used most commonly ( 33% ) , meditation was used in 15% , and progressive relaxation was employed in 10%7 ) . in another study in 2010 in north jordan , 56% of children ( n=176 ) with neurologic illnesses used cam for their care2 ) . regarding the most commonly used methods , prayer / reciting the quran was used in 77% , massage with olive oil in 32% , religious healers in 30% , and consumption of honey products in 29% . among the reasons provided for the use of cam , religious beliefs were the most common ( 68% ) , and no patients described distrust in conventional medicine2 ) . other significant factors connected to the use of cam included belief in its effects , speech delay , father 's age greater than 30 years , and mother 's education less than high school2 ) . additionally , parents ' experience or hearing of successful cases from acquaintances or mass media was found to be other reasons for using cam8 ) . side effects were rare8 ) . in a study by soo et al.8 ) regarding the use of cam in a pediatric neurology clinic , 44% of patients ( n=105 ) were found to use cam . in regard to the most commonly used cam modalities , chiropractic manipulation was used in 15% , dietary therapy was used in 12% , and homeopathy , herbal remedies , and prayer healing were each used in 8% . the difference in this study compared to other studies is that the parents ' sociodemographic characteristics were not significantly associated with the use of cam . pediatric health - related quality of life was also not a significant factor8 ) . in total , 59% of patients who used cam experienced benefits . the total median cost of cam compared with conventional medicines was not different ( $ 31.70 vs. $ 50.00 per month)8 ) . in a study on cam use in pediatric neurology , the overall rates of cam use at two centers were 78% ( n=151 ) and 48% ( n=55 ) , respectively6 ) . as for the most commonly used cam products , multivitamins were used in 84% , vitamin c was used in 37% , homeopathic remedies were used in 24% , and fish oil / omega-3s were employed in 22%6 ) . regarding the most commonly used cam practices , massage was used in 47% , and 37% used chiropractic methods , 18% used faith - healing , and aromatherapy , homeopathy , and relaxation were each used in 16%6 ) . over half of patients disclosed to their physicians their use of cam in addition to conventional medicine6 ) . in a study examining the use of traditional herbal medicines in korean elementary school children , 65% ( n=905 ) of children were found to be taking herbal medicines12 ) . in a 2008 study in korea , 65 of 378 children ( 17% ) with epilepsy were found to be taking herbal medicines4 ) . in our study in 2014 , 19% ( n=398 ) of children with epilepsy used traditional korean medicine ( tkm ) ( acupuncture , moxibustion , herbal medicine)10 ) . these results suggest that tkm is not the favored method for attaining seizure control and that patients are careful about using tkm to treat epilepsy . in addition , 60% of children with epilepsy were receiving other types of cam10 ) . according to a study by lee et al.4 ) , patients with psychosomatic disorders used herbal medicine significantly more frequently , whereas in a study by kim et al.13 ) , neurologically normal patients used it slightly more frequently ; however , the difference did not have statistical significance . in our study , among receivers of combination treatments besides tkm , tkm was used more frequently in those receiving language , music , or art therapy than in those undergoing physiotherapy or other combination therapies10 ) . this finding suggests that the use of tkm is associated more with the purpose of treatment of cognitive function than motor functions , and parents of children with epilepsy may believe that tkm has an effect on the former rather than the latter10 ) . additionally , regarding significant factors , in the group of patients using a greater number of antiepileptic drugs and having low seizure - free rates , the use of tkm was more frequent10 ) . regarding the effect of cam on epilepsy , inconsistent results had been reported in previous studies . recent cochrane reviews show that some studies on the use of acupuncture and chinese traditional herbal medicine did not succeed in providing clear evidence proving the effects of acupuncture and herbal medicine for epilepsy patients1415 ) . according to the study by lee et al.4 ) , 18% of respondents using herbal medicine reported its effectiveness for seizure control , but the rest of the respondents reported no effectiveness or even worsening of symptoms , although many patients ( 33% ) agreed with using herbal medicine . it was also reported that most patients did not feel that cam was superior to general epilepsy treatments , although 32% of them continued using cam16 ) . it is presumed that this persistent usage is due to people 's beliefs that herbal medicines are safe ( not dangerous ) and help to improve health and enhance cognitive function10 ) . in spite of these beliefs , however , according to the report of kuan et al.16 ) , the most frequent adverse effect of cam was aggravated seizures . it is presumed that this is due to the effects of seizure - inducing materials of herbal medicine or drug - drug interactions . regarding the frequency of cam use for epileptic patients in various countries , 49% ( n=403 ) of taiwanese were found to use cam , 44% used it in arizona , 37% ( n=265 ) of nigerians used cam , and 32% ( n=1,000 ) in india and 24% ( n=92 ) in ohio used cam516171819 ) . regarding commonly used forms of cam for treatment of epileptic patients , prayer and stress relief were used in the west , whereas traditional herbal medicines were employed both in the east and in africa516171819 ) . in us , the preferred modalities were biological therapies and mind - body practices7 ) . in canada , the commonly used methods were chiropractic manipulations , dietary therapy , multivitamins , and massage68 ) . however , in north jordan , prayer / reciting the quran was the most commonly used modality2 ) . in korea these differences in use of cam between countries may be owing to their different cultures , religions , and traditions10 ) . the characteristics of the use of cam in children with neurologic diseases are summarized in table 2 . according to the study by barnes et al.9 ) , almost 40% ( n=23,393 ) of us adults used cam in 2007 and the most commonly used modalities were natural products ( neither vitamin nor mineral ) ( 18% ) and deep breathing exercises ( 13% . ) . cam was used more frequently in american adults with common neurological conditions ( migraines , regular headaches , strokes , back pain with sciatica , seizures , dementia or memory loss ) than in those without such conditions ( 44% [ n=6,587 ] vs. 33% [ n=16,806])20 ) . the frequency of use of each cam modality was higher in adults with common neurological conditions than in those without20 ) . the most common modalities were mind / body therapies , and the least common were alternative medical systems20 ) . the reasons why those with common neurological conditions used cam more frequently than those without such conditions included provider 's recommendations or little effect achieved with conventional medicine or a cost that was too high20 ) . about 50% of adults with common neurological conditions did not report their use of cam to their doctors20 ) . factors that significantly influenced the use of cam in those with common neurological conditions included higher educational level than high school education , positive history of anxiety , living in a western area , being a former smoker , and drinking alcohol lightly20 ) . in the previous studies on the use of cam in various neurologic conditions , compared to the frequency of use of cam in children with common neurologic diseases , the frequency of use in adults with neurologic diseases was similar267810 ) . for korean adults with other diseases , several studies showed the following findings regarding cam use : 60%70% of patients with stroke used cam , 53% of those with cancer used it and 53% of patients with asthma and preferred modalities of cam use in children and adults had diverse features , and these results may be influenced by individuals ' different cultures and traditions of use of cam . the factor most commonly related to use of cam in children with neurologic conditions was parental cam use , and it could be presumed that the use of cam in children may be due to such usage running in the family , as well as belief in or experience with the efficacy of cam . in adults with neurologic conditions , increased educational level was the most common factor associated with use of cam202123 ) , and this result may relate to economic level . characteristics of the use of cam in adults with neurologic diseases are summarized in table 3 . in a systematic review on the use of cam in pediatric cancer patients , the prevalence of use of cam ( since the diagnosis of cancer ) the most common cam modalities were herbal medicines , diets / nutrition , and faith - healing28 ) . the frequent reasons given for the use of cam were to help cure or fight the child 's cancer , relief of symptom , and support of ongoing use of conventional medicine28 ) . there were few factors related to patients ' sociodemographic characteristics associated with use of cam , and this result was different from the results of several other studies28 ) . according to the study by barnes et al.9 ) , for pediatric patients with specific diseases , the proportions were 2% in insomnia , 3% in attention deficit hyperactivity disorder ( adhd ) , 5% in other musculoskeletal diseases , 5% in stress or anxiety , 7% in head or chest colds , and 7% in back or neck pain9 ) . from these results , it is presumed that patients in these cases use cam to manage symptoms , to maintain and manage health , or to prevent illness , rather than to treat specific diseases28 ) . many studies , however , reported a higher frequency of cam use in pediatric patients with chronic diseases , such as asthma , autism , adhd , cancer , and sickle cell anemia . medical symptoms such as nausea and abdominal pain rather than certain medical diagnoses were found to be some of the factors associated with the use of cam28 ) . in the study by oshikoya et al.29 ) , the researchers interviewed parents of children with epilepsy ( n=122 ) , asthma ( n=78 ) , or sickle cell anemia ( n=118 ) . three hundred three types of cam were used by 99 patients ( 31% ) ( epilepsy , 38% ; sickle cell anemia , 36% ; asthma , 25% ) . the most commonly used cams were biological products ( 58% ) , while alternative medical systems ( 27% ) as well as mind / body interventions ( 14% ) were also employed . eighty - five parents ( 86% ) were going to report the use of cam to their physicians , but had not yet done so . in 7% of the patients , levy and hyman30 ) investigated the use of cam for children with autistic spectrum disorder ( asd ) . about 50%75% ( n=50112 ) of children with autism were found to use cam31 ) . patients with comorbid intellectual disability may have a higher frequency of use of cam . in the study by levy et al.32 ) , one - third of children requested for examination of asd were found to have already used cam , even before diagnosis . the most frequently used form of cam for children with asd were mind / body practices30 ) . in children with inflammatory bowel disease , adverse effect was the significant predictor of cam use33 ) . in cases of children with asthma , the significant predictors of use of cam were older age and worse control of symptoms . also , more medications and more medical visits as well as more side effects were significant predictors in using cam34 ) a study in italy showed that the most common reason for the use of cam was a fear of side effects of conventional therapy for routine illness35 ) . in the study by hanson et al.36 ) , 75% of parents of children with asd chose the use of cam because they felt cam was perceived to be safe and it did not have side effect or they experienced side effects of conventional therapy previously . the purposes of the use of cam in children with asd are treatment of major symptoms of asd , improvement of attention , enhancement of relaxation , treatment of gastrointestinal symptoms , regulation of sleep and promotion of general health31 ) . the characteristics of the use of cam in children with other chronic diseases are summarized in table 4 . in a recent study on patterns of cam use in pediatric patients with common neurologic conditions ( headaches , migraines , seizures ) , pediatric patients with common neurological conditions were found to use cam significantly more compared to children without these conditions ( 24% [ n=586 ] vs.13% [ n=7,083])7 ) . pediatric patients with neurologic conditions used biological therapies with a similar frequency and mind / body practices with a significantly higher frequency compared to other pediatric patients using cam . among mind / body practices , deep breathing was used most commonly ( 33% ) , meditation was used in 15% , and progressive relaxation was employed in 10%7 ) . in another study in 2010 in north jordan , 56% of children ( n=176 ) with neurologic illnesses used cam for their care2 ) . regarding the most commonly used methods , prayer / reciting the quran was used in 77% , massage with olive oil in 32% , religious healers in 30% , and consumption of honey products in 29% . among the reasons provided for the use of cam , religious beliefs were the most common ( 68% ) , and no patients described distrust in conventional medicine2 ) . other significant factors connected to the use of cam included belief in its effects , speech delay , father 's age greater than 30 years , and mother 's education less than high school2 ) . additionally , parents ' experience or hearing of successful cases from acquaintances or mass media was found to be other reasons for using cam8 ) . side effects were rare8 ) . in a study by soo et al.8 ) regarding the use of cam in a pediatric neurology clinic , 44% of patients ( n=105 ) were found to use cam . in regard to the most commonly used cam modalities , chiropractic manipulation was used in 15% , dietary therapy was used in 12% , and homeopathy , herbal remedies , and prayer healing were each used in 8% . the difference in this study compared to other studies is that the parents ' sociodemographic characteristics were not significantly associated with the use of cam . pediatric health - related quality of life was also not a significant factor8 ) . in total , 59% of patients who used cam experienced benefits . the total median cost of cam compared with conventional medicines was not different ( $ 31.70 vs. $ 50.00 per month)8 ) . in a study on cam use in pediatric neurology , the overall rates of cam use at two centers were 78% ( n=151 ) and 48% ( n=55 ) , respectively6 ) . as for the most commonly used cam products , multivitamins were used in 84% , vitamin c was used in 37% , homeopathic remedies were used in 24% , and fish oil / omega-3s were employed in 22%6 ) . regarding the most commonly used cam practices , massage was used in 47% , and 37% used chiropractic methods , 18% used faith - healing , and aromatherapy , homeopathy , and relaxation were each used in 16%6 ) . over half of patients disclosed to their physicians their use of cam in addition to conventional medicine6 ) . in a study examining the use of traditional herbal medicines in korean elementary school children , 65% ( n=905 ) of children were found to be taking herbal medicines12 ) . in a 2008 study in korea , 65 of 378 children ( 17% ) with epilepsy were found to be taking herbal medicines4 ) . in our study in 2014 , 19% ( n=398 ) of children with epilepsy used traditional korean medicine ( tkm ) ( acupuncture , moxibustion , herbal medicine)10 ) . these results suggest that tkm is not the favored method for attaining seizure control and that patients are careful about using tkm to treat epilepsy . in addition , 60% of children with epilepsy were receiving other types of cam10 ) . according to a study by lee et al.4 ) , patients with psychosomatic disorders used herbal medicine significantly more frequently , whereas in a study by kim et al.13 ) , neurologically normal patients used it slightly more frequently ; however , the difference did not have statistical significance . in our study , among receivers of combination treatments besides tkm , tkm was used more frequently in those receiving language , music , or art therapy than in those undergoing physiotherapy or other combination therapies10 ) . this finding suggests that the use of tkm is associated more with the purpose of treatment of cognitive function than motor functions , and parents of children with epilepsy may believe that tkm has an effect on the former rather than the latter10 ) . additionally , regarding significant factors , in the group of patients using a greater number of antiepileptic drugs and having low seizure - free rates , the use of tkm was more frequent10 ) . regarding the effect of cam on epilepsy , inconsistent results had been reported in previous studies . recent cochrane reviews show that some studies on the use of acupuncture and chinese traditional herbal medicine did not succeed in providing clear evidence proving the effects of acupuncture and herbal medicine for epilepsy patients1415 ) . according to the study by lee et al.4 ) , 18% of respondents using herbal medicine reported its effectiveness for seizure control , but the rest of the respondents reported no effectiveness or even worsening of symptoms , although many patients ( 33% ) agreed with using herbal medicine . it was also reported that most patients did not feel that cam was superior to general epilepsy treatments , although 32% of them continued using cam16 ) . it is presumed that this persistent usage is due to people 's beliefs that herbal medicines are safe ( not dangerous ) and help to improve health and enhance cognitive function10 ) . in spite of these beliefs , however , according to the report of kuan et al.16 ) , the most frequent adverse effect of cam was aggravated seizures . it is presumed that this is due to the effects of seizure - inducing materials of herbal medicine or drug - drug interactions . regarding the frequency of cam use for epileptic patients in various countries , 49% ( n=403 ) of taiwanese were found to use cam , 44% used it in arizona , 37% ( n=265 ) of nigerians used cam , and 32% ( n=1,000 ) in india and 24% ( n=92 ) in ohio used cam516171819 ) . regarding commonly used forms of cam for treatment of epileptic patients , prayer and stress relief were used in the west , whereas traditional herbal medicines were employed both in the east and in africa516171819 ) . in us , the preferred modalities were biological therapies and mind - body practices7 ) . in canada , the commonly used methods were chiropractic manipulations , dietary therapy , multivitamins , and massage68 ) . however , in north jordan , prayer / reciting the quran was the most commonly used modality2 ) . in korea these differences in use of cam between countries may be owing to their different cultures , religions , and traditions10 ) . the characteristics of the use of cam in children with neurologic diseases are summarized in table 2 . according to the study by barnes et al.9 ) , almost 40% ( n=23,393 ) of us adults used cam in 2007 and the most commonly used modalities were natural products ( neither vitamin nor mineral ) ( 18% ) and deep breathing exercises ( 13% . ) . cam was used more frequently in american adults with common neurological conditions ( migraines , regular headaches , strokes , back pain with sciatica , seizures , dementia or memory loss ) than in those without such conditions ( 44% [ n=6,587 ] vs. 33% [ n=16,806])20 ) . the frequency of use of each cam modality was higher in adults with common neurological conditions than in those without20 ) . the most common modalities were mind / body therapies , and the least common were alternative medical systems20 ) . the reasons why those with common neurological conditions used cam more frequently than those without such conditions included provider 's recommendations or little effect achieved with conventional medicine or a cost that was too high20 ) . about 50% of adults with common neurological conditions did not report their use of cam to their doctors20 ) . factors that significantly influenced the use of cam in those with common neurological conditions included higher educational level than high school education , positive history of anxiety , living in a western area , being a former smoker , and drinking alcohol lightly20 ) . in the previous studies on the use of cam in various neurologic conditions , the prevalence of use of cam ranged from 19% to 43%212223 ) . compared to the frequency of use of cam in children with common neurologic diseases , the frequency of use in adults with neurologic diseases was similar267810 ) . for korean adults with other diseases , several studies showed the following findings regarding cam use : 60%70% of patients with stroke used cam , 53% of those with cancer used it and 53% of patients with asthma and 48% of patients with psychiatric disorders used herbal medicines24252627 ) . preferred modalities of cam use in children and adults had diverse features , and these results may be influenced by individuals ' different cultures and traditions of use of cam . the factor most commonly related to use of cam in children with neurologic conditions was parental cam use , and it could be presumed that the use of cam in children may be due to such usage running in the family , as well as belief in or experience with the efficacy of cam . in adults with neurologic conditions , increased educational level was the most common factor associated with use of cam202123 ) , and this result may relate to economic level . characteristics of the use of cam in adults with neurologic diseases are summarized in table 3 . in a systematic review on the use of cam in pediatric cancer patients , the prevalence of use of cam ( since the diagnosis of cancer ) ranged from 6% to 91% ( n=2,871 ) . the most common cam modalities were herbal medicines , diets / nutrition , and faith - healing28 ) . the frequent reasons given for the use of cam were to help cure or fight the child 's cancer , relief of symptom , and support of ongoing use of conventional medicine28 ) . there were few factors related to patients ' sociodemographic characteristics associated with use of cam , and this result was different from the results of several other studies28 ) . according to the study by barnes et al.9 ) , for pediatric patients with specific diseases , the use of cam was not highly prevalent . the proportions were 2% in insomnia , 3% in attention deficit hyperactivity disorder ( adhd ) , 5% in other musculoskeletal diseases , 5% in stress or anxiety , 7% in head or chest colds , and 7% in back or neck pain9 ) . from these results , it is presumed that patients in these cases use cam to manage symptoms , to maintain and manage health , or to prevent illness , rather than to treat specific diseases28 ) . many studies , however , reported a higher frequency of cam use in pediatric patients with chronic diseases , such as asthma , autism , adhd , cancer , and sickle cell anemia . medical symptoms such as nausea and abdominal pain rather than certain medical diagnoses were found to be some of the factors associated with the use of cam28 ) . in the study by oshikoya et al.29 ) , the researchers interviewed parents of children with epilepsy ( n=122 ) , asthma ( n=78 ) , or sickle cell anemia ( n=118 ) . three hundred three types of cam were used by 99 patients ( 31% ) ( epilepsy , 38% ; sickle cell anemia , 36% ; asthma , 25% ) . the most commonly used cams were biological products ( 58% ) , while alternative medical systems ( 27% ) as well as mind / body interventions ( 14% ) were also employed . eighty - five parents ( 86% ) were going to report the use of cam to their physicians , but had not yet done so . in 7% of the patients , levy and hyman30 ) investigated the use of cam for children with autistic spectrum disorder ( asd ) . about 50%75% ( n=50112 ) of children with autism were found to use cam31 ) . patients with comorbid intellectual disability may have a higher frequency of use of cam . in the study by levy et al.32 ) , one - third of children requested for examination of asd were found to have already used cam , even before diagnosis . the most frequently used form of cam for children with asd were mind / body practices30 ) . in children with inflammatory bowel disease , adverse effect was the significant predictor of cam use33 ) . in cases of children with asthma , the significant predictors of use of cam were older age and worse control of symptoms . also , more medications and more medical visits as well as more side effects were significant predictors in using cam34 ) . a study in italy showed that the most common reason for the use of cam was a fear of side effects of conventional therapy for routine illness35 ) . in the study by hanson et al.36 ) , 75% of parents of children with asd chose the use of cam because they felt cam was perceived to be safe and it did not have side effect or they experienced side effects of conventional therapy previously . the purposes of the use of cam in children with asd are treatment of major symptoms of asd , improvement of attention , enhancement of relaxation , treatment of gastrointestinal symptoms , regulation of sleep and promotion of general health31 ) . the characteristics of the use of cam in children with other chronic diseases are summarized in table 4 . the use of cam in children with neurologic diseases is common , ranging from 24%78% and it is more frequent than the use in healthy children . preferred modalities of cam differed according to patients ' country of residence and due to their different cultures and traditions , with examples such as prayer / reciting the quran in jordan , biologic therapies , and mind / body therapies in the us , and tkm in korea . the most common factor significantly associated with the use of cam was parental cam use in most studies . the reasons for using cam included religious beliefs , parents ' personal experience , or exposure to success stories from the media and acquaintances . the frequency of cam use in children and adults with neurologic diseases is higher than that in those without these conditions . preferred modalities of cam in adults with common neurological conditions were diverse , including megavitamins and mind / body therapy ( prayer and chiropractic care ) . the most common factor significantly associated with use of cam in adults with neurologic diseases was higher educational level . among children and adults with neurologic diseases , less than half of patients who used cam told their physicians about this usage . thus , physicians need to ask patients about their use of cam to allow further careful treatment . additionally , further study is needed to determine the evidence - based efficacy of the use of cam in children with common neurologic diseases .

complementary and alternative medicine ( cam ) is a phrase used to describe additional health care methods such as mind / body practices and natural products not regarded as treatments by conventional medicine . the use of cam in children with common neurologic diseases is more frequent than its use in healthy children ( 24%78% vs. 12% ) . however , less than half of patients report such use to their physicians . the preferred modalities of cam vary in different countries due to their different cultures and traditions . the most common factor significantly associated with the use of cam is parental cam use in most studies . the frequency of the use of cam in children and adults with neurologic diseases is similar , and both rates are higher than the rates in those without these conditions . the preferred modalities of cam in adults are diverse , and megavitamins and mind / body therapy ( prayer and chiropractic care ) are included . the most common factor significantly associated with the use of cam in adults with neurologic diseases is high educational level . physicians need to be concerned with patients ' use of cam and provide correct information about cam so that patients may make the right decisions . further study is needed to determine the evidence - based efficacy of cam use in children with common neurologic diseases .

Introduction Characteristics of the use of CAM 1. Characteristics of the use of CAM in children with neurologic diseases 2. Characteristics of the use of CAM in adults with common neurologic conditions 3. The use of CAM in children with other chronic diseases Conclusions

complementary and alternative medicine ( cam ) is an additional health care system , comprising mind / body practices and natural products that are not regarded as part of conventional medicine12 ) . conventional medicine is used together when complementary medicine is applied , and alternative medicine is employed instead of conventional medicine1 ) . natural products include herbs , minerals , vitamins , and probiotics , and mind / body practices include acupuncture , massage therapy , meditation , movement therapies , relaxation techniques , spinal manipulation , and others1 ) . physicians should be aware of their patients ' use of cam and provide accurate information about cam to enable patients to make the right decisions . this review aims to investigate the characteristics of the use of cam in children with neurologic diseases such as epilepsy , migraine , cerebral palsy , and congenital malformations in order to improve management through analysis of several studies of cam use in children with chronic neurologic conditions2678910 ) . further , this review will discuss the differences in the use of cam between children and adults with neurologic diseases , and between patients with neurologic and nonneurologic conditions . barnes et al.9 ) reported that approximately 12% of children ( total sample size [ n]=9,417 ) used cam in the united states ( us ) , and mccann and newell3 ) reported that 12% of healthy children ( n=25 ) used cam in the united kingdom ( uk ) . they also reported that 40% ( n=75 ) of children with chronic health conditions used cam in the uk , and this proportion is much higher than that in healthy children3 ) . in the study by barnes et al.9 ) , the most common modalities used were natural products ( neither vitamin nor mineral ) and osteopathic or chiropractic manipulation . regarding significant factors , parental use of cam was associated with cam use in children compared to children whose parents did not use cam ( 24% vs. 5% ) . parental use of cam was also found to be a strongly associated factor in a study by birdee et al.11 ) . additionally , for both adults and children , worry about cost was found to be one of the factors related to the use of cam and delaying using conventional medicine , and patients were more likely to use cam when expenses were a concern than when expenses were not a problem for conventional medicine9 ) . other factors significantly associated with pediatric use of cam , in the study of birdee et al.11 ) , were adolescent age rather than that of toddler or infant , parent with a college education , more frequent use of prescription medication , stress or anxiety , musculoskeletal conditions , dermatologic conditions , and sinus infection . in a recent study on patterns of cam use in pediatric patients with common neurologic conditions ( headaches , migraines , seizures ) , pediatric patients with common neurological conditions were found to use cam significantly more compared to children without these conditions ( 24% [ n=586 ] vs.13% [ n=7,083])7 ) . pediatric patients with neurologic conditions used biological therapies with a similar frequency and mind / body practices with a significantly higher frequency compared to other pediatric patients using cam . among mind / body practices , deep breathing was used most commonly ( 33% ) , meditation was used in 15% , and progressive relaxation was employed in 10%7 ) . among the reasons provided for the use of cam , religious beliefs were the most common ( 68% ) , and no patients described distrust in conventional medicine2 ) . other significant factors connected to the use of cam included belief in its effects , speech delay , father 's age greater than 30 years , and mother 's education less than high school2 ) . in a study by soo et al.8 ) regarding the use of cam in a pediatric neurology clinic , 44% of patients ( n=105 ) were found to use cam . the difference in this study compared to other studies is that the parents ' sociodemographic characteristics were not significantly associated with the use of cam . in a study on cam use in pediatric neurology , the overall rates of cam use at two centers were 78% ( n=151 ) and 48% ( n=55 ) , respectively6 ) . over half of patients disclosed to their physicians their use of cam in addition to conventional medicine6 ) . in a study examining the use of traditional herbal medicines in korean elementary school children , 65% ( n=905 ) of children were found to be taking herbal medicines12 ) . this finding suggests that the use of tkm is associated more with the purpose of treatment of cognitive function than motor functions , and parents of children with epilepsy may believe that tkm has an effect on the former rather than the latter10 ) . in spite of these beliefs , however , according to the report of kuan et al.16 ) , the most frequent adverse effect of cam was aggravated seizures . regarding the frequency of cam use for epileptic patients in various countries , 49% ( n=403 ) of taiwanese were found to use cam , 44% used it in arizona , 37% ( n=265 ) of nigerians used cam , and 32% ( n=1,000 ) in india and 24% ( n=92 ) in ohio used cam516171819 ) . in us , the preferred modalities were biological therapies and mind - body practices7 ) . in korea these differences in use of cam between countries may be owing to their different cultures , religions , and traditions10 ) . the characteristics of the use of cam in children with neurologic diseases are summarized in table 2 . according to the study by barnes et al.9 ) , almost 40% ( n=23,393 ) of us adults used cam in 2007 and the most commonly used modalities were natural products ( neither vitamin nor mineral ) ( 18% ) and deep breathing exercises ( 13% . ) cam was used more frequently in american adults with common neurological conditions ( migraines , regular headaches , strokes , back pain with sciatica , seizures , dementia or memory loss ) than in those without such conditions ( 44% [ n=6,587 ] vs. 33% [ n=16,806])20 ) . the frequency of use of each cam modality was higher in adults with common neurological conditions than in those without20 ) . the most common modalities were mind / body therapies , and the least common were alternative medical systems20 ) . the reasons why those with common neurological conditions used cam more frequently than those without such conditions included provider 's recommendations or little effect achieved with conventional medicine or a cost that was too high20 ) . about 50% of adults with common neurological conditions did not report their use of cam to their doctors20 ) . factors that significantly influenced the use of cam in those with common neurological conditions included higher educational level than high school education , positive history of anxiety , living in a western area , being a former smoker , and drinking alcohol lightly20 ) . in the previous studies on the use of cam in various neurologic conditions , compared to the frequency of use of cam in children with common neurologic diseases , the frequency of use in adults with neurologic diseases was similar267810 ) . for korean adults with other diseases , several studies showed the following findings regarding cam use : 60%70% of patients with stroke used cam , 53% of those with cancer used it and 53% of patients with asthma and preferred modalities of cam use in children and adults had diverse features , and these results may be influenced by individuals ' different cultures and traditions of use of cam . the factor most commonly related to use of cam in children with neurologic conditions was parental cam use , and it could be presumed that the use of cam in children may be due to such usage running in the family , as well as belief in or experience with the efficacy of cam . in adults with neurologic conditions , increased educational level was the most common factor associated with use of cam202123 ) , and this result may relate to economic level . characteristics of the use of cam in adults with neurologic diseases are summarized in table 3 . in a systematic review on the use of cam in pediatric cancer patients , the prevalence of use of cam ( since the diagnosis of cancer ) the most common cam modalities were herbal medicines , diets / nutrition , and faith - healing28 ) . the frequent reasons given for the use of cam were to help cure or fight the child 's cancer , relief of symptom , and support of ongoing use of conventional medicine28 ) . there were few factors related to patients ' sociodemographic characteristics associated with use of cam , and this result was different from the results of several other studies28 ) . many studies , however , reported a higher frequency of cam use in pediatric patients with chronic diseases , such as asthma , autism , adhd , cancer , and sickle cell anemia . medical symptoms such as nausea and abdominal pain rather than certain medical diagnoses were found to be some of the factors associated with the use of cam28 ) . the most commonly used cams were biological products ( 58% ) , while alternative medical systems ( 27% ) as well as mind / body interventions ( 14% ) were also employed . eighty - five parents ( 86% ) were going to report the use of cam to their physicians , but had not yet done so . in 7% of the patients , levy and hyman30 ) investigated the use of cam for children with autistic spectrum disorder ( asd ) . patients with comorbid intellectual disability may have a higher frequency of use of cam . the most frequently used form of cam for children with asd were mind / body practices30 ) . also , more medications and more medical visits as well as more side effects were significant predictors in using cam34 ) a study in italy showed that the most common reason for the use of cam was a fear of side effects of conventional therapy for routine illness35 ) . in the study by hanson et al.36 ) , 75% of parents of children with asd chose the use of cam because they felt cam was perceived to be safe and it did not have side effect or they experienced side effects of conventional therapy previously . the purposes of the use of cam in children with asd are treatment of major symptoms of asd , improvement of attention , enhancement of relaxation , treatment of gastrointestinal symptoms , regulation of sleep and promotion of general health31 ) . the characteristics of the use of cam in children with other chronic diseases are summarized in table 4 . in a recent study on patterns of cam use in pediatric patients with common neurologic conditions ( headaches , migraines , seizures ) , pediatric patients with common neurological conditions were found to use cam significantly more compared to children without these conditions ( 24% [ n=586 ] vs.13% [ n=7,083])7 ) . pediatric patients with neurologic conditions used biological therapies with a similar frequency and mind / body practices with a significantly higher frequency compared to other pediatric patients using cam . among mind / body practices , deep breathing was used most commonly ( 33% ) , meditation was used in 15% , and progressive relaxation was employed in 10%7 ) . among the reasons provided for the use of cam , religious beliefs were the most common ( 68% ) , and no patients described distrust in conventional medicine2 ) . other significant factors connected to the use of cam included belief in its effects , speech delay , father 's age greater than 30 years , and mother 's education less than high school2 ) . in a study by soo et al.8 ) regarding the use of cam in a pediatric neurology clinic , 44% of patients ( n=105 ) were found to use cam . the difference in this study compared to other studies is that the parents ' sociodemographic characteristics were not significantly associated with the use of cam . over half of patients disclosed to their physicians their use of cam in addition to conventional medicine6 ) . in a study examining the use of traditional herbal medicines in korean elementary school children , 65% ( n=905 ) of children were found to be taking herbal medicines12 ) . this finding suggests that the use of tkm is associated more with the purpose of treatment of cognitive function than motor functions , and parents of children with epilepsy may believe that tkm has an effect on the former rather than the latter10 ) . additionally , regarding significant factors , in the group of patients using a greater number of antiepileptic drugs and having low seizure - free rates , the use of tkm was more frequent10 ) . in spite of these beliefs , however , according to the report of kuan et al.16 ) , the most frequent adverse effect of cam was aggravated seizures . regarding the frequency of cam use for epileptic patients in various countries , 49% ( n=403 ) of taiwanese were found to use cam , 44% used it in arizona , 37% ( n=265 ) of nigerians used cam , and 32% ( n=1,000 ) in india and 24% ( n=92 ) in ohio used cam516171819 ) . in us , the preferred modalities were biological therapies and mind - body practices7 ) . in korea these differences in use of cam between countries may be owing to their different cultures , religions , and traditions10 ) . the characteristics of the use of cam in children with neurologic diseases are summarized in table 2 . according to the study by barnes et al.9 ) , almost 40% ( n=23,393 ) of us adults used cam in 2007 and the most commonly used modalities were natural products ( neither vitamin nor mineral ) ( 18% ) and deep breathing exercises ( 13% . ) cam was used more frequently in american adults with common neurological conditions ( migraines , regular headaches , strokes , back pain with sciatica , seizures , dementia or memory loss ) than in those without such conditions ( 44% [ n=6,587 ] vs. 33% [ n=16,806])20 ) . the frequency of use of each cam modality was higher in adults with common neurological conditions than in those without20 ) . the most common modalities were mind / body therapies , and the least common were alternative medical systems20 ) . the reasons why those with common neurological conditions used cam more frequently than those without such conditions included provider 's recommendations or little effect achieved with conventional medicine or a cost that was too high20 ) . about 50% of adults with common neurological conditions did not report their use of cam to their doctors20 ) . factors that significantly influenced the use of cam in those with common neurological conditions included higher educational level than high school education , positive history of anxiety , living in a western area , being a former smoker , and drinking alcohol lightly20 ) . in the previous studies on the use of cam in various neurologic conditions , the prevalence of use of cam ranged from 19% to 43%212223 ) . compared to the frequency of use of cam in children with common neurologic diseases , the frequency of use in adults with neurologic diseases was similar267810 ) . for korean adults with other diseases , several studies showed the following findings regarding cam use : 60%70% of patients with stroke used cam , 53% of those with cancer used it and 53% of patients with asthma and 48% of patients with psychiatric disorders used herbal medicines24252627 ) . preferred modalities of cam use in children and adults had diverse features , and these results may be influenced by individuals ' different cultures and traditions of use of cam . the factor most commonly related to use of cam in children with neurologic conditions was parental cam use , and it could be presumed that the use of cam in children may be due to such usage running in the family , as well as belief in or experience with the efficacy of cam . in adults with neurologic conditions , increased educational level was the most common factor associated with use of cam202123 ) , and this result may relate to economic level . characteristics of the use of cam in adults with neurologic diseases are summarized in table 3 . in a systematic review on the use of cam in pediatric cancer patients , the prevalence of use of cam ( since the diagnosis of cancer ) ranged from 6% to 91% ( n=2,871 ) . the most common cam modalities were herbal medicines , diets / nutrition , and faith - healing28 ) . the frequent reasons given for the use of cam were to help cure or fight the child 's cancer , relief of symptom , and support of ongoing use of conventional medicine28 ) . there were few factors related to patients ' sociodemographic characteristics associated with use of cam , and this result was different from the results of several other studies28 ) . according to the study by barnes et al.9 ) , for pediatric patients with specific diseases , the use of cam was not highly prevalent . many studies , however , reported a higher frequency of cam use in pediatric patients with chronic diseases , such as asthma , autism , adhd , cancer , and sickle cell anemia . medical symptoms such as nausea and abdominal pain rather than certain medical diagnoses were found to be some of the factors associated with the use of cam28 ) . the most commonly used cams were biological products ( 58% ) , while alternative medical systems ( 27% ) as well as mind / body interventions ( 14% ) were also employed . eighty - five parents ( 86% ) were going to report the use of cam to their physicians , but had not yet done so . in 7% of the patients , levy and hyman30 ) investigated the use of cam for children with autistic spectrum disorder ( asd ) . patients with comorbid intellectual disability may have a higher frequency of use of cam . the most frequently used form of cam for children with asd were mind / body practices30 ) . a study in italy showed that the most common reason for the use of cam was a fear of side effects of conventional therapy for routine illness35 ) . in the study by hanson et al.36 ) , 75% of parents of children with asd chose the use of cam because they felt cam was perceived to be safe and it did not have side effect or they experienced side effects of conventional therapy previously . the purposes of the use of cam in children with asd are treatment of major symptoms of asd , improvement of attention , enhancement of relaxation , treatment of gastrointestinal symptoms , regulation of sleep and promotion of general health31 ) . the characteristics of the use of cam in children with other chronic diseases are summarized in table 4 . the use of cam in children with neurologic diseases is common , ranging from 24%78% and it is more frequent than the use in healthy children . preferred modalities of cam differed according to patients ' country of residence and due to their different cultures and traditions , with examples such as prayer / reciting the quran in jordan , biologic therapies , and mind / body therapies in the us , and tkm in korea . the most common factor significantly associated with the use of cam was parental cam use in most studies . the frequency of cam use in children and adults with neurologic diseases is higher than that in those without these conditions . preferred modalities of cam in adults with common neurological conditions were diverse , including megavitamins and mind / body therapy ( prayer and chiropractic care ) . the most common factor significantly associated with use of cam in adults with neurologic diseases was higher educational level . among children and adults with neurologic diseases , less than half of patients who used cam told their physicians about this usage . thus , physicians need to ask patients about their use of cam to allow further careful treatment . additionally , further study is needed to determine the evidence - based efficacy of the use of cam in children with common neurologic diseases .

children with chronic health conditions use cam more frequently3 ) , but it was found that only around 30% of patients with epilepsy and 50% of children with chronic health conditions who use cam reported this behavior to their physicians456 ) . this review aims to investigate the characteristics of the use of cam in children with neurologic diseases such as epilepsy , migraine , cerebral palsy , and congenital malformations in order to improve management through analysis of several studies of cam use in children with chronic neurologic conditions2678910 ) . further , this review will discuss the differences in the use of cam between children and adults with neurologic diseases , and between patients with neurologic and nonneurologic conditions . barnes et al.9 ) reported that approximately 12% of children ( total sample size [ n]=9,417 ) used cam in the united states ( us ) , and mccann and newell3 ) reported that 12% of healthy children ( n=25 ) used cam in the united kingdom ( uk ) . they also reported that 40% ( n=75 ) of children with chronic health conditions used cam in the uk , and this proportion is much higher than that in healthy children3 ) . regarding significant factors , parental use of cam was associated with cam use in children compared to children whose parents did not use cam ( 24% vs. 5% ) . additionally , for both adults and children , worry about cost was found to be one of the factors related to the use of cam and delaying using conventional medicine , and patients were more likely to use cam when expenses were a concern than when expenses were not a problem for conventional medicine9 ) . other factors significantly associated with pediatric use of cam , in the study of birdee et al.11 ) , were adolescent age rather than that of toddler or infant , parent with a college education , more frequent use of prescription medication , stress or anxiety , musculoskeletal conditions , dermatologic conditions , and sinus infection . in a recent study on patterns of cam use in pediatric patients with common neurologic conditions ( headaches , migraines , seizures ) , pediatric patients with common neurological conditions were found to use cam significantly more compared to children without these conditions ( 24% [ n=586 ] vs.13% [ n=7,083])7 ) . pediatric patients with neurologic conditions used biological therapies with a similar frequency and mind / body practices with a significantly higher frequency compared to other pediatric patients using cam . among mind / body practices , deep breathing was used most commonly ( 33% ) , meditation was used in 15% , and progressive relaxation was employed in 10%7 ) . regarding the most commonly used methods , prayer / reciting the quran was used in 77% , massage with olive oil in 32% , religious healers in 30% , and consumption of honey products in 29% . among the reasons provided for the use of cam , religious beliefs were the most common ( 68% ) , and no patients described distrust in conventional medicine2 ) . other significant factors connected to the use of cam included belief in its effects , speech delay , father 's age greater than 30 years , and mother 's education less than high school2 ) . in regard to the most commonly used cam modalities , chiropractic manipulation was used in 15% , dietary therapy was used in 12% , and homeopathy , herbal remedies , and prayer healing were each used in 8% . the difference in this study compared to other studies is that the parents ' sociodemographic characteristics were not significantly associated with the use of cam . the total median cost of cam compared with conventional medicines was not different ( $ 31.70 vs. $ 50.00 per month)8 ) . in a study on cam use in pediatric neurology , the overall rates of cam use at two centers were 78% ( n=151 ) and 48% ( n=55 ) , respectively6 ) . as for the most commonly used cam products , multivitamins were used in 84% , vitamin c was used in 37% , homeopathic remedies were used in 24% , and fish oil / omega-3s were employed in 22%6 ) . regarding the most commonly used cam practices , massage was used in 47% , and 37% used chiropractic methods , 18% used faith - healing , and aromatherapy , homeopathy , and relaxation were each used in 16%6 ) . in a study examining the use of traditional herbal medicines in korean elementary school children , 65% ( n=905 ) of children were found to be taking herbal medicines12 ) . in our study in 2014 , 19% ( n=398 ) of children with epilepsy used traditional korean medicine ( tkm ) ( acupuncture , moxibustion , herbal medicine)10 ) . according to a study by lee et al.4 ) , patients with psychosomatic disorders used herbal medicine significantly more frequently , whereas in a study by kim et al.13 ) , neurologically normal patients used it slightly more frequently ; however , the difference did not have statistical significance . in our study , among receivers of combination treatments besides tkm , tkm was used more frequently in those receiving language , music , or art therapy than in those undergoing physiotherapy or other combination therapies10 ) . this finding suggests that the use of tkm is associated more with the purpose of treatment of cognitive function than motor functions , and parents of children with epilepsy may believe that tkm has an effect on the former rather than the latter10 ) . additionally , regarding significant factors , in the group of patients using a greater number of antiepileptic drugs and having low seizure - free rates , the use of tkm was more frequent10 ) . recent cochrane reviews show that some studies on the use of acupuncture and chinese traditional herbal medicine did not succeed in providing clear evidence proving the effects of acupuncture and herbal medicine for epilepsy patients1415 ) . according to the study by lee et al.4 ) , 18% of respondents using herbal medicine reported its effectiveness for seizure control , but the rest of the respondents reported no effectiveness or even worsening of symptoms , although many patients ( 33% ) agreed with using herbal medicine . in spite of these beliefs , however , according to the report of kuan et al.16 ) , the most frequent adverse effect of cam was aggravated seizures . regarding the frequency of cam use for epileptic patients in various countries , 49% ( n=403 ) of taiwanese were found to use cam , 44% used it in arizona , 37% ( n=265 ) of nigerians used cam , and 32% ( n=1,000 ) in india and 24% ( n=92 ) in ohio used cam516171819 ) . regarding commonly used forms of cam for treatment of epileptic patients , prayer and stress relief were used in the west , whereas traditional herbal medicines were employed both in the east and in africa516171819 ) . according to the study by barnes et al.9 ) , almost 40% ( n=23,393 ) of us adults used cam in 2007 and the most commonly used modalities were natural products ( neither vitamin nor mineral ) ( 18% ) and deep breathing exercises ( 13% . ) cam was used more frequently in american adults with common neurological conditions ( migraines , regular headaches , strokes , back pain with sciatica , seizures , dementia or memory loss ) than in those without such conditions ( 44% [ n=6,587 ] vs. 33% [ n=16,806])20 ) . factors that significantly influenced the use of cam in those with common neurological conditions included higher educational level than high school education , positive history of anxiety , living in a western area , being a former smoker , and drinking alcohol lightly20 ) . in the previous studies on the use of cam in various neurologic conditions , compared to the frequency of use of cam in children with common neurologic diseases , the frequency of use in adults with neurologic diseases was similar267810 ) . for korean adults with other diseases , several studies showed the following findings regarding cam use : 60%70% of patients with stroke used cam , 53% of those with cancer used it and 53% of patients with asthma and preferred modalities of cam use in children and adults had diverse features , and these results may be influenced by individuals ' different cultures and traditions of use of cam . the factor most commonly related to use of cam in children with neurologic conditions was parental cam use , and it could be presumed that the use of cam in children may be due to such usage running in the family , as well as belief in or experience with the efficacy of cam . in adults with neurologic conditions , increased educational level was the most common factor associated with use of cam202123 ) , and this result may relate to economic level . in a systematic review on the use of cam in pediatric cancer patients , the prevalence of use of cam ( since the diagnosis of cancer ) the most common cam modalities were herbal medicines , diets / nutrition , and faith - healing28 ) . the frequent reasons given for the use of cam were to help cure or fight the child 's cancer , relief of symptom , and support of ongoing use of conventional medicine28 ) . there were few factors related to patients ' sociodemographic characteristics associated with use of cam , and this result was different from the results of several other studies28 ) . according to the study by barnes et al.9 ) , for pediatric patients with specific diseases , the proportions were 2% in insomnia , 3% in attention deficit hyperactivity disorder ( adhd ) , 5% in other musculoskeletal diseases , 5% in stress or anxiety , 7% in head or chest colds , and 7% in back or neck pain9 ) . many studies , however , reported a higher frequency of cam use in pediatric patients with chronic diseases , such as asthma , autism , adhd , cancer , and sickle cell anemia . in the study by oshikoya et al.29 ) , the researchers interviewed parents of children with epilepsy ( n=122 ) , asthma ( n=78 ) , or sickle cell anemia ( n=118 ) . in 7% of the patients , levy and hyman30 ) investigated the use of cam for children with autistic spectrum disorder ( asd ) . in cases of children with asthma , the significant predictors of use of cam were older age and worse control of symptoms . also , more medications and more medical visits as well as more side effects were significant predictors in using cam34 ) a study in italy showed that the most common reason for the use of cam was a fear of side effects of conventional therapy for routine illness35 ) . in the study by hanson et al.36 ) , 75% of parents of children with asd chose the use of cam because they felt cam was perceived to be safe and it did not have side effect or they experienced side effects of conventional therapy previously . the purposes of the use of cam in children with asd are treatment of major symptoms of asd , improvement of attention , enhancement of relaxation , treatment of gastrointestinal symptoms , regulation of sleep and promotion of general health31 ) . in a recent study on patterns of cam use in pediatric patients with common neurologic conditions ( headaches , migraines , seizures ) , pediatric patients with common neurological conditions were found to use cam significantly more compared to children without these conditions ( 24% [ n=586 ] vs.13% [ n=7,083])7 ) . pediatric patients with neurologic conditions used biological therapies with a similar frequency and mind / body practices with a significantly higher frequency compared to other pediatric patients using cam . regarding the most commonly used methods , prayer / reciting the quran was used in 77% , massage with olive oil in 32% , religious healers in 30% , and consumption of honey products in 29% . among the reasons provided for the use of cam , religious beliefs were the most common ( 68% ) , and no patients described distrust in conventional medicine2 ) . other significant factors connected to the use of cam included belief in its effects , speech delay , father 's age greater than 30 years , and mother 's education less than high school2 ) . in regard to the most commonly used cam modalities , chiropractic manipulation was used in 15% , dietary therapy was used in 12% , and homeopathy , herbal remedies , and prayer healing were each used in 8% . the difference in this study compared to other studies is that the parents ' sociodemographic characteristics were not significantly associated with the use of cam . in a study on cam use in pediatric neurology , the overall rates of cam use at two centers were 78% ( n=151 ) and 48% ( n=55 ) , respectively6 ) . as for the most commonly used cam products , multivitamins were used in 84% , vitamin c was used in 37% , homeopathic remedies were used in 24% , and fish oil / omega-3s were employed in 22%6 ) . regarding the most commonly used cam practices , massage was used in 47% , and 37% used chiropractic methods , 18% used faith - healing , and aromatherapy , homeopathy , and relaxation were each used in 16%6 ) . in a study examining the use of traditional herbal medicines in korean elementary school children , 65% ( n=905 ) of children were found to be taking herbal medicines12 ) . in our study in 2014 , 19% ( n=398 ) of children with epilepsy used traditional korean medicine ( tkm ) ( acupuncture , moxibustion , herbal medicine)10 ) . according to a study by lee et al.4 ) , patients with psychosomatic disorders used herbal medicine significantly more frequently , whereas in a study by kim et al.13 ) , neurologically normal patients used it slightly more frequently ; however , the difference did not have statistical significance . in our study , among receivers of combination treatments besides tkm , tkm was used more frequently in those receiving language , music , or art therapy than in those undergoing physiotherapy or other combination therapies10 ) . this finding suggests that the use of tkm is associated more with the purpose of treatment of cognitive function than motor functions , and parents of children with epilepsy may believe that tkm has an effect on the former rather than the latter10 ) . additionally , regarding significant factors , in the group of patients using a greater number of antiepileptic drugs and having low seizure - free rates , the use of tkm was more frequent10 ) . recent cochrane reviews show that some studies on the use of acupuncture and chinese traditional herbal medicine did not succeed in providing clear evidence proving the effects of acupuncture and herbal medicine for epilepsy patients1415 ) . according to the study by lee et al.4 ) , 18% of respondents using herbal medicine reported its effectiveness for seizure control , but the rest of the respondents reported no effectiveness or even worsening of symptoms , although many patients ( 33% ) agreed with using herbal medicine . regarding the frequency of cam use for epileptic patients in various countries , 49% ( n=403 ) of taiwanese were found to use cam , 44% used it in arizona , 37% ( n=265 ) of nigerians used cam , and 32% ( n=1,000 ) in india and 24% ( n=92 ) in ohio used cam516171819 ) . regarding commonly used forms of cam for treatment of epileptic patients , prayer and stress relief were used in the west , whereas traditional herbal medicines were employed both in the east and in africa516171819 ) . according to the study by barnes et al.9 ) , almost 40% ( n=23,393 ) of us adults used cam in 2007 and the most commonly used modalities were natural products ( neither vitamin nor mineral ) ( 18% ) and deep breathing exercises ( 13% . ) cam was used more frequently in american adults with common neurological conditions ( migraines , regular headaches , strokes , back pain with sciatica , seizures , dementia or memory loss ) than in those without such conditions ( 44% [ n=6,587 ] vs. 33% [ n=16,806])20 ) . factors that significantly influenced the use of cam in those with common neurological conditions included higher educational level than high school education , positive history of anxiety , living in a western area , being a former smoker , and drinking alcohol lightly20 ) . in the previous studies on the use of cam in various neurologic conditions , the prevalence of use of cam ranged from 19% to 43%212223 ) . compared to the frequency of use of cam in children with common neurologic diseases , the frequency of use in adults with neurologic diseases was similar267810 ) . for korean adults with other diseases , several studies showed the following findings regarding cam use : 60%70% of patients with stroke used cam , 53% of those with cancer used it and 53% of patients with asthma and 48% of patients with psychiatric disorders used herbal medicines24252627 ) . the factor most commonly related to use of cam in children with neurologic conditions was parental cam use , and it could be presumed that the use of cam in children may be due to such usage running in the family , as well as belief in or experience with the efficacy of cam . in adults with neurologic conditions , increased educational level was the most common factor associated with use of cam202123 ) , and this result may relate to economic level . in a systematic review on the use of cam in pediatric cancer patients , the prevalence of use of cam ( since the diagnosis of cancer ) ranged from 6% to 91% ( n=2,871 ) . the frequent reasons given for the use of cam were to help cure or fight the child 's cancer , relief of symptom , and support of ongoing use of conventional medicine28 ) . there were few factors related to patients ' sociodemographic characteristics associated with use of cam , and this result was different from the results of several other studies28 ) . the proportions were 2% in insomnia , 3% in attention deficit hyperactivity disorder ( adhd ) , 5% in other musculoskeletal diseases , 5% in stress or anxiety , 7% in head or chest colds , and 7% in back or neck pain9 ) . many studies , however , reported a higher frequency of cam use in pediatric patients with chronic diseases , such as asthma , autism , adhd , cancer , and sickle cell anemia . in the study by oshikoya et al.29 ) , the researchers interviewed parents of children with epilepsy ( n=122 ) , asthma ( n=78 ) , or sickle cell anemia ( n=118 ) . in cases of children with asthma , the significant predictors of use of cam were older age and worse control of symptoms . in the study by hanson et al.36 ) , 75% of parents of children with asd chose the use of cam because they felt cam was perceived to be safe and it did not have side effect or they experienced side effects of conventional therapy previously . the purposes of the use of cam in children with asd are treatment of major symptoms of asd , improvement of attention , enhancement of relaxation , treatment of gastrointestinal symptoms , regulation of sleep and promotion of general health31 ) . preferred modalities of cam differed according to patients ' country of residence and due to their different cultures and traditions , with examples such as prayer / reciting the quran in jordan , biologic therapies , and mind / body therapies in the us , and tkm in korea .

during acute inflammation , leukocytes , mostly neutrophils , are recruited to the afflicted site by a well - defined and dynamic multistep process which includes leukocyte tethering , rolling , adhesion , and transmigration out of the vasculature [ 13 ] . in vivo , neutrophil recruitment is complex and regulated by a variety of molecules and signalling cascades triggered by the cross - talk between neutrophils and endothelium . neutrophil rolling is mediated by selectins , and adhesion is mediated by 2 integrins while emigration is regulated by the interactions between integrins , pecam-1 , and junctional adhesion molecules and their respective ligands . recently , an additional step of neutrophil recruitment termed intraluminal crawling was described , a process which is molecularly distinct from adhesion . intraluminal crawling enables neutrophils to reach optimal emigration sites at endothelial junctions independently of hemodynamic forces [ 6 , 7 ] . the 2 integrin lfa-1 mediates firm adhesion of neutrophils to endothelial cells while the subsequent step of intraluminal crawling occurs as a result of interactions between mac-1 and endothelial icam-1 . blocking of lfa-1 and mac-1 in monocytes and icam-1 and icam-2 on endothelial cells was shown to prevent the intraluminal locomotion of monocytes to endothelial cell junctions and ensuing diapedesis . signalling mechanisms that regulate intraluminal crawling and subsequent transendothelial migration of leukocytes are not completely understood . p38 mitogen - activated protein kinase ( mapk ) signalling regulates a wide array of inflammatory processes , cell proliferation , differentiation , survival , and invasion [ 9 , 10 ] . p38 mapk signalling participates in the expression and function of inflammatory cytokines such as tnf , il-1 , il-2 , il-6 , il-7 , and il-8 . pharmacological inhibition of p38 mapk signalling was previously shown to ameliorate inflammatory disorders such as asthma , rheumatoid arthritis , inflammatory bowel disease , stroke , systemic lupus erythematosus , and autoimmune diseases [ 1217 ] , thus serving as a promising therapeutic target . previous studies have documented the role of p38 mapk in neutrophil function during different steps of neutrophil recruitment . cytokines and inflammatory mediators such as tnf , lps and fmlp , and chemokine kc / cxcl1 were shown to phosphorylate p38 mapk during inflammation [ 1921 ] . pharmacological inhibition of p38 mapk was found to impair neutrophil chemotaxis and transendothelial migration induced by the chemokine kc . in this latter study , the number of emigrated neutrophils in each grid of defined distance from the observed venule was measured as neutrophil chemotaxis parameter . however , the dynamic motile behavioural changes of chemotaxing neutrophils such as velocity of migration and chemotaxis index were not measured , and the role of p38 mapk in these changes was not determined . in addition , whether there is a role of p38 mapk in intraluminal crawling of neutrophils has not been reported . sb203580 is a widely used selective inhibitor of the p38 and p38 isoforms and binds to the active site of p38 mapk in an atp - competitive manner . sb203580 can further block the translocation of p38 mapk and its downstream substrate mapk activated protein kinase 2 from the nucleus to cytosol . mounting evidence suggests that sb203580 is effective in a variety of in vitro and in vivo models of inflammation characterized by attenuated production of proinflammatory cytokines such as tnf and il-1 . the use of real - time intravital microscopy and time - lapsed video photography analysis makes it possible to determine simultaneously multiple leukocyte recruitment parameters of rolling , adhesion , emigration , intraluminal crawling velocity , transmigration time , detachment time , migration velocity , chemotaxis velocity , and chemotaxis index in tissue [ 5 , 24 ] . in the present study , we explored the effects of the p38 mapk inhibitor sb203580 on various parameters of neutrophil recruitment induced by chemotactic gradient of kc in vivo . c57bl/6 mice were purchased from charles river canada ( saint - constant , qc , canada ) . this study was carried out with the approval of animal protocols from the university committee on animal care and supply ( ucacs ) at the university of saskatchewan following the standards of the canadian association of animal care . injection of 10 mg / kg xylazine ( bayer , toronto , on , canada ) and 200 mg / kg ketamine hydrochloride ( rogar , montreal , qc , canada ) . the mouse cremaster muscle preparation was used to study neutrophil behaviour in microcirculation and tissue as described previously [ 2426 ] . the cremaster muscle was superfused with 37c warmed bicarbonate - buffered saline ( ph 7.4 ; containing in mm 133.9 nacl , 4.7 kcl , 1.2 mgso4 , and 20 nahco3 ) . an upright microscope ( model eclipse ci - s , nikon ) with a lucplfln 20x objective lens was connected to a ccd color video camera ( dc-220 , dage ) for bright - field intravital microscopy . for the induction of neutrophil recruitment , an agarose gel at 1 mm size containing the optimal concentration of cxc chemokine kc / cxcl1 ( 0.5 m ; peprotech , dollard des ormeaux , qc , canada ) was placed on the surface of the cremaster muscle in a preselected area 350 m from and parallel to the observed postcapillary venule . after placing a coverslip , the cremaster muscle was superfused with bicarbonate - buffered saline at a very slow rate ( 10 l / min ) for allowing formation of chemoattractant gradient . throughout the experiment , neutrophil behaviour and hemodynamic changes in the selected cremasteric postcapillary venule ( 2540 m diameter ) were visualized , projected on a tv monitor , recorded at real time on a dvd recorder before ( for time 0 min ) and after addition of kc - gel ( recorded for 60 or 90 min ) . during recording , all efforts were made to adjust and keep the microscope focused on the adhering , crawling , transmigrating , and chemotaxing neutrophil inside the venule and in the muscle tissue . the number of rolling , adherent , and emigrated neutrophils during offline playback analysis of the recorded video was determined in the cremasteric microvasculature as described previously [ 2426 ] . the inhibitor sb203580 ( emd , billenca , ma ) was used to experimentally suppress the activity of p38 mapk . sb203580 was dissolved in dmso at 10 mm and stored in aliquots at 20c . to determine the role of p38 mapk in neutrophil crawling and transmigration , sb203580 ( 100 nm ) was superfused 30 min prior to the placement of kc - containing gel and sb203580 superfusion remained for the duration of kc treatment ( 60 min ) . to analyze the role of p38 mapk in neutrophil migration and chemotaxis in cremasteric tissue after transendothelial migration , sb203580 was superfused 30 min after the placement of kc - containing gel when at least 8 emigrated neutrophils were identified in perivascular tissue and remained perfused for additional 60 min . sb203580 administered intravenously before this kc treatment completely inhibited adhesion and emigration induced by kc ( data not shown ) . for in vitro experiments , sb203580 ( 10 m ) was added to the medium 30 min prior to stimulation of neutrophils with kc . in all experiments , the same amount of dmso as in the sb203580-treated group was used in the control group without sb203580 and was not found to affect any responses . using imagej software , leukocyte intraluminal crawling , transmigration , and chemotaxis in cremasteric vasculature were analysed using the time - lapsed movie converted from the real - time video recording of the experiment as described previously [ 5 , 7 , 24 ] . the following recruitment parameters were quantified from tracking and analyzing at least 40 cells for each treatment group.percentage of adherent leukocytes that crawled : the number of adherent and then crawled leukocytes divided by the total number of adherent leukocytes ( % ) . crawling distance : the total distance the leukocyte crawled from the initial site of adhesion to the transmigration site ( m ) . crawling time : duration of leukocytes undergoing intraluminal crawling ( min).velocity of crawling : the crawling distance in the vessel lumen divided by crawling time ( m / min).percentage of crawling leukocytes along the blood flow : the number of crawling leukocytes with their final crawling location in the direction of blood flow divided by the total crawling leukocytes ( % ) .directional crawling index : the ratio of the crawling distance in the direction toward the kc gel to the total crawling distance that the cell crawled in the lumen of the venule.percentage of crawling leukocytes that transmigrated : the number of crawled and then transmigrated leukocytes divided by the total number of crawling leukocytes ( % ) . transmigration time : from the time the leukocyte stopped crawling and started to transmigrate across endothelium to the time the whole leukocyte body was just outside the venule ( min).detachment time : from the time the leukocyte body was just outside the venule after its transmigration to the time when the leukocyte migrated away and lost contact to the venule ( min).migration distance : the sum of the distance that the leukocyte moved from the start point after detachment from the venule to the end point of the migration in tissue at 60 or 90 min of kc treatment or to the end point of the field of view ( m ) . velocity of migration : migration distance in tissue divided by migration time ( m / min).chemotaxis index in tissue : the ratio of total chemotaxis distance , that is , the distance in the direction toward the kc - gel , to the total migration distance that the cell moved in tissue . percentage of adherent leukocytes that crawled : the number of adherent and then crawled leukocytes divided by the total number of adherent leukocytes ( % ) . crawling distance : the total distance the leukocyte crawled from the initial site of adhesion to the transmigration site ( m ) . crawling time : duration of leukocytes undergoing intraluminal crawling ( min ) . velocity of crawling : the crawling distance in the vessel lumen divided by crawling time ( m / min ) . percentage of crawling leukocytes along the blood flow : the number of crawling leukocytes with their final crawling location in the direction of blood flow divided by the total crawling leukocytes ( % ) . directional crawling index : the ratio of the crawling distance in the direction toward the kc gel to the total crawling distance that the cell crawled in the lumen of the venule . percentage of crawling leukocytes that transmigrated : the number of crawled and then transmigrated leukocytes divided by the total number of crawling leukocytes ( % ) . transmigration time : from the time the leukocyte stopped crawling and started to transmigrate across endothelium to the time the whole leukocyte body was just outside the venule ( min ) . detachment time : from the time the leukocyte body was just outside the venule after its transmigration to the time when the leukocyte migrated away and lost contact to the venule ( min ) . migration distance : the sum of the distance that the leukocyte moved from the start point after detachment from the venule to the end point of the migration in tissue at 60 or 90 min of kc treatment or to the end point of the field of view ( m ) . velocity of migration : migration distance in tissue divided by migration time ( m / min ) . chemotaxis index in tissue : the ratio of total chemotaxis distance , that is , the distance in the direction toward the kc - gel , to the total migration distance that the cell moved in tissue . femurs and tibias from mice were isolated and the marrow flushed with ice - cold ca - free and mg - free phosphate - buffered saline ( pbs ) solution . neutrophils were isolated using a percoll ( ge healthcare , uppsala , sweden ) gradient ( 72% , 64% , and 52% ) centrifugation at 1060 g for 30 min as described previously and subsequently were washed with pbs . following lysis of red blood cells , bone - marrow - derived neutrophils were incubated at 37c for 30 min in the presence or absence of 10 m sb203580 in vitro . the cells were stimulated with kc ( 5 nm for 10 min ) to upregulate mac-1 or lfa-1 expression . aliquots of the neutrophil suspension ( 10/ml ) were washed in ice - cold pbs containing 0.5% bsa , stained with a fluorescent anti - mac-1 antibody ( anti - mouse cd11b fitc ; clone m1/70 ; ebioscience , san diego , ca ) or its respective isotype control ( rat igg2b fitc ; ebioscience ) or fluorescent anti - lfa-1 antibody ( anti - mouse cd11a fitc ; clone m17/4 ; ebioscience ) or its respective isotype control ( rat igg2a fitc ; ebioscience ) , and incubated for 30 min at 4c . the samples were then centrifuged ( 1200 rpm , 3 min , 4c ) and washed twice with ice - cold pbs containing 0.5% bsa and were analysed in the fl-1 channel of an epics xl flow cytometer ( beckman coulter , miami , fl ) with an excitation wavelength of 488 nm and an emission wavelength of 530 nm . n denotes the number of mice studied in each group or the number of mice used to derive bone marrow neutrophils for in vitro studies . statistical analysis was made using student 's t - test , and values of p < 0.05 were considered statistically significant . previous studies have shown and we have confirmed in our model system that using neutrophil - selective chemoattractants such as cxc chemokine kc or mip-2 , more than 95% of the adherent or emigrated leukocytes were found to be neutrophils [ 5 , 21 , 26 ] . therefore , in this study we use terms of leukocytes and neutrophils interchangeably when dealing with the cell type in different recruitment parameters . as a first step , the effect of sb203580 on the early neutrophil recruitment process of rolling was analyzed using intravital microscopy . to this end , superfusion of murine cremaster muscles with sb203580 ( 100 nm ) for 30 min prior to and for 1 h following the placement of kc gel did not significantly modify the rolling flux and rolling velocity of neutrophils as compared to the control saline - superfused cremaster muscle ( p > 0.05 ; figures 1(a ) and 1(b ) ) . to analyse the subsequent steps of neutrophil recruitment in the same cremaster muscles , we further quantitatively visualized and determined the number of adherent and emigrated neutrophils . as depicted in figures 2(a ) and 2(c ) , the number of adherent neutrophils had a tendency of reduction at 30 min and decreased significantly at 60 min after stimulation with kc in sb203580-superfused cremaster muscle as compared to saline superfusion . similarly , the number of emigrated neutrophil was not reduced until 60 min kc treatment in sb203580-treated cremaster muscles as compared to the saline - superfusion control ( figures 2(b ) and 2(c ) ) . interestingly , the same treatment with sb203580 reduced leukocyte adhesion by 32% and leukocyte emigration by 50% at 60 min after kc treatment . this suggests that there is a different effect of sb203580 on leukocyte adhesion and emigration and there may be additional inhibition by the inhibitor on other recruitment steps such as intraluminal crawling and transendothelial migration that caused more substantial suppression in emigration . next , we assessed the effects of pharmacologically inhibiting p38 mapk on neutrophil intraluminal crawling and transendothelial migration . to address this , the intermediate step between adhesion and emigration , the intraluminal crawling that follows kc - triggered adhesion , was studied using time - lapsed video microscopy . as shown in figure 3(a ) , the percentage of adherent neutrophils that proceeded to intraluminal crawling was significantly reduced upon treatment with sb203580 . interestingly , treatment with sb203580 did not significantly modify the total distance traversed by crawling neutrophils and the time taken ( figures 3(b ) and 3(c ) ) . accordingly , the velocity of crawling was not significantly altered upon treatment with sb203580 ( figure 3(d ) ) . in addition , neither the directionality of intraluminal crawling relative to blood flow nor the directional crawling index was significantly different in the two groups ( figures 3(e ) and 3(f ) ) . a decrease in the number of emigrated neutrophils could have resulted from impaired transendothelial migration following intraluminal crawling . to test this possibility , we further determined various parameters of transendothelial migration . clearly , the percentage of crawling neutrophils that proceeded to transendothelial migration was significantly lower in sb203580-superfused than in only saline - superfused control cremaster muscle ( figure 4(a ) ) . determination of transmigration time revealed that neutrophils in sb203580-superfused cremaster muscle required significantly longer time as compared to the control group reflecting impaired migration of neutrophils across the endothelial layer ( figure 4(b ) ) . we further analysed the time required for detachment of neutrophils from the endothelial cells prior to their chemotaxis . as illustrated in figure 4(c ) , the detachment time in sb203580-superfused cremaster muscle tended to be higher than the control , an effect not reaching statistical significance . as mac-1 expression on neutrophil surface is important for the intraluminal crawling and subsequent transendothelial migration of neutrophils , we further analysed whether or not sb203580 modulated the expression of mac-1 in vitro . to this end , stimulation with kc resulted in significantly increased expression of mac-1 on neutrophils . treatment with sb203580 ( 10 m ) virtually abrogated kc - triggered upregulation of mac-1 expression ( figures 5(a ) and 5(b ) ) . we also found that the treatment of neutrophils with kc in vitro did not change the expression level of lfa-1 , and sb203580 ( 10 m ) did not affect the expression level of lfa-1 after stimulation with kc ( data not shown ) . the reduced intraluminal crawling and transendothelial migration upon sb203580 treatment would subsequently result in a decrease in the migration and chemotaxis of emigrated neutrophils in cremasteric tissue . to have some chemotaxing neutrophils in the tissue , sb203580 was , therefore , superfused on the cremaster muscle after their transmigration into the tissue ; that is , superfusion of cremaster muscle with sb203580 was started at 30 min after kc - gel addition when about 8 or more leukocytes were identified being just emigrated in perivascular tissue , and sb203580 remained perfused for 60 min in the presence of kc - gel . stimulation of kc enhanced rolling velocity , rolling flux , and the number of adherent and emigrated neutrophils . neutrophil rolling velocity and rolling flux at 90 min following kc treatment ( 64.0 4.8 m / sec and 92.6 4.6 cells / min , resp . ; n = 3 ) were not significantly modified by the presence of 100 nm sb203580 , added at 30 min after kc stimulation ( 55.3.0 6.5 m / sec and 76.6 10.9 cells / min , resp . ; similarly , neutrophils adhesion and emigration at 90 min following kc treatment ( 12.3 0.1 cells/100-m venule and 57.6 0.7 cells/235 208 m field , resp . ; n = 3 ) were not significantly modified by superfusion with 100 nm sb203580 started at 30 min following kc stimulation ( 10.3 0.8 m / sec and 55.0 5.0 cells / min , resp . ; n = 3 ) . after observing no significant changes in leukocyte rolling , adhesion , and emigration with sb203580 after kc treatment , we then employed time - lapsed video analysis to determine the parameters of neutrophil migration and chemotaxis towards chemoattractant kc gel in cremasteric tissue . administration of sb203580 modestly but significantly attenuated the velocity of neutrophil migration as compared to saline control ( figure 6(a ) ) . similarly , as illustrated in figure 6(b ) , neutrophil chemotaxis index was significantly decreased in sb203580-superfused cremasteric tissue in mice when compared with chemotaxis index in saline - superfused control mice . this suggests that pharmacological inhibition of p38 mapk by sb203580 reduced neutrophil migration and more substantially neutrophil chemotaxis in extravascular tissue . in this study , we demonstrate the effects of the p38 mapk inhibitor sb203580 on the modulation of different steps of neutrophil recruitment . we found that in vivo treatment with sb203580 does not appreciably alter the parameters of neutrophil intraluminal crawling but significantly modifies cellular functions during transendothelial migration and chemotaxis in tissue . our results reveal an important role of p38 mapk in various steps of neutrophil recruitment particularly the initiation of intraluminal crawling and transmigration , transmigration time , and migration and chemotaxis in tissue . acquiring the knowledge of the functional role of p38 mapk in leukocyte recruitment is in large part dependent on the pharmacological inhibition of the kinase by sb203580 [ 21 , 2933 ] . however , more recent studies highlight that the limited specificity of sb203580 precludes safe conclusions in regards to the precise role of p38 mapk in the regulation of leukocyte recruitment [ 34 , 35 ] . earlier studies documented that sb203580 does not inhibit other kinases of the mapk family at relatively high molar concentrations ( 100 m ) . it is obvious that due to the lack of cellular specificity in vivo , the inhibition of endothelial p38 mapk by sb203580 treatment may not be ruled out . to the best of our knowledge , this is the first study that systematically analysed the effect of sb203580 on each step of neutrophil recruitment cascade in vivo . administration of sb203580 prior to the stimulation with kc resulted in significantly lower adhesion and emigration of neutrophils . however , administration of sb203580 30 min following stimulation with kc dissipated the differences in adhesion and emigration . our emigration data are in agreement with previous reports where both p38 mapk inhibitors sb203580 and skf86002 significantly reduced the number of emigrated cells . we observed a robust decrease in the percentage of crawling neutrophils that transmigrated in response to kc as the result of sb203580 treatment . in contrast to the previous report , we observed a slight but significant reduction in the number of neutrophils adhering to the microvasculature . in view of this discrepancy , previous studies support our findings that p38 mapk plays a role in neutrophil adhesion [ 3740 ] , an effect that is in part mediated by downregulation of 2 integrin expression . the transition from adhesion to emigration is effectively accomplished by intraluminal crawling of neutrophils from the initial site of adhesion to the junctional extravasation site in endothelium . although the efficiency of adherent leukocytes to crawl was slightly but significantly diminished in the presence of sb203580 , the crawling directionality along the blood flow , the total crawling distance , time , and velocity were not apparently affected . a recent report revealed that vav1 , a guanine exchange factor for the rho family gtpases rac and cdc42 , is a key regulator of actin cytoskeletal organization during intraluminal crawling . the mammalian - actin binding protein 1 ( mabp1 ) was also shown to participate in intraluminal crawling of neutrophils under conditions of physiological shear stress . both vav1 and mabp1 are downstream molecules of the spleen tyrosine kinase ( syk ) that plays a crucial role in 2 integrin - mediated signalling [ 42 , 43 ] . by the same token , syk - mediated signalling was shown to be essential for p38 mapk activation [ 44 , 45 ] . it is possible that kc activates p38 mapk through syk and regulates intraluminal crawling of neutrophils in our model . the chemokine kc elicits mac-1 expression which mediates intraluminal crawling [ 5 , 46 ] . we observed that upregulation of mac-1 expression after stimulation with kc was significantly abated in the presence of sb203580 . sb203580 treatment may also impair the transition of adherent leukocytes to the crawling phase without modulating the dynamics of neutrophil crawling per se . interestingly , mac-1 was previously shown to be downregulated in tnf-stimulated and fmlp - stimulated neutrophils treated with sb203580 [ 47 , 48 ] . similarly , the upregulation of mac-1 expression on neutrophils elicited by the bacterial m1 proteins in vivo was significantly abated by the p38 mapk inhibitors sb239063 and skf86002 . however , the authors showed that both sb239063 and skf86002 did not significantly alter mac-1 expression in vitro after stimulation with bacterial m1 protein . it is tempting to speculate that ramifications of p38 mapk inhibition in vivo include dysregulated expression of endothelial junctional adhesion molecules and neutrophil integrins that may contribute to the marked decrease in neutrophil transmigration . previous studies have highlighted the role of p38 mapk in neutrophil chemotaxis [ 19 , 21 ] . cara and coworkers utilized a grid model to quantify the number of chemotaxing neutrophils in several partitions between the vessel and chemoattractant - containing gel . the authors reported a significant reduction in the number of chemotaxing neutrophils in partitions closer to the chemoattractant - containing gel . in line with their findings , our data reveal that inhibition of p38 mapk decreases the chemotaxis index and velocity of migration which may contribute to the overall impairment of neutrophil chemotaxis by sb203580 as reported previously . taken together , our results suggest that the pharmacological p38 mapk inhibitor sb203580 affects neutrophil recruitment induced by the cxc chemokine kc by modulating various important neutrophil recruitment steps such as neutrophil transendothelial migration and chemotaxis in tissue . although sb203580 significantly reduced the number of adherent neutrophils that proceeded to crawling , the various functional parameters of intraluminal crawling were not substantially altered , an effect that may be attributed to the modulation of 2 integrin mac-1 expression on neutrophils .

p38 mitogen - activated protein kinase ( mapk ) signalling is critical in the pathophysiology of a variety of inflammatory processes . leukocyte recruitment to the site of inflammation is a multistep process governed by specific signalling cascades . after adhesion in the lumen , many leukocytes crawl to optimal sites at endothelial junctions and transmigrate to extravascular tissue in a mac-1-dependent manner . the signalling mechanisms that regulate postadhesion steps of intraluminal crawling , transmigration , and chemotaxis in tissue remain incompletely understood . the present study explored the effect of p38 mapk inhibitor sb203580 on various parameters of neutrophil recruitment triggered by chemokine kc ( cxcl1 ) gradient . neutrophil - endothelial interactions in microvasculature of murine cremaster muscle were determined using intravital microscopy and time - lapsed video analysis . sb203580 ( 100 nm ) did not change leukocyte rolling but significantly attenuated neutrophil adhesion , emigration , and transmigration and impaired the initiation of neutrophil crawling and transmigration . in response to kc chemotactic gradient , sb203580 significantly reduced the velocity of migration and chemotaxis index of neutrophils in tissue . the upregulation of mac-1 expression in neutrophils stimulated by kc was significantly blunted by sb203580 in vitro . collectively , our findings demonstrate that pharmacological suppression of p38 mapk significantly impairs multiple steps of neutrophil recruitment in vivo .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

during acute inflammation , leukocytes , mostly neutrophils , are recruited to the afflicted site by a well - defined and dynamic multistep process which includes leukocyte tethering , rolling , adhesion , and transmigration out of the vasculature [ 13 ] . in vivo , neutrophil recruitment is complex and regulated by a variety of molecules and signalling cascades triggered by the cross - talk between neutrophils and endothelium . recently , an additional step of neutrophil recruitment termed intraluminal crawling was described , a process which is molecularly distinct from adhesion . intraluminal crawling enables neutrophils to reach optimal emigration sites at endothelial junctions independently of hemodynamic forces [ 6 , 7 ] . the 2 integrin lfa-1 mediates firm adhesion of neutrophils to endothelial cells while the subsequent step of intraluminal crawling occurs as a result of interactions between mac-1 and endothelial icam-1 . signalling mechanisms that regulate intraluminal crawling and subsequent transendothelial migration of leukocytes are not completely understood . p38 mitogen - activated protein kinase ( mapk ) signalling regulates a wide array of inflammatory processes , cell proliferation , differentiation , survival , and invasion [ 9 , 10 ] . p38 mapk signalling participates in the expression and function of inflammatory cytokines such as tnf , il-1 , il-2 , il-6 , il-7 , and il-8 . pharmacological inhibition of p38 mapk signalling was previously shown to ameliorate inflammatory disorders such as asthma , rheumatoid arthritis , inflammatory bowel disease , stroke , systemic lupus erythematosus , and autoimmune diseases [ 1217 ] , thus serving as a promising therapeutic target . previous studies have documented the role of p38 mapk in neutrophil function during different steps of neutrophil recruitment . cytokines and inflammatory mediators such as tnf , lps and fmlp , and chemokine kc / cxcl1 were shown to phosphorylate p38 mapk during inflammation [ 1921 ] . pharmacological inhibition of p38 mapk was found to impair neutrophil chemotaxis and transendothelial migration induced by the chemokine kc . however , the dynamic motile behavioural changes of chemotaxing neutrophils such as velocity of migration and chemotaxis index were not measured , and the role of p38 mapk in these changes was not determined . in addition , whether there is a role of p38 mapk in intraluminal crawling of neutrophils has not been reported . sb203580 is a widely used selective inhibitor of the p38 and p38 isoforms and binds to the active site of p38 mapk in an atp - competitive manner . sb203580 can further block the translocation of p38 mapk and its downstream substrate mapk activated protein kinase 2 from the nucleus to cytosol . mounting evidence suggests that sb203580 is effective in a variety of in vitro and in vivo models of inflammation characterized by attenuated production of proinflammatory cytokines such as tnf and il-1 . the use of real - time intravital microscopy and time - lapsed video photography analysis makes it possible to determine simultaneously multiple leukocyte recruitment parameters of rolling , adhesion , emigration , intraluminal crawling velocity , transmigration time , detachment time , migration velocity , chemotaxis velocity , and chemotaxis index in tissue [ 5 , 24 ] . in the present study , we explored the effects of the p38 mapk inhibitor sb203580 on various parameters of neutrophil recruitment induced by chemotactic gradient of kc in vivo . the cremaster muscle was superfused with 37c warmed bicarbonate - buffered saline ( ph 7.4 ; containing in mm 133.9 nacl , 4.7 kcl , 1.2 mgso4 , and 20 nahco3 ) . for the induction of neutrophil recruitment , an agarose gel at 1 mm size containing the optimal concentration of cxc chemokine kc / cxcl1 ( 0.5 m ; peprotech , dollard des ormeaux , qc , canada ) was placed on the surface of the cremaster muscle in a preselected area 350 m from and parallel to the observed postcapillary venule . during recording , all efforts were made to adjust and keep the microscope focused on the adhering , crawling , transmigrating , and chemotaxing neutrophil inside the venule and in the muscle tissue . the number of rolling , adherent , and emigrated neutrophils during offline playback analysis of the recorded video was determined in the cremasteric microvasculature as described previously [ 2426 ] . the inhibitor sb203580 ( emd , billenca , ma ) was used to experimentally suppress the activity of p38 mapk . to determine the role of p38 mapk in neutrophil crawling and transmigration , sb203580 ( 100 nm ) was superfused 30 min prior to the placement of kc - containing gel and sb203580 superfusion remained for the duration of kc treatment ( 60 min ) . to analyze the role of p38 mapk in neutrophil migration and chemotaxis in cremasteric tissue after transendothelial migration , sb203580 was superfused 30 min after the placement of kc - containing gel when at least 8 emigrated neutrophils were identified in perivascular tissue and remained perfused for additional 60 min . sb203580 administered intravenously before this kc treatment completely inhibited adhesion and emigration induced by kc ( data not shown ) . for in vitro experiments , sb203580 ( 10 m ) was added to the medium 30 min prior to stimulation of neutrophils with kc . using imagej software , leukocyte intraluminal crawling , transmigration , and chemotaxis in cremasteric vasculature were analysed using the time - lapsed movie converted from the real - time video recording of the experiment as described previously [ 5 , 7 , 24 ] . crawling distance : the total distance the leukocyte crawled from the initial site of adhesion to the transmigration site ( m ) . crawling time : duration of leukocytes undergoing intraluminal crawling ( min).velocity of crawling : the crawling distance in the vessel lumen divided by crawling time ( m / min).percentage of crawling leukocytes along the blood flow : the number of crawling leukocytes with their final crawling location in the direction of blood flow divided by the total crawling leukocytes ( % ) .directional crawling index : the ratio of the crawling distance in the direction toward the kc gel to the total crawling distance that the cell crawled in the lumen of the venule.percentage of crawling leukocytes that transmigrated : the number of crawled and then transmigrated leukocytes divided by the total number of crawling leukocytes ( % ) . transmigration time : from the time the leukocyte stopped crawling and started to transmigrate across endothelium to the time the whole leukocyte body was just outside the venule ( min).detachment time : from the time the leukocyte body was just outside the venule after its transmigration to the time when the leukocyte migrated away and lost contact to the venule ( min).migration distance : the sum of the distance that the leukocyte moved from the start point after detachment from the venule to the end point of the migration in tissue at 60 or 90 min of kc treatment or to the end point of the field of view ( m ) . velocity of migration : migration distance in tissue divided by migration time ( m / min).chemotaxis index in tissue : the ratio of total chemotaxis distance , that is , the distance in the direction toward the kc - gel , to the total migration distance that the cell moved in tissue . crawling distance : the total distance the leukocyte crawled from the initial site of adhesion to the transmigration site ( m ) . velocity of crawling : the crawling distance in the vessel lumen divided by crawling time ( m / min ) . directional crawling index : the ratio of the crawling distance in the direction toward the kc gel to the total crawling distance that the cell crawled in the lumen of the venule . migration distance : the sum of the distance that the leukocyte moved from the start point after detachment from the venule to the end point of the migration in tissue at 60 or 90 min of kc treatment or to the end point of the field of view ( m ) . velocity of migration : migration distance in tissue divided by migration time ( m / min ) . chemotaxis index in tissue : the ratio of total chemotaxis distance , that is , the distance in the direction toward the kc - gel , to the total migration distance that the cell moved in tissue . following lysis of red blood cells , bone - marrow - derived neutrophils were incubated at 37c for 30 min in the presence or absence of 10 m sb203580 in vitro . previous studies have shown and we have confirmed in our model system that using neutrophil - selective chemoattractants such as cxc chemokine kc or mip-2 , more than 95% of the adherent or emigrated leukocytes were found to be neutrophils [ 5 , 21 , 26 ] . as a first step , the effect of sb203580 on the early neutrophil recruitment process of rolling was analyzed using intravital microscopy . to this end , superfusion of murine cremaster muscles with sb203580 ( 100 nm ) for 30 min prior to and for 1 h following the placement of kc gel did not significantly modify the rolling flux and rolling velocity of neutrophils as compared to the control saline - superfused cremaster muscle ( p > 0.05 ; figures 1(a ) and 1(b ) ) . to analyse the subsequent steps of neutrophil recruitment in the same cremaster muscles , we further quantitatively visualized and determined the number of adherent and emigrated neutrophils . this suggests that there is a different effect of sb203580 on leukocyte adhesion and emigration and there may be additional inhibition by the inhibitor on other recruitment steps such as intraluminal crawling and transendothelial migration that caused more substantial suppression in emigration . next , we assessed the effects of pharmacologically inhibiting p38 mapk on neutrophil intraluminal crawling and transendothelial migration . to address this , the intermediate step between adhesion and emigration , the intraluminal crawling that follows kc - triggered adhesion , was studied using time - lapsed video microscopy . as shown in figure 3(a ) , the percentage of adherent neutrophils that proceeded to intraluminal crawling was significantly reduced upon treatment with sb203580 . accordingly , the velocity of crawling was not significantly altered upon treatment with sb203580 ( figure 3(d ) ) . in addition , neither the directionality of intraluminal crawling relative to blood flow nor the directional crawling index was significantly different in the two groups ( figures 3(e ) and 3(f ) ) . a decrease in the number of emigrated neutrophils could have resulted from impaired transendothelial migration following intraluminal crawling . to test this possibility , we further determined various parameters of transendothelial migration . determination of transmigration time revealed that neutrophils in sb203580-superfused cremaster muscle required significantly longer time as compared to the control group reflecting impaired migration of neutrophils across the endothelial layer ( figure 4(b ) ) . as mac-1 expression on neutrophil surface is important for the intraluminal crawling and subsequent transendothelial migration of neutrophils , we further analysed whether or not sb203580 modulated the expression of mac-1 in vitro . treatment with sb203580 ( 10 m ) virtually abrogated kc - triggered upregulation of mac-1 expression ( figures 5(a ) and 5(b ) ) . we also found that the treatment of neutrophils with kc in vitro did not change the expression level of lfa-1 , and sb203580 ( 10 m ) did not affect the expression level of lfa-1 after stimulation with kc ( data not shown ) . the reduced intraluminal crawling and transendothelial migration upon sb203580 treatment would subsequently result in a decrease in the migration and chemotaxis of emigrated neutrophils in cremasteric tissue . to have some chemotaxing neutrophils in the tissue , sb203580 was , therefore , superfused on the cremaster muscle after their transmigration into the tissue ; that is , superfusion of cremaster muscle with sb203580 was started at 30 min after kc - gel addition when about 8 or more leukocytes were identified being just emigrated in perivascular tissue , and sb203580 remained perfused for 60 min in the presence of kc - gel . after observing no significant changes in leukocyte rolling , adhesion , and emigration with sb203580 after kc treatment , we then employed time - lapsed video analysis to determine the parameters of neutrophil migration and chemotaxis towards chemoattractant kc gel in cremasteric tissue . administration of sb203580 modestly but significantly attenuated the velocity of neutrophil migration as compared to saline control ( figure 6(a ) ) . similarly , as illustrated in figure 6(b ) , neutrophil chemotaxis index was significantly decreased in sb203580-superfused cremasteric tissue in mice when compared with chemotaxis index in saline - superfused control mice . this suggests that pharmacological inhibition of p38 mapk by sb203580 reduced neutrophil migration and more substantially neutrophil chemotaxis in extravascular tissue . in this study , we demonstrate the effects of the p38 mapk inhibitor sb203580 on the modulation of different steps of neutrophil recruitment . we found that in vivo treatment with sb203580 does not appreciably alter the parameters of neutrophil intraluminal crawling but significantly modifies cellular functions during transendothelial migration and chemotaxis in tissue . our results reveal an important role of p38 mapk in various steps of neutrophil recruitment particularly the initiation of intraluminal crawling and transmigration , transmigration time , and migration and chemotaxis in tissue . acquiring the knowledge of the functional role of p38 mapk in leukocyte recruitment is in large part dependent on the pharmacological inhibition of the kinase by sb203580 [ 21 , 2933 ] . however , more recent studies highlight that the limited specificity of sb203580 precludes safe conclusions in regards to the precise role of p38 mapk in the regulation of leukocyte recruitment [ 34 , 35 ] . it is obvious that due to the lack of cellular specificity in vivo , the inhibition of endothelial p38 mapk by sb203580 treatment may not be ruled out . to the best of our knowledge , this is the first study that systematically analysed the effect of sb203580 on each step of neutrophil recruitment cascade in vivo . administration of sb203580 prior to the stimulation with kc resulted in significantly lower adhesion and emigration of neutrophils . our emigration data are in agreement with previous reports where both p38 mapk inhibitors sb203580 and skf86002 significantly reduced the number of emigrated cells . we observed a robust decrease in the percentage of crawling neutrophils that transmigrated in response to kc as the result of sb203580 treatment . in contrast to the previous report , we observed a slight but significant reduction in the number of neutrophils adhering to the microvasculature . in view of this discrepancy , previous studies support our findings that p38 mapk plays a role in neutrophil adhesion [ 3740 ] , an effect that is in part mediated by downregulation of 2 integrin expression . the transition from adhesion to emigration is effectively accomplished by intraluminal crawling of neutrophils from the initial site of adhesion to the junctional extravasation site in endothelium . although the efficiency of adherent leukocytes to crawl was slightly but significantly diminished in the presence of sb203580 , the crawling directionality along the blood flow , the total crawling distance , time , and velocity were not apparently affected . a recent report revealed that vav1 , a guanine exchange factor for the rho family gtpases rac and cdc42 , is a key regulator of actin cytoskeletal organization during intraluminal crawling . the mammalian - actin binding protein 1 ( mabp1 ) was also shown to participate in intraluminal crawling of neutrophils under conditions of physiological shear stress . it is possible that kc activates p38 mapk through syk and regulates intraluminal crawling of neutrophils in our model . the chemokine kc elicits mac-1 expression which mediates intraluminal crawling [ 5 , 46 ] . we observed that upregulation of mac-1 expression after stimulation with kc was significantly abated in the presence of sb203580 . sb203580 treatment may also impair the transition of adherent leukocytes to the crawling phase without modulating the dynamics of neutrophil crawling per se . similarly , the upregulation of mac-1 expression on neutrophils elicited by the bacterial m1 proteins in vivo was significantly abated by the p38 mapk inhibitors sb239063 and skf86002 . however , the authors showed that both sb239063 and skf86002 did not significantly alter mac-1 expression in vitro after stimulation with bacterial m1 protein . it is tempting to speculate that ramifications of p38 mapk inhibition in vivo include dysregulated expression of endothelial junctional adhesion molecules and neutrophil integrins that may contribute to the marked decrease in neutrophil transmigration . previous studies have highlighted the role of p38 mapk in neutrophil chemotaxis [ 19 , 21 ] . the authors reported a significant reduction in the number of chemotaxing neutrophils in partitions closer to the chemoattractant - containing gel . in line with their findings , our data reveal that inhibition of p38 mapk decreases the chemotaxis index and velocity of migration which may contribute to the overall impairment of neutrophil chemotaxis by sb203580 as reported previously . taken together , our results suggest that the pharmacological p38 mapk inhibitor sb203580 affects neutrophil recruitment induced by the cxc chemokine kc by modulating various important neutrophil recruitment steps such as neutrophil transendothelial migration and chemotaxis in tissue . although sb203580 significantly reduced the number of adherent neutrophils that proceeded to crawling , the various functional parameters of intraluminal crawling were not substantially altered , an effect that may be attributed to the modulation of 2 integrin mac-1 expression on neutrophils .

during acute inflammation , leukocytes , mostly neutrophils , are recruited to the afflicted site by a well - defined and dynamic multistep process which includes leukocyte tethering , rolling , adhesion , and transmigration out of the vasculature [ 13 ] . in vivo , neutrophil recruitment is complex and regulated by a variety of molecules and signalling cascades triggered by the cross - talk between neutrophils and endothelium . neutrophil rolling is mediated by selectins , and adhesion is mediated by 2 integrins while emigration is regulated by the interactions between integrins , pecam-1 , and junctional adhesion molecules and their respective ligands . the 2 integrin lfa-1 mediates firm adhesion of neutrophils to endothelial cells while the subsequent step of intraluminal crawling occurs as a result of interactions between mac-1 and endothelial icam-1 . blocking of lfa-1 and mac-1 in monocytes and icam-1 and icam-2 on endothelial cells was shown to prevent the intraluminal locomotion of monocytes to endothelial cell junctions and ensuing diapedesis . p38 mitogen - activated protein kinase ( mapk ) signalling regulates a wide array of inflammatory processes , cell proliferation , differentiation , survival , and invasion [ 9 , 10 ] . p38 mapk signalling participates in the expression and function of inflammatory cytokines such as tnf , il-1 , il-2 , il-6 , il-7 , and il-8 . pharmacological inhibition of p38 mapk signalling was previously shown to ameliorate inflammatory disorders such as asthma , rheumatoid arthritis , inflammatory bowel disease , stroke , systemic lupus erythematosus , and autoimmune diseases [ 1217 ] , thus serving as a promising therapeutic target . previous studies have documented the role of p38 mapk in neutrophil function during different steps of neutrophil recruitment . cytokines and inflammatory mediators such as tnf , lps and fmlp , and chemokine kc / cxcl1 were shown to phosphorylate p38 mapk during inflammation [ 1921 ] . however , the dynamic motile behavioural changes of chemotaxing neutrophils such as velocity of migration and chemotaxis index were not measured , and the role of p38 mapk in these changes was not determined . in addition , whether there is a role of p38 mapk in intraluminal crawling of neutrophils has not been reported . sb203580 is a widely used selective inhibitor of the p38 and p38 isoforms and binds to the active site of p38 mapk in an atp - competitive manner . sb203580 can further block the translocation of p38 mapk and its downstream substrate mapk activated protein kinase 2 from the nucleus to cytosol . mounting evidence suggests that sb203580 is effective in a variety of in vitro and in vivo models of inflammation characterized by attenuated production of proinflammatory cytokines such as tnf and il-1 . the use of real - time intravital microscopy and time - lapsed video photography analysis makes it possible to determine simultaneously multiple leukocyte recruitment parameters of rolling , adhesion , emigration , intraluminal crawling velocity , transmigration time , detachment time , migration velocity , chemotaxis velocity , and chemotaxis index in tissue [ 5 , 24 ] . in the present study , we explored the effects of the p38 mapk inhibitor sb203580 on various parameters of neutrophil recruitment induced by chemotactic gradient of kc in vivo . this study was carried out with the approval of animal protocols from the university committee on animal care and supply ( ucacs ) at the university of saskatchewan following the standards of the canadian association of animal care . injection of 10 mg / kg xylazine ( bayer , toronto , on , canada ) and 200 mg / kg ketamine hydrochloride ( rogar , montreal , qc , canada ) . for the induction of neutrophil recruitment , an agarose gel at 1 mm size containing the optimal concentration of cxc chemokine kc / cxcl1 ( 0.5 m ; peprotech , dollard des ormeaux , qc , canada ) was placed on the surface of the cremaster muscle in a preselected area 350 m from and parallel to the observed postcapillary venule . throughout the experiment , neutrophil behaviour and hemodynamic changes in the selected cremasteric postcapillary venule ( 2540 m diameter ) were visualized , projected on a tv monitor , recorded at real time on a dvd recorder before ( for time 0 min ) and after addition of kc - gel ( recorded for 60 or 90 min ) . during recording , all efforts were made to adjust and keep the microscope focused on the adhering , crawling , transmigrating , and chemotaxing neutrophil inside the venule and in the muscle tissue . the number of rolling , adherent , and emigrated neutrophils during offline playback analysis of the recorded video was determined in the cremasteric microvasculature as described previously [ 2426 ] . to determine the role of p38 mapk in neutrophil crawling and transmigration , sb203580 ( 100 nm ) was superfused 30 min prior to the placement of kc - containing gel and sb203580 superfusion remained for the duration of kc treatment ( 60 min ) . to analyze the role of p38 mapk in neutrophil migration and chemotaxis in cremasteric tissue after transendothelial migration , sb203580 was superfused 30 min after the placement of kc - containing gel when at least 8 emigrated neutrophils were identified in perivascular tissue and remained perfused for additional 60 min . in all experiments , the same amount of dmso as in the sb203580-treated group was used in the control group without sb203580 and was not found to affect any responses . using imagej software , leukocyte intraluminal crawling , transmigration , and chemotaxis in cremasteric vasculature were analysed using the time - lapsed movie converted from the real - time video recording of the experiment as described previously [ 5 , 7 , 24 ] . the following recruitment parameters were quantified from tracking and analyzing at least 40 cells for each treatment group.percentage of adherent leukocytes that crawled : the number of adherent and then crawled leukocytes divided by the total number of adherent leukocytes ( % ) . crawling distance : the total distance the leukocyte crawled from the initial site of adhesion to the transmigration site ( m ) . crawling time : duration of leukocytes undergoing intraluminal crawling ( min).velocity of crawling : the crawling distance in the vessel lumen divided by crawling time ( m / min).percentage of crawling leukocytes along the blood flow : the number of crawling leukocytes with their final crawling location in the direction of blood flow divided by the total crawling leukocytes ( % ) .directional crawling index : the ratio of the crawling distance in the direction toward the kc gel to the total crawling distance that the cell crawled in the lumen of the venule.percentage of crawling leukocytes that transmigrated : the number of crawled and then transmigrated leukocytes divided by the total number of crawling leukocytes ( % ) . transmigration time : from the time the leukocyte stopped crawling and started to transmigrate across endothelium to the time the whole leukocyte body was just outside the venule ( min).detachment time : from the time the leukocyte body was just outside the venule after its transmigration to the time when the leukocyte migrated away and lost contact to the venule ( min).migration distance : the sum of the distance that the leukocyte moved from the start point after detachment from the venule to the end point of the migration in tissue at 60 or 90 min of kc treatment or to the end point of the field of view ( m ) . velocity of migration : migration distance in tissue divided by migration time ( m / min).chemotaxis index in tissue : the ratio of total chemotaxis distance , that is , the distance in the direction toward the kc - gel , to the total migration distance that the cell moved in tissue . percentage of adherent leukocytes that crawled : the number of adherent and then crawled leukocytes divided by the total number of adherent leukocytes ( % ) . crawling distance : the total distance the leukocyte crawled from the initial site of adhesion to the transmigration site ( m ) . percentage of crawling leukocytes along the blood flow : the number of crawling leukocytes with their final crawling location in the direction of blood flow divided by the total crawling leukocytes ( % ) . directional crawling index : the ratio of the crawling distance in the direction toward the kc gel to the total crawling distance that the cell crawled in the lumen of the venule . detachment time : from the time the leukocyte body was just outside the venule after its transmigration to the time when the leukocyte migrated away and lost contact to the venule ( min ) . migration distance : the sum of the distance that the leukocyte moved from the start point after detachment from the venule to the end point of the migration in tissue at 60 or 90 min of kc treatment or to the end point of the field of view ( m ) . chemotaxis index in tissue : the ratio of total chemotaxis distance , that is , the distance in the direction toward the kc - gel , to the total migration distance that the cell moved in tissue . femurs and tibias from mice were isolated and the marrow flushed with ice - cold ca - free and mg - free phosphate - buffered saline ( pbs ) solution . aliquots of the neutrophil suspension ( 10/ml ) were washed in ice - cold pbs containing 0.5% bsa , stained with a fluorescent anti - mac-1 antibody ( anti - mouse cd11b fitc ; clone m1/70 ; ebioscience , san diego , ca ) or its respective isotype control ( rat igg2b fitc ; ebioscience ) or fluorescent anti - lfa-1 antibody ( anti - mouse cd11a fitc ; clone m17/4 ; ebioscience ) or its respective isotype control ( rat igg2a fitc ; ebioscience ) , and incubated for 30 min at 4c . previous studies have shown and we have confirmed in our model system that using neutrophil - selective chemoattractants such as cxc chemokine kc or mip-2 , more than 95% of the adherent or emigrated leukocytes were found to be neutrophils [ 5 , 21 , 26 ] . therefore , in this study we use terms of leukocytes and neutrophils interchangeably when dealing with the cell type in different recruitment parameters . as a first step , the effect of sb203580 on the early neutrophil recruitment process of rolling was analyzed using intravital microscopy . to this end , superfusion of murine cremaster muscles with sb203580 ( 100 nm ) for 30 min prior to and for 1 h following the placement of kc gel did not significantly modify the rolling flux and rolling velocity of neutrophils as compared to the control saline - superfused cremaster muscle ( p > 0.05 ; figures 1(a ) and 1(b ) ) . to analyse the subsequent steps of neutrophil recruitment in the same cremaster muscles , we further quantitatively visualized and determined the number of adherent and emigrated neutrophils . as depicted in figures 2(a ) and 2(c ) , the number of adherent neutrophils had a tendency of reduction at 30 min and decreased significantly at 60 min after stimulation with kc in sb203580-superfused cremaster muscle as compared to saline superfusion . similarly , the number of emigrated neutrophil was not reduced until 60 min kc treatment in sb203580-treated cremaster muscles as compared to the saline - superfusion control ( figures 2(b ) and 2(c ) ) . this suggests that there is a different effect of sb203580 on leukocyte adhesion and emigration and there may be additional inhibition by the inhibitor on other recruitment steps such as intraluminal crawling and transendothelial migration that caused more substantial suppression in emigration . next , we assessed the effects of pharmacologically inhibiting p38 mapk on neutrophil intraluminal crawling and transendothelial migration . to address this , the intermediate step between adhesion and emigration , the intraluminal crawling that follows kc - triggered adhesion , was studied using time - lapsed video microscopy . as shown in figure 3(a ) , the percentage of adherent neutrophils that proceeded to intraluminal crawling was significantly reduced upon treatment with sb203580 . in addition , neither the directionality of intraluminal crawling relative to blood flow nor the directional crawling index was significantly different in the two groups ( figures 3(e ) and 3(f ) ) . clearly , the percentage of crawling neutrophils that proceeded to transendothelial migration was significantly lower in sb203580-superfused than in only saline - superfused control cremaster muscle ( figure 4(a ) ) . determination of transmigration time revealed that neutrophils in sb203580-superfused cremaster muscle required significantly longer time as compared to the control group reflecting impaired migration of neutrophils across the endothelial layer ( figure 4(b ) ) . as mac-1 expression on neutrophil surface is important for the intraluminal crawling and subsequent transendothelial migration of neutrophils , we further analysed whether or not sb203580 modulated the expression of mac-1 in vitro . we also found that the treatment of neutrophils with kc in vitro did not change the expression level of lfa-1 , and sb203580 ( 10 m ) did not affect the expression level of lfa-1 after stimulation with kc ( data not shown ) . to have some chemotaxing neutrophils in the tissue , sb203580 was , therefore , superfused on the cremaster muscle after their transmigration into the tissue ; that is , superfusion of cremaster muscle with sb203580 was started at 30 min after kc - gel addition when about 8 or more leukocytes were identified being just emigrated in perivascular tissue , and sb203580 remained perfused for 60 min in the presence of kc - gel . after observing no significant changes in leukocyte rolling , adhesion , and emigration with sb203580 after kc treatment , we then employed time - lapsed video analysis to determine the parameters of neutrophil migration and chemotaxis towards chemoattractant kc gel in cremasteric tissue . administration of sb203580 modestly but significantly attenuated the velocity of neutrophil migration as compared to saline control ( figure 6(a ) ) . in this study , we demonstrate the effects of the p38 mapk inhibitor sb203580 on the modulation of different steps of neutrophil recruitment . we found that in vivo treatment with sb203580 does not appreciably alter the parameters of neutrophil intraluminal crawling but significantly modifies cellular functions during transendothelial migration and chemotaxis in tissue . our results reveal an important role of p38 mapk in various steps of neutrophil recruitment particularly the initiation of intraluminal crawling and transmigration , transmigration time , and migration and chemotaxis in tissue . acquiring the knowledge of the functional role of p38 mapk in leukocyte recruitment is in large part dependent on the pharmacological inhibition of the kinase by sb203580 [ 21 , 2933 ] . however , more recent studies highlight that the limited specificity of sb203580 precludes safe conclusions in regards to the precise role of p38 mapk in the regulation of leukocyte recruitment [ 34 , 35 ] . it is obvious that due to the lack of cellular specificity in vivo , the inhibition of endothelial p38 mapk by sb203580 treatment may not be ruled out . to the best of our knowledge , this is the first study that systematically analysed the effect of sb203580 on each step of neutrophil recruitment cascade in vivo . we observed a robust decrease in the percentage of crawling neutrophils that transmigrated in response to kc as the result of sb203580 treatment . in contrast to the previous report , we observed a slight but significant reduction in the number of neutrophils adhering to the microvasculature . in view of this discrepancy , previous studies support our findings that p38 mapk plays a role in neutrophil adhesion [ 3740 ] , an effect that is in part mediated by downregulation of 2 integrin expression . the transition from adhesion to emigration is effectively accomplished by intraluminal crawling of neutrophils from the initial site of adhesion to the junctional extravasation site in endothelium . although the efficiency of adherent leukocytes to crawl was slightly but significantly diminished in the presence of sb203580 , the crawling directionality along the blood flow , the total crawling distance , time , and velocity were not apparently affected . a recent report revealed that vav1 , a guanine exchange factor for the rho family gtpases rac and cdc42 , is a key regulator of actin cytoskeletal organization during intraluminal crawling . both vav1 and mabp1 are downstream molecules of the spleen tyrosine kinase ( syk ) that plays a crucial role in 2 integrin - mediated signalling [ 42 , 43 ] . we observed that upregulation of mac-1 expression after stimulation with kc was significantly abated in the presence of sb203580 . similarly , the upregulation of mac-1 expression on neutrophils elicited by the bacterial m1 proteins in vivo was significantly abated by the p38 mapk inhibitors sb239063 and skf86002 . it is tempting to speculate that ramifications of p38 mapk inhibition in vivo include dysregulated expression of endothelial junctional adhesion molecules and neutrophil integrins that may contribute to the marked decrease in neutrophil transmigration . in line with their findings , our data reveal that inhibition of p38 mapk decreases the chemotaxis index and velocity of migration which may contribute to the overall impairment of neutrophil chemotaxis by sb203580 as reported previously . taken together , our results suggest that the pharmacological p38 mapk inhibitor sb203580 affects neutrophil recruitment induced by the cxc chemokine kc by modulating various important neutrophil recruitment steps such as neutrophil transendothelial migration and chemotaxis in tissue . although sb203580 significantly reduced the number of adherent neutrophils that proceeded to crawling , the various functional parameters of intraluminal crawling were not substantially altered , an effect that may be attributed to the modulation of 2 integrin mac-1 expression on neutrophils .

after more than 40 years of clinical use , levodopa ( ld ) remains the gold standard regarding symptomatic efficacy in the drug treatment of parkinson s disease ( pd).1 compared with other available dopaminergic therapies , dopamine replacement with ld is associated with the greatest improvement in motor function , as assessed by reduced scores in the unified parkinson s disease rating scale [ updrs]).25 in addition , responsiveness to ld ( required to exceed 25%30% reduction in the motor part of the updrs ) is a diagnostic criterion for pd.6 in clinical practice , ld slows the progression of disability as assessed by the hoehn and yahr staging system,7 and is associated with a reduction in mortality.8,9 importantly , ld is one of the best tolerated drugs to treat pd , particularly in the elderly population.10 however , long - term treatment with ld is often complicated by the development of various types of motor response oscillations over the day as well as drug - induced dyskinesia , a complication characterized by erratic involuntary movements . such treatment - related motor complications eventually develop in the majority of patients and are found in about one - third of patients after only two years of exposure.11 once established , motor complications are difficult to treat and can develop into a significant source of disability . in extreme cases , treatment - induced dyskinesias may completely annihilate the therapeutic benefit initially gained from the drug . concerns about the potential induction of long - term motor complications have led many physicians to use ld in a restricted manner and reserve it as a second - line strategy . this approach has gained wide acceptance following clinical trials in early pd , showing significantly reduced risks of developing motor complications with dopamine agonists ( das ) as compared with ld monotherapy.25 while these trials have indeed established the potential of das to delay the onset of motor complications , they have also consistently demonstrated the superior symptomatic efficacy of ld , with a need for ld supplementation within the first 23 years in most patients started on a da.2 despite such evidence from clinical trials of the need for ld supplementation to maintain symptomatic control in early pd , in general clinical practice , ld is often withheld beyond the time when symptomatic control with das has become insufficient.12 this results , in part , from patient perceptions . information gleaned from physicians or the media may alert patients to the risk of dyskinesia associated with ld . the alarming nature of dyskinesia can , in turn , lead to a phenomenon labeled dopa - phobia.12 these concerns have even been extrapolated to using single doses of ld in challenge tests , in order to prime the striatum putatively for subsequent dyskinetic responses to dopaminergic therapies.13 increasing evidence now suggests that motor complications ( particularly dyskinesia ) associated with sustained ld therapy are a result of discontinuous and intermittent delivery of ld to the brain , resulting in nonphysiologic pulsatile stimulation of striatal dopamine receptors . thus , the short half - life ( 90 minutes ) of immediate - release ld formulations is thought to be the key factor in the pathogenesis of motor complications , rather than their induction being an intrinsic property of the ld molecule.1 this review will summarize the available evidence regarding the use of ld to treat pd and will also address the issue of ld delivery as a critical factor for the drug s propensity to induce motor complications . there have been a number of large - scale , long - term , ld - controlled monotherapy trials of das in early parkinson s disease on which strong conclusions about the relative effect size of ld compared with das can be based . the four - year randomized comparison of the agonist pramipexole versus levodopa on motor complications of parkinson s disease ( calm - pd ) trial , compared initial treatment with pramipexole ( 0.5 mg three times daily ) versus ld - carbidopa ( 100/25 mg three times daily ) , followed by open - label ld supplementation as required.2 the primary outcome measure was the time to first occurrence of dopaminergic complications , which included wearing - off ( the re - emergence of pd symptoms due to the diminishing effect of ld ) , dyskinesias , on off fluctuations ( unpredictable fluctuations varying between symptoms being well controlled [ on ] to uncontrolled [ off ] ) , and freezing . although initial treatment with pramipexole resulted in lower incidences of dyskinesia and wearing - off compared with initial treatment with ld , symptom control , as assessed by the updrs , was superior in patients treated with ld . from baseline to month 48 , there was a worsening from baseline of 1.3 13.3 ( mean standard deviation [ sd ] ) points in updrs motor scores in the pramipexole group compared with an improvement of 3.4 12.3 points in the ld group ( treatment difference of 4.9 points , p = 0.001 ) as shown in figure 1a . interestingly , even though physicians in this trial had the option to use open - label ld supplementation to enhance symptomatic control , the group differences observed in the updrs motor and activities of daily living components between patients randomized to pramipexole or ld remained relatively uniform throughout the four years of the study . it is not definitely established why the updrs scores of the pramipexole group never caught up with the ld group , despite the option of open - label ld and other antiparkinsonian therapies , but it might be related to the lower dose of ld used in the supplemented pramipexole patients ( 434 498 mg / day ) compared with those on ld monotherapy ( 702 461 mg / day ) . indeed , the calculated ld equivalent dose used in the supplemented pramipexole patients falls in the range of 468584 mg / day , which is notably less than the 702 461 mg / day dose used in patients initiated on ld monotherapy . this indicates that combined treatment with a lower dose of ld and a da is not equivalent to higher dose ld monotherapy in terms of symptomatic control , calling into question the concept of ld - equivalent doses of das . in the five - year 056 study , which compared the safety and efficacy of ropinirole with that of ld , the primary efficacy measure was the occurrence of dyskinesia.3 ropinirole was initiated at 0.25 mg three times daily and increased weekly , as necessary , up to a maximum dose of 8 mg three times daily . levodopa was initiated at a dose of 50 mg once daily and increased weekly , as necessary , to a maximum of 400 mg three times daily . as in the calm - pd study , if symptoms were not adequately controlled by the assigned study medication , patients could receive supplementary ld , administered in an open - label fashion . in this study , 84% of all patients initiated on ropinirole monotherapy either required ld supplementation ( 427 221 mg / day ) or dropped out of the study . although this study demonstrated a reduced incidence of dyskinesia for ropinirole versus ld ( 20% versus 45% , respectively ) , this benefit was once again found to be at the expense of symptomatic control . in those patients who completed the study , there was a minimal improvement from baseline of 0.8 10.1 points in updrs motor scores in the ropinirole group , contrasting with an improvement of 4.8 8.3 points in the ld group ( figure 1a ) . similar results confirming the superior efficacy of ld ( in terms of updrs motor scores ) were also observed in the two - year real - pet ( requip as early therapy versus l - dopa positron emission tomography ) study of ld versus ropinirole monotherapy.14 the clinical relevance of such differences in updrs scores has been debated , but is vividly illustrated by the increasing rates of supplementation with open - label ld over the course of these double - blind comparative trials . at four years follow - up in the calm - pd trial , 72% of patients randomized to pramipexole monotherapy had required add - on ld to maintain symptomatic control , and this figure was 66% after five years in the 056 study with ropinirole ( figure 1b).25 the term ld rescue , commonly used in this context , aptly describes the role of ld as the most efficacious drug at hand to control motor symptoms when other drugs begin to fail . the recently published pelmopet ( pergolide versus l - dopa monotherapy and positron emission tomography ) study5 employed a strict pergolide ( 0.755.0 mg / day ) and ld ( 1501200 mg / day ) monotherapy design in which no rescue therapy was allowed . at one year , there was a relatively small difference in favor of ld in the mean change from baseline in updrs motor scores ( 3.2 points in the pergolide treatment group compared with 5.2 points in the ld treatment group ) . however , after three years of monotherapy , patients receiving pergolide had deteriorated below baseline by 2.8 9.8 points , whereas patients receiving ld were still improved by 2.8 7.8 points . taken together , these ld - controlled trials of da monotherapy in early pd clearly show the need for ld supplementation to maintain symptomatic control . current recommendations to initiate dopaminergic therapy in early pd with a da , or even a monoamine oxidase inhibitor , are chiefly based on concerns about the evolution of motor complications characteristic of sustained ld therapy , most notably drug - induced dyskinesias . while the monotherapy trials cited above have clearly established a reduced dyskinesia risk with da monotherapy , they have also shown that this is exclusively due to the delay in starting patients on ld . indeed , pd patients initiated on a da in these trials eventually required supplemental ld in order to maintain symptomatic control , and developed dyskinesias at an identical rate ( albeit with a delay ) to those started on ld , leading to steady increases in dyskinesia rates from this point onwards.15 therefore , the question remains as to whether the initial benefit of reduced motor complication rates can be maintained in the longer term when virtually all patients will be on combined drug treatment with das , ld , and possibly other agents . while none of the aforementioned comparative trials has been designed to assess outcomes under double - blind conditions for longer than five years , longer term , open - label , follow - up data are available . the parkinson study group recently reported on the six - year outcomes of patient groups initially randomized to receive monotherapy with pramipexole or ld in the calm - pd trial . results of this study showed a persistent , statistically significant difference in overall dyskinesia rates of about 37% in those randomized to initial ld compared with 20% in those initiated on pramipexole ( p = 0.004).16 despite this , 90% of the population followed were on ld by six years.16 similarly , 10-year outcomes reported by lees et al17 comparing initial monotherapy with bromocriptine versus ld , along with hauser et al18 from the 056 ropinirole study , also demonstrated a reduced overall incidence of dyskinesias with das in the long term . after 10 years , 45% of patients who were initiated with bromocriptine had dyskinesias compared with 54% of those started on ld ( corresponding to an incidence rate for first dyskinesia occurrence of 145.3 versus 105.9 per 1000 patient - years after starting treatment with ld or da , respectively).17 similar figures were reported for the 10-year follow - up of patients of the 056 study of ropinirole versus ld ( 52.4% versus 77.8% , p = 0.0457).19 while these results convincingly show long - term reductions in the incidence of dyskinesia with das compared with ld , rates of dyskinesia do not necessarily reflect differences in dyskinesia - related disability between treatments . hauser et al20 were the first to demonstrate differential functional significance of dyskinesias by asking patients to rate their involuntary movements as nontroublesome in a modified on / off diary . when patients were subsequently asked to rate their recorded on or off times as functionally good time , whereas 84.9% of off time and 89.9% of on time with troublesome dyskinesia was considered time.20 in this respect , it is interesting to note that , at 10 and 14 years , respectively , neither the ropinirole nor bromocriptine studies found significant differences between treatment arms in the emergence of disabling dyskinesia.15,17,21 this finding was also observed in the calm - pd study at four years , where the rate of disabling dyskinesias was low in patients treated with either pramipexole or ld.16 in addition , the recent stride - pd ( stalevo reduction in dyskinesia evaluation ) trial comparing time to dyskinesia development in patients treated with either standard ld or a fixed combination of ld , carbidopa , and entacapone showed a less than 10% incidence of disabling dyskinesias in both groups.22 furthermore , patient surveys have also indicated that patients with dyskinesias are less concerned about their dyskinesias than those who have not yet experienced dyskinesias , and that more than 80% of these patients prefer having dyskinesia over their pd symptoms.23 since patients often tolerate mild dyskinesia well , the risk of dyskinesia should not cause physicians to delay ld initiation in patients whose pd symptoms can not be sufficiently controlled with other treatments , regardless of patient age . given these findings , the decision to initiate ld must be tailored to the patient s needs and should include proper counseling about the impact of dyskinesia and current options to minimize their incidence , including cautious ld dosing , or to treat them once they are present . in routine clinical practice , the benefits and risks of any anti - pd therapy must be weighed before prescribing the medication . ld therapy is generally well tolerated , and acute side effects include nausea , vomiting , and hypotension.24,25 as such , ld is generally started at a low dose to minimize these risks . with chronic use , the most common complications include wearing - off and dyskinesia , which can be troublesome for the patient ( figure 2).26 however , while the use of das is not associated with motor complications , a different array of side effects , including hallucinations , somnolence , and edema , are observed more commonly with these anti - pd medications ( figure 2 ) . dopamine agonists have also been linked to impulse - control disorders , such as pathologic gambling , hypersexuality , binge eating , or pathologic shopping , with about 13.7%17.1% of patients on da therapy showing signs of such disorders.2729 while some of the adverse events associated with da therapy are often perceived to be less bothersome for patients than ld - induced motor complications , others such as impulse - control disorders and sleep attacks , can have serious consequences for both the patient and their social relationships such that the advantages of delaying ld - associated risks by treatment with das may be negated . therefore , the choice of therapy should be an individualized decision that takes into account the differential risk profiles of the various da replacement strategies . another perceived risk of ld that still causes concern among many neurologists is related to its effect on disease progression . in the early 1990s , a number of in vitro studies demonstrated that high doses of ld can be toxic to dopaminergic neurons in cell culture,3032 causing some pd specialists to recommend withholding ld for as long as possible.33 since that time , data have accumulated showing that ld may also have protective effects for cultured dopamine neurons , depending on experimental conditions , such as presence or absence of glia34,35 or ascorbic acid,36,37 as well as ld dose used.38 in addition , many in vivo studies could not find evidence of ld - induced neurodegeneration in normal rodents,39 primates,40 and nonparkinsonian humans,41 and some have even reported neuroprotective effects of ld on midbrain dopaminergic neurons.42,43 of particular interest are pathology reports from patients with essential tremor or dopa - responsive dystonia who had been chronically exposed to large amounts of ld over many years . none of these patients have shown evidence of substantia nigra degeneration at autopsy.8,9,44 after almost 40 years of established clinical use , the parkinson study group recently conducted the first high - quality , randomized , placebo - controlled trial of ld to define better the effects of ld monotherapy on clinical progression in early pd.45 the elldopa ( earlier versus later levodopa ) study included 361 patients with early pd who were randomized to receive ld carbidopa at a daily dose of 150/37.5 mg , 300/75 mg , or 600/150 mg , respectively , or a matching placebo for a period of 40 weeks . the primary outcome was the difference in updrs scores between treatment groups at week 42 after withdrawal of treatment for two weeks . this endpoint was chosen to detect any potential underlying effect of active treatment on pd progression , with the assumption that a two - week washout period would remove all symptomatic ld effects , and any remaining differences in updrs scores between groups by this time would reflect treatment effects on disease progression . in addition , assessments of striatal dopamine transporter density using iodine-123-labeled 2--carboxymethoxy-3--(4-iodophenyl)tropane ( [ i ] -cit ) single photon emission computed tomography were performed at baseline and at the end of study as a surrogate measure of progression of nigrostriatal terminal dysfunction . after a two - week washout period , the updrs motor scores in each group of ld - treated patients were still significantly improved compared with patients on placebo . this not only seems to exclude any evidence for negative effects of ld on the progression of pd but , on the contrary , suggests that treatment with ld results in a decline of updrs scores over time as compared with placebo . nevertheless , there remains a possibility that a two - week washout could be insufficient for symptomatic effects of ld to wear off completely , thus precluding firm conclusions about the disease - modifying efficacy of ld from this study . adding to this uncertainty , there was a significantly greater decline of striatal -cit binding in the high - dose arm of this trial compared with placebo in patients with abnormal scans at baseline . interpretation of this finding is again confounded by the possibility of regulatory effects of ld on dopamine transporter binding or expression . integrating the currently available evidence from experimental studies and clinical trials , there is very little reason to assume that ld might hasten the clinical progression of pd , and withholding the drug because of such fears from patients in clinical need of optimized symptomatic control is not warranted . accordingly , us and european pd practice guidelines consistently recommend early use of ld in patients requiring initiation of dopaminergic treatment when the perceived dyskinesia risk is low , as is the case in the elderly.46 the mechanism of action of ld is related to its activity as a prodrug for central dopamine and involves a number of critical steps , including gastrointestinal absorption , passage across the blood brain barrier , neuronal uptake , and conversion to dopamine via enzymatic action of aromatic amino acid decarboxylase ( aadc ) ( figure 3 ) , and eventually synaptic release of dopamine thus generated from exogenous ld . this sequence of events , needed for ld to exert its antiparkinsonian effect , is subject to a number of interfering processes , which can contribute to dose failures and long - term complications.47 these include delayed gastric emptying and altered absorption of ld due to a competitive effect with ingested proteins at the level of amino acid transporters located in the gastrointestinal tract and the blood two major peripheral ld metabolic pathways , driven by the enzymes aadc and catechol - o - methyl transferase , significantly deplete the amount of ld reaching the brain . in addition , the short half - life ( 3696 minutes ) of ld is associated with fluctuating ld plasma levels , which eventually translate into fluctuating levels of synaptic dopamine derived from exogenous ld.47 consequently , for optimal benefit , ld has to be administered as multiple daily doses , but conventional three times daily regimens have not been found to be sufficient to establish constant plasma levels.48 in the earlier stages of the disease , oscillations in plasma levels are not clearly associated with fluctuations in motor function , presumably due to central buffering via intraneuronal storage in surviving nigrostriatal terminals , providing continuous stimulation even in the context of discontinuous exogenous delivery ( figure 4 ) . however , with progressive loss of nigrostriatal terminals and accompanying changes in the central pharmacodynamics of ld , the clinical response to individual doses becomes progressively short - lived , resulting in wearing - off and on - off -type motor fluctuations . these motor fluctuations can be completely abolished by continuous intravenous infusions of ld,48 supporting the concept that providing a less pulsatile , more continuous , striatal dopamine receptor stimulation may be critical to restoring physiological motor processing in the striato - pallido - thalamo - cortical network in pd.4953 the issue of continuous drug delivery is also relevant to the current understanding of mechanisms underlying the development of ld - induced dyskinesia . in animal models of pd , administration of d1 or d2 agonists with short half - lives is associated with dyskinetic responses,5457 while exposure to long - acting agonists does not induce dyskinesia.5860 the same differences have also been observed in studies comparing pulsatile versus continuous delivery of the same dopaminergic agent.61,62 such results are consistent with clinical studies of continuous infusions of das , such as apomorphine63,64 or lisuride,65 which were found to downregulate pre - existing ld - induced dyskinesia . indeed , when given as continuous intraduodenal infusions , marked reductions in dyskinesia have been reported for ld itself49,53,66,67 and the gel preparation of ld ( duodopa , solvay pharmaceuticals gmbh).68,69 although pulsatile stimulation may not be sufficient to explain the mechanisms underlying the induction of dyskinesia completely , such observations highlight the need to optimize ld delivery in pd . the use of intravenous infusions is not feasible for chronic treatment , and intrajejunal infusion strategies are currently limited by high costs and the need for percutaneous gastrostomy . other routes of ld delivery , eg , transdermal or transnasal , are currently under investigation but no nonenteral system has yet reached the market.5860,70,71 previous attempts to improve oral delivery have included the development of sustained - release preparations of ld , but unfortunately randomized controlled studies have failed to reveal any difference between such formulations and standard ld with respect to long - term dyskinesia risk.7274 sustained - release ld preparations exhibit erratic absorption patterns and unpredictable plasma levels,67 resulting in dose failures as well as a delay in producing a clinical benefit.75 as such , the unpredictable absorption of these agents may not abolish high peak and low trough ld plasma levels that are associated with the development of dyskinesia . likewise , controlled trials have not clearly established superiority of sustained - release ld in terms of control of motor fluctuations.76 by contrast , prolonging the ld half - life via adjunctive treatment with a catechol - o - methyltransferase inhibitor , such as entacapone or tolcapone , has been found to be efficacious in reducing daily off time in a number of well - performed , randomized , controlled trials in pd patients with wearing - off.7780 the established efficacy of entacapone in pd patients with wearing - off has raised the possibility that it may also be effective in reducing the risk of dyskinesias . however , results of the recent stride - pd trial demonstrated that , in patients with early pd who are not experiencing wearing - off , a four times daily dose of a ld formulation comprising ld , carbidopa , and entacapone is not superior to conventional ld in delaying dyskinesias.22 the study investigators have proposed that entacapone may not have been administered frequently enough to achieve smooth levels of ld in the plasma.22 as such , current research is focusing on the development of new ld formulations and delivery systems , such as novel controlled - release and transdermal preparations , which may improve delivery of ld in order to achieve smoother ld plasma levels . it remains to be seen whether these new ld formulations will prove effective in minimizing the risk of drug - induced motor complications . despite all recent advances in the medical management of pd , ld has remained the therapeutic gold standard in controlling the cardinal motor features of this illness . there is no evidence to support withholding ld for fear of hastening the progression of pd , although current three times daily regimens of standard oral ld carry a definite risk of inducing potentially disabling drug - induced involuntary movements . while dyskinetic responses are common with sustained ld therapy , the proportion of patients actually developing disabling and severe dyskinesia has been below 10% in a recent four - year randomized trial2 and below 20% in a 10-year follow - up series.17 this should be taken into account when the physician discusses the potential use of ld with the patient , particularly in patients who require enhanced symptomatic control despite optimized treatment with das . moreover , when making individual decisions on how to initiate dopaminergic treatment , the weighing of relative risks and benefits of starting with a da or ld should not only consider dyskinesia risks but also risks for other side effects , including daytime somnolence , impulse - control disorders and , in the case of ergot - derived das , cardiac valvulopathy and other forms of potentially life - threatening fibrosis.27 in addition , individual needs for magnitude and speed of symptomatic improvement must be balanced against potential side effects . future endeavors will focus on optimizing delivery of ld in order to expand and improve the treatment options of pd .

after more than 40 years of clinical use , levodopa ( ld ) remains the gold standard of symptomatic efficacy in the drug treatment of parkinson s disease ( pd ) . compared with other available dopaminergic therapies , dopamine replacement with ld is associated with the greatest improvement in motor function . long - term treatment with ld is , however , often complicated by the development of various types of motor response oscillations over the day , as well as drug - induced dyskinesias . motor fluctuations can be improved by the addition of drugs such as entacapone or monoamine oxidase inhibitors , which extend the half - life of levodopa or dopamine , respectively . however , dyskinesia control still represents a major challenge . as a result , many neurologists have become cautious when prescribing therapy with ld . this review summarizes the available evidence regarding the use of ld to treat pd and will also address the issue of ld delivery as a critical factor for the drug s propensity to induce motor complications .

Introduction Efficacy of levodopa in early Parkinsons disease Delaying dyskinesias using initial monotherapy with dopamine agonists Tolerability and safety issues Levodopa and progression of Parkinsons disease Challenge of optimizing levodopa delivery Conclusions

after more than 40 years of clinical use , levodopa ( ld ) remains the gold standard regarding symptomatic efficacy in the drug treatment of parkinson s disease ( pd).1 compared with other available dopaminergic therapies , dopamine replacement with ld is associated with the greatest improvement in motor function , as assessed by reduced scores in the unified parkinson s disease rating scale [ updrs]).25 in addition , responsiveness to ld ( required to exceed 25%30% reduction in the motor part of the updrs ) is a diagnostic criterion for pd.6 in clinical practice , ld slows the progression of disability as assessed by the hoehn and yahr staging system,7 and is associated with a reduction in mortality.8,9 importantly , ld is one of the best tolerated drugs to treat pd , particularly in the elderly population.10 however , long - term treatment with ld is often complicated by the development of various types of motor response oscillations over the day as well as drug - induced dyskinesia , a complication characterized by erratic involuntary movements . such treatment - related motor complications eventually develop in the majority of patients and are found in about one - third of patients after only two years of exposure.11 once established , motor complications are difficult to treat and can develop into a significant source of disability . in extreme cases , treatment - induced dyskinesias may completely annihilate the therapeutic benefit initially gained from the drug . concerns about the potential induction of long - term motor complications have led many physicians to use ld in a restricted manner and reserve it as a second - line strategy . this approach has gained wide acceptance following clinical trials in early pd , showing significantly reduced risks of developing motor complications with dopamine agonists ( das ) as compared with ld monotherapy.25 while these trials have indeed established the potential of das to delay the onset of motor complications , they have also consistently demonstrated the superior symptomatic efficacy of ld , with a need for ld supplementation within the first 23 years in most patients started on a da.2 despite such evidence from clinical trials of the need for ld supplementation to maintain symptomatic control in early pd , in general clinical practice , ld is often withheld beyond the time when symptomatic control with das has become insufficient.12 this results , in part , from patient perceptions . the alarming nature of dyskinesia can , in turn , lead to a phenomenon labeled dopa - phobia.12 these concerns have even been extrapolated to using single doses of ld in challenge tests , in order to prime the striatum putatively for subsequent dyskinetic responses to dopaminergic therapies.13 increasing evidence now suggests that motor complications ( particularly dyskinesia ) associated with sustained ld therapy are a result of discontinuous and intermittent delivery of ld to the brain , resulting in nonphysiologic pulsatile stimulation of striatal dopamine receptors . thus , the short half - life ( 90 minutes ) of immediate - release ld formulations is thought to be the key factor in the pathogenesis of motor complications , rather than their induction being an intrinsic property of the ld molecule.1 this review will summarize the available evidence regarding the use of ld to treat pd and will also address the issue of ld delivery as a critical factor for the drug s propensity to induce motor complications . there have been a number of large - scale , long - term , ld - controlled monotherapy trials of das in early parkinson s disease on which strong conclusions about the relative effect size of ld compared with das can be based . the four - year randomized comparison of the agonist pramipexole versus levodopa on motor complications of parkinson s disease ( calm - pd ) trial , compared initial treatment with pramipexole ( 0.5 mg three times daily ) versus ld - carbidopa ( 100/25 mg three times daily ) , followed by open - label ld supplementation as required.2 the primary outcome measure was the time to first occurrence of dopaminergic complications , which included wearing - off ( the re - emergence of pd symptoms due to the diminishing effect of ld ) , dyskinesias , on off fluctuations ( unpredictable fluctuations varying between symptoms being well controlled [ on ] to uncontrolled [ off ] ) , and freezing . although initial treatment with pramipexole resulted in lower incidences of dyskinesia and wearing - off compared with initial treatment with ld , symptom control , as assessed by the updrs , was superior in patients treated with ld . it is not definitely established why the updrs scores of the pramipexole group never caught up with the ld group , despite the option of open - label ld and other antiparkinsonian therapies , but it might be related to the lower dose of ld used in the supplemented pramipexole patients ( 434 498 mg / day ) compared with those on ld monotherapy ( 702 461 mg / day ) . indeed , the calculated ld equivalent dose used in the supplemented pramipexole patients falls in the range of 468584 mg / day , which is notably less than the 702 461 mg / day dose used in patients initiated on ld monotherapy . this indicates that combined treatment with a lower dose of ld and a da is not equivalent to higher dose ld monotherapy in terms of symptomatic control , calling into question the concept of ld - equivalent doses of das . in the five - year 056 study , which compared the safety and efficacy of ropinirole with that of ld , the primary efficacy measure was the occurrence of dyskinesia.3 ropinirole was initiated at 0.25 mg three times daily and increased weekly , as necessary , up to a maximum dose of 8 mg three times daily . similar results confirming the superior efficacy of ld ( in terms of updrs motor scores ) were also observed in the two - year real - pet ( requip as early therapy versus l - dopa positron emission tomography ) study of ld versus ropinirole monotherapy.14 the clinical relevance of such differences in updrs scores has been debated , but is vividly illustrated by the increasing rates of supplementation with open - label ld over the course of these double - blind comparative trials . at four years follow - up in the calm - pd trial , 72% of patients randomized to pramipexole monotherapy had required add - on ld to maintain symptomatic control , and this figure was 66% after five years in the 056 study with ropinirole ( figure 1b).25 the term ld rescue , commonly used in this context , aptly describes the role of ld as the most efficacious drug at hand to control motor symptoms when other drugs begin to fail . at one year , there was a relatively small difference in favor of ld in the mean change from baseline in updrs motor scores ( 3.2 points in the pergolide treatment group compared with 5.2 points in the ld treatment group ) . however , after three years of monotherapy , patients receiving pergolide had deteriorated below baseline by 2.8 9.8 points , whereas patients receiving ld were still improved by 2.8 7.8 points . current recommendations to initiate dopaminergic therapy in early pd with a da , or even a monoamine oxidase inhibitor , are chiefly based on concerns about the evolution of motor complications characteristic of sustained ld therapy , most notably drug - induced dyskinesias . indeed , pd patients initiated on a da in these trials eventually required supplemental ld in order to maintain symptomatic control , and developed dyskinesias at an identical rate ( albeit with a delay ) to those started on ld , leading to steady increases in dyskinesia rates from this point onwards.15 therefore , the question remains as to whether the initial benefit of reduced motor complication rates can be maintained in the longer term when virtually all patients will be on combined drug treatment with das , ld , and possibly other agents . after 10 years , 45% of patients who were initiated with bromocriptine had dyskinesias compared with 54% of those started on ld ( corresponding to an incidence rate for first dyskinesia occurrence of 145.3 versus 105.9 per 1000 patient - years after starting treatment with ld or da , respectively).17 similar figures were reported for the 10-year follow - up of patients of the 056 study of ropinirole versus ld ( 52.4% versus 77.8% , p = 0.0457).19 while these results convincingly show long - term reductions in the incidence of dyskinesia with das compared with ld , rates of dyskinesia do not necessarily reflect differences in dyskinesia - related disability between treatments . when patients were subsequently asked to rate their recorded on or off times as functionally good time , whereas 84.9% of off time and 89.9% of on time with troublesome dyskinesia was considered time.20 in this respect , it is interesting to note that , at 10 and 14 years , respectively , neither the ropinirole nor bromocriptine studies found significant differences between treatment arms in the emergence of disabling dyskinesia.15,17,21 this finding was also observed in the calm - pd study at four years , where the rate of disabling dyskinesias was low in patients treated with either pramipexole or ld.16 in addition , the recent stride - pd ( stalevo reduction in dyskinesia evaluation ) trial comparing time to dyskinesia development in patients treated with either standard ld or a fixed combination of ld , carbidopa , and entacapone showed a less than 10% incidence of disabling dyskinesias in both groups.22 furthermore , patient surveys have also indicated that patients with dyskinesias are less concerned about their dyskinesias than those who have not yet experienced dyskinesias , and that more than 80% of these patients prefer having dyskinesia over their pd symptoms.23 since patients often tolerate mild dyskinesia well , the risk of dyskinesia should not cause physicians to delay ld initiation in patients whose pd symptoms can not be sufficiently controlled with other treatments , regardless of patient age . with chronic use , the most common complications include wearing - off and dyskinesia , which can be troublesome for the patient ( figure 2).26 however , while the use of das is not associated with motor complications , a different array of side effects , including hallucinations , somnolence , and edema , are observed more commonly with these anti - pd medications ( figure 2 ) . dopamine agonists have also been linked to impulse - control disorders , such as pathologic gambling , hypersexuality , binge eating , or pathologic shopping , with about 13.7%17.1% of patients on da therapy showing signs of such disorders.2729 while some of the adverse events associated with da therapy are often perceived to be less bothersome for patients than ld - induced motor complications , others such as impulse - control disorders and sleep attacks , can have serious consequences for both the patient and their social relationships such that the advantages of delaying ld - associated risks by treatment with das may be negated . in the early 1990s , a number of in vitro studies demonstrated that high doses of ld can be toxic to dopaminergic neurons in cell culture,3032 causing some pd specialists to recommend withholding ld for as long as possible.33 since that time , data have accumulated showing that ld may also have protective effects for cultured dopamine neurons , depending on experimental conditions , such as presence or absence of glia34,35 or ascorbic acid,36,37 as well as ld dose used.38 in addition , many in vivo studies could not find evidence of ld - induced neurodegeneration in normal rodents,39 primates,40 and nonparkinsonian humans,41 and some have even reported neuroprotective effects of ld on midbrain dopaminergic neurons.42,43 of particular interest are pathology reports from patients with essential tremor or dopa - responsive dystonia who had been chronically exposed to large amounts of ld over many years . none of these patients have shown evidence of substantia nigra degeneration at autopsy.8,9,44 after almost 40 years of established clinical use , the parkinson study group recently conducted the first high - quality , randomized , placebo - controlled trial of ld to define better the effects of ld monotherapy on clinical progression in early pd.45 the elldopa ( earlier versus later levodopa ) study included 361 patients with early pd who were randomized to receive ld carbidopa at a daily dose of 150/37.5 mg , 300/75 mg , or 600/150 mg , respectively , or a matching placebo for a period of 40 weeks . this not only seems to exclude any evidence for negative effects of ld on the progression of pd but , on the contrary , suggests that treatment with ld results in a decline of updrs scores over time as compared with placebo . accordingly , us and european pd practice guidelines consistently recommend early use of ld in patients requiring initiation of dopaminergic treatment when the perceived dyskinesia risk is low , as is the case in the elderly.46 the mechanism of action of ld is related to its activity as a prodrug for central dopamine and involves a number of critical steps , including gastrointestinal absorption , passage across the blood brain barrier , neuronal uptake , and conversion to dopamine via enzymatic action of aromatic amino acid decarboxylase ( aadc ) ( figure 3 ) , and eventually synaptic release of dopamine thus generated from exogenous ld . this sequence of events , needed for ld to exert its antiparkinsonian effect , is subject to a number of interfering processes , which can contribute to dose failures and long - term complications.47 these include delayed gastric emptying and altered absorption of ld due to a competitive effect with ingested proteins at the level of amino acid transporters located in the gastrointestinal tract and the blood two major peripheral ld metabolic pathways , driven by the enzymes aadc and catechol - o - methyl transferase , significantly deplete the amount of ld reaching the brain . in addition , the short half - life ( 3696 minutes ) of ld is associated with fluctuating ld plasma levels , which eventually translate into fluctuating levels of synaptic dopamine derived from exogenous ld.47 consequently , for optimal benefit , ld has to be administered as multiple daily doses , but conventional three times daily regimens have not been found to be sufficient to establish constant plasma levels.48 in the earlier stages of the disease , oscillations in plasma levels are not clearly associated with fluctuations in motor function , presumably due to central buffering via intraneuronal storage in surviving nigrostriatal terminals , providing continuous stimulation even in the context of discontinuous exogenous delivery ( figure 4 ) . however , with progressive loss of nigrostriatal terminals and accompanying changes in the central pharmacodynamics of ld , the clinical response to individual doses becomes progressively short - lived , resulting in wearing - off and on - off -type motor fluctuations . these motor fluctuations can be completely abolished by continuous intravenous infusions of ld,48 supporting the concept that providing a less pulsatile , more continuous , striatal dopamine receptor stimulation may be critical to restoring physiological motor processing in the striato - pallido - thalamo - cortical network in pd.4953 the issue of continuous drug delivery is also relevant to the current understanding of mechanisms underlying the development of ld - induced dyskinesia . in animal models of pd , administration of d1 or d2 agonists with short half - lives is associated with dyskinetic responses,5457 while exposure to long - acting agonists does not induce dyskinesia.5860 the same differences have also been observed in studies comparing pulsatile versus continuous delivery of the same dopaminergic agent.61,62 such results are consistent with clinical studies of continuous infusions of das , such as apomorphine63,64 or lisuride,65 which were found to downregulate pre - existing ld - induced dyskinesia . other routes of ld delivery , eg , transdermal or transnasal , are currently under investigation but no nonenteral system has yet reached the market.5860,70,71 previous attempts to improve oral delivery have included the development of sustained - release preparations of ld , but unfortunately randomized controlled studies have failed to reveal any difference between such formulations and standard ld with respect to long - term dyskinesia risk.7274 sustained - release ld preparations exhibit erratic absorption patterns and unpredictable plasma levels,67 resulting in dose failures as well as a delay in producing a clinical benefit.75 as such , the unpredictable absorption of these agents may not abolish high peak and low trough ld plasma levels that are associated with the development of dyskinesia . likewise , controlled trials have not clearly established superiority of sustained - release ld in terms of control of motor fluctuations.76 by contrast , prolonging the ld half - life via adjunctive treatment with a catechol - o - methyltransferase inhibitor , such as entacapone or tolcapone , has been found to be efficacious in reducing daily off time in a number of well - performed , randomized , controlled trials in pd patients with wearing - off.7780 the established efficacy of entacapone in pd patients with wearing - off has raised the possibility that it may also be effective in reducing the risk of dyskinesias . however , results of the recent stride - pd trial demonstrated that , in patients with early pd who are not experiencing wearing - off , a four times daily dose of a ld formulation comprising ld , carbidopa , and entacapone is not superior to conventional ld in delaying dyskinesias.22 the study investigators have proposed that entacapone may not have been administered frequently enough to achieve smooth levels of ld in the plasma.22 as such , current research is focusing on the development of new ld formulations and delivery systems , such as novel controlled - release and transdermal preparations , which may improve delivery of ld in order to achieve smoother ld plasma levels . it remains to be seen whether these new ld formulations will prove effective in minimizing the risk of drug - induced motor complications . while dyskinetic responses are common with sustained ld therapy , the proportion of patients actually developing disabling and severe dyskinesia has been below 10% in a recent four - year randomized trial2 and below 20% in a 10-year follow - up series.17 this should be taken into account when the physician discusses the potential use of ld with the patient , particularly in patients who require enhanced symptomatic control despite optimized treatment with das . moreover , when making individual decisions on how to initiate dopaminergic treatment , the weighing of relative risks and benefits of starting with a da or ld should not only consider dyskinesia risks but also risks for other side effects , including daytime somnolence , impulse - control disorders and , in the case of ergot - derived das , cardiac valvulopathy and other forms of potentially life - threatening fibrosis.27 in addition , individual needs for magnitude and speed of symptomatic improvement must be balanced against potential side effects .

after more than 40 years of clinical use , levodopa ( ld ) remains the gold standard regarding symptomatic efficacy in the drug treatment of parkinson s disease ( pd).1 compared with other available dopaminergic therapies , dopamine replacement with ld is associated with the greatest improvement in motor function , as assessed by reduced scores in the unified parkinson s disease rating scale [ updrs]).25 in addition , responsiveness to ld ( required to exceed 25%30% reduction in the motor part of the updrs ) is a diagnostic criterion for pd.6 in clinical practice , ld slows the progression of disability as assessed by the hoehn and yahr staging system,7 and is associated with a reduction in mortality.8,9 importantly , ld is one of the best tolerated drugs to treat pd , particularly in the elderly population.10 however , long - term treatment with ld is often complicated by the development of various types of motor response oscillations over the day as well as drug - induced dyskinesia , a complication characterized by erratic involuntary movements . such treatment - related motor complications eventually develop in the majority of patients and are found in about one - third of patients after only two years of exposure.11 once established , motor complications are difficult to treat and can develop into a significant source of disability . this approach has gained wide acceptance following clinical trials in early pd , showing significantly reduced risks of developing motor complications with dopamine agonists ( das ) as compared with ld monotherapy.25 while these trials have indeed established the potential of das to delay the onset of motor complications , they have also consistently demonstrated the superior symptomatic efficacy of ld , with a need for ld supplementation within the first 23 years in most patients started on a da.2 despite such evidence from clinical trials of the need for ld supplementation to maintain symptomatic control in early pd , in general clinical practice , ld is often withheld beyond the time when symptomatic control with das has become insufficient.12 this results , in part , from patient perceptions . the alarming nature of dyskinesia can , in turn , lead to a phenomenon labeled dopa - phobia.12 these concerns have even been extrapolated to using single doses of ld in challenge tests , in order to prime the striatum putatively for subsequent dyskinetic responses to dopaminergic therapies.13 increasing evidence now suggests that motor complications ( particularly dyskinesia ) associated with sustained ld therapy are a result of discontinuous and intermittent delivery of ld to the brain , resulting in nonphysiologic pulsatile stimulation of striatal dopamine receptors . thus , the short half - life ( 90 minutes ) of immediate - release ld formulations is thought to be the key factor in the pathogenesis of motor complications , rather than their induction being an intrinsic property of the ld molecule.1 this review will summarize the available evidence regarding the use of ld to treat pd and will also address the issue of ld delivery as a critical factor for the drug s propensity to induce motor complications . the four - year randomized comparison of the agonist pramipexole versus levodopa on motor complications of parkinson s disease ( calm - pd ) trial , compared initial treatment with pramipexole ( 0.5 mg three times daily ) versus ld - carbidopa ( 100/25 mg three times daily ) , followed by open - label ld supplementation as required.2 the primary outcome measure was the time to first occurrence of dopaminergic complications , which included wearing - off ( the re - emergence of pd symptoms due to the diminishing effect of ld ) , dyskinesias , on off fluctuations ( unpredictable fluctuations varying between symptoms being well controlled [ on ] to uncontrolled [ off ] ) , and freezing . from baseline to month 48 , there was a worsening from baseline of 1.3 13.3 ( mean standard deviation [ sd ] ) points in updrs motor scores in the pramipexole group compared with an improvement of 3.4 12.3 points in the ld group ( treatment difference of 4.9 points , p = 0.001 ) as shown in figure 1a . interestingly , even though physicians in this trial had the option to use open - label ld supplementation to enhance symptomatic control , the group differences observed in the updrs motor and activities of daily living components between patients randomized to pramipexole or ld remained relatively uniform throughout the four years of the study . it is not definitely established why the updrs scores of the pramipexole group never caught up with the ld group , despite the option of open - label ld and other antiparkinsonian therapies , but it might be related to the lower dose of ld used in the supplemented pramipexole patients ( 434 498 mg / day ) compared with those on ld monotherapy ( 702 461 mg / day ) . indeed , the calculated ld equivalent dose used in the supplemented pramipexole patients falls in the range of 468584 mg / day , which is notably less than the 702 461 mg / day dose used in patients initiated on ld monotherapy . in the five - year 056 study , which compared the safety and efficacy of ropinirole with that of ld , the primary efficacy measure was the occurrence of dyskinesia.3 ropinirole was initiated at 0.25 mg three times daily and increased weekly , as necessary , up to a maximum dose of 8 mg three times daily . in those patients who completed the study , there was a minimal improvement from baseline of 0.8 10.1 points in updrs motor scores in the ropinirole group , contrasting with an improvement of 4.8 8.3 points in the ld group ( figure 1a ) . similar results confirming the superior efficacy of ld ( in terms of updrs motor scores ) were also observed in the two - year real - pet ( requip as early therapy versus l - dopa positron emission tomography ) study of ld versus ropinirole monotherapy.14 the clinical relevance of such differences in updrs scores has been debated , but is vividly illustrated by the increasing rates of supplementation with open - label ld over the course of these double - blind comparative trials . at four years follow - up in the calm - pd trial , 72% of patients randomized to pramipexole monotherapy had required add - on ld to maintain symptomatic control , and this figure was 66% after five years in the 056 study with ropinirole ( figure 1b).25 the term ld rescue , commonly used in this context , aptly describes the role of ld as the most efficacious drug at hand to control motor symptoms when other drugs begin to fail . at one year , there was a relatively small difference in favor of ld in the mean change from baseline in updrs motor scores ( 3.2 points in the pergolide treatment group compared with 5.2 points in the ld treatment group ) . indeed , pd patients initiated on a da in these trials eventually required supplemental ld in order to maintain symptomatic control , and developed dyskinesias at an identical rate ( albeit with a delay ) to those started on ld , leading to steady increases in dyskinesia rates from this point onwards.15 therefore , the question remains as to whether the initial benefit of reduced motor complication rates can be maintained in the longer term when virtually all patients will be on combined drug treatment with das , ld , and possibly other agents . results of this study showed a persistent , statistically significant difference in overall dyskinesia rates of about 37% in those randomized to initial ld compared with 20% in those initiated on pramipexole ( p = 0.004).16 despite this , 90% of the population followed were on ld by six years.16 similarly , 10-year outcomes reported by lees et al17 comparing initial monotherapy with bromocriptine versus ld , along with hauser et al18 from the 056 ropinirole study , also demonstrated a reduced overall incidence of dyskinesias with das in the long term . after 10 years , 45% of patients who were initiated with bromocriptine had dyskinesias compared with 54% of those started on ld ( corresponding to an incidence rate for first dyskinesia occurrence of 145.3 versus 105.9 per 1000 patient - years after starting treatment with ld or da , respectively).17 similar figures were reported for the 10-year follow - up of patients of the 056 study of ropinirole versus ld ( 52.4% versus 77.8% , p = 0.0457).19 while these results convincingly show long - term reductions in the incidence of dyskinesia with das compared with ld , rates of dyskinesia do not necessarily reflect differences in dyskinesia - related disability between treatments . when patients were subsequently asked to rate their recorded on or off times as functionally good time , whereas 84.9% of off time and 89.9% of on time with troublesome dyskinesia was considered time.20 in this respect , it is interesting to note that , at 10 and 14 years , respectively , neither the ropinirole nor bromocriptine studies found significant differences between treatment arms in the emergence of disabling dyskinesia.15,17,21 this finding was also observed in the calm - pd study at four years , where the rate of disabling dyskinesias was low in patients treated with either pramipexole or ld.16 in addition , the recent stride - pd ( stalevo reduction in dyskinesia evaluation ) trial comparing time to dyskinesia development in patients treated with either standard ld or a fixed combination of ld , carbidopa , and entacapone showed a less than 10% incidence of disabling dyskinesias in both groups.22 furthermore , patient surveys have also indicated that patients with dyskinesias are less concerned about their dyskinesias than those who have not yet experienced dyskinesias , and that more than 80% of these patients prefer having dyskinesia over their pd symptoms.23 since patients often tolerate mild dyskinesia well , the risk of dyskinesia should not cause physicians to delay ld initiation in patients whose pd symptoms can not be sufficiently controlled with other treatments , regardless of patient age . with chronic use , the most common complications include wearing - off and dyskinesia , which can be troublesome for the patient ( figure 2).26 however , while the use of das is not associated with motor complications , a different array of side effects , including hallucinations , somnolence , and edema , are observed more commonly with these anti - pd medications ( figure 2 ) . dopamine agonists have also been linked to impulse - control disorders , such as pathologic gambling , hypersexuality , binge eating , or pathologic shopping , with about 13.7%17.1% of patients on da therapy showing signs of such disorders.2729 while some of the adverse events associated with da therapy are often perceived to be less bothersome for patients than ld - induced motor complications , others such as impulse - control disorders and sleep attacks , can have serious consequences for both the patient and their social relationships such that the advantages of delaying ld - associated risks by treatment with das may be negated . in the early 1990s , a number of in vitro studies demonstrated that high doses of ld can be toxic to dopaminergic neurons in cell culture,3032 causing some pd specialists to recommend withholding ld for as long as possible.33 since that time , data have accumulated showing that ld may also have protective effects for cultured dopamine neurons , depending on experimental conditions , such as presence or absence of glia34,35 or ascorbic acid,36,37 as well as ld dose used.38 in addition , many in vivo studies could not find evidence of ld - induced neurodegeneration in normal rodents,39 primates,40 and nonparkinsonian humans,41 and some have even reported neuroprotective effects of ld on midbrain dopaminergic neurons.42,43 of particular interest are pathology reports from patients with essential tremor or dopa - responsive dystonia who had been chronically exposed to large amounts of ld over many years . none of these patients have shown evidence of substantia nigra degeneration at autopsy.8,9,44 after almost 40 years of established clinical use , the parkinson study group recently conducted the first high - quality , randomized , placebo - controlled trial of ld to define better the effects of ld monotherapy on clinical progression in early pd.45 the elldopa ( earlier versus later levodopa ) study included 361 patients with early pd who were randomized to receive ld carbidopa at a daily dose of 150/37.5 mg , 300/75 mg , or 600/150 mg , respectively , or a matching placebo for a period of 40 weeks . accordingly , us and european pd practice guidelines consistently recommend early use of ld in patients requiring initiation of dopaminergic treatment when the perceived dyskinesia risk is low , as is the case in the elderly.46 the mechanism of action of ld is related to its activity as a prodrug for central dopamine and involves a number of critical steps , including gastrointestinal absorption , passage across the blood brain barrier , neuronal uptake , and conversion to dopamine via enzymatic action of aromatic amino acid decarboxylase ( aadc ) ( figure 3 ) , and eventually synaptic release of dopamine thus generated from exogenous ld . this sequence of events , needed for ld to exert its antiparkinsonian effect , is subject to a number of interfering processes , which can contribute to dose failures and long - term complications.47 these include delayed gastric emptying and altered absorption of ld due to a competitive effect with ingested proteins at the level of amino acid transporters located in the gastrointestinal tract and the blood two major peripheral ld metabolic pathways , driven by the enzymes aadc and catechol - o - methyl transferase , significantly deplete the amount of ld reaching the brain . in addition , the short half - life ( 3696 minutes ) of ld is associated with fluctuating ld plasma levels , which eventually translate into fluctuating levels of synaptic dopamine derived from exogenous ld.47 consequently , for optimal benefit , ld has to be administered as multiple daily doses , but conventional three times daily regimens have not been found to be sufficient to establish constant plasma levels.48 in the earlier stages of the disease , oscillations in plasma levels are not clearly associated with fluctuations in motor function , presumably due to central buffering via intraneuronal storage in surviving nigrostriatal terminals , providing continuous stimulation even in the context of discontinuous exogenous delivery ( figure 4 ) . these motor fluctuations can be completely abolished by continuous intravenous infusions of ld,48 supporting the concept that providing a less pulsatile , more continuous , striatal dopamine receptor stimulation may be critical to restoring physiological motor processing in the striato - pallido - thalamo - cortical network in pd.4953 the issue of continuous drug delivery is also relevant to the current understanding of mechanisms underlying the development of ld - induced dyskinesia . in animal models of pd , administration of d1 or d2 agonists with short half - lives is associated with dyskinetic responses,5457 while exposure to long - acting agonists does not induce dyskinesia.5860 the same differences have also been observed in studies comparing pulsatile versus continuous delivery of the same dopaminergic agent.61,62 such results are consistent with clinical studies of continuous infusions of das , such as apomorphine63,64 or lisuride,65 which were found to downregulate pre - existing ld - induced dyskinesia . indeed , when given as continuous intraduodenal infusions , marked reductions in dyskinesia have been reported for ld itself49,53,66,67 and the gel preparation of ld ( duodopa , solvay pharmaceuticals gmbh).68,69 although pulsatile stimulation may not be sufficient to explain the mechanisms underlying the induction of dyskinesia completely , such observations highlight the need to optimize ld delivery in pd . other routes of ld delivery , eg , transdermal or transnasal , are currently under investigation but no nonenteral system has yet reached the market.5860,70,71 previous attempts to improve oral delivery have included the development of sustained - release preparations of ld , but unfortunately randomized controlled studies have failed to reveal any difference between such formulations and standard ld with respect to long - term dyskinesia risk.7274 sustained - release ld preparations exhibit erratic absorption patterns and unpredictable plasma levels,67 resulting in dose failures as well as a delay in producing a clinical benefit.75 as such , the unpredictable absorption of these agents may not abolish high peak and low trough ld plasma levels that are associated with the development of dyskinesia . likewise , controlled trials have not clearly established superiority of sustained - release ld in terms of control of motor fluctuations.76 by contrast , prolonging the ld half - life via adjunctive treatment with a catechol - o - methyltransferase inhibitor , such as entacapone or tolcapone , has been found to be efficacious in reducing daily off time in a number of well - performed , randomized , controlled trials in pd patients with wearing - off.7780 the established efficacy of entacapone in pd patients with wearing - off has raised the possibility that it may also be effective in reducing the risk of dyskinesias . however , results of the recent stride - pd trial demonstrated that , in patients with early pd who are not experiencing wearing - off , a four times daily dose of a ld formulation comprising ld , carbidopa , and entacapone is not superior to conventional ld in delaying dyskinesias.22 the study investigators have proposed that entacapone may not have been administered frequently enough to achieve smooth levels of ld in the plasma.22 as such , current research is focusing on the development of new ld formulations and delivery systems , such as novel controlled - release and transdermal preparations , which may improve delivery of ld in order to achieve smoother ld plasma levels . while dyskinetic responses are common with sustained ld therapy , the proportion of patients actually developing disabling and severe dyskinesia has been below 10% in a recent four - year randomized trial2 and below 20% in a 10-year follow - up series.17 this should be taken into account when the physician discusses the potential use of ld with the patient , particularly in patients who require enhanced symptomatic control despite optimized treatment with das . moreover , when making individual decisions on how to initiate dopaminergic treatment , the weighing of relative risks and benefits of starting with a da or ld should not only consider dyskinesia risks but also risks for other side effects , including daytime somnolence , impulse - control disorders and , in the case of ergot - derived das , cardiac valvulopathy and other forms of potentially life - threatening fibrosis.27 in addition , individual needs for magnitude and speed of symptomatic improvement must be balanced against potential side effects .

the tumor suppressor function of p53 is compromised in essentially all human cancers . in about half of human cancers , tp53 , the gene encoding p53 protein , is mutated or deleted , and this inactivates the tumor suppressor function of p53 . in the remaining 50% , p53 retains its wild - type status but its function is effectively inhibited by the murine double minute 2 ( mdm2 ) protein through a direct protein protein interaction . through direct binding , the mdm2 protein blocks the transactivation domain of p53 , transports p53 from the nucleus to the cytoplasm , and ubiquitinates p53 for proteasomal degradation . small - molecule inhibitors designed to block the mdm2p53 interaction ( hereafter called mdm2 inhibitors ) can liberate the tumor suppressor function of p53 and may have a promising therapeutic potential for cancer treatment . intense research efforts have resulted in advancement of several potent , selective , non - peptide mdm2 inhibitors ( 15 ) , shown in figure 1 , into clinical development . our laboratory has designed and optimized spirooxindoles as a new class of mdm2 inhibitors and has advanced compound 2 ( mi-77301 ) into clinical development . one shortcoming of 2 and its earlier analogues that we have found is that they slowly isomerize in solution . to overcome this stability issue , we recently reported the design of new spirooxindoles containing two identical substituents at c-2 of the pyrrolidine ring , which can undergo a rapid and irreversible conversion to a single diastereoisomer . in the present study , we report an extensive structure activity relationship ( sar ) study for this class of mdm2 inhibitors . this study has led to the discovery of 60 ( aa-115/apg-115 ) , a highly potent , chemically stable and efficacious mdm2 inhibitor , which has entered clinical development for cancer treatment ( clinicaltrials.gov identifier for 60 : nct02935907 ) . our exploration started with lead molecule 6(21 ) ( figure 2a ) that binds to mdm2 with ki = 2.9 nm , inhibits the growth of the sjsa-1 cancer cell line with ic50 = 190 nm , and achieves strong tumor growth inhibition but not complete tumor regression in an sjsa-1 osteosarcoma xenograft model in mice . in a search for superior compounds ( a ) chemical structure of compound 6 and five regions ( a e ) where chemical modifications were made . ( b ) cocrystal structure of 2 ( light blue spheres , pdb code 5trf ) and model of the binding mode of 6 ( green sticks ) in complex with mdm2 . to guide our chemical modifications , we modeled the structure of compound 6 in a complex with mdm2 based upon the cocrystal structure of compound 2 complexed with human mdm2 ( figure 2b ) . our model shows that the oxindolephenyl , 3-chloro-2-fluorophenyl , and cyclohexyl groups of 6 project into the trp23 , leu26 , and phe19 p53 binding pockets of the mdm2 protein and the 4-hydroxyl of the n - cyclohexylcarboxamide group forms a hydrogen bond with lys94 of mdm2 ( figure 2b ) . guided by this model , we performed extensive modifications on five regions of the molecule ( labeled as sites a we began by replacing the 4-hydroxycyclohexyl group at site d in compound 6 with a methyl group , giving compound 7 . since compounds 6 and 7 have similar binding affinities to mdm2 , we have used either compound 6 or 7 as the template for modifications of site a ( table 1 ) . replacement of the cyclohexyl group at site a with 4-piperidinyl group led to compound 8 , which has no appreciable binding up to 2 m to mdm2 . methylation or acetylation of the amine group in 8 resulted in compound 9 or 10 , respectively , and these also have very weak affinities for mdm2 . replacement of the cyclohexyl group at site a in 6 with 4-tetrahydro-2h - pyran generated compound 11 , which has a moderate binding affinity to mdm2 ( ki = 75.9 nm ) . introduction of two fluorine substituents at the 4-position of the cyclohexyl group in compound 6 resulted in compound 12 , which has a good affinity to mdm2 ( ki = 9.8 nm ) but is 4 times less potent than 6 . however , introduction of two methyl groups at the same 4-position in 6 yielded compound 15 , which has a ki value of < 1 nm to mdm2 and is several times more potent than 6 . replacement of the cyclohexyl group with a cyclobutyl group generated compound 13 , which is 6 times less potent than 6 . installation of methyl groups at the 3-position of the cyclobutyl ring led to compound 14 , which is equally potent as compound 6 and 7 times more potent than compound 13 . hence , our sar data for site a clearly show that a hydrophobic group is highly desirable for achieving a strong binding affinity to mdm2 , and cyclohexyl , 4,4-dimethylcyclohexyl , and 3,3-cyclobutyl groups are the most preferred . next , we explored modifications of the 3-chloro-2-fluorophenyl and the oxindolephenyl substituents ( sites b and c in figure 2a , respectively ) . to improve solubility , we inserted one or more nitrogen atoms into the aryl rings , producing compounds 16 , 17 , 18 , and 19 ( table 2 ) . insertion of a nitrogen into the 3-chloro-2-fluorophenyl substituent ( ring b ) gave 16 , which has a ki of 77 nm , 26 times weaker than 6 . interestingly , although the trp23 pocket is hydrophobic in nature , compounds 17 , 18 , and 19 containing a pyridinyl or a pyrimidinyl oxindole group ( ring c ) are all quite potent with ki = 1314 nm . our data , however , are consistent with a previous study , which showed that a compound containing a chloropyridyl oxindole group is a potent mdm2 inhibitor . this previous study also showed that replacing the oxindole with a thienopyrrolone led to potent mdm2 inhibitors . we therefore synthesized compound 20 containing a thienopyrrolone , which has a ki of 6.5 nm and is 2-fold less potent than 6 . we found that the thiophene group in 20 can be oxidized during the oxidative removal of the chiral auxiliary , preventing further investigation of this compound . our modeling of 6 and the cocrystal structure of 2 ( figure 2b ) showed that the carbonyl of the amide ( site e ) forms a hydrogen bond with his96 of the mdm2 protein but the amino group does not interact with the mdm2 protein . we evaluated a series of five - membered heteroaromatic rings as the amide bioisoteres ( table 3 ) . among these replacements , we found that heteroaromatic rings with carboxylic acid substituents , e.g. , 21 , 22 , 26 , and 27 have the best binding affinities with ki values between 1 and 3 nm . unfortunately , these compounds show weak cell growth inhibitory activity ( ic50 > 2 m , table 3 ) in the sjsa-1 cancer cells , probably because the negatively charged acid group in these compounds decreases their cell permeability . we therefore converted the acid group in compound 21 to an amide but found that the resulting compound 28 is 20 times less potent than 21 . our modeling of 6 and the cocrystal structure of 2 also showed that the 4-hydroxyl on the cyclohexylcarboxamide substituent of 6 forms a hydrogen bond with lys94 of the mdm2 protein ( figure 2b ) . we synthesized a series of compounds containing either a terminal carboxylic acid or a sulfone to investigate the effect on binding ( compounds 2938 ) ( table 4 ) . in particular , compounds 31 , 32 , and 33 bind to mdm2 with ki < 1 nm and are more than 3 times more potent than 6 . value of one time testing . we tested the cell growth inhibitory activity of compounds 2938 in the sjsa-1 cell line , and the data are summarized in table 4 . among this series of compounds , 32 , 33 , and 38 have the best antiproliferative activity with ic50 values of 0.22 , 0.15 , and 0.24 m , respectively . we further evaluated compound 33 for its pharmacodynamic ( pd ) effect in the sjsa-1 xenograft tissue . our pd data showed that a single , oral administration of 33 at 100 mg / kg is very effective in inducing upregulation of mdm2 , p53 , and p21 proteins at both 3 and 6 h time - points in the sjsa-1 tumor tissue , indicating strong activation of p53 ( figure 3a ) . upon the basis of its promising pd data , we evaluated compound 33 for its antitumor efficacy in the sjsa-1 xenograft model ( figure 3b , c ) . while compound 33 can effectively retard tumor growth , it failed to achieve tumor regression . during the course of our research , we also observed that compound 33 can decompose in solution , particularly in cell culture media ( panels a and b in scheme 1 ) . to analyze the potential structural features affecting the stability of 33 , we determined the stability of compound 31 with a flexible butanoic acid group , compound 6 with a 4-hydroxylcyclohexyl group , and 33 . compound 6 had negligible ( < 1% ) decomposition ( panel c in scheme 1 ) . on the other hand , compound 31 showed 18% decomposition after 2 days in cell culture media , which is greater than that observed for compound 33 ( panel d in scheme 1 ) . on the basis of these results , we proposed that the carboxylic group assists in the decomposition of 33 . ( a ) pharmacodynamics ( pd ) effect of 33 in sjsa-1 tumors in mice . mice were given a single , oral dose of the vehicle , or 33 at 100 mg / kg and sacrificed at indicated time points . west blotting was performed on harvested tumor tissue to probe mdm2 , p53 , p21 , papr , cleaved parp ( cl - papr ) proteins . ( b ) antitumor activity of 33 in the sjsa-1 osteosarcoma tumor xenograft model in mice . compound 33 was administered via oral gavage at 100 mg / kg daily for 14 days . compound 33 was incubated in ch3cn / h2o , meoh / h2o , or cell culture media , and the % composition of 33 in the solutions was determined daily for 2 days by uplc ( ultraperformance liquid chromatography ) and plotted . ( b , c , d ) uplc / ms spectrum of 33 , 6 , and 31 , respectively , after incubation for 2 days in cell culture media . we next performed further chemical modifications of 33 with the goal to improve its chemical stability , cellular potency , and in vivo efficacy . we investigated if replacement of the cyclohexyl group with an even more rigid aryl group can prevent the decomposition . a series of compounds containing an n - arylcarboxamide substituent were synthesized and tested ( table 5 ) . these compounds all display very high binding affinities to mdm2 , superior to that of 6 and equivalent to that of 33 . the cell growth inhibition of sjsa-1 cells by these compounds was determined ( table 5 ) . compounds 39 ( mi-1061 ) and 44 displayed the best cellular activity among the compounds in this series and are 3 times more potent than compound 33 in inhibition of cell growth in the sjsa-1 cell line . furthermore , 39 was capable of achieving partial tumor regression in the sjsa-1 xenograft model as shown in our previous report . we next combined all the favorable modifications on sites a and d and designed and synthesized compound 54 ( scheme 2 ) . compound 54 has a very high binding affinity to mdm2 ( ic50 = 2.4 nm , ki < 1 nm , scheme 2 ) and potently inhibits cell growth of sjsa-1 cells with ic50 = 13 nm ( scheme 2 ) . we assessed the antitumor activity of compound 54 in the sjsa-1 xenograft model in mice , with compound 2 included as the control ( figure 4 ) . compound 54 effectively inhibits tumor growth in a dose - dependent manner compared to the vehicle control . while compound 54 at 50 mg / kg daily dosing effectively retards tumor growth , at 100 mg / kg it achieves a maximum of 87% tumor regression during the treatment . however , while compound 2 at 100 mg / kg achieves complete tumor regression , compound 54 fails to do so . ( a ) antitumor activity of compound 54 , in comparison to compound 2 in the sjsa-1 osteosarcoma tumor xenograft model in mice . compound 54 was administered via oral gavage at 50 or 100 mg / kg daily for 14 days and compound 2 was administered via oral gavage at 100 mg / kg daily for 14 days . pharmacokinetic ( pk ) studies in rats ( table 6 ) showed that with oral administration , compounds 39 and 54 achieve considerably lower cmax and auc values than compound 2 , suggesting that further improvement of the oral pk for compounds 39 and 54 is needed in order to achieve stronger in vivo antitumor activity . since 39 and 54 achieve comparable tumor regression in the sjsa-1 xenograft model and 39 is less hydrophobic than 54 , we have selected compound 39 for further optimization of its pk properties and in vivo efficacy . in our attempt to further improve the oral pk properties of compound 39 while retaining its high binding affinity to mdm2 and cellular potency , we investigated the replacement of the benzoic acid with nonclassical benzoic acid mimetics such as a bicyclo[1.1.1]pentane-1-carboxylic acid or a bicyclo[2.2.2]octane-1-carboxylic acid . these efforts led to the design and synthesis of compounds 55 and 56 ( scheme 3 ) . compound 55 binds to mdm2 with a high affinity ( ic50 = 6.4 nm , ki < 1 nm ) but is 5 times less potent than compound 39 in inhibition of cell growth in the sjsa-1 cell line . in comparison , compound 56 has a high binding affinity to mdm2 ( ic50 = 3.7 nm , ki < 1 nm ) and is as potent as compound 39 in inhibition of cell growth in the sjsa-1 cell line ( scheme 3 and table 7 ) . value from one experiment . a pk study in rats showed that 56 has a higher plasma exposure than compound 2 based upon both the cmax ( 8234 vs 4547 hg / l ) . however , our pd experiment showed that oral administration of compound 56 at 100 mg / kg induces only modest p53 activation and no cleavage of parp and caspase-3 in the sjsa-1 xenograft tumor tissue in mice ( figure 6a ) . consistent with the pd data , compound 56 at 100 mg / kg administered daily for 14 days demonstrates only a very moderate tumor growth inhibition in the sjsa-1 xenograft model in mice ( figure 5a ) . hence , the antitumor activity of 56 is much inferior to that achieved by 2 , 39 , and 54 . because of its excellent in vitro cellular potency and excellent plasma exposure , the modest p53 activation and antitumor activity achieved by compound 56 suggests that this compound has a poor penetration into the xenograft tumor tissue . accordingly , we investigated strategies with which to improve the tissue penetration while retaining high binding affinity to mdm2 , potent cellular activity , and good oral bioavailability of compound 56 . ( a ) antitumor activity of compounds 39 and 56 in the sjsa-1 osteosarcoma xenograft model in mice . we investigated three strategies to improve the tissue penetration : ( 1 ) decreasing the lipophilicity of the molecule ; ( 2 ) reducing the acidity of carboxylic acids ; and ( 3 ) increasing the basicity of the nitrogen atom in the pyrrolidine core . we therefore synthesized compound 57 with a 4,4-difluorocyclohexyl group at carbon-2 of the pyrrolidine core ( table 7 ) . for the second strategy , we replaced the carboxylic acid group with an acyl sulfonamide bioisostere , which resulted in 58 ( table 7 ) . for the third strategy , we installed a methyl or ethyl group on the nitrogen of the pyrrolidine core , which led to 59 and 60 , respectively ( table 7 ) . compound 57 binds to mdm2 with a high affinity but is less potent than 56 and is 2 times less potent than 56 in inhibition of cell growth in the sjsa-1 cell line ( table 7 ) . compound 58 is 2 times less potent than 56 in binding to mdm2 and is 4 times less potent than 56 in inhibition of cell growth in the sjsa-1 cell line ( table 7 ) . the pd study showed that oral administration of 57 or 58 at 100 mg / kg only has a modest effect on activation of p53 and induction of cleavage of parp and caspase-3 ( figure 6b ) . pharmacodynamic ( pd ) analysis of the effect of mdm2 inhibitors in sjsa-1 tumors . mice bearing sjsa-1 tumors were dosed with a single oral dose of 56 , 57 , 58 , 59 , 60 and 2 at 100 mg / kg and the tumors were harvested at 6 and 24 hours for western blot analysis . ( a , b ) pd of 56 , 57 and 58 show modest activation of p53 as seen with the low levels of p53 , mdm2 , and p21 ; ( c ) compounds 59 , 60 and 2 show robust activation of p53 as seen with increased levels of p53 , mdm2 , ubiquitinated mdm2 ( ub - mmd2 ) and p21 , and induce strong apoptosis as seen with increased levels of cleaved parp ( cl - papr ) and cleaved caspase-3 ( cl - casp3 ) . in comparison , compounds 59 and 60 bind to mdm2 with a high binding affinity ( ki < 1 nm , table 7 ) . both compounds potently inhibit cell growth in the sjsa-1 cell line with ic50 values of 70 and 60 nm , respectively , and are thus as potent as compound 56 ( table 7 ) . pd experiments showed that a single , oral dose of 59 and 60 at 100 mg / kg achieves strong p53 activation , with the effect persisting for 24 h in the sjsa-1 xenograft tumor tissue ( figure 6c ) . compounds 59 and 60 also achieve strong apoptosis induction in the sjsa-1 xenograft tumor , based upon cleavage of parp and caspase-3 ( figure 6c ) . we next tested 59 and 60 for the antitumor activity in the sjsa-1 xenograft model ( figure 7 ) . compound 59 or 60 orally administered at 30 mg / kg daily for 14 days effectively inhibits tumor growth . compound 59 or 60 orally administered at 100 mg / kg daily for 14 days effectively achieves complete and long - lasting tumor regression . in comparison , compound 56 at 100 mg / kg inhibits tumor growth only moderately in the same experiment ( figure 7a ) . compounds 56 , 59 , and 60 cause no or minimal weight loss during the entire experiment ( figure 7b ) . ( a ) antitumor activity of compound 59 , 60 in comparison to 56 in the sjsa-1 osteosarcoma xenograft model in mice . compound 59 and 60 were administered at 30 mg / kg or 100 mg / kg daily for 14 days and 56 at 100 mg / kg daily for 14 days . our pk studies in rats with oral administration showed that 59 and 60 achieve a cmax value similar to that of 2 but have a 2 times higher auc than compound 2 ( table 6 ) . we evaluated compounds 56 , 59 , and 60 for their activity and selectivity in a number of human cancer cell lines of different tumor types ( table 8) . consistent with their mechanism of action , these three compounds potently inhibit cell growth in cancer cell lines with wild - type p53 and display outstanding selectivity in cancer cell lines with deleted p53 . compound 60 is the most potent compound , achieving ic50 values of 38 , 18 , and 104 nm in the rs4;11 acute leukemia , lncap prostate cancer , and hct116 colon cancer cell lines , respectively . we next evaluated compound 60 for its antitumor activity in the rs4;11 acute leukemia xenograft model in mice . compound 60 effectively inhibits tumor growth in a dose - dependent manner and achieves partial tumor regression at 100 mg / kg , daily , oral administration for 14 days , or at 200 mg / kg weekly dosing for 3 weeks ( figure 8) . ( a ) antitumor activity of compound 60 in the rs4 ; 11 acute lymphoblastic leukemia xenograft model in mice . compound 60 was administered at 50 mg / kg , 75 mg / kg , or 100 mg / kg daily for 14 days and 200 mg / kg weekly for 3 weeks . we evaluated the chemical stability of compound 60 in three different solutions and found that this compound is very stable in solutions for a period of 7 days ( figure 9 ) . in comparison , compound 2 slowly isomerizes in solutions and retains only 9396% purity after 7 days ( figure 9 ) . stability comparison of compounds 2 and 60 in ( a ) 1:1 ch3cn to h2o ; ( b ) 1:1 meoh to h2o ; ( c ) cell culture media . to explore achiral substituents in carbon-2 of the pyrrolidine core , the compounds in table 1 were synthesized using the method outlined in scheme 4 . the azomethine ylide generated in situ by reaction of the chiral amine ( 62 ) with the cyclic ketone ( 63 ) was allowed to react with 61 in a 1,3-dipolar cycloaddition reaction , generating the chiral spirooxindoles ( 6a15a ) . reaction of this morpholine ( 6a15a ) with an aliphatic amine led to the open - ring compound ( 6b15b ) . upon oxidation with ceric ammonium nitrate ( can ) , 6c15c were formed as a mixture of two diastereoisomers . aging in acetonitrile / water resulted in formation of the single diastereoisomers 615 . reagents and conditions : ( a ) toluene , reflux , 2 h ; ( b ) r1nh2 , thf , reflux overnight ; ( c ) ch3cn / h2o ( 1:1 ) , can , 15 min ; ( d ) ch3cn / h2o , overnight . scheme 5 shows the method by which the b and c aryl rings were replaced . first the intermediates 16b20b were synthesized by condensation of the pyrrolidones ( 16a20a ) with an arylaldehyde . subsequently , the compounds ( 1620 ) in table 2 were obtained by using the intermediates ( 16b20b ) , which are equivalent to 61 , in the synthetic method outlined in scheme 4 . compounds containing various five - membered heteroaromatic rings as bioisostere replacements for the carboxamide group e were synthesized as outlined in schemes 6 and 7 . the n - methylpyrrolidine ( 65 ) was generated by reductive amination of 64 with paraformaldehyde and nabh(oac)3 . hydrolysis of the methyl ester ( 65 ) was followed by conversion of the acid ( 66 ) to the corresponding carboxamide ( 67 ) . refluxing of 67 with lawesson s reagent produced the thioamide ( 68 ) which was reacted with an -bromoketone in a hantzsch thiazole synthesis reaction , generating the thiazoles 69 and 70 . compound 21 was further elaborated by amide coupling , generating the carboxamide ( 28 ) . reagents and conditions : ( a ) dcm / acoh ( 1:1 ) , paraformaldehyde , nabh(oac)3 ; ( b ) thf / meoh / h2o , liohh2o ; ( c ) dce , cdi , diea , 50 c 30 min , then nh4oh ; ( d ) dcm , lawesson s reagent , reflux overnight ; ( e ) ( 1 ) thf , brch2cor , et3n , reflux overnight , ( 2 ) dme , tfaa , pyridine , 20 c to rt , 30 min ; ( f ) thf / meoh / h2o ( 1:1:1 ) , naoh , rt , prep - hplc . reagents and conditions : ( a ) dce , cdi , diea , dmap , 50 c for 30 min , then 71 , 74 , 76 , 78 , and reflux ; ( b ) ( 1 ) thf , deoxo - fluor , 20 c for 30 min , ( 2 ) brccl3 , dbu at rt overnight ; ( c ) thf / h2o ( 2:1 ) , liohh2o , rt for 30 min , then preparative hplc ; ( d ) pyridine , 100 c for 4 h ; ( e ) dcm , pph3 , i2 , et3n , rt ; ( f ) dcm , lawesson s reagent , reflux overnight . several five - membered heteroaromatic rings were formed from the carboxylic acid ( 66 ) . the carboxylic acid ( 66 ) was activated with cdi ( 1,1-carbonyldiimidazole ) , and this was followed by addition of commercially available compounds 71 , 74 , 76 , 78 , resulting in the formation of intermediates 72 , 75 , 77 , 79 , respectively . cyclization to the respective five - membered heteroaromatic compounds ( 73,23,24,25 , 80 ) was accomplished under the conditions outlined in scheme 7 . the carboxylic acid compounds 22 and 26 were produced by hydrolysis of the esters in compounds 73 and 80 , respectively . the compounds in tables 4 and 5 were obtained by the synthesis outlined in scheme 8 . activation of the carboxylic acid ( 29 ) by hatu ( 1-[bis(dimethylamino)methylene]-1h-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate ) followed by addition of an alkylamine gave the n - alkylcarboxamide intermediates 30 , 36 , 38 , 3135 , 37 , 55 , and 56 . when n - arylamines were used , hatu failed to accomplish the coupling , but the coupling was successful when the acid was activated with ph2pocl and produced the n - arylcarboxamides 3948 , 50 , and 51 . basic hydrolysis of the esters produced the carboxylic acid compounds listed in tables 4 and 5 . further elaboration of the acids ( 56 to give 58 ; 39 to give 49 and 52 ; and 44 to give 53 ) was accomplished by cdi activation of the carboxylic acid followed by reaction with the respective amine . reagents and conditions : ( a ) dcm , hatu , diea , alkylamine ; ( b ) thf / meoh / h2o ( 1:1:1 ) , liohh2o for alkyl esters or naoh for aryl esters ; ( c ) dcm , ph2pocl , diea 30 min , then arylamine and dmap ; ( d ) dce , cdi , diea , dmap , 50 c for 30 min , then r2nh2 and reflux . reductive amination of 56 with paraformaldehyde or acetaldehyde produced 59 and 60 as shown in scheme 9 . reagents and conditions : ( a ) dcm / acoh ( 1:1 ) , paraformaldehyde or acetaldehyde , nabh(oac)3 , rt overnight . in this study , we have performed an extensive sar study on spirooxindoles containing two substituents at the carbon-2 of the pyrrolidine core as mdm2 inhibitors . extensive modifications in five different regions of the molecule have led to discovery of a number of potent and highly efficacious mdm2 inhibitors . in particular , compound 60 has a very high binding affinity to mdm2 ( ki < 1 nm ) , potently activates wild - type p53 , inhibits cell growth with low nanomolar ic50 values in human cancer cell lines carrying wild - type p53 , and demonstrates an outstanding cellular selectivity over human cancer cell lines with deleted p53 . compound 60 is very stable in solutions , has excellent oral pharmacokinetics , and effectively activates p53 in the sjsa-1 xenograft tumor tissue in mice following a single oral administration . significantly , 60 achieves complete and long - lasting regression of the sjsa-1 xenograft tumors in mice and demonstrates strong antitumor activity in the rs4;11 acute leukemia model at well tolerated dose schedules . compound 60 has been advanced into phase i clinical development for the treatment of human cancer . unless otherwise stated , all commercial reagents were used as supplied without further purification and all reactions were performed under a nitrogen atmosphere in dry solvents under anhydrous conditions . nmr spectra were obtained on either a bruker 300 ultrashield spectrometer at a h frequency of 300 mhz and c frequency of 75 mhz or bruker 400 ascend spectrometer at a h frequency of 400 mhz and c frequency of 100 mhz . chemical shifts ( ) are reported in parts per million ( ppm ) relative to an internal standard . the final products were purified on a preparative hplc ( waters 2545 , quaternary gradient module ) with a sunfire prep c18 obd 5 m , 50 mm 100 mm reverse phase column . the mobile phase was a gradient of solvent a ( h2o with 0.1% tfa ) and solvent b ( meoh with 0.1% tfa or mecn with 0.1% tfa ) at a flow rate of 40 ml / min and 1%/2 min increase of solvent b or a flow rate of 60 ml / min and 1%/min increase of solvent b. all final compounds have purity of 95% as determined by waters acquity uplc using reverse phase column ( sunfire , c18 - 5 m , 4.6 mm 150 mm ) and a solvent gradient of a ( h2o with 0.1% of tfa ) and solvent d ( meoh with 0.1% of tfa ) or a ( h2o with 0.1% of tfa ) and solvent b ( ch3cn with 0.1% of tfa ) . esi mass spectrum analysis was performed on a thermo - scientific lcq fleet mass spectrometer . no pains liability was found in any of the compounds presented as determined by analysis in the online filter smartsfilter ( http://pasilla.health.unm.edu/tomcat/biocomp/smartsfilter ) . compounds 6 , 13 , 29 , 39 and intermediate 64 were reported previously . compounds 720 were synthesized according to our reported method which is described below for the synthesis of compound 6 . in a round - bottom flask , ( e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one ( 500 mg , 1.62 mmol ) , ( 5r,6s)-5,6-diphenyl-2-morpholinone ( 492 mg , 1.94 mmol ) , and cyclohexanone ( 4.86 mmol ) were suspended in toluene ( 10 ml ) and heated at reflux for 2 h at 140 c . then the reaction was allowed to cool to room temperature and the solvent was removed by rotoevaporation . the crude product was purified by column chromatography ( the compound was eluted with 100% dcm ) to give 665 mg ( 64% yield ) of 6a as a pale yellow solid . trans-4-aminocyclohexanol ( 475 mg , 4.13 mmol ) was added to a solution of 6a ( 530 mg , 0.826 mmol ) in thf ( 20 ml ) and heated at reflux overnight . then the reaction was cooled to room temperature and the solvent was removed by rotoevaporation . the crude 6b was purified by column chromatography to produce 224 mg of 6b . cerium ammonium nitrate ( 324 mg , 0.592 mmol ) was added to a solution of the resulting 6b ( 224 mg , 0.296 mmol ) in mecn ( 6 ml ) and stirred for 5 min at room temperature , then h2o ( 6 ml ) was added . after stirring the reaction for an additional 10 min , it was quenched with saturated sodium bicarbonate the etoac solution was dried over sodium sulfate , filtered through celite and the solvent was removed by rotoevaporation to produce crude 6c as a mixture of diastereisomers . the crude material was dissolved in a 3:1 mixture of meoh / h2o that was acidified with tfa and aged . the combined fractions of the pure compound were concentrated and then redissolved in a minimum amount of mecn , h2o was added , and the solution was frozen and lyophilized to produce the tfa salt of 6 ( 116 mg , 70% yield ) as a white powder . h nmr ( 300 mhz , cd3od ) ppm 8.29 ( br s , 1h ) , 7.63 ( t , 1h , j = 7.2 hz ) , 7.51 ( dd , 1h , j = 2.4 , 8.2 hz ) , 7.39 ( t , 1h , j = 7.6 hz ) , 7.217.07 ( m , 2h ) , 6.77 ( d , 1h , j = 1.5 hz ) , 5.13 ( d , 1h , j = 10.9 hz ) , 4.83 ( d , 1h , j = 11.0 hz ) , 2.83 ( d , 1h , j = 8.3 hz ) , 2.73 ( s , 3h ) , 2.17 ( d , 1h , j = 15.3 hz ) , 2.041.68 ( m , 5h ) , 1.52 ( q , 1h , j = 14.6 hz ) , 1.311.09 ( m , 2h ) ; esi - ms m / z 476.25 ( m + 1 ) . starting with tert - butyl 4-oxopiperidine-1-carboxylate in place of cyclohexanone , compound 8-boc ( compound 8 with n - boc piperidine ) was prepared according to the synthetic method described for the preparation of 6 . after 30 min the trifluoroacetic acid was removed and the crude was purified by hplc to give 8 ( tfa salt ) . h nmr ( 300 mhz , cd3od ) ppm 8.24 ( br s , 1h ) , 7.64 ( t , 1h , j = 7.2 hz ) , 7.49 ( dd , 1h , j = 2.2 , 8.1 hz ) , 7.27 ( t , 1h , j = 7.3 hz ) , 7.137.03 ( m , 2h ) , 6.68 ( s , 1h ) , 4.79 ( d , 1h , j = 9.6 hz ) , 4.64 ( d , 1h , j = 9.6 hz ) , 3.70 ( t , 1h , j = 13.1 hz ) , 3.443.18 ( m , 3h ) , 2.77 ( d , 3h , j = 4.3 hz ) , 2.39 ( d , 1h , j = 14.5 hz ) , 2.101.88 ( m , 2h ) , 1.501.26 ( m , 1h ) . esi - ms m / z 477.17 ( m + 1 ) . starting with 1-methylpiperidin-4-one in place of cyclohexanone , compound 9 was prepared according to the synthetic method described for the preparation of 6 . h nmr ( 300 mhz , cd3od ) ppm 8.25 ( br s , 1h ) , 7.62 ( t , 1h , j = 7.3 hz ) , 7.47 ( dd , 1h , j = 2.1 , 8.2 hz ) , 7.25 ( t , 1h , j = 7.5 hz ) , 7.127.01 ( m , 2h ) , 6.77 ( d , 1h , j = 1.6 hz ) , 4.76 ( d , 1h , j = 9.5 hz ) , 4.62 ( d , 1h , j = 9.5 hz ) , 3.75 ( t , 1h , j = 12.5 hz ) , 3.523.40 ( m , 2h ) , 3.243.12 ( m , 1h ) , 2.86 ( s , 3h ) , 2.76 ( d , 3h , j = 4.0 hz ) , 2.40 ( d , 1h , j = 14.4 hz ) , 2.121.86 ( m , 2h ) , 1.541.34 ( m , 1h ) ; esi - ms m / z 491.08 ( m + 1 ) . starting with 1-acetylpiperidin-4-one in place of cyclohexanone , compound 10 was prepared according to the synthetic method described for the preparation of 6 . h nmr ( 300 mhz , cd3od ) ppm 7.63 ( t , 1h , j = 7.8 hz ) , 7.47 ( dd , 1h , j = 2.6 , 8.2 hz ) , 7.31 ( t , 1h , j = 8.3 hz ) , 7.157.04 ( m , 2h ) , 6.75 ( d , 1h , j = 1.7 hz ) , 4.77 ( d , 1h , j = 10.1 hz ) , 4.61 ( d , 0.5h , j = 16.4 hz , rotamer ) , 4.43 ( d , 0.5h , j = 11.9 hz , rotamer ) , 4.00 ( d , 0.5h , j = 13.1 hz , rotamer ) , 3.85 ( d , 0.5h , j = 12.6 hz , rotamer ) , 3.813.68 ( m , 1h ) , 2.76 ( s , 3h ) , 2.562.40 ( m , 1h ) , 2.161.76 ( m , 5h ) , 1.451.11 ( m , 2h ) ; esi - ms m / z 519.17 ( m + 1 ) . starting with tetrahydro-4h - pyran-4-one in place of cyclohexanone , compound 11 was prepared according to the synthetic method described for the preparation of 6 . h nmr ( 300 mhz , cd3od ) ppm 8.19 ( d , j = 7.9 hz 1h ) , 7.63 ( ddd , 1h , j = 1.5 , 6.5 , 7.9 hz ) , 7.51 ( dd , 1h , j = 2.3 , 8.2 hz ) , 7.37 ( t , 1h , j = 8.3 hz ) , 7.197.07 ( m , 2h ) , 6.80 ( d , 1h , j = 1.9 hz ) , 5.02 ( d , 1h , j = 10.8 hz ) , 4.74 ( d , 1h , j = 10.8 hz ) , 4.113.93 ( m , 2h ) , 3.87 ( dd , 1h , j = 3.9 , 12.4 hz ) , 3.693.55 ( m , 2h ) , 3.503.38 ( m , 1h ) , 2.62 ( d , 1h , j = 13.2 hz ) , 2.262.12 ( m , 1h ) , 2.041.73 ( m , 4h ) , 1.701.17 ( m , 5h ) , 1.08 ( ddd , 1h , j = 3.5 , 12.7 , 24.0 hz ) ; esi - ms m / z 562.67 ( m + 1 ) . starting with 4,4-difluorocyclohexan-1-one in place of cyclohexanone , compound 12 was prepared according to the synthetic method described for the preparation of 6 . h nmr ( 300 mhz , cd3od ) ppm 8.12 ( d , 1h , j = 8.1 hz ) , 7.62 ( t , 1h , j = 7.2 hz ) , 7.49 ( dd , 1h , j = 2.3 , 8.2 hz ) , 7.33 ( t , 1h , j = 8.3 hz ) , 7.167.05 ( m , 2h ) , 6.78 ( d , 1h , j = 1.9 hz ) , 4.77 ( d , 1h , j = 10.3 hz ) , 3.703.41 ( m , 2h ) , 2.741.64 ( m , 11h ) , 1.481.21 ( m , 4h ) , 1.181.02 ( m , 1h ) ; esi - ms m / z 596.75 ( m + 1 ) . starting with 3,3-dimethylcyclobutan-1-one in place of cyclohexanone , compound 14 was prepared according to the synthetic method described for the preparation of 6 . h nmr ( 400 mhz , cd3od ) ppm 7.61 ( dd , 1h , j = 1.9 , 8.2 hz ) , 7.52 ( ddd , 1h , j = 1.5 , 6.4 , 7.9 hz ) , 7.39 ( ddd , 1h , j = 1.5 , 7.3 , 8.6 hz ) , 7.18711 ( m , 2h ) , 6.89 ( d , 1h , j = 1.9 hz ) , 4.92 ( d , 1h , j = 10.9 hz ) , 4.46 ( d , 1h , j = 10.9 hz ) , 3.683.58 ( m , 1h ) , 3.503.39 ( m , 1h ) , 2.78 ( dd , 2h , j = 14.5 , 39.1 hz ) , 2.37 ( d , 1h , j = 14.2 hz ) , 1.951.76 ( m , 3h ) , 1.691.59 ( m , 1h ) , 1.381.17 ( m , 7h ) , 0.98 ( ddd , 1h , j = 3.6 , 12.9 , j = 24.3 hz ) , 0.54 ( s , 3h ) ; esi - ms m / z 560.25 ( m + h ) . starting with 4,4-dimethylcyclohexan-1-one in place of cyclohexanone , compound 15 was prepared according to the synthetic method described for the preparation of 6 . h nmr ( 400 mhz , cd3od ) ppm 7.62 ( t , 1h , j = 7.9 hz ) , 7.48 ( dd , 1h , j = 1.4 , 7.8 hz ) , 7.357.25 ( m , 1h ) , 7.157.04 ( m , 2h ) , 6.78 ( d , 1h , j = 1.7 hz ) , 4.73 ( d , 1h , j = 9.9 hz ) , 3.673.57 ( m , 1h ) , 3.523.43 ( m , 1h ) , 2.081.64 ( m , 8h ) , 1.581.42 ( m , 2h ) , 1.411.20 ( m , 6h ) , 0.98 ( s , 3h ) , 0.73 ( s , 3h ) ; esi - ms m / z 588.25 ( m + h ) . starting with ( e)-6-chloro-3-((5-chloropyridin-3-yl)methylene)indolin-2-one ( 16b ) in place of ( e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one ( 61 ) , compound 16 was prepared according to the synthetic method described for the preparation of 6 . h nmr ( 300 mhz , cd3od ) ppm 8.45 ( d , 1h , j = 2.1 hz ) , 8.24 ( d , 1h , j = 1.7 hz ) , 7.89 ( s , 1h ) , 7.60 ( d , 1h , j = 8.2 hz ) , 7.14 ( dd , 1h , j = 1.8 , 8.2 hz ) , 6.78 ( d , 1h , j = 1.8 hz ) , 5.10 ( d , 1h , j = 10.9 hz ) , 4.47 ( d , 1h , j = 10.9 hz ) , 3.733.57 ( m , 1h ) , 3.503.36 ( m , 1h ) , 2.83 ( d , 1h , j = 12.5 hz ) , 2.17 ( d , 1h , j = 14.3 hz ) , 2.031.70 ( m , 8h ) , 1.701.13 ( m , 7h ) , 1.080.88 ( m , 1h ) ; esi - ms m / z 543.75 ( m + h ) . starting with ( e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)-1,3-dihydro-2h - pyrrolo[3,2-c]pyridin-2-one ( 17b ) in place of ( e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one ( 61 ) , compound 17 was prepared according to the synthetic method described for the preparation of 6 . h nmr ( 400 mhz , cd3od ) ppm 8.32 ( s , 1h ) , 7.58 ( t , 1h , j = 6.7 hz ) , 7.367.27 ( m , 1h ) , 7.11 ( t , 1h , j = 8.6 hz ) , 6.81 ( s , 1h ) 3.673.57 ( m , 1h ) , 3.553.45 ( m , 1h ) , 2.011.51 ( m , 10h ) , 1.421.00 ( m , 8h ) ; esi - ms m / z 561.25 ( m + h ) . starting with ( e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)-1,3-dihydro-2h - pyrrolo[2,3-b]pyridin-2-one ( 18b ) in place of ( e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one ( 61 ) , compound 18 was prepared according to the synthetic method described for the preparation of 6 . h nmr ( 300 mhz , cd3od ) ppm 7.89 ( dd , 1h , j = 2.14 , 7.84 hz ) , 7.61 ( t , 1h , j = 7.43 hz ) , 7.40 ( t , 1h , j = 7.14 hz ) , 7.17 ( t , 1h , j = 8.40 hz ) , 7.12 ( d , 1h , j = 7.96 hz ) , 5.074.94 ( m , 1h ) , 4.78 ( d , 1h , j = 10.91 hz ) , 3.713.57 ( m , 1h ) , 3.513.39 ( m , 1h ) , 2.892.66 ( m , 1h ) , 2.172.02 ( m , 1h ) , 1.991.43 ( m , 10h ) , 1.431.11 ( m , 5h ) , 1.090.93 ( m , 1h ) ; esi - ms m / z 561.58 ( m + h ) . starting with ( e)-2-chloro-5-(3-chloro-2-fluorobenzylidene)-5,7-dihydro-6h - pyrrolo[2,3-d]pyrimidin-6-one ( 19b ) in place of ( e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one ( 61 ) , compound 19 was prepared according to the synthetic method described for the preparation of 6 . h nmr ( 400 mhz , cd3od ) ppm 8.55 ( d , 1h , j = 2.2 hz ) , 7.55 ( t , 1h , j = 7.8 hz ) , 7.39 ( t , 1h , j = 7.0 hz ) , 7.16 ( t , 1h , j = 7.9 hz ) , 3.673.56 ( m , 1h ) , 3.533.43 ( m , 1h ) , 1.991.48 ( m , 10h ) , 1.421.04 ( m , 8h ) ; esi - ms m / z 562.33 ( m + h ) . starting with ( z)-2-chloro-6-(3-chloro-2-fluorobenzylidene)-4,6-dihydro-5h - thieno[3,2-b]pyrrol-5-one ( 20b ) in place of ( e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one ( 61 ) , compound 20 was prepared according to the synthetic method described for the preparation of 6 . h nmr ( 400 mhz , cd3od ) ppm 7.40 ( td , 2h , j = 6.8 , 15.1 hz ) , 7.15 ( t , 1h , j = 8.0 hz ) , 6.71 ( s , 1h ) , 4.58 ( d , 1h , j = 10.3 hz ) , 4.45 ( d , 1h , j = 10.4 hz ) , 3.683.57 ( m , 1h ) , 3.533.43 ( m , 1h ) , 2.16 ( d , 1h , j = 11.4 hz ) , 2.001.79 ( m , 4h ) , 1.771.46 ( m , 7h ) , 1.401.07 ( m , 6h ) ; esi - ms m / z 566.25 ( m + h ) . ethyl 3-bromo-2-oxopropanoate ( 48 mg , 0.244 mmol ) and triethylamine ( 0.033 ml , 0.244 mmol ) , were added to a solution of 68 ( 30 mg , 0.061 mmol ) in thf ( 3 ml ) and refluxed overnight the crude product was redissolved in dme ( 3 ml ) , and to this solution , at 20 c , was added trifluoracetic anhydride ( 0.025 ml , 0.183 mmol ) and pyridine ( 0.025 ml , 0.305 mmol ) , and then the reaction was allowed to warm to rt . after 30 min , saturated sodium bicarbonate solution was added and the reaction was extracted with etoac . the etoac solution was dried over sodium sulfate , filtered , and purified by column chromatography to produce 18 mg of compound 69 . lithium hydroxide monohydrate ( 50 mg , 1.19 mmol ) was added to a solution of 69 ( 18 mg , 0.031 mmol ) in thf / meoh / h2o ( 1:1:1 , 3 ml ) . after 2 h the reaction was quenched with ammonium chloride , and brine was added and extracted with etoac . the etoac solution was dried over sodium sulfate , filtered , concentrated and the crude was purified by preparative hplc to produce compound 21 ( tfa salt ) . h nmr ( 300 mhz , cd3od ) ppm 8.34 ( s , 1h ) , 7.70 ( t , 1h , j = 6.8 hz ) , 7.53 ( d , 1h , j = 7.1 hz ) , 7.27 ( t , 1h , j = 7.5 hz ) , 7.137.03 ( m , 2h ) , 6.73 ( d , 1h , j = 1.8 hz ) , 5.785.59 ( m , 1h ) , 4.814.68 ( m , 1h ) , 3.10 ( s , 3h ) , 2.512.38 ( m , 1h ) , 2.25 ( d , 1h , j = 15.1 hz ) , 2.131.94 ( m , 1h ) , 1.871.10 ( m , 7h ) ; esi - ms m / z 560.33 ( m + h ) . cdi ( 3 equiv ) , diea ( 5 equiv ) , dmap ( 1 equiv ) were added to a suspension of compound 66 ( 1 equiv ) in 1,2-dichloroethane and heated at 50 c . after 30 min , the respective amine ( 5 equiv ) was added and the reaction was refluxed overnight . then the solvent was removed and the crude product was purified to produce the carboxamide . compound 72 was produced according to general procedure a. at 20 c , deoxo - fluor ( 50 l ) was added to a solution of 72 ( 51 mg ) in thf ( 3 ml ) . after 30 min bromotrichloromethane ( 500 l ) and dbu ( 400 l ) were added and the reaction was allowed to warm to room temperature . after 5 h the reaction was quenched with saturated sodium bicarbonate ( stirred for 5 min ) and then extracted with etoac . the etoac solution was dried over sodium sulfate , filtered , and purified by column chromatography to produce 34 mg of compound 73 . lithium hydroxide monohydrate ( 60 mg , 1.43 mmol ) was added to a solution of 73 ( 34 mg , 0.061 mmol ) in thf / h2o ( 1:1 , 4 ml ) . after 2 h the reaction was quenched with tfa , concentrated and the crude was redissolved in 3:1 meoh / h2o and purified by hplc to produce 22 ( tfa salt ) as a white powder . h nmr ( 400 mhz , cd3od ) ppm 8.50 ( s , 1h ) , 7.627.53 ( m , 2h ) , 7.24 ( t , 1h , j = 7.3 hz ) , 7.07 ( dd , 1h , j = 1.7 , 8.2 hz ) , 7.03 ( t , 1h , j = 8.1 hz ) , 6.73 ( d , 1h , j = 1.8 hz ) , 5.34 ( d , 1h , j = 10.3 hz ) , 5.02 ( d , 1h , j = 11.6 hz ) , 2.97 ( s , 3h ) , 2.33 ( d , 1h , j = 13.1 hz ) , 2.15 ( d , 1h , j = 14.1 hz ) , 1.951.85 ( m , 1h ) , 1.741.44 ( m , 5h ) , 1.381.26 ( m , 1h ) , 1.191.05 ( m , 1h ) ; esi - ms m / z 544.42 ( m + h ) . starting with 50 mg of compound 66 , compound 75 was obtained using general procedure a. crude 75 was dissolved in pyridine ( 5 ml ) and heated to 100 c . after 5 h the solution was cooled and the solvent was removed by rotoevaporation and then the crude product was purified by hplc to produce 23 ( tfa salt ) as a white powder . h nmr ( 400 mhz , cd3od ) ppm 7.60 ( ddd , 1h , j = 1.5 , 6.4 , 7.9 hz ) , 7.50 ( dd , 1h , j = 3.0 , 8.2 hz ) , 7.25 ( ddd , 1h , j = 1.6 , 7.2 , j = 8.6 hz ) , 7.077.01 ( m , 2h ) , 6.72 ( d , 1h , j = 1.9 hz ) , 5.30 ( d , 1h , j = 11.0 hz ) , 4.98 ( d , 1h , j = 11.1 hz ) , 2.99 ( s , 3h ) , 2.32 ( s , 3h ) , 2.24 ( d , 1h , j = 13.2 hz ) , 2.192.10 ( m , 1h ) , 1.861.47 ( m , 6h ) , 1.21 ( ddd , 1h , j = 5.0 , 12.6 hz , 13.9 hz ) , 1.151.02 ( m , 1h ) ; esi - ms m / z 514.92 ( m + h ) . starting with 77 mg of compound 66 , 63 mg of 77 was obtained using general procedure a. a solution of 77 ( 30 mg , 0.056 mmol ) in dcm ( 3 ml ) was slowly added to a solution of pph3 ( 30 mg , 0.112 mmol ) , et3n ( 31 l ) , and iodine ( 28 mg , 0.112 mmol ) in dcm ( 1 ml ) . the etoac solution was dried over sodium sulfate , filtered , concentrated and the crude product was purified by hplc to produce compound 24 ( tfa salt ) as a white powder . h nmr ( 400 mhz , cd3od ) ppm 7.60 ( t , 1h , j = 7.9 hz ) , 7.51 ( dd , 1h , j = 2.9 , 8.2 hz ) , 7.25 ( t , 1h , j = 8.3 hz ) , 7.087.00 ( m , 2h ) , 6.72 ( d , 1h , j = 1.9 hz ) , 5.29 ( d , 1h , j = 11.1 hz ) , 4.95 ( d , 1h , j = 10.8 hz ) , 2.95 ( s , 3h ) , 2.52 ( s , 3h ) , 2.24 ( d , 1h , j = 13.8 hz ) , 2.15 ( dd , 1h , j = 2.3 , 14.1 hz ) , 1.861.02 ( m , 8h ) ; esi lawesson s reagent ( 45 mg , 0.112 mmol ) was added to a solution of 77 ( 30 mg , 0.056 mmol ) in dcm and refluxed . after standing overnight the solvent was removed and the crude was purified by hplc to produce compound 25 ( tfa salt ) as a white powder . h nmr ( 400 mhz , cd3od ) ppm 7.69 ( t , 1h , j = 7.9 hz ) , 7.46 ( dd , 1h , j = 2.8 , 8.2 hz ) , 7.25 ( t , 1h , j = 8.4 hz ) , 7.08 ( dt , 1h , j = 1.1 , 8.1 hz ) , 7.03 ( dd , 1h , j = 2.0 , 8.2 hz ) , 6.72 ( d , 1h , j = 1.9 hz ) , 5.50 ( d , 1h , j = 10.2 hz ) , 4.63 ( d , 1h , j = 11.0 hz ) , 3.00 ( s , 3h ) , 2.71 ( s , 3h ) , 2.27 ( d , 1h , j = 13.0 hz ) , 2.232.15 ( m , 1h ) , 1.88 ( t , 1h , j = 11.6 hz ) , 1.831.60 ( m , 4h ) , 1.601.49 ( m , 1h ) , 1.311.07 ( m , 2h ) ; esi - ms m / z 531.17 ( m + h ) . starting with 44 mg of compound 66 , 25 mg of 79 was obtained using general procedure a. cyclization to generate 80 ( 13 mg ) was accomplished according to the same procedure used to obtain 24 . lithium hydroxide monohydrate ( 30 mg , 0.715 mmol ) was added to a solution of 80 ( 13 mg , 0.023 mmol ) in thf / h2o ( 1:1 , 2 ml ) . after 2 h the reaction was quenched with tfa , concentrated and the crude was redissolved in 3:1 meoh / h2o and purified by hplc to produce 26 ( tfa salt ) as a white powder . h nmr ( 400 mhz , cd3od ) ppm 7.60 ( t , 1h , j = 7.2 hz ) , 7.52 ( dd , 1h , j = 2.7 , 8.1 hz ) , 7.21 ( t , 1h , j = 8.3 hz ) , 7.076.98 ( m , 2h ) , 6.70 ( d , 1h , j = 1.9 hz ) , 5.07 ( d , 1h , j = 10.8 hz ) , 4.91 ( d , 1h , j = 11.7 hz ) , 2.83 ( s , 3h ) , 2.59 ( s , 3h ) , 2.312.23 ( m , 1h ) , 2.122.04 ( m , 1h ) , 1.851.02 ( m , 8h ) ; esi - ms m / z 558.75 h nmr ( 400 mhz , cd3od ) ppm 7.65 ( t , 1h , j = 6.7 hz ) , 7.57 ( d , 1h , j = 7.1 hz ) , 7.42 ( s , 1h ) , 7.27 ( t , 1h , j = 8.2 hz ) , 7.137.04 ( m , 2h ) , 6.75 ( d , 1h , j = 1.9 hz ) , 5.925.70 ( m , 1h ) , 3.78 ( s , 2h ) , 3.20 ( s , 3h ) , 2.582.44 ( m , 1h ) , 2.26 ( d , 1h , j = 13.8 hz ) , 2.222.08 ( m , 1h ) , 1.831.43 ( m , 5h ) , 1.381.27 ( m , 1h ) , 1.251.13 ( m , 1h ) ; esi - ms m / z 574.08 ( m + h ) . starting with acid 21 in place of 66 , compound 28 was produced using general procedure a. crude 28 was purified by hplc to produce 28 as its tfa salt . h nmr ( 400 mhz , cd3od ) ppm 8.11 ( s , 1h ) , 7.75 ( t , 1h , j = 7.2 hz ) , 7.42 ( d , 1h , j = 8.1 hz ) , 7.26 ( t , 1h , j = 8.46 hz ) , 7.09 ( t , 1h , j = 7.83 ) , 7.01 ( dd , 1h , j = 1.7 , 8.1 hz ) , 6.71 ( d , 1h , j = 1.8 hz ) , 3.00 ( s , 3h ) , 2.362.16 ( m , 2h ) , 2.092.00 ( m , 1h ) , 1.911.52 ( m , 5h ) , 1.381.24 ( m , 1h ) , 1.211.06 ( m , 1h ) ; esi - ms m / z 559.42 hatu ( 2 equiv ) and diea ( 4 equiv ) were added to a suspension of acid compound 29 ( 1 equiv ) in dcm . after 12 h , the reaction was concentrated and purified by column chromatography to produce alkyl amide compounds . liohh2o ( 3 equiv ) was added to a solution of ester ( 1 equiv ) in thf / meoh / h2o ( 1:1:1 ) . after 2 h , tfa was added and the solution was concentrated , redissolved in meoh / h2o ( 3:1 ) , and purified by reverse phase preparative hplc . the pure fractions were combined and lyophilized to give the carboxylic acid products ( tfa salt ) as a white powder . ph2pocl ( 3 equiv ) and diea ( 5 equiv ) were added to a suspension of compound 29 ( 1 equiv ) in dcm . after 30 min , arylamine ( 4 equiv ) and dmap ( 1 equiv ) were added . after 12 h , the reaction was concentrated and purified by column chromatography to produce arylamide compounds . naoh ( 3 equiv ) was added to a solution of ester ( 1 equiv ) in thf / meoh / h2o ( 1:1:1 ) . after 2 h , tfa was added and the reaction was concentrated , redissolved in meoh / h2o ( 3:1 ) , and purified by reverse phase preparative hplc . the pure fractions were combined and lyophilized to give the carboxylic acid products as the tfa salt as a white powder . starting with compound 29 in place of 66 , compound 30 was obtained according to general procedure a. h nmr ( 300 mhz , cd3od ) ppm 7.61 ( t , 1h , j = 7.5 hz ) , 7.53 ( dd , 1h , j = 2.1 , 8.2 hz ) , 7.33 ( t , 1h , j = 8.0 hz ) , 7.177.06 ( m , 2h ) , 6.76 ( d , 1h , j = 1.7 hz ) , 4.98 ( d , 1h , j = 10.4 hz ) , 3.09 ( s , 3h ) , 2.60 ( d , 1h , j = 13.7 hz ) , 2.09 ( d , 1h , j = 16.1 hz ) , 2.011.43 ( m , 6h ) , 1.341.06 ( m , 2h ) ; esi - ms m / z 540.08 31 was obtained according to general procedure b followed by general procedure c. h nmr ( 300 mhz , cd3od ) ppm 7.64 ( t , 1h , j = 7.1 hz ) , 7.51 ( dd , 1h , j = 2.2 , 8.3 hz ) , 7.39 ( t , 1h , j = 7.5 hz ) , 7.17 ( t , 1h , j = 8.1 hz ) , 7.11 ( dd , 1h , j = 1.6 , 8.2 hz ) , 6.78 ( d , 1h , j = 1.6 hz ) , 5.12 ( d , 1h , j = 11.0 hz ) , 4.80 ( d , 1h , j = 11.1 hz ) , 3.213.04 ( m , 1h ) , 2.81 ( d , 1h , j = 7.4 hz ) , 2.17 ( d , 1h , j = 12.0 hz ) , 2.06 ( t , 2h , j = 7.4 hz ) , 2.011.84 ( m , 3h ) , 1.841.40 ( m , 5h ) , 1.321.12 ( m , 2h ) ; esi - ms m / z 548.42 32 was obtained by general procedure b followed by general procedure c. h nmr ( 300 mhz , cd3od ) ppm 7.65 ( t , 1h , j = 7.0 hz ) , 7.49 ( dd , 1h , j = 1.7 , 8.0 hz ) , 7.40 ( t , 1h , j = 7.3 hz ) , 7.18 ( t , 1h , j = 8.0 hz ) , 7.11 ( d , 1h , j = 8.3 hz ) , 6.79 ( s , 1h ) , 5.06 ( d , j = 10.8 , 1h ) , 4.79 ( d , 1h , j = 10.8 hz ) , 4.47 ( p , 1h , j = 8.1 hz ) , 2.962.71 ( m , 2h ) , 2.632.38 ( m , 2h ) , 2.292.08 ( m , 2h ) , 2.051.83 ( m , 4h ) , 1.76 ( d , 2h , j = 16.2 hz ) , 1.621.39 ( m , 1h ) , 1.331.11 ( m , 2h ) esi - ms m / z 560.50 ( m + 1 ) . 33 was obtained by general procedure b followed by general procedure c. h nmr ( 300 mhz , cd3od ) ppm 7.64 ( t , 1h , j = 7.1 hz ) , 7.49 ( d , 1h , j = 8.1 hz ) , 7.39 ( t , 1h , j = 7.5 hz ) , 7.17 ( t , 1h , j = 8.1 hz ) , 7.11 ( d , 1h , j = 8.1 hz ) , 6.79 ( s , 1h ) , 5.08 ( d , 1h , j = 11.1 hz ) , 4.79 ( d , 1h , j = 11.1 hz ) , 3.723.54 ( m , 1h ) , 2.84 ( d , 1h , j = 8.4 hz ) , 2.252.07 ( m , 2h ) , 2.041.84 ( m , 6h ) , 1.77 ( d , 2h , j = 12.0 hz ) , 1.63 ( d , 1h , j = 13.0 hz ) , 1.561.34 ( m , 3h ) , 1.321.11 ( m , 3h ) , 0.990.82 ( m , 1h ) ; c nmr ( 75 mhz , cd3od ) ppm 179.01 , 178.21 , 167.66 , 157.76 ( d , jc f = 248.87 hz ) , 145.27 , 137.07 , 132.35 , 129.50 , 128.91 , 126.48 ( d , jc f = 4.74 hz ) , 123.39 , 122.45 , 122.31 , 122.05 , 121.87 , 111.77 , 73.24 , 67.76 , 62.25 , 46.72 , 46.68 , 43.15 , 32.33 , 32.09 , 32.07 , 31.37 , 28.79 , 28.72 , 25.35 , 23.11 , 21.70 ; esi - ms m / z 588.33 ( m + 1 ) . 34 was obtained according to general procedure b followed by general procedure c. h nmr ( 300 mhz , cd3od ) ppm 7.67 ( t , 1h , j = 6.61 hz ) , 7.49 ( dd , 1h , j = 2.0 , 8.1 hz ) , 7.37 ( t , 1h , j = 7.5 hz ) , 7.16 ( t , 1h , j = 7.9 hz ) , 7.10 ( dd , 1h , j = 1.7 , 8.3 hz ) , 6.79 ( d , 1h , j = 1.7 hz ) , 5.20 ( d , 1h , j = 11.3 hz ) , 4.76 ( d , 1h , j = 11.2 hz ) , 3.893.75 ( m , 1h ) , 2.84 ( d , 1h , j = 9.1 hz ) , 2.412.29 ( m , 1h ) , 2.20 ( d , 1h , j = 15.3 hz ) , 2.031.12 ( m , 16h ) ; esi - ms m / z 588.50 35 was obtained according to general procedure b followed by general procedure c. h nmr ( 300 mhz , cd3od ) ppm 8.368.26 ( m , 1h ) , 7.67 ( t , 1h , j = 6.8 hz ) , 7.52 ( d , 1h , j = 8.2 hz ) , 7.42 ( t , 1h , j = 7.5 hz ) , 7.20 ( t , 1h , d j = 8.0 hz ) , 7.12 ( dd , 1h , j = 1.4 , 8.2 hz ) , 6.78 ( d , 1h , j = 1.5 hz ) , 5.14 ( d , 1h , j = 11.1 hz ) , 4.78 ( d , 1h , j = 11.3 hz ) , 3.483.34 ( m , 1h ) , 2.902.64 ( m , 2h ) , 2.19 ( d , 1h , j = 11.3 hz ) , 2.091.70 ( m , 8h ) , 1.611.11 ( m , 8h ) , 0.790.59 ( m , 2h ) ; esi - ms m / z 602.58 ( m + 1 ) . 36 was obtained according to general procedure b. h nmr ( 300 mhz , cd3od ) ppm 7.64 ( t , 1h , j = 7.1 hz ) , 7.49 ( dd , 1h , j = 2.1 , 8.1 hz ) , 7.41 ( t , 1h , j = 7.5 hz ) , 7.18 ( t , 1h , j = 9.9 hz ) , 7.11 ( dd , 1h , j = 1.6 , 8.2 hz ) , 6.79 ( d , 1h , j = 1.6 hz ) , 5.10 ( d , 1h , j = 11.1 hz ) , 4.80 ( d , 1h , j = 11.2 hz ) , 4.103.94 ( m , 1h ) , 3.272.91 ( m , 3h ) , 2.882.74 ( m , 2h ) , 2.351.64 ( m , 10h ) , 1.52 ( q , 1h , j = 13.9 hz ) , 1.311.11 ( m , 2h ) ; esi - ms m / z 594.50 ( m + 1 ) . 37 was obtained according to general procedure b followed by general procedure c. h nmr ( 300 mhz , cd3od ) ppm 7.66 ( t , 1h , j = 7.1 hz ) , 7.48 ( dd , 1h , j = 2.1 , 8.2 hz ) , 7.39 ( t , 1h , j = 7.3 hz ) , 7.17 ( t , 1h , j = 8.0 hz ) , 7.10 ( dd , 1h , j = 1.6 , 8.1 hz ) , 6.79 ( d , 1h , j = 1.7 hz ) , 5.12 ( d , 1h , j = 11.0 hz ) , 4.80 ( d , 1h , j = 10.9 hz ) , 4.053.89 ( m , 2h ) , 3.793.39 ( m , 2h ) , 3.293.04 ( m , 2h ) , 2.79 ( d , 1h , j = 9.4 hz ) , 2.17 ( d , 2h , j = 9.4 hz ) , 2.031.42 ( m , 8h ) , 1.421.33 ( m , 2h ) , 1.311.10 ( m , 2h ) ; esi - ms m / z 603.67 ( m + 1 ) . 38 was obtained according to general procedure b. h nmr ( 300 mhz , cd3od ) ppm 7.64 ( t , 1h , j = 7.2 hz ) , 7.50 ( dd , 1h , j = 1.8 , 8.2 hz ) , 7.40 ( t , 1h , j = 7.7 hz ) , 7.17 ( t , 1h , j = 8.1 hz ) , 7.11 ( dd , 1h , j = 1.7 , 8.2 hz ) , 6.79 ( s , 1h ) , 5.10 ( d , 1h , j = 10.6 hz ) , 4.644.36 ( m , 2h ) , 4.294.11 ( m , 1h ) , 4.093.56 ( m , 2h ) , 2.79 ( d , 1h , j = 9.8 hz ) , 2.16 ( d , 1h , j = 14.9 hz ) , 2.011.68 ( m , 8h ) , 1.641.37 ( m , 1h ) , 1.341.11 ( m , 2h ) ; esi - ms m / z 560.08 ( m + 1 ) . 40 was obtained according to general procedure d followed by general procedure e. h nmr ( 300 mhz , cd3od ) ppm 8.14 ( s , 1h ) , 7.847.67 ( m , 3h ) , 7.55 ( dd , 1h , j = 1.9 , 8.2 hz ) , 7.487.34 ( m , 2h ) , 7.19 ( t , 1h , j = 8.0 hz ) , 7.12 ( dd , 1h , j = 1.6 , 8.2 hz ) , 6.79 ( d , 1h , j = 1.6 hz ) , 5.29 ( d , 1h , j = 11.0 hz ) , 4.97 ( d , 1h , j = 10.7 hz ) , 2.962.84 ( m , 1h ) , 2.18 ( d , j = 14.0 hz 1h ) , 2.081.85 ( m , 3h ) , 1.78 ( d , 2h , j = 12.2 hz ) , 1.631.42 ( m , 1h ) , 1.351.13 ( m , 2h ) ; esi - ms m / z 582.58 41 was obtained according to general procedure b followed by general procedure e. h nmr ( 300 mhz , cd3od ) ppm 7.87 ( d , 2h , j = 8.2 hz ) , 7.66 ( t , 1h , j = 7.0 hz ) , 7.50 ( dd , 1h , j = 2.3 , 8.3 hz ) , 7.40 ( t , 1h , j = 7.6 hz ) , 7.227.08 ( m , 2h ) , 7.04 ( d , 2h , j = 8.2 hz ) , 6.78 ( d , 1h , j = 1.6 hz ) , 5.20 ( d , 1h , j = 11.2 hz ) , 4.80 ( d , 1h , j = 11.1 hz ) , 4.66 ( d , 1h , j = 15.3 hz ) , 4.20 ( d , 1h , j = 15.3 hz ) , 2.83 ( d , 1h , j = 10.0 hz ) , 2.20 ( d , 1h , j = 15.8 hz ) , 2.041.85 ( m , 3h ) , 1.77 ( d , 2h , j = 11.9 hz ) , 1.52 ( q , 1h , j = 13.7 hz ) , 1.331.10 ( m , 2h ) ; esi - ms m / z 596.33 ( m + 1 ) . 42 was obtained according to general procedure d followed by general procedure e. h nmr ( 400 mhz , cd3od ) ppm 7.70 ( t , 1h , j = 7.8 hz ) , 7.52 ( dd , 1h , j = 2.5 , 8.2 hz ) , 7.47 ( d , 2h , j = 8.6 hz ) , 7.35 ( t , 1h , j = 8.1 hz ) , 7.24 ( d , 2h , j = 8.6 hz ) , 7.16 ( t , 1h , j = 8.0 hz ) , 7.10 ( dd , 1h , j = 1.9 , 8.2 hz ) , 6.78 ( d , 1h , j = 1.9 hz ) , 5.20 ( d , 1h , j = 9.8 hz ) , 4.93 ( d , 1h , j = 10.8 hz ) , 3.57 ( s , 2h ) , 2.872.69 ( m , 1h ) , 2.13 ( d , 1h , j = 13.3 hz ) , 1.991.70 ( m , 5h ) , 1.641.48 ( m , 1h ) , 1.271.12 ( m , 2h ) ; esi - ms m / z 596.42 ( m + h ) . 43 was obtained according to general procedure d followed by general procedure e. h nmr ( 300 mhz , cd3od ) ppm 7.83 ( d , 1h , j = 8.51 hz ) , 7.70 ( t , 1h , j = 6.92 hz ) , 7.62 ( s , 1h ) , 7.52 ( dd , 1h , j = 2.17 , 8.08 hz ) , 7.35 ( t , 1h , j = 7.08 hz ) , 7.207.02 ( m , 3h ) , 6.78 ( d , 1h , j = 1.71 hz ) , 5.245.09 ( m , 1h ) , 4.94 ( d , 1h , j = 10.49 hz ) , 3.89 ( s , 3h ) , 2.802.58 ( m , 1h ) , 2.09 ( d , 1h , j = 14.12 hz ) , 2.011.47 ( m , 6h ) , 1.261.09 ( m , 2h ) ; esi - ms m / z 612.42 ( m + h ) . 44 was obtained according to general procedure d followed by general procedure e. h nmr ( 300 mhz , cd3od ) ppm 8.26 ( d , 1h , j = 8.6 hz ) , 7.79 ( t , 1h , j = 7.1 hz ) , 7.66 ( dd , 1h , j = 1.4 , 8.4 hz ) , 7.60 ( s , 1h ) , 7.51 ( dd , 1h , j = 2.2 , 8.3 hz ) , 7.40 ( t , 1h , j = 8.2 hz ) , 7.21 ( t , 1h , j = 8.6 hz ) , 7.10 ( dd , 1h , j = 1.8 , 8.1 hz ) , 6.79 ( d , 1h , j = 1.7 hz ) , 5.495.23 ( m , 1h ) , 3.80 ( s , 3h ) , 2.682.47 ( m , 1h ) , 2.211.52 ( m , 7h ) , 1.351.07 ( m , 2h ) ; esi - ms m / z 612.17 ( m + 1 ) . 45 was obtained according to general procedure d followed by general procedure e. h nmr ( 300 mhz , cd3od ) ppm 7.90 ( t , 1h , j = 8.4 hz ) , 7.747.61 ( m , 2h ) , 7.55 ( dd , 1h , j = 2.5 , 8.2 hz ) , 7.38 ( t , 1h , j = 7.2 hz ) , 7.26 ( dd , 1h , j = 1.7 , 8.6 hz ) , 7.19 ( t , 1h , j = 8.2 hz ) , 7.12 ( dd , 1h , j = 1.8 , 8.2 hz ) , 6.80 ( d , 1h , j = 1.7 hz ) , 5.31 ( d , 1h , j = 10.8 hz ) , 4.97 ( d , 1h , j = 10.8 hz ) , 2.942.84 ( m , 1h ) , 2.20 ( d , 1h , j = 15.7 hz ) , 2.061.85 ( m , 3h ) , 1.79 ( d , 2h , j = 10.9 hz ) , 1.651.43 ( m , 1h ) , 1.341.11 ( m , 2h ) ; esi - ms m / z 600.42 ( m + h ) . 46 was obtained according to general procedure d followed by general procedure e. h nmr ( 300 mhz , cd3od ) ppm 8.21 ( t , 1h , j = 8.0 hz ) , 7.917.79 ( m , 1h ) , 7.797.67 ( m , 2h ) , 7.52 ( dd , 1h , j = 2.4 , 8.2 hz ) , 7.37 ( t , 1h , j = 7.3 hz ) , 7.257.06 ( m , 2h ) , 6.79 ( d , 1h , j = 1.7 hz ) , 5.41 ( d , 1h , j = 9.4 hz ) , 2.812.67 ( m , 1h ) , 2.14 ( d , 1h , j = 14.7 hz ) , 2.001.84 ( m , 3h ) , 1.841.70 ( m , 2h ) , 1.681.47 ( m , 1h ) , 1.411.09 ( m , 2h ) ; esi - ms m / z 600.83 ( m + h ) . 47 was obtained according to general procedure d followed by general procedure e. h nmr ( 300 mhz , cd3od ) ppm 8.80 ( s , 1h ) , 8.27 ( d , 1h , j = 8.8 hz ) , 8.15 ( d , 1h , j = 8.7 hz ) , 7.71 ( t , 1h , j = 7.2 hz ) , 7.55 ( d , 1h , j = 8.7 hz ) , 7.37 ( t , 1h , j = 7.1 hz ) , 7.18 ( t , 1h , j = 7.9 hz ) , 7.12 ( d , 1h , j = 8.1 hz ) , 6.80 ( s , 1h ) , 5.30 ( d , 1h , j = 11.1 hz ) , 5.00 ( d , 1h , j = 10.8 hz ) , 2.902.75 ( m , 1h ) , 2.16 ( d , 1h , j = 16.9 hz ) , 2.061.84 ( m , 3h ) , 1.78 ( d , 2h , j = 13.2 hz ) , 1.661.42 ( m , 1h ) , 1.321.11 ( m , 2h ) ; esi - ms m / z 583.96 ( m + 1 ) . 48 was obtained according to general procedure d followed by general procedure e. h nmr ( 300 mhz , cd3od ) ppm 7.66 ( t , 1h , j = 7.23 hz ) , 7.49 ( dd , 1h , j = 2.46 , 8.24 hz ) , 7.31 ( t , 1h , j = 7.44 hz ) , 7.22 ( d , 1h , j = 3.60 hz ) , 7.167.04 ( m , 2h ) , 6.76 ( d , 1h , j = 1.68 hz ) , 6.51 ( d , 1h , j = 3.53 hz ) , 5.064.90 ( m , 2h ) , 2.532.37 ( m , 1h ) , 2.061.50 ( m , 7h ) , 1.251.00 ( m , 2h ) ; esi - ms m / z 572.42 ( m + h ) . starting with compound 39 in place of 66 , compound 49 was obtained according to general procedure a. h nmr ( 300 mhz , cd3od ) ppm 7.89 ( d , 2h , j = 8.8 hz ) , 7.787.66 ( m , 3h ) , 7.52 ( dd , 1h , j = 2.5 , 8.4 hz ) , 7.36 ( t , 1h , j = 7.8 hz ) , 7.17 ( t , 1h , j = 7.6 hz ) , 7.11 ( dd , 1h , j = 1.7 , 8.2 hz ) , 6.79 ( d , 1h , j = 1.7 hz ) , 5.315.15 ( m , 1h ) , 3.35 ( s , 3h ) , 2.892.70 ( m , 1h ) , 2.13 ( d , 1h , j = 13.6 hz ) , 2.031.70 ( m , 5h ) , 1.681.46 ( m , 1h ) , 1.351.12 ( m , 2h ) ; esi - ms m / z 659.67 50 was obtained according to general procedure d. h nmr ( 300 mhz , cd3od ) ppm 7.607.50 ( m , 3h ) , 7.39 ( dd , 1h , j = 2.24 , 8.22 hz ) , 7.30 ( t , 1h , j = 6.96 hz ) , 7.127.02 ( m , 2h ) , 6.73 ( d , 1h , j = 1.59 hz ) , 6.64 ( d , 2h , j = 8.68 hz ) , 4.80 ( d , 1h , j = 10.19 hz ) , 4.62 ( d , 1h , j = 10.28 hz ) , 2.24 ( d , 1h , j = 13.09 hz ) , 1.94 ( d , 1h , j = 13.01 hz ) , 1.881.41 ( m , 6h ) , 1.200.93 ( m , 2h ) ; esi - ms m / z 617.17 ( m + h ) . 51 was obtained according to general procedure d. h nmr ( 300 mhz , cd3od ) ppm 7.91 ( d , 2h , j = 8.80 hz ) , 7.82 ( d , 2h , j = 8.85 hz ) , 7.70 ( t , 1h , j = 6.71 hz ) , 7.52 ( dd , 1h , j = 2.42 , 8.17 hz ) , 7.35 ( t , 1h , j = 7.58 hz ) , 7.16 ( t , 1h , j = 8.05 hz ) , 7.10 ( dd , 1h , j = 1.84 , 8.21 hz ) , 6.78 ( d , 1h , j = 1.80 hz ) , 5.20 ( d , 1h , j = 14.03 hz ) , 4.94 ( d , 1h , j = 10.48 hz ) , 3.09 ( s , 3h ) , 2.802.63 ( m , 1h ) , 2.11 ( d , 1h , j = 13.18 hz ) , 2.011.46 ( m , 6h ) , 1.311.08 ( m , 2h ) ; esi - ms m / z 616.58 ( m + h ) . starting with compound 39 in place of 66 , compound 52 was obtained according to general procedure a. h nmr ( 400 mhz , cd3od ) ppm 7.85 ( d , 2h , j = 8.9,hz ) , 7.70 ( t , 1h , j = 7.9 hz ) , 7.65 ( d , 2h , j = 8.8 hz ) , 7.51 ( dd , 1h , j = 2.4 , 8.2 hz ) , 7.34 ( t , 1h , j = 7.1 hz ) , 7.15 ( t , 1h , j = 8.0 hz ) , 7.09 ( dd , 1h , j = 1.9 , 8.2 hz ) , 6.78 ( d , 1h , j = 1.9 hz ) , 5.225.10 ( m , 1h ) , 4.93 ( d , 1h , j = 9.6 hz ) , 2.812.58 ( m , 1h ) , 2.09 ( d , 1h , j = 12.4 hz ) , 1.991.67 ( m , 5h ) , 1.671.50 ( m , 1h ) , 1.281.10 ( m , 2h ) ; esi - ms m / z 581.33 ( m + h ) . starting with compound 44 in place of 66 , compound 53 was obtained according to general procedure a. h nmr ( 400 mhz , cd3od ) ppm 8.24 ( d , 1h , j = 8.4 hz ) , 7.75 ( t , 1h , j = 7.0 hz ) , 7.557.45 ( m , 3h ) , 7.35 ( t , 1h , j = 7.0 hz ) , 7.15 ( t , 1h , j = 7.9 hz ) , 7.06 ( dd , 1h , j = 1.9 , 8.2 hz ) , 6.76 ( d , 1h , j = 1.9 hz ) , 5.345.10 ( m , 1h ) , 3.85 ( s , 3h ) , 2.572.31 ( m , 1h ) , 2.181.96 ( m , 2h ) , 1.911.57 ( m , 5h ) , 1.251.05 ( m , 2h ) ; esi - ms m / z 611.25 ( m + h ) . 54 was obtained according to general procedure d followed by general procedure e. h nmr ( 400 mhz , cd3od ) ppm 7.98 ( d , 2h , j = 8.7 hz ) , 7.70 ( t , 1h , j = 7.7 hz ) , 7.64 ( d , 2h , j = 8.7 hz ) , 7.57 ( dd , 1h , j = 2.4 , 8.2 hz ) , 7.38 ( t , 1h , j = 8.2 hz ) , 7.19 ( t , 1h , j = 8.1 hz ) , 7.13 ( dd , 1h , j = 1.9 , 8.2 hz ) , 6.81 ( d , 1h , j = 1.9 hz ) , 5.29 ( d , 1h , j = 10.5 hz ) , 4.97 ( d , 1h , j = 10.8 hz ) , 2.75 ( d , 1h , j = 13.6 hz ) , 2.13 ( dt , 1h , j = 4.2 , 14.0 hz ) , 2.04 ( d , 1h , j = 13.4 hz ) , 1.87 ( dt , 1h , j = 3.4 , 14.2 hz ) , 1.64 ( d , 1h , j = 14.7 hz ) , 1.531.34 ( m , 3h ) , 1.02 ( s , 3h ) , 0.77 ( s , 3h ) ; c nmr ( 100 mhz , cd3od ) ppm 178.48 , 169.09 , 168.51 , 157.71 ( d , jc f = 249.3 hz ) , 145.30 , 142.77 , 136.94 , 132.18 , 131.88(2c ) , 129.50 , 128.91 , 128.25 , 126.32 ( d , jc f = 4.6 hz ) , 123.29 , 122.77 , 122.28 ( d , jc f = 19.1 hz ) , 120.51(2c ) , 111.75 , 72.59 , 68.33 , 62.99 , 46.84 , 35.75 , 34.45 , 32.21 , 30.00 , 28.07 , 27.96 , 23.63 ; esi - ms m / z 610.25 ( m + h ) . h nmr ( 300 mhz , cd3od ) ppm 7.61 ( t , 1h , j = 6.55 hz ) , 7.49 ( dd , 1h , j = 2.34 , 8.20 hz ) , 7.39 ( t , 1h , j = 6.90 hz ) , 7.15 ( t , 1h , j = 8.53 hz ) , 7.10 ( dd , 1h , j = 1.94 , 8.22 hz ) , 6.78 ( d , 1h , j = 1.88 hz ) , 4.98 ( d , 1h , j = 10.87 hz ) , 4.78 ( d , 1h , j = 10.92 hz ) , 2.842.71 ( m , 1h ) , 2.26 ( s , 6h ) , 2.14 ( d , 1h , j = 13.90 hz ) , 2.021.67 ( m , 5h ) , 1.601.38 ( m , 1h ) , 1.311.10 ( m , 2h ) ; esi - ms m / z 572.25 ( m + h ) . hatu ( 616 mg , 1.62 mmol ) and diea ( 0.550 ml , 3.24 mmol ) were added to a suspension of acid 29 ( 500 mg , 1.08 mmol ) in dcm ( 15 ml ) and stirred . after 10 min , methyl 4-aminobicyclo[2.2.2]octane-1-carboxylate ( 396 mg , 2.16 mmol ) and dmap ( 132 mg , 1.08 mmol ) were added to the reaction . after overnight , the solvent was removed in vacuo and the crude was purified by column chromatography to give 549 mg of interemediate 56-ester . liohh2o ( 110 mg , 2.62 mmol ) and sodium hydroxide ( 105 mg , 2.62 mmol ) were added to a solution of interemidiate 56-ester ( 549 mg , 0.873 mmol ) dissolved in a mixture of thf ( 3 ml ) , h2o ( 3 ml ) , and meoh ( 3 ml ) . after the hydrolysis was complete , as determined by tlc , the reaction was quenched with tfa ( 3 ml ) and stirred . after 5 min , the solution was concentrated in vacuo ( not to dryness ) and the resulting oil was redissolved in ch3cn and h2o ( 1:1 ) and the solution was purified by preparative hplc . the purified fractions were combined , concentrated in vacuo , redissolved in h2o , frozen , and lyophilized to give 56 ( tfa salt ) as a white powder . h nmr ( 300 mhz , cd3od ) ppm 7.63 ( t , 1h , j = 6.84 hz ) , 7.45 ( d , 1h , j = 6.76 hz ) , 7.35 ( t , 1h , j = 7.21 hz ) , 7.187.04 ( m , 2h ) , 6.77 ( dd , 1h , j = 1.26 hz ) , 4.68 ( d , 1h , j = 10.61 hz ) , 2.732.48 ( m , 1h ) , 2.161.98 ( m , 1h ) , 1.981.43 ( m , 18h ) , 1.271.02 ( m , 2h ) ; c nmr ( 100 mhz , cd3od ) ppm 180.79 , 178.22 , 167.86 , 157.78 ( d , jc f = 248.9 hz ) , 145.23 , 137.07 , 132.32 , 129.39 , 128.99 , 126.45 ( d , jc f = 4.9 hz ) , 123.39 , 122.50 , 122.17 , 122.15 ( d , jc f = 19.3 hz ) , 111.77 , 73.22 , 67.72 , 62.58 , 52.82 , 46.96 ( d , jc f = 3.5 hz ) , 38.96 , 32.17 , 31.36 , 30.41(3c ) , 29.45(3c ) , 25.35 , 23.08 , 21.74 ; esi - ms m / z 614.92 ( m + h ) . h nmr ( 300 mhz , cd3od ) ppm 7.71 ( s , 1h ) , 7.63 ( t , 1h , j = 6.61 hz ) , 7.50 ( dd , 1h , j = 2.08 , 8.18 hz ) , 7.36 ( t , 1h , j = 7.54 hz ) , 7.187.05 ( m , 2h ) , 6.79 ( d , 1h , j = 1.83 hz ) 4.96 ( d , 1h , j = 10.48 hz ) , 4.71 ( d , 1h , j = 10.51 hz ) , 2.78 ( d , 1h , j = 14.25 hz ) , 2.591.91 ( m , 6h ) , 1.911.70 ( m , 12h ) , 1.531.33 ( m , 1h ) ; esi - ms m / z 650.92 ( m + h ) . cdi ( 49 mg , 0.303 mmol ) , diea ( 88 l , 0.505 mmol ) , and dmap ( cat . ) were added to a solution of 56 ( 62 mg , 0.101 mmol ) in 1,2-dichloroethane ( 10 ml ) , and the reaction was heated to 40 c . after 20 min , methanesulfonamide ( 96 mg , 1.01 mmol ) was added and the reaction was refluxed . after overnight , the sovent was removed in vacuo and the crude was purified by prepartive hplc to give 58 ( tfa salt ) as a white solid . h nmr ( 300 mhz , cd3od ) ppm 7.64 ( t , 1h , j = 7.23 hz ) , 7.45 ( dd , 1h , j = 1.93 , 8.22 hz ) , 7.36 ( t , 1h , j = 7.23 hz ) , 7.187.04 ( m , 2h ) , 6.77 ( d , 1h , j = 1.66 hz ) , 4.69 ( d , 1h , j = 10.70 hz ) , 3.19 ( s , 3h ) , 2.752.52 ( m , 1h ) , 2.211.99 ( m , 1h ) , 1.991.44 ( m , 17h ) , 1.411.27 ( m , 1h ) , 1.271.03 ( m , 2h ) ; esi - ms m / z 691.42 paraformaldehyde ( 15 mg , 0.506 mmol ) was added to a solution of compound 56 ( 20 mg , 0.028 mmol ) dissolved in acoh ( 1 ml ) . after 15 min sodium triacetoxyborohydride ( 59 mg , 0.28 mmol ) was added , and after reacting overnight , the reaction was quenched with saturated ammonium chloride solution and extracted with etoac . the etoac solvent was evaporated in vacuo , and the resulting oil was redissolved in a solution of mecn and h2o ( 1:1 with 0.1% tfa ) and purified by preparative hplc . the pure 59 fractions were combined , concentrate in vacuo , redissolved in h2o ( with minimum amount of mecn ) , frozen , and lyophilized to give 59 as the tfa salt as a white powder . h nmr ( 300 mhz , cd3od ) ppm 7.94 ( s , 1h ) , 7.617.52 ( m , 2h ) , 7.40 ( t , 1h , j = 7.32 hz ) , 7.197.08 ( m , 2h ) , 6.78 ( d , 1h , j = 1.56 hz ) , 4.99 ( d , 1h , j = 11.86 hz ) , 4.63 ( d , 1h , j = 12.06 hz ) , 3.27 ( s , 3h ) , 2.612.48 ( m , 1h ) , 2.322.14 ( m , 2h ) , 1.881.40 ( m , 18h ) , 1.371.12 ( m , 1h ) ; c nmr ( 100 mhz , cd3od ) ppm 180.74 , 178.21 , 166.55 , 157.74 ( d , jc f = 249.6 hz ) , 144.90 , 137.11 , 132.39 , 129.80 , 129.09 , 126.30 ( d , jc f = 4.7 hz ) , 124.00 , 123.43 , 122.31 ( d , jc f = 13.6 hz ) , 111.77 , 77.98 , 73.06 , 67.67 , 52.98 , 46.56 , 40.34 , 38.97 , 34.28 , 30.38(3c ) , 29.44(3c ) , 28.97 , 24.95 , 23.57 , 22.07 ; esi - ms m / z 628.83 ( m + h ) . h nmr ( 300 mhz , cd3od ) ppm 7.63 ( t , 1h , j = 7.04 hz ) , 7.567.48 ( m , 2h ) , 7.42 ( t , 1h , j = 7.39 hz ) , 7.18 ( t , 1h , j = 7.96 hz ) , 7.10 ( d , 1h , j = 8.06 hz ) , 6.79 ( s , 1h ) , 5.084.96 ( m , 1h ) , 4.57 ( d , 1h , j = 11.85 hz ) , 4.183.99 ( m , 1h ) , 3.873.69 ( m , 1h ) , 2.702.54 ( m , 1h ) , 2.362.13 ( m , 2h ) , 1.941.45 ( m , 18h ) , 1.39 ( t , 3h , j = 6.65 hz ) , 1.321.14 ( m , 1h ) ; c nmr ( 100 mhz , cd3od ) ppm 180.95 , 180.06 , 177.01 , 157.71 ( d , jc f = 247.1 hz ) , 144.91 , 135.39 , 130.37 , 129.74 ( d , jc f = 19.7 hz ) , 110.69 , 74.84 , 72.58 , 71.05 , 51.51 , 48.09 , 47.36 , 39.17 , 37.56 , 30.84(3c ) , 29.61(3c ) , 29.25 , 26.37 , 24.25 , 23.14 , 17.04 ; esi - ms m / z 642.50 ( m + h ) . intermediate 64(21 ) ( 1.0 g , 2.09 mmol ) was dissolved in a mixture of dcm ( 10 ml ) and acoh ( 10 ml ) . paraformaldehyde ( 1.13 g , 37.62 mmol ) was added , and after 10 min nabh(oac)3 ( 4.43 g , 20.9 mmol ) was added in portions . after 4 h , as determined by tlc , the reaction was complete . saturated nh4cl ( aq ) was added to the reaction which was then extracted with etoac . the etoac solution was washed with naoh ( 1 m ) and brine , then dried over na2so4 , filtered , concentrated , and purified by column chromatography to give 764 mg of intermediate 65 . sodium hydroxide ( 320 mg , 8.0 mmol ) was added to a solution of intermediate 65 ( 764 mg , 1.6 mmol ) dissolved in a mixture of thf ( 3 ml ) , h2o ( 3 ml ) , and meoh ( 3 ml ) , and the solution was heated to 4550 c . after hydrolysis was complete , as determined by tlc , the reaction was cooled , neutralized with 2 m hcl , and extracted with etoac . the etoac solution was dried over na2so4 , filtered , and concentrated to give the acid intermediate 66 as a solid that was used without further purification . cdi ( 315 mg , 1.94 mmol ) and diea ( 0.450 ml , 2.59 mmol ) were added to a solution of 66 ( 300 mg , 0.648 mmol ) in thf ( 15 ml ) and heated to 50 c . the etoac solution was dried over sodium sulfate , filtered , concentrated , and purified by column chromatography to give 240 mg of compound 67 . reagent ( 102 mg , 0.252 mmol ) was added to a solution of compound 67 ( 60 mg , 0.126 mmol ) in dcm ( 3 ml ) and refluxed overnight . then the reaction was cooled , the solvent was removed , and the crude was column chromatographed to produce 60 mg of 68 . h nmr ( 400 mhz , cd3od ) ppm 7.66 ( ddd , 1h , j = 1.4 , 6.4 , 7.9 hz ) , 7.53 ( dd , 1h , j = 2.5 , 8.3 hz ) , 7.38 ( t , 1h , j = 7.3 hz ) , 7.15 ( t , 1h , j = 8.0 hz ) , 7.10 ( dd , 1h , j = 1.9 , 8.2 hz ) , 6.77 ( d , 1h , j = 1.9 hz ) , 5.48 ( br s , 1h ) , 4.71 ( d , 1h , j = 11.2 hz ) , 3.37 ( s , 3h ) , 2.38 ( d , 1h , j = 14.2 hz ) , 2.192.02 ( m , 1h ) , 1.991.85 ( m , 1h ) , 1.831.61 ( m , 4h ) , 1.501.36 ( m , 1h ) , 1.351.16 ( m , 2h ) ; esi - ms m / z 492.67 ( m + h ) . these assays were performed according to the previously reported procedures . all the in vivo studies were performed under an animal protocol ( pro00005315 ) approved by the university committee on use and care of animals of the university of michigan , in accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health . docking studies were performed using our previously reported structure of mdm2 in complex with 2 ( pdb code 5trf ) with the gold program ( version 5.2 ) . for each genetic algorithm ( ga ) run , a maximum number of 200 000 operations were performed on a population of five islands of 100 individuals . operator weights for crossover , mutation , and migration were set to 95 , 95 , and 10 , respectively , and the chemscore fitness function was used to evaluate the docked conformations . docking was terminated after 10 runs for each ligand , and the top 10 conformations were saved for analysis of the predicted binding modes .

we previously reported the design of spirooxindoles with two identical substituents at the carbon-2 of the pyrrolidine core as potent mdm2 inhibitors . in this paper we describe an extensive structure activity relationship study of this class of mdm2 inhibitors , which led to the discovery of 60 ( aa-115/apg-115 ) . compound 60 has a very high affinity to mdm2 ( ki < 1 nm ) , potent cellular activity , and an excellent oral pharmacokinetic profile . compound 60 is capable of achieving complete and long - lasting tumor regression in vivo and is currently in phase i clinical trials for cancer treatment .

Introduction Results and Discussion Chemistry Conclusion Experimental Section

small - molecule inhibitors designed to block the mdm2p53 interaction ( hereafter called mdm2 inhibitors ) can liberate the tumor suppressor function of p53 and may have a promising therapeutic potential for cancer treatment . intense research efforts have resulted in advancement of several potent , selective , non - peptide mdm2 inhibitors ( 15 ) , shown in figure 1 , into clinical development . our laboratory has designed and optimized spirooxindoles as a new class of mdm2 inhibitors and has advanced compound 2 ( mi-77301 ) into clinical development . to overcome this stability issue , we recently reported the design of new spirooxindoles containing two identical substituents at c-2 of the pyrrolidine ring , which can undergo a rapid and irreversible conversion to a single diastereoisomer . in the present study , we report an extensive structure activity relationship ( sar ) study for this class of mdm2 inhibitors . this study has led to the discovery of 60 ( aa-115/apg-115 ) , a highly potent , chemically stable and efficacious mdm2 inhibitor , which has entered clinical development for cancer treatment ( clinicaltrials.gov identifier for 60 : nct02935907 ) . our exploration started with lead molecule 6(21 ) ( figure 2a ) that binds to mdm2 with ki = 2.9 nm , inhibits the growth of the sjsa-1 cancer cell line with ic50 = 190 nm , and achieves strong tumor growth inhibition but not complete tumor regression in an sjsa-1 osteosarcoma xenograft model in mice . our model shows that the oxindolephenyl , 3-chloro-2-fluorophenyl , and cyclohexyl groups of 6 project into the trp23 , leu26 , and phe19 p53 binding pockets of the mdm2 protein and the 4-hydroxyl of the n - cyclohexylcarboxamide group forms a hydrogen bond with lys94 of mdm2 ( figure 2b ) . replacement of the cyclohexyl group at site a with 4-piperidinyl group led to compound 8 , which has no appreciable binding up to 2 m to mdm2 . replacement of the cyclohexyl group at site a in 6 with 4-tetrahydro-2h - pyran generated compound 11 , which has a moderate binding affinity to mdm2 ( ki = 75.9 nm ) . introduction of two fluorine substituents at the 4-position of the cyclohexyl group in compound 6 resulted in compound 12 , which has a good affinity to mdm2 ( ki = 9.8 nm ) but is 4 times less potent than 6 . however , introduction of two methyl groups at the same 4-position in 6 yielded compound 15 , which has a ki value of < 1 nm to mdm2 and is several times more potent than 6 . installation of methyl groups at the 3-position of the cyclobutyl ring led to compound 14 , which is equally potent as compound 6 and 7 times more potent than compound 13 . hence , our sar data for site a clearly show that a hydrophobic group is highly desirable for achieving a strong binding affinity to mdm2 , and cyclohexyl , 4,4-dimethylcyclohexyl , and 3,3-cyclobutyl groups are the most preferred . our data , however , are consistent with a previous study , which showed that a compound containing a chloropyridyl oxindole group is a potent mdm2 inhibitor . this previous study also showed that replacing the oxindole with a thienopyrrolone led to potent mdm2 inhibitors . we therefore synthesized compound 20 containing a thienopyrrolone , which has a ki of 6.5 nm and is 2-fold less potent than 6 . we found that the thiophene group in 20 can be oxidized during the oxidative removal of the chiral auxiliary , preventing further investigation of this compound . in particular , compounds 31 , 32 , and 33 bind to mdm2 with ki < 1 nm and are more than 3 times more potent than 6 . these compounds all display very high binding affinities to mdm2 , superior to that of 6 and equivalent to that of 33 . furthermore , 39 was capable of achieving partial tumor regression in the sjsa-1 xenograft model as shown in our previous report . compound 54 has a very high binding affinity to mdm2 ( ic50 = 2.4 nm , ki < 1 nm , scheme 2 ) and potently inhibits cell growth of sjsa-1 cells with ic50 = 13 nm ( scheme 2 ) . since 39 and 54 achieve comparable tumor regression in the sjsa-1 xenograft model and 39 is less hydrophobic than 54 , we have selected compound 39 for further optimization of its pk properties and in vivo efficacy . in our attempt to further improve the oral pk properties of compound 39 while retaining its high binding affinity to mdm2 and cellular potency , we investigated the replacement of the benzoic acid with nonclassical benzoic acid mimetics such as a bicyclo[1.1.1]pentane-1-carboxylic acid or a bicyclo[2.2.2]octane-1-carboxylic acid . these efforts led to the design and synthesis of compounds 55 and 56 ( scheme 3 ) . compound 55 binds to mdm2 with a high affinity ( ic50 = 6.4 nm , ki < 1 nm ) but is 5 times less potent than compound 39 in inhibition of cell growth in the sjsa-1 cell line . in comparison , compound 56 has a high binding affinity to mdm2 ( ic50 = 3.7 nm , ki < 1 nm ) and is as potent as compound 39 in inhibition of cell growth in the sjsa-1 cell line ( scheme 3 and table 7 ) . accordingly , we investigated strategies with which to improve the tissue penetration while retaining high binding affinity to mdm2 , potent cellular activity , and good oral bioavailability of compound 56 . we investigated three strategies to improve the tissue penetration : ( 1 ) decreasing the lipophilicity of the molecule ; ( 2 ) reducing the acidity of carboxylic acids ; and ( 3 ) increasing the basicity of the nitrogen atom in the pyrrolidine core . we therefore synthesized compound 57 with a 4,4-difluorocyclohexyl group at carbon-2 of the pyrrolidine core ( table 7 ) . for the third strategy , we installed a methyl or ethyl group on the nitrogen of the pyrrolidine core , which led to 59 and 60 , respectively ( table 7 ) . compound 57 binds to mdm2 with a high affinity but is less potent than 56 and is 2 times less potent than 56 in inhibition of cell growth in the sjsa-1 cell line ( table 7 ) . compound 58 is 2 times less potent than 56 in binding to mdm2 and is 4 times less potent than 56 in inhibition of cell growth in the sjsa-1 cell line ( table 7 ) . pharmacodynamic ( pd ) analysis of the effect of mdm2 inhibitors in sjsa-1 tumors . in comparison , compounds 59 and 60 bind to mdm2 with a high binding affinity ( ki < 1 nm , table 7 ) . both compounds potently inhibit cell growth in the sjsa-1 cell line with ic50 values of 70 and 60 nm , respectively , and are thus as potent as compound 56 ( table 7 ) . compound 59 or 60 orally administered at 100 mg / kg daily for 14 days effectively achieves complete and long - lasting tumor regression . compound 60 is the most potent compound , achieving ic50 values of 38 , 18 , and 104 nm in the rs4;11 acute leukemia , lncap prostate cancer , and hct116 colon cancer cell lines , respectively . to explore achiral substituents in carbon-2 of the pyrrolidine core , the compounds in table 1 were synthesized using the method outlined in scheme 4 . reaction of this morpholine ( 6a15a ) with an aliphatic amine led to the open - ring compound ( 6b15b ) . subsequently , the compounds ( 1620 ) in table 2 were obtained by using the intermediates ( 16b20b ) , which are equivalent to 61 , in the synthetic method outlined in scheme 4 . hydrolysis of the methyl ester ( 65 ) was followed by conversion of the acid ( 66 ) to the corresponding carboxamide ( 67 ) . reagents and conditions : ( a ) dce , cdi , diea , dmap , 50 c for 30 min , then 71 , 74 , 76 , 78 , and reflux ; ( b ) ( 1 ) thf , deoxo - fluor , 20 c for 30 min , ( 2 ) brccl3 , dbu at rt overnight ; ( c ) thf / h2o ( 2:1 ) , liohh2o , rt for 30 min , then preparative hplc ; ( d ) pyridine , 100 c for 4 h ; ( e ) dcm , pph3 , i2 , et3n , rt ; ( f ) dcm , lawesson s reagent , reflux overnight . in this study , we have performed an extensive sar study on spirooxindoles containing two substituents at the carbon-2 of the pyrrolidine core as mdm2 inhibitors . extensive modifications in five different regions of the molecule have led to discovery of a number of potent and highly efficacious mdm2 inhibitors . in particular , compound 60 has a very high binding affinity to mdm2 ( ki < 1 nm ) , potently activates wild - type p53 , inhibits cell growth with low nanomolar ic50 values in human cancer cell lines carrying wild - type p53 , and demonstrates an outstanding cellular selectivity over human cancer cell lines with deleted p53 . compound 60 is very stable in solutions , has excellent oral pharmacokinetics , and effectively activates p53 in the sjsa-1 xenograft tumor tissue in mice following a single oral administration . significantly , 60 achieves complete and long - lasting regression of the sjsa-1 xenograft tumors in mice and demonstrates strong antitumor activity in the rs4;11 acute leukemia model at well tolerated dose schedules . compound 60 has been advanced into phase i clinical development for the treatment of human cancer . starting with ( e)-6-chloro-3-((5-chloropyridin-3-yl)methylene)indolin-2-one ( 16b ) in place of ( e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one ( 61 ) , compound 16 was prepared according to the synthetic method described for the preparation of 6 . starting with ( e)-2-chloro-5-(3-chloro-2-fluorobenzylidene)-5,7-dihydro-6h - pyrrolo[2,3-d]pyrimidin-6-one ( 19b ) in place of ( e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one ( 61 ) , compound 19 was prepared according to the synthetic method described for the preparation of 6 . starting with ( z)-2-chloro-6-(3-chloro-2-fluorobenzylidene)-4,6-dihydro-5h - thieno[3,2-b]pyrrol-5-one ( 20b ) in place of ( e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one ( 61 ) , compound 20 was prepared according to the synthetic method described for the preparation of 6 . ethyl 3-bromo-2-oxopropanoate ( 48 mg , 0.244 mmol ) and triethylamine ( 0.033 ml , 0.244 mmol ) , were added to a solution of 68 ( 30 mg , 0.061 mmol ) in thf ( 3 ml ) and refluxed overnight the crude product was redissolved in dme ( 3 ml ) , and to this solution , at 20 c , was added trifluoracetic anhydride ( 0.025 ml , 0.183 mmol ) and pyridine ( 0.025 ml , 0.305 mmol ) , and then the reaction was allowed to warm to rt . starting with 77 mg of compound 66 , 63 mg of 77 was obtained using general procedure a. a solution of 77 ( 30 mg , 0.056 mmol ) in dcm ( 3 ml ) was slowly added to a solution of pph3 ( 30 mg , 0.112 mmol ) , et3n ( 31 l ) , and iodine ( 28 mg , 0.112 mmol ) in dcm ( 1 ml ) . h nmr ( 400 mhz , cd3od ) ppm 7.69 ( t , 1h , j = 7.9 hz ) , 7.46 ( dd , 1h , j = 2.8 , 8.2 hz ) , 7.25 ( t , 1h , j = 8.4 hz ) , 7.08 ( dt , 1h , j = 1.1 , 8.1 hz ) , 7.03 ( dd , 1h , j = 2.0 , 8.2 hz ) , 6.72 ( d , 1h , j = 1.9 hz ) , 5.50 ( d , 1h , j = 10.2 hz ) , 4.63 ( d , 1h , j = 11.0 hz ) , 3.00 ( s , 3h ) , 2.71 ( s , 3h ) , 2.27 ( d , 1h , j = 13.0 hz ) , 2.232.15 ( m , 1h ) , 1.88 ( t , 1h , j = 11.6 hz ) , 1.831.60 ( m , 4h ) , 1.601.49 ( m , 1h ) , 1.311.07 ( m , 2h ) ; esi - ms m / z 531.17 ( m + h ) . h nmr ( 400 mhz , cd3od ) ppm 7.60 ( t , 1h , j = 7.2 hz ) , 7.52 ( dd , 1h , j = 2.7 , 8.1 hz ) , 7.21 ( t , 1h , j = 8.3 hz ) , 7.076.98 ( m , 2h ) , 6.70 ( d , 1h , j = 1.9 hz ) , 5.07 ( d , 1h , j = 10.8 hz ) , 4.91 ( d , 1h , j = 11.7 hz ) , 2.83 ( s , 3h ) , 2.59 ( s , 3h ) , 2.312.23 ( m , 1h ) , 2.122.04 ( m , 1h ) , 1.851.02 ( m , 8h ) ; esi - ms m / z 558.75 h nmr ( 400 mhz , cd3od ) ppm 7.65 ( t , 1h , j = 6.7 hz ) , 7.57 ( d , 1h , j = 7.1 hz ) , 7.42 ( s , 1h ) , 7.27 ( t , 1h , j = 8.2 hz ) , 7.137.04 ( m , 2h ) , 6.75 ( d , 1h , j = 1.9 hz ) , 5.925.70 ( m , 1h ) , 3.78 ( s , 2h ) , 3.20 ( s , 3h ) , 2.582.44 ( m , 1h ) , 2.26 ( d , 1h , j = 13.8 hz ) , 2.222.08 ( m , 1h ) , 1.831.43 ( m , 5h ) , 1.381.27 ( m , 1h ) , 1.251.13 ( m , 1h ) ; esi - ms m / z 574.08 ( m + h ) . after 2 h , tfa was added and the solution was concentrated , redissolved in meoh / h2o ( 3:1 ) , and purified by reverse phase preparative hplc . after 2 h , tfa was added and the reaction was concentrated , redissolved in meoh / h2o ( 3:1 ) , and purified by reverse phase preparative hplc . cdi ( 49 mg , 0.303 mmol ) , diea ( 88 l , 0.505 mmol ) , and dmap ( cat . ) were added to a solution of 56 ( 62 mg , 0.101 mmol ) in 1,2-dichloroethane ( 10 ml ) , and the reaction was heated to 40 c . the pure 59 fractions were combined , concentrate in vacuo , redissolved in h2o ( with minimum amount of mecn ) , frozen , and lyophilized to give 59 as the tfa salt as a white powder . sodium hydroxide ( 320 mg , 8.0 mmol ) was added to a solution of intermediate 65 ( 764 mg , 1.6 mmol ) dissolved in a mixture of thf ( 3 ml ) , h2o ( 3 ml ) , and meoh ( 3 ml ) , and the solution was heated to 4550 c . all the in vivo studies were performed under an animal protocol ( pro00005315 ) approved by the university committee on use and care of animals of the university of michigan , in accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health .

through direct binding , the mdm2 protein blocks the transactivation domain of p53 , transports p53 from the nucleus to the cytoplasm , and ubiquitinates p53 for proteasomal degradation . small - molecule inhibitors designed to block the mdm2p53 interaction ( hereafter called mdm2 inhibitors ) can liberate the tumor suppressor function of p53 and may have a promising therapeutic potential for cancer treatment . to overcome this stability issue , we recently reported the design of new spirooxindoles containing two identical substituents at c-2 of the pyrrolidine ring , which can undergo a rapid and irreversible conversion to a single diastereoisomer . in the present study , we report an extensive structure activity relationship ( sar ) study for this class of mdm2 inhibitors . this study has led to the discovery of 60 ( aa-115/apg-115 ) , a highly potent , chemically stable and efficacious mdm2 inhibitor , which has entered clinical development for cancer treatment ( clinicaltrials.gov identifier for 60 : nct02935907 ) . our exploration started with lead molecule 6(21 ) ( figure 2a ) that binds to mdm2 with ki = 2.9 nm , inhibits the growth of the sjsa-1 cancer cell line with ic50 = 190 nm , and achieves strong tumor growth inhibition but not complete tumor regression in an sjsa-1 osteosarcoma xenograft model in mice . to guide our chemical modifications , we modeled the structure of compound 6 in a complex with mdm2 based upon the cocrystal structure of compound 2 complexed with human mdm2 ( figure 2b ) . our model shows that the oxindolephenyl , 3-chloro-2-fluorophenyl , and cyclohexyl groups of 6 project into the trp23 , leu26 , and phe19 p53 binding pockets of the mdm2 protein and the 4-hydroxyl of the n - cyclohexylcarboxamide group forms a hydrogen bond with lys94 of mdm2 ( figure 2b ) . guided by this model , we performed extensive modifications on five regions of the molecule ( labeled as sites a we began by replacing the 4-hydroxycyclohexyl group at site d in compound 6 with a methyl group , giving compound 7 . replacement of the cyclohexyl group at site a with 4-piperidinyl group led to compound 8 , which has no appreciable binding up to 2 m to mdm2 . replacement of the cyclohexyl group at site a in 6 with 4-tetrahydro-2h - pyran generated compound 11 , which has a moderate binding affinity to mdm2 ( ki = 75.9 nm ) . introduction of two fluorine substituents at the 4-position of the cyclohexyl group in compound 6 resulted in compound 12 , which has a good affinity to mdm2 ( ki = 9.8 nm ) but is 4 times less potent than 6 . hence , our sar data for site a clearly show that a hydrophobic group is highly desirable for achieving a strong binding affinity to mdm2 , and cyclohexyl , 4,4-dimethylcyclohexyl , and 3,3-cyclobutyl groups are the most preferred . next , we explored modifications of the 3-chloro-2-fluorophenyl and the oxindolephenyl substituents ( sites b and c in figure 2a , respectively ) . interestingly , although the trp23 pocket is hydrophobic in nature , compounds 17 , 18 , and 19 containing a pyridinyl or a pyrimidinyl oxindole group ( ring c ) are all quite potent with ki = 1314 nm . we found that the thiophene group in 20 can be oxidized during the oxidative removal of the chiral auxiliary , preventing further investigation of this compound . our modeling of 6 and the cocrystal structure of 2 ( figure 2b ) showed that the carbonyl of the amide ( site e ) forms a hydrogen bond with his96 of the mdm2 protein but the amino group does not interact with the mdm2 protein . unfortunately , these compounds show weak cell growth inhibitory activity ( ic50 > 2 m , table 3 ) in the sjsa-1 cancer cells , probably because the negatively charged acid group in these compounds decreases their cell permeability . our modeling of 6 and the cocrystal structure of 2 also showed that the 4-hydroxyl on the cyclohexylcarboxamide substituent of 6 forms a hydrogen bond with lys94 of the mdm2 protein ( figure 2b ) . we tested the cell growth inhibitory activity of compounds 2938 in the sjsa-1 cell line , and the data are summarized in table 4 . among this series of compounds , 32 , 33 , and 38 have the best antiproliferative activity with ic50 values of 0.22 , 0.15 , and 0.24 m , respectively . our pd data showed that a single , oral administration of 33 at 100 mg / kg is very effective in inducing upregulation of mdm2 , p53 , and p21 proteins at both 3 and 6 h time - points in the sjsa-1 tumor tissue , indicating strong activation of p53 ( figure 3a ) . upon the basis of its promising pd data , we evaluated compound 33 for its antitumor efficacy in the sjsa-1 xenograft model ( figure 3b , c ) . during the course of our research , we also observed that compound 33 can decompose in solution , particularly in cell culture media ( panels a and b in scheme 1 ) . to analyze the potential structural features affecting the stability of 33 , we determined the stability of compound 31 with a flexible butanoic acid group , compound 6 with a 4-hydroxylcyclohexyl group , and 33 . compound 33 was incubated in ch3cn / h2o , meoh / h2o , or cell culture media , and the % composition of 33 in the solutions was determined daily for 2 days by uplc ( ultraperformance liquid chromatography ) and plotted . compounds 39 ( mi-1061 ) and 44 displayed the best cellular activity among the compounds in this series and are 3 times more potent than compound 33 in inhibition of cell growth in the sjsa-1 cell line . compound 54 has a very high binding affinity to mdm2 ( ic50 = 2.4 nm , ki < 1 nm , scheme 2 ) and potently inhibits cell growth of sjsa-1 cells with ic50 = 13 nm ( scheme 2 ) . we assessed the antitumor activity of compound 54 in the sjsa-1 xenograft model in mice , with compound 2 included as the control ( figure 4 ) . ( a ) antitumor activity of compound 54 , in comparison to compound 2 in the sjsa-1 osteosarcoma tumor xenograft model in mice . pharmacokinetic ( pk ) studies in rats ( table 6 ) showed that with oral administration , compounds 39 and 54 achieve considerably lower cmax and auc values than compound 2 , suggesting that further improvement of the oral pk for compounds 39 and 54 is needed in order to achieve stronger in vivo antitumor activity . since 39 and 54 achieve comparable tumor regression in the sjsa-1 xenograft model and 39 is less hydrophobic than 54 , we have selected compound 39 for further optimization of its pk properties and in vivo efficacy . in our attempt to further improve the oral pk properties of compound 39 while retaining its high binding affinity to mdm2 and cellular potency , we investigated the replacement of the benzoic acid with nonclassical benzoic acid mimetics such as a bicyclo[1.1.1]pentane-1-carboxylic acid or a bicyclo[2.2.2]octane-1-carboxylic acid . in comparison , compound 56 has a high binding affinity to mdm2 ( ic50 = 3.7 nm , ki < 1 nm ) and is as potent as compound 39 in inhibition of cell growth in the sjsa-1 cell line ( scheme 3 and table 7 ) . a pk study in rats showed that 56 has a higher plasma exposure than compound 2 based upon both the cmax ( 8234 vs 4547 hg / l ) . however , our pd experiment showed that oral administration of compound 56 at 100 mg / kg induces only modest p53 activation and no cleavage of parp and caspase-3 in the sjsa-1 xenograft tumor tissue in mice ( figure 6a ) . consistent with the pd data , compound 56 at 100 mg / kg administered daily for 14 days demonstrates only a very moderate tumor growth inhibition in the sjsa-1 xenograft model in mice ( figure 5a ) . because of its excellent in vitro cellular potency and excellent plasma exposure , the modest p53 activation and antitumor activity achieved by compound 56 suggests that this compound has a poor penetration into the xenograft tumor tissue . accordingly , we investigated strategies with which to improve the tissue penetration while retaining high binding affinity to mdm2 , potent cellular activity , and good oral bioavailability of compound 56 . we investigated three strategies to improve the tissue penetration : ( 1 ) decreasing the lipophilicity of the molecule ; ( 2 ) reducing the acidity of carboxylic acids ; and ( 3 ) increasing the basicity of the nitrogen atom in the pyrrolidine core . for the third strategy , we installed a methyl or ethyl group on the nitrogen of the pyrrolidine core , which led to 59 and 60 , respectively ( table 7 ) . compound 58 is 2 times less potent than 56 in binding to mdm2 and is 4 times less potent than 56 in inhibition of cell growth in the sjsa-1 cell line ( table 7 ) . the pd study showed that oral administration of 57 or 58 at 100 mg / kg only has a modest effect on activation of p53 and induction of cleavage of parp and caspase-3 ( figure 6b ) . mice bearing sjsa-1 tumors were dosed with a single oral dose of 56 , 57 , 58 , 59 , 60 and 2 at 100 mg / kg and the tumors were harvested at 6 and 24 hours for western blot analysis . ( a , b ) pd of 56 , 57 and 58 show modest activation of p53 as seen with the low levels of p53 , mdm2 , and p21 ; ( c ) compounds 59 , 60 and 2 show robust activation of p53 as seen with increased levels of p53 , mdm2 , ubiquitinated mdm2 ( ub - mmd2 ) and p21 , and induce strong apoptosis as seen with increased levels of cleaved parp ( cl - papr ) and cleaved caspase-3 ( cl - casp3 ) . pd experiments showed that a single , oral dose of 59 and 60 at 100 mg / kg achieves strong p53 activation , with the effect persisting for 24 h in the sjsa-1 xenograft tumor tissue ( figure 6c ) . our pk studies in rats with oral administration showed that 59 and 60 achieve a cmax value similar to that of 2 but have a 2 times higher auc than compound 2 ( table 6 ) . we evaluated compounds 56 , 59 , and 60 for their activity and selectivity in a number of human cancer cell lines of different tumor types ( table 8) . consistent with their mechanism of action , these three compounds potently inhibit cell growth in cancer cell lines with wild - type p53 and display outstanding selectivity in cancer cell lines with deleted p53 . compound 60 is the most potent compound , achieving ic50 values of 38 , 18 , and 104 nm in the rs4;11 acute leukemia , lncap prostate cancer , and hct116 colon cancer cell lines , respectively . compound 60 effectively inhibits tumor growth in a dose - dependent manner and achieves partial tumor regression at 100 mg / kg , daily , oral administration for 14 days , or at 200 mg / kg weekly dosing for 3 weeks ( figure 8) . we evaluated the chemical stability of compound 60 in three different solutions and found that this compound is very stable in solutions for a period of 7 days ( figure 9 ) . to explore achiral substituents in carbon-2 of the pyrrolidine core , the compounds in table 1 were synthesized using the method outlined in scheme 4 . the azomethine ylide generated in situ by reaction of the chiral amine ( 62 ) with the cyclic ketone ( 63 ) was allowed to react with 61 in a 1,3-dipolar cycloaddition reaction , generating the chiral spirooxindoles ( 6a15a ) . reagents and conditions : ( a ) dce , cdi , diea , dmap , 50 c for 30 min , then 71 , 74 , 76 , 78 , and reflux ; ( b ) ( 1 ) thf , deoxo - fluor , 20 c for 30 min , ( 2 ) brccl3 , dbu at rt overnight ; ( c ) thf / h2o ( 2:1 ) , liohh2o , rt for 30 min , then preparative hplc ; ( d ) pyridine , 100 c for 4 h ; ( e ) dcm , pph3 , i2 , et3n , rt ; ( f ) dcm , lawesson s reagent , reflux overnight . the carboxylic acid ( 66 ) was activated with cdi ( 1,1-carbonyldiimidazole ) , and this was followed by addition of commercially available compounds 71 , 74 , 76 , 78 , resulting in the formation of intermediates 72 , 75 , 77 , 79 , respectively . when n - arylamines were used , hatu failed to accomplish the coupling , but the coupling was successful when the acid was activated with ph2pocl and produced the n - arylcarboxamides 3948 , 50 , and 51 . in particular , compound 60 has a very high binding affinity to mdm2 ( ki < 1 nm ) , potently activates wild - type p53 , inhibits cell growth with low nanomolar ic50 values in human cancer cell lines carrying wild - type p53 , and demonstrates an outstanding cellular selectivity over human cancer cell lines with deleted p53 . significantly , 60 achieves complete and long - lasting regression of the sjsa-1 xenograft tumors in mice and demonstrates strong antitumor activity in the rs4;11 acute leukemia model at well tolerated dose schedules . the mobile phase was a gradient of solvent a ( h2o with 0.1% tfa ) and solvent b ( meoh with 0.1% tfa or mecn with 0.1% tfa ) at a flow rate of 40 ml / min and 1%/2 min increase of solvent b or a flow rate of 60 ml / min and 1%/min increase of solvent b. all final compounds have purity of 95% as determined by waters acquity uplc using reverse phase column ( sunfire , c18 - 5 m , 4.6 mm 150 mm ) and a solvent gradient of a ( h2o with 0.1% of tfa ) and solvent d ( meoh with 0.1% of tfa ) or a ( h2o with 0.1% of tfa ) and solvent b ( ch3cn with 0.1% of tfa ) . the combined fractions of the pure compound were concentrated and then redissolved in a minimum amount of mecn , h2o was added , and the solution was frozen and lyophilized to produce the tfa salt of 6 ( 116 mg , 70% yield ) as a white powder . ethyl 3-bromo-2-oxopropanoate ( 48 mg , 0.244 mmol ) and triethylamine ( 0.033 ml , 0.244 mmol ) , were added to a solution of 68 ( 30 mg , 0.061 mmol ) in thf ( 3 ml ) and refluxed overnight the crude product was redissolved in dme ( 3 ml ) , and to this solution , at 20 c , was added trifluoracetic anhydride ( 0.025 ml , 0.183 mmol ) and pyridine ( 0.025 ml , 0.305 mmol ) , and then the reaction was allowed to warm to rt . starting with 77 mg of compound 66 , 63 mg of 77 was obtained using general procedure a. a solution of 77 ( 30 mg , 0.056 mmol ) in dcm ( 3 ml ) was slowly added to a solution of pph3 ( 30 mg , 0.112 mmol ) , et3n ( 31 l ) , and iodine ( 28 mg , 0.112 mmol ) in dcm ( 1 ml ) . liohh2o ( 110 mg , 2.62 mmol ) and sodium hydroxide ( 105 mg , 2.62 mmol ) were added to a solution of interemidiate 56-ester ( 549 mg , 0.873 mmol ) dissolved in a mixture of thf ( 3 ml ) , h2o ( 3 ml ) , and meoh ( 3 ml ) . sodium hydroxide ( 320 mg , 8.0 mmol ) was added to a solution of intermediate 65 ( 764 mg , 1.6 mmol ) dissolved in a mixture of thf ( 3 ml ) , h2o ( 3 ml ) , and meoh ( 3 ml ) , and the solution was heated to 4550 c . all the in vivo studies were performed under an animal protocol ( pro00005315 ) approved by the university committee on use and care of animals of the university of michigan , in accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health .

this is a complex organ highly regulated by several external systems ( central nervous , neurohormonal ) and by an important internal system . investigating its contractile properties has always been an important , but challenging goal . in 1866 , carl ludwig and elias cyon , decided to take up the challenge to develop the first ex - vivo beating whole heart system . they used this system to keep an excised and perfused frog heart beating for a relatively long period of time ( i.e. several minutes ) whilst isolated from the body . this major discovery has motivated other investigators to develop a variety of excised whole heart experimental apparatus . to date , the langendorff heart is by far the most famous heart perfusion setup that is still being used in laboratories . by using his system , oscar langendorff , in the 19 century , was able to maintain a perfused mammalian heart in a complete isolated state for several hours . by taking advantage of a similar experimental system , otto frank in germany ( 1895 ) and ernest starling ( 1918 ) in england , discovered a crucial mechanism by which the left ventricle senses variations in its filling volume ( i.e. pre - load ) and responds by increasing its stroke volume . this was the first experiment that established a direct relationship between the end - diastolic volume and the end - systolic pressure and has been known , as the frank - starling law of the heart . since then , numerous studies have shown alterations of this relationship to be associated with heart failure . based on these observations , the frank - starling law of the heart constitutes a crucial intrinsic mechanism by which the heart maintains its normal function on a beat - to - beat basis . the technological development has been a pre - requisite in the understanding of cardiac regulation , as demonstrated by langendorff heart system . since the success of the whole heart perfusion apparatus , researchers have developed other experimental systems to explore different myocardium components from trabeculae to single myofibrils . it is of note however , that myocardium mechanical properties significance depends on the sample being used in a given experiment . for instance , a single myofibril preparation is preferred for actin - myosin interaction kinetics measurement . this is because calcium binding to the thin filament significantly reduces the apparent diffusion rate of this ion . therefore , due to the small dimension of the single myofibril , contraction kinetics are not biased by calcium diffusion . trabeculae and papillary muscles , on the other hand , are more appropriate for force - frequency relationship , and slow force response studies . moreover , each preparation type requires a particular hardware setup in respect to its physiological working range . in the past decades , investigators have developed different experimental systems adapted to the various types of cardiac specimens ( trabeculae muscles , papillary muscles , single cardiac cells , and single myofibrils ) . many of them were unsatisfactory , too damaging for the tissue and too difficult to handle and were finally given up . only a few techniques are now used around the world to investigate cardiac contractile properties and these will be discussed in this review . towards this aim , we will be focusing on length dependent activation as an example of a biophysical parameter that can be studied on these types of preparations . according to frank - starling 's law , under healthy conditions , increasing venous return , and therefore the filling pressure of the lv , results in increased stroke volume . figure 1 shows a typical pressure - volume relationship ( fig . 1 ; solid curve ) . following increased end - diastolic volume , the volume of blood ejected is increased beat - by - beat , this curve is shifted to the right and enlarged ( fig . 1 ; dashed curve ) . interestingly , single cardiac cells , single myofibril , trabeculae and papillary muscles all show a positive correlation between sarcomere length and generated tension named length dependent activation ( lda ) . since lda supports the frank - starling law of the heart , unraveling the cellular and sub - cellular mechanisms involved in its regulation may help better understand the frank - starling law alterations observed during physiological and pathophysiological stresses . in this review article , we propose to provide an overview of some experimental approaches employed to investigate biophysical properties of myocardium samples . we will particularly emphasize on how researchers have progressed from studying the frank - starling law at the tissue level ( i.e. trabeculae and papillary muscles ) , to single cell contractile biology , and finally to lda and activation / relaxation dynamics at the single myofibril level . in the intact heart , the parameters measured in vivo such as pressure and volume relate to the parameters of force , length or velocity measured in less complex preparations such as cardiac multicellular preparations . papillary muscles consist of major muscular trunks from which an average of six cordae tendinae project ( fig . trabeculae muscles are divided into two types : the trabeculae carneae and the trabeculae septomarginalis . in the ventricle the trabeculae carneae cover the inside of the ventricle and are the structures used for research purposes of mechanical investigation . accordingly , in this review , the word trabeculae will be referring to the trabeculae carneae . trabeculae and papillary muscles offer a complete cardiac multicellular structure where one can measure the developed active and passive properties under different conditions . in these preparations , lda can be estimated by acquiring developed tension while varying sarcomere length . due to muscle thickness and non - uniformity , researchers had difficulties in acquiring accurate and dynamic sarcomere length in these preparations . in the mid-1970 's , krueger and pollack employed the laser diffraction technique to achieve proper dynamic sarcomere length measurement on isolated rat papillary muscles . in the early 80 's , based on krueger and pollack 's technique , ter keurs ' group used small trabeculae muscles isolated from the right ventricle of rat heart to estimate lda . in these experiments , trabeculae were mounted on spring - loaded stainless steel clips and force measured with a capacitive force transducer . the entire experimental setup was built on top of a modified inverted microscope to allow simultaneous microscopic and laser diffraction measurement . since these muscles were thick and do not transmit light , a laser diffraction technique was used to estimate sarcomere length . when the muscle is exposed to the laser , it produces a particular diffraction pattern . with a known calibration factor , the intensity distribution of the first diffraction order is used to estimate sarcomere length . the developed isometric twitching force was then correlated to both sarcomere length and extracellular calcium concentration . with this technical approach many new mechanisms were revealed such as the first evidence that calcium release from sarcoplasmic reticulum and calcium binding to the myofilaments could both be responsible for lda regulation . later , allen and kurihara showed that lda is composed of an immediate response ( few milliseconds ) followed by a slower response ( several minutes ) if the stretch is maintained . the early response is due to increase is myofilament calcium sensitivity and the slow force response is due to increased sarcoplasmic reticulum ( sr ) calcium release . since then , the laser diffraction technique has been combined with other experimental techniques to explore additional mechanisms . for instance , along with the x - ray diffraction technique , this experimental procedure has been used to explore the myofilament structural arrangement at different sl 's . with this approach researchers obtained information about the lattice spacing between actin and myosin filaments and even the position of the myosin head in the sarcomere . for instance , based on the fact that a cardiac cell has a constant volume , it has long been thought that stretch would induce cell compression , reduction in interfilament lattice spacing that could increase acto - myosin formation probability and , consequently , contraction . using the x - ray diffraction technique , irving et al . observed that although lattice spacing decreases with length it does not correlate with the extent of developed force . during contraction , at a given sl , the extent of force generated by the myocardium is correlated with the number of active thin filament units ( i.e. troponins ) ; the number of strongly bound cross - bridges ; and to a feedback combination between thin and thick filaments . knowing that each myosin head consumes one atp molecule per cycle , by measuring atp consumption , one can estimate the energy cost for each active contraction . light source and pmt ( photomultiplier tube ) as part of the enzyme - coupled optical unit . the updated system was capable of performing simultaneous measurements of force , sarcomere length , and energy consumption on skinned ( i.e. permeabilized ) trabeculae and papillary muscles . this experimental setup is used to measure how many atp molecules are converted to adp molecules per contraction . the most direct way to estimate this reaction kinetics is to measure the nadh oxidation into nad+ . this oxidation is coupled with conversion of one molecule of atp to one molecule of adp and one inorganic phosphate . knowing that nadh absorbs light at 340 nm , and nad+ does not , one can practically measure the amount of nadh molecules being converted into nad+ by measuring light absorbance at 340 nm . accordingly , this approach revealed that stretch improves weak to strong forces generating cross - bridge formations improving thus , the developed tension . multicellular preparations provided important information for cardiac biophysical contraction , particularly in relation to the role of the collagen extracellular matrix and also for the sub - molecular structure during contraction . however as summarized by brady and garnier , interpretation of data from these preparations is limited by their complicated structure , non - uniformity and attachment - induced alteration . these complexities led to the development of cellular and molecular approaches providing a more simplified system . attaching a single cardiomyocyte ( 20 m thick and 200 m long ) at both ends to tiny needle tips connected to mechanical transducers has been a significant challenge for researchers . the membrane of the cardiomyocytes is very fragile and attaching it to stainless steel needles or glass pipettes can easily cause irreversible damage to the membrane 's structure and/or to the contractile apparatus . moreover , cardiomyocytes are stretch / stress sensitive due to stretch - activated channels ( sac ) . in intact cardiomyocytes , damages or activation of sacs result in perturbation of intracellular calcium homeostasis leading to spontaneous contractions and potentially to cell death . next , we discuss experimental systems developed to measure biophysical parameters on both intact and skinned cardiomyocytes . single intact cardiomyocytes offer a unique stand - alone system that would tolerate variations in physiological calcium concentration . due to their cellular integrity , single intact cardiac cells are useful in cases where simultaneous measurement of electrical , metabolic , and mechanical properties is needed ( see for review ) . in the late 80s and early 90s , different groups tried to attach a single intact cardiomyocyte while preserving its sarcolemmal integrity . these techniques used different strategies : suction micropipettes , silicon glue , impalement pipettes . at that time , the most advanced technique was developed by garnier and le guennec . using carbon fibers , they established an easy to reproduce , yet efficient , method of attaching and stretching a single intact mammalian cardiac cell . later , le guennec 's colleagues employed this technique to simultaneously measure electrical , calcium and mechanical properties of a single intact cell at different cell lengths . below is a brief description of the experimental system used . attachment was performed by approaching a cell membrane with two carbon fibers with different pre - determined compliances . the more compliant fiber ( 80 m/n ) was used to report the developed active and passive forces . whereas , the less compliant ( 4 m/n ) fiber was used for cell positioning and mechanical stretching . this experimental procedure has been commonly known as the carbon fibers technique and has been successfully used by others to investigate biophysical properties of single intact cardiac cells . in this system , the force developed by the cardiomyocyte was determined by following the position of the compliant carbon fiber by optical contrast ; knowing the compliance ( distance / force ) , the force can be estimated . sugiura et al . have published a detailed protocol for single cardiomyocyte attachment procedures using the carbon fiber technique . this technique has been used to study lda in intact myocytes . to this aim , special attention to the adhesion efficiency 3 ) , it is not strong enough to stretch the cell up to the required length for lda evaluation ( 20 % from slack length ) . rather , the cells start detaching at 10 to 15 % stretch from slack length . this may be due to different weaknesses including : the attachment cell - fiber capacity itself that is too weak to support such forces , the stretch - induced damages on the membrane at the level of the attachment , or to stretch activated channels and calcium entry . consequently , researchers have put more effort into investigation of other attachment alternatives to counter balance these weaknesses . recently , a biological adhesive known as myotak has been used to attach intact cardiac and skeletal myocytes . this technique uses glass micro - rods coated with the myotak to attach single cardiac cells . when compared to the carbon fibers technique , . this may be due to the low compliance of glass micro - rods along with better signal processing units used with the myotak and the stronger adhesion between the cell membrane and the micro - rods . it is worth noting however , that although efficient , this technique is relatively new and suffers from a lower success rate . despite the advantages and inconveniences of both methods , various studies have employed them to investigate the effect of stretch on cellular contractility . confirming results in multicellular preparations , stretch of isolated myocytes has been shown to induce an immediate increase of passive and active tensions , the latter being attributed to improvement of myofilament responsiveness to calcium . given that , the early force response appears to be independent of calcium transient amplitude . however , it would be interesting to investigate whether calcium homeostasis is involved in the slow force response observed on multicellular specimens discussed earlier . unfortunately , due to the limitations previously discussed , researchers have not been able to record forces over a longer period of time ( i.e. several minutes ) on a single intact cardiomyocyte at higher sl . indeed , in the case of weak attachment at long sarcomere length ( 2.1 m ) , each active twitch causes the cell to slightly detach from the supporting tips . this phenomenon can be clearly visible on the force recording that shows sudden force drops . future improvement of single cell attachment procedures will certainly help investigating contraction properties over longer periods of time and at longer sarcomere length . in an intact preparation the amplitude of contraction is influenced by several factors such as action potential properties , calcium homeostasis , myofilament properties , intracellular ph , and reactive oxygen species production . to limit the influence of these parameters on contraction , the myocytes has been transformed and permeabilized to control the intracellular environment of the myofilaments . to this aim , researchers have developed the skinned cell experimental procedure ( fig . first approaches used to remove the cell membrane were based on a challenging mechanical procedure . this technique consists of approaching the sarcolemma with pulled glass pipettes , and to gently tear it off the cell ; hence , the challenging aspect . it consists of incubating myocardium specimens in a solution containing a detergent ( i.e. triton x-100 ) . given its reproducibility and ease , the later approach has gained popularity in the field . once skinned , the cardiomyocyte is exposed to custom - made intracellular solutions , containing various known calcium concentration , to activate the myofilaments and steady state developed tensions were measured . the resulted tension-[ca ] relationship can be fitted to a hill equation and the myofilament active properties estimated . to attach a skinned cardiomyocyte to a force transducer and motor , the first is expanding polyurethane foam called great stuff manufactured by the dow chemical company that may not be so great for the cell due to the release of toxic solvents during the polymerization phase . the other is silicone - based glue manufactured by dow corning corp classically in acetic acid solvent used as aquarium sealant . finally , the group of vassort has developed a non - injurious approach based on , uv - curing , optical adhesive . experiments based on skinned cardiomyocytes attachment are used to estimate myofilament passive and active properties such as calcium sensitivity , maximal active force , and hill coefficient . myofilament sensitivity to calcium is derived from the pca50 ( -log [ ca ] ) which is the calcium concentrations producing half maximal active tension . by evaluating the pca50 at short ( 1.9 m ) and long ( 2.3 m ) sarcomere length pca50 at short sl . in skinned cardiac cells , pca50 is the main parameter upon which lda is estimated . increased lda is manifested by a leftward shift of the force - pca curve following stretch to lower calcium concentrations and upward to higher generated force ( fig . experimentations performed on a single cardiac cell ( intact and skinned ) were helpful to specifically study a region within the lv thus unraveling specific mechanical properties across the left ventricular wall . in addition to the spatial heterogeneity ( i.e. fiber orientation ) , a mechanical heterogeneity spreads across the left ventricle from the epicardium ( i.e. the lv outer layer ) to the endocardium ( i.e. the lv inner layer ) ( fig . 6 ; solid line ) . using a similar experimental system , previous studies have demonstrated that alterations of this transmural mechanical heterogeneity are associated with heart failure . interestingly , heart failure eliminates this heterogeneity mainly by altering the endocardium contractility ( fig . this property can be used by researchers to elaborate on treatment that specifically targets the altered endocardium ( fig . although highly specific in terms of biophysical mechanics investigation , single skinned cardiomyocytes can not answer all the questions related to the myofilament contractility . for instance , questions such as how fast the myofilaments activate / deactivate can not be accurately addressed with this type of preparation . the main barrier to this is the calcium diffusion rate that induces a bias on the kinetics data . accordingly , an isolated single myofibril is preferred to perform myofilament activation / deactivation kinetics . a cardiomyocyte is composed of a multiple myofibrils that contracts together to produce global cell contraction . due to their size , myofibrils are ideal to measure rapid myofilaments activation / deactivation kinetics ( i.e. in a millisecond range ) . recently , mateja and detombe showed , in a mammalian single myofibril , that the rapid phase of myofilament activation develops within 5 milliseconds of calcium infusion . this experiment would n't be feasible on a single cardiomyocyte due to longer calcium diffusion phenomena . , the myofibril appears as a uniform alternation of dark ( a - band ) and light ( i - band ) bands that result in a striated appearance . the alternation between the a and i bands is used to estimate sarcomere length . due to their size and force ranges , researchers have developed specific experimental systems to isolate and collect mechanical information on mammalian myofibrils . similar to single cardiomyocytes , one tip is employed to apply mechanical strains ( low compliance ) the other tip is used to report developed force ( high compliance ) . each barrel contains either activation ( high calcium ) or relaxation ( low calcium ) solutions ( see blue and red flow jets on fig . 8) . the myofibril is then exposed to either solution at a given time . the rapid solution switching , together with the double barrel pipette , help reduce any unwanted limitation on myofilament contraction kinetics that might be due to calcium ion diffusion . by applying this experimental protocol , the acquired force will be mostly the result of mechanical properties inherent within the contractile apparatus itself . hence , most early hf treatments are based on -adrenergic receptor blockers and angiotensin converting enzyme ( ace ) inhibitors . these agents , although efficient to some extent , suffer from their non - specific actions . indeed , heart failure might originate from specific sub - cellular components such as the contractile machinery ( see for review ) . development of experimental systems has helped investigators conduct studies on the specific impacts of heart failure relating to myofilament biophysical properties . accordingly , researchers found that contractile protein mutations , post - transcriptional contractile protein modifications ( i.e. phosphorylation , nitrosylation , glutathionylation , and oxidation ) , and isoform expression may all induce organ - level heart failure through myofilament apparatus alteration ( see for review ) . consequently , researchers have oriented their efforts toward the discovery of drugs that specifically improve altered myofilament contractility during heart failure . thus , a whole new class of drugs known as calcium sensitizers has been discovered . examples of these drugs include levosimendan ; pimobendan ; emd-57033 ; mci-154 ; and sr33805 . most of these drugs have been tested using similar systems to the one described in this review and have been shown to improve myocardium function by specifically altering myofilament sensitivity to calcium , hence , the term calcium sensitizers . experimental system developments are important steps in fundamental disease mechanisms understanding as well as drug discovery . in this review , we provided an overview of some experimental systems employed to investigate biophysical properties of myocardium samples . we particularly emphasized on how researchers have progressed from studying the frank - starling law at the whole organ level , to single cell contractile biology , and finally to length dependent activation and activation / relaxation dynamics at the single myofibril level . by employing these systems , one can study contractile properties in various regions of the lv muscle , and this has led to the discovery of a gradient of contractility across the left ventricle wall . as discussed earlier , cardiomyocytes isolated from the inner layer of the lv ( i.e. endocardium(endo ) ) have different biophysical properties as the ones isolated from the outer layer ( i.e. epicardium ( epi ) ) . that established a contractile heterogeneity that can only be evaluated at the single cardiac cell level . interestingly , during heart failure , these contractile heterogeneities disappear mainly because of reduced lda in the endo layer . these findings may be of great help in specific drug synthesis to specifically improve the endo altered lda . consequently , the contraction heterogeneity across the left ventricle free wall can be restored and heart failure improved . similarly , we have previously shown that physiological treatment ( i.e. exercise training ) , improves global heart pump function by specifically restoring altered lda in endo layer . this constitutes a proof of concept showing that the heart pump dysfunction can , indeed , be reversed by specifically acting upon the altered endo layer . to conduct cutting edge scientific research , medical investigators and engineers must always push the limits of what can be measured and how these measurements can be accomplished . this review shows how researchers have progressed from studying the frank - starling law on the whole organ , to length dependent activation on a single myofibril . despite their importance in disease diagnostic , cardiac biophysical exploration systems still suffer from their luck of popularity . due to their complexity and time requirement it would be of great interest to orient these systems development toward a more clinical approach .

the heart is subject to multiple sources of stress . to maintain its normal function , and successfully overcome these stresses , heart muscle is equipped with fine - tuned regulatory mechanisms . some of these mechanisms are inherent within the myocardium itself and are known as intrinsic mechanisms . over a century ago , otto frank and ernest starling described an intrinsic mechanism by which the heart , even ex vivo , regulates its function on a beat - to - beat basis . according to this phenomenon , the higher the ventricular filling is , the bigger the stroke volume . thus , the frank - starling law establishes a direct relationship between the diastolic and systolic function of the heart . to observe this biophysical phenomenon and to investigate it , technologic development has been a pre - requisite to scientific knowledge . it allowed for example to observe , at the cellular level , a frank - starling like mechanism and has been termed : length dependent activation ( lda).in this review , we summarize some experimental systems that have been developed and are currently still in use to investigate cardiac biophysical properties from the whole heart down to the single myofibril . as a scientific support , investigation of the frank - starling mechanism will be used as a case study .

Introduction Multicellular preparation (trabeculae and papillary muscles) Single cardiomyocyte preparations Intact cardiomyocyte Mechanically and chemically permeabilized (i.e. skinned) cells Single myofibril Clinical implications Concluding remarks

in 1866 , carl ludwig and elias cyon , decided to take up the challenge to develop the first ex - vivo beating whole heart system . several minutes ) whilst isolated from the body . this major discovery has motivated other investigators to develop a variety of excised whole heart experimental apparatus . to date , the langendorff heart is by far the most famous heart perfusion setup that is still being used in laboratories . by using his system , oscar langendorff , in the 19 century , was able to maintain a perfused mammalian heart in a complete isolated state for several hours . by taking advantage of a similar experimental system , otto frank in germany ( 1895 ) and ernest starling ( 1918 ) in england , discovered a crucial mechanism by which the left ventricle senses variations in its filling volume ( i.e. pre - load ) and responds by increasing its stroke volume . this was the first experiment that established a direct relationship between the end - diastolic volume and the end - systolic pressure and has been known , as the frank - starling law of the heart . based on these observations , the frank - starling law of the heart constitutes a crucial intrinsic mechanism by which the heart maintains its normal function on a beat - to - beat basis . the technological development has been a pre - requisite in the understanding of cardiac regulation , as demonstrated by langendorff heart system . since the success of the whole heart perfusion apparatus , researchers have developed other experimental systems to explore different myocardium components from trabeculae to single myofibrils . it is of note however , that myocardium mechanical properties significance depends on the sample being used in a given experiment . for instance , a single myofibril preparation is preferred for actin - myosin interaction kinetics measurement . this is because calcium binding to the thin filament significantly reduces the apparent diffusion rate of this ion . therefore , due to the small dimension of the single myofibril , contraction kinetics are not biased by calcium diffusion . trabeculae and papillary muscles , on the other hand , are more appropriate for force - frequency relationship , and slow force response studies . moreover , each preparation type requires a particular hardware setup in respect to its physiological working range . in the past decades , investigators have developed different experimental systems adapted to the various types of cardiac specimens ( trabeculae muscles , papillary muscles , single cardiac cells , and single myofibrils ) . many of them were unsatisfactory , too damaging for the tissue and too difficult to handle and were finally given up . only a few techniques are now used around the world to investigate cardiac contractile properties and these will be discussed in this review . towards this aim , we will be focusing on length dependent activation as an example of a biophysical parameter that can be studied on these types of preparations . according to frank - starling 's law , under healthy conditions , increasing venous return , and therefore the filling pressure of the lv , results in increased stroke volume . figure 1 shows a typical pressure - volume relationship ( fig . 1 ; solid curve ) . following increased end - diastolic volume , the volume of blood ejected is increased beat - by - beat , this curve is shifted to the right and enlarged ( fig . interestingly , single cardiac cells , single myofibril , trabeculae and papillary muscles all show a positive correlation between sarcomere length and generated tension named length dependent activation ( lda ) . since lda supports the frank - starling law of the heart , unraveling the cellular and sub - cellular mechanisms involved in its regulation may help better understand the frank - starling law alterations observed during physiological and pathophysiological stresses . in this review article , we propose to provide an overview of some experimental approaches employed to investigate biophysical properties of myocardium samples . we will particularly emphasize on how researchers have progressed from studying the frank - starling law at the tissue level ( i.e. trabeculae and papillary muscles ) , to single cell contractile biology , and finally to lda and activation / relaxation dynamics at the single myofibril level . in the intact heart , the parameters measured in vivo such as pressure and volume relate to the parameters of force , length or velocity measured in less complex preparations such as cardiac multicellular preparations . in the ventricle the trabeculae carneae cover the inside of the ventricle and are the structures used for research purposes of mechanical investigation . accordingly , in this review , the word trabeculae will be referring to the trabeculae carneae . trabeculae and papillary muscles offer a complete cardiac multicellular structure where one can measure the developed active and passive properties under different conditions . in the early 80 's , based on krueger and pollack 's technique , ter keurs ' group used small trabeculae muscles isolated from the right ventricle of rat heart to estimate lda . since these muscles were thick and do not transmit light , a laser diffraction technique was used to estimate sarcomere length . when the muscle is exposed to the laser , it produces a particular diffraction pattern . with a known calibration factor , the intensity distribution of the first diffraction order is used to estimate sarcomere length . the developed isometric twitching force was then correlated to both sarcomere length and extracellular calcium concentration . with this technical approach many new mechanisms were revealed such as the first evidence that calcium release from sarcoplasmic reticulum and calcium binding to the myofilaments could both be responsible for lda regulation . since then , the laser diffraction technique has been combined with other experimental techniques to explore additional mechanisms . for instance , along with the x - ray diffraction technique , this experimental procedure has been used to explore the myofilament structural arrangement at different sl 's . with this approach researchers obtained information about the lattice spacing between actin and myosin filaments and even the position of the myosin head in the sarcomere . using the x - ray diffraction technique , irving et al . during contraction , at a given sl , the extent of force generated by the myocardium is correlated with the number of active thin filament units ( i.e. troponins ) ; the number of strongly bound cross - bridges ; and to a feedback combination between thin and thick filaments . knowing that each myosin head consumes one atp molecule per cycle , by measuring atp consumption , one can estimate the energy cost for each active contraction . light source and pmt ( photomultiplier tube ) as part of the enzyme - coupled optical unit . the updated system was capable of performing simultaneous measurements of force , sarcomere length , and energy consumption on skinned ( i.e. permeabilized ) trabeculae and papillary muscles . this experimental setup is used to measure how many atp molecules are converted to adp molecules per contraction . knowing that nadh absorbs light at 340 nm , and nad+ does not , one can practically measure the amount of nadh molecules being converted into nad+ by measuring light absorbance at 340 nm . accordingly , this approach revealed that stretch improves weak to strong forces generating cross - bridge formations improving thus , the developed tension . multicellular preparations provided important information for cardiac biophysical contraction , particularly in relation to the role of the collagen extracellular matrix and also for the sub - molecular structure during contraction . however as summarized by brady and garnier , interpretation of data from these preparations is limited by their complicated structure , non - uniformity and attachment - induced alteration . these complexities led to the development of cellular and molecular approaches providing a more simplified system . attaching a single cardiomyocyte ( 20 m thick and 200 m long ) at both ends to tiny needle tips connected to mechanical transducers has been a significant challenge for researchers . the membrane of the cardiomyocytes is very fragile and attaching it to stainless steel needles or glass pipettes can easily cause irreversible damage to the membrane 's structure and/or to the contractile apparatus . moreover , cardiomyocytes are stretch / stress sensitive due to stretch - activated channels ( sac ) . next , we discuss experimental systems developed to measure biophysical parameters on both intact and skinned cardiomyocytes . single intact cardiomyocytes offer a unique stand - alone system that would tolerate variations in physiological calcium concentration . due to their cellular integrity , single intact cardiac cells are useful in cases where simultaneous measurement of electrical , metabolic , and mechanical properties is needed ( see for review ) . in the late 80s and early 90s , different groups tried to attach a single intact cardiomyocyte while preserving its sarcolemmal integrity . these techniques used different strategies : suction micropipettes , silicon glue , impalement pipettes . at that time , the most advanced technique was developed by garnier and le guennec . later , le guennec 's colleagues employed this technique to simultaneously measure electrical , calcium and mechanical properties of a single intact cell at different cell lengths . below is a brief description of the experimental system used . attachment was performed by approaching a cell membrane with two carbon fibers with different pre - determined compliances . whereas , the less compliant ( 4 m/n ) fiber was used for cell positioning and mechanical stretching . this experimental procedure has been commonly known as the carbon fibers technique and has been successfully used by others to investigate biophysical properties of single intact cardiac cells . in this system , the force developed by the cardiomyocyte was determined by following the position of the compliant carbon fiber by optical contrast ; knowing the compliance ( distance / force ) , the force can be estimated . sugiura et al . this technique has been used to study lda in intact myocytes . to this aim , special attention to the adhesion efficiency 3 ) , it is not strong enough to stretch the cell up to the required length for lda evaluation ( 20 % from slack length ) . rather , the cells start detaching at 10 to 15 % stretch from slack length . this may be due to different weaknesses including : the attachment cell - fiber capacity itself that is too weak to support such forces , the stretch - induced damages on the membrane at the level of the attachment , or to stretch activated channels and calcium entry . consequently , researchers have put more effort into investigation of other attachment alternatives to counter balance these weaknesses . recently , a biological adhesive known as myotak has been used to attach intact cardiac and skeletal myocytes . when compared to the carbon fibers technique , . this may be due to the low compliance of glass micro - rods along with better signal processing units used with the myotak and the stronger adhesion between the cell membrane and the micro - rods . despite the advantages and inconveniences of both methods , various studies have employed them to investigate the effect of stretch on cellular contractility . confirming results in multicellular preparations , stretch of isolated myocytes has been shown to induce an immediate increase of passive and active tensions , the latter being attributed to improvement of myofilament responsiveness to calcium . given that , the early force response appears to be independent of calcium transient amplitude . however , it would be interesting to investigate whether calcium homeostasis is involved in the slow force response observed on multicellular specimens discussed earlier . unfortunately , due to the limitations previously discussed , researchers have not been able to record forces over a longer period of time ( i.e. several minutes ) on a single intact cardiomyocyte at higher sl . indeed , in the case of weak attachment at long sarcomere length ( 2.1 m ) , each active twitch causes the cell to slightly detach from the supporting tips . this phenomenon can be clearly visible on the force recording that shows sudden force drops . future improvement of single cell attachment procedures will certainly help investigating contraction properties over longer periods of time and at longer sarcomere length . in an intact preparation the amplitude of contraction is influenced by several factors such as action potential properties , calcium homeostasis , myofilament properties , intracellular ph , and reactive oxygen species production . to limit the influence of these parameters on contraction , the myocytes has been transformed and permeabilized to control the intracellular environment of the myofilaments . to this aim , researchers have developed the skinned cell experimental procedure ( fig . first approaches used to remove the cell membrane were based on a challenging mechanical procedure . this technique consists of approaching the sarcolemma with pulled glass pipettes , and to gently tear it off the cell ; hence , the challenging aspect . triton x-100 ) . given its reproducibility and ease , the later approach has gained popularity in the field . once skinned , the cardiomyocyte is exposed to custom - made intracellular solutions , containing various known calcium concentration , to activate the myofilaments and steady state developed tensions were measured . the resulted tension-[ca ] relationship can be fitted to a hill equation and the myofilament active properties estimated . to attach a skinned cardiomyocyte to a force transducer and motor , the first is expanding polyurethane foam called great stuff manufactured by the dow chemical company that may not be so great for the cell due to the release of toxic solvents during the polymerization phase . the other is silicone - based glue manufactured by dow corning corp classically in acetic acid solvent used as aquarium sealant . finally , the group of vassort has developed a non - injurious approach based on , uv - curing , optical adhesive . experiments based on skinned cardiomyocytes attachment are used to estimate myofilament passive and active properties such as calcium sensitivity , maximal active force , and hill coefficient . myofilament sensitivity to calcium is derived from the pca50 ( -log [ ca ] ) which is the calcium concentrations producing half maximal active tension . increased lda is manifested by a leftward shift of the force - pca curve following stretch to lower calcium concentrations and upward to higher generated force ( fig . experimentations performed on a single cardiac cell ( intact and skinned ) were helpful to specifically study a region within the lv thus unraveling specific mechanical properties across the left ventricular wall . in addition to the spatial heterogeneity ( i.e. fiber orientation ) , a mechanical heterogeneity spreads across the left ventricle from the epicardium ( i.e. the lv outer layer ) to the endocardium ( i.e. the lv inner layer ) ( fig . 6 ; solid line ) . interestingly , heart failure eliminates this heterogeneity mainly by altering the endocardium contractility ( fig . this property can be used by researchers to elaborate on treatment that specifically targets the altered endocardium ( fig . although highly specific in terms of biophysical mechanics investigation , single skinned cardiomyocytes can not answer all the questions related to the myofilament contractility . for instance , questions such as how fast the myofilaments activate / deactivate can not be accurately addressed with this type of preparation . the main barrier to this is the calcium diffusion rate that induces a bias on the kinetics data . accordingly , an isolated single myofibril is preferred to perform myofilament activation / deactivation kinetics . a cardiomyocyte is composed of a multiple myofibrils that contracts together to produce global cell contraction . recently , mateja and detombe showed , in a mammalian single myofibril , that the rapid phase of myofilament activation develops within 5 milliseconds of calcium infusion . this experiment would n't be feasible on a single cardiomyocyte due to longer calcium diffusion phenomena . , the myofibril appears as a uniform alternation of dark ( a - band ) and light ( i - band ) bands that result in a striated appearance . the alternation between the a and i bands is used to estimate sarcomere length . due to their size and force ranges , researchers have developed specific experimental systems to isolate and collect mechanical information on mammalian myofibrils . similar to single cardiomyocytes , one tip is employed to apply mechanical strains ( low compliance ) the other tip is used to report developed force ( high compliance ) . each barrel contains either activation ( high calcium ) or relaxation ( low calcium ) solutions ( see blue and red flow jets on fig . the myofibril is then exposed to either solution at a given time . the rapid solution switching , together with the double barrel pipette , help reduce any unwanted limitation on myofilament contraction kinetics that might be due to calcium ion diffusion . by applying this experimental protocol , the acquired force will be mostly the result of mechanical properties inherent within the contractile apparatus itself . indeed , heart failure might originate from specific sub - cellular components such as the contractile machinery ( see for review ) . development of experimental systems has helped investigators conduct studies on the specific impacts of heart failure relating to myofilament biophysical properties . accordingly , researchers found that contractile protein mutations , post - transcriptional contractile protein modifications ( i.e. phosphorylation , nitrosylation , glutathionylation , and oxidation ) , and isoform expression may all induce organ - level heart failure through myofilament apparatus alteration ( see for review ) . consequently , researchers have oriented their efforts toward the discovery of drugs that specifically improve altered myofilament contractility during heart failure . thus , a whole new class of drugs known as calcium sensitizers has been discovered . examples of these drugs include levosimendan ; pimobendan ; emd-57033 ; mci-154 ; and sr33805 . most of these drugs have been tested using similar systems to the one described in this review and have been shown to improve myocardium function by specifically altering myofilament sensitivity to calcium , hence , the term calcium sensitizers . in this review , we provided an overview of some experimental systems employed to investigate biophysical properties of myocardium samples . we particularly emphasized on how researchers have progressed from studying the frank - starling law at the whole organ level , to single cell contractile biology , and finally to length dependent activation and activation / relaxation dynamics at the single myofibril level . by employing these systems , one can study contractile properties in various regions of the lv muscle , and this has led to the discovery of a gradient of contractility across the left ventricle wall . as discussed earlier , cardiomyocytes isolated from the inner layer of the lv ( i.e. endocardium(endo ) ) have different biophysical properties as the ones isolated from the outer layer ( i.e. that established a contractile heterogeneity that can only be evaluated at the single cardiac cell level . these findings may be of great help in specific drug synthesis to specifically improve the endo altered lda . consequently , the contraction heterogeneity across the left ventricle free wall can be restored and heart failure improved . similarly , we have previously shown that physiological treatment ( i.e. exercise training ) , improves global heart pump function by specifically restoring altered lda in endo layer . this constitutes a proof of concept showing that the heart pump dysfunction can , indeed , be reversed by specifically acting upon the altered endo layer . this review shows how researchers have progressed from studying the frank - starling law on the whole organ , to length dependent activation on a single myofibril . despite their importance in disease diagnostic , cardiac biophysical exploration systems still suffer from their luck of popularity .

this is a complex organ highly regulated by several external systems ( central nervous , neurohormonal ) and by an important internal system . in 1866 , carl ludwig and elias cyon , decided to take up the challenge to develop the first ex - vivo beating whole heart system . this major discovery has motivated other investigators to develop a variety of excised whole heart experimental apparatus . to date , the langendorff heart is by far the most famous heart perfusion setup that is still being used in laboratories . by using his system , oscar langendorff , in the 19 century , was able to maintain a perfused mammalian heart in a complete isolated state for several hours . by taking advantage of a similar experimental system , otto frank in germany ( 1895 ) and ernest starling ( 1918 ) in england , discovered a crucial mechanism by which the left ventricle senses variations in its filling volume ( i.e. this was the first experiment that established a direct relationship between the end - diastolic volume and the end - systolic pressure and has been known , as the frank - starling law of the heart . since then , numerous studies have shown alterations of this relationship to be associated with heart failure . based on these observations , the frank - starling law of the heart constitutes a crucial intrinsic mechanism by which the heart maintains its normal function on a beat - to - beat basis . the technological development has been a pre - requisite in the understanding of cardiac regulation , as demonstrated by langendorff heart system . since the success of the whole heart perfusion apparatus , researchers have developed other experimental systems to explore different myocardium components from trabeculae to single myofibrils . it is of note however , that myocardium mechanical properties significance depends on the sample being used in a given experiment . for instance , a single myofibril preparation is preferred for actin - myosin interaction kinetics measurement . this is because calcium binding to the thin filament significantly reduces the apparent diffusion rate of this ion . therefore , due to the small dimension of the single myofibril , contraction kinetics are not biased by calcium diffusion . trabeculae and papillary muscles , on the other hand , are more appropriate for force - frequency relationship , and slow force response studies . in the past decades , investigators have developed different experimental systems adapted to the various types of cardiac specimens ( trabeculae muscles , papillary muscles , single cardiac cells , and single myofibrils ) . many of them were unsatisfactory , too damaging for the tissue and too difficult to handle and were finally given up . only a few techniques are now used around the world to investigate cardiac contractile properties and these will be discussed in this review . towards this aim , we will be focusing on length dependent activation as an example of a biophysical parameter that can be studied on these types of preparations . according to frank - starling 's law , under healthy conditions , increasing venous return , and therefore the filling pressure of the lv , results in increased stroke volume . following increased end - diastolic volume , the volume of blood ejected is increased beat - by - beat , this curve is shifted to the right and enlarged ( fig . interestingly , single cardiac cells , single myofibril , trabeculae and papillary muscles all show a positive correlation between sarcomere length and generated tension named length dependent activation ( lda ) . since lda supports the frank - starling law of the heart , unraveling the cellular and sub - cellular mechanisms involved in its regulation may help better understand the frank - starling law alterations observed during physiological and pathophysiological stresses . in this review article , we propose to provide an overview of some experimental approaches employed to investigate biophysical properties of myocardium samples . we will particularly emphasize on how researchers have progressed from studying the frank - starling law at the tissue level ( i.e. trabeculae and papillary muscles ) , to single cell contractile biology , and finally to lda and activation / relaxation dynamics at the single myofibril level . in the intact heart , the parameters measured in vivo such as pressure and volume relate to the parameters of force , length or velocity measured in less complex preparations such as cardiac multicellular preparations . papillary muscles consist of major muscular trunks from which an average of six cordae tendinae project ( fig . trabeculae muscles are divided into two types : the trabeculae carneae and the trabeculae septomarginalis . in the ventricle the trabeculae carneae cover the inside of the ventricle and are the structures used for research purposes of mechanical investigation . accordingly , in this review , the word trabeculae will be referring to the trabeculae carneae . trabeculae and papillary muscles offer a complete cardiac multicellular structure where one can measure the developed active and passive properties under different conditions . due to muscle thickness and non - uniformity , researchers had difficulties in acquiring accurate and dynamic sarcomere length in these preparations . in the mid-1970 's , krueger and pollack employed the laser diffraction technique to achieve proper dynamic sarcomere length measurement on isolated rat papillary muscles . in the early 80 's , based on krueger and pollack 's technique , ter keurs ' group used small trabeculae muscles isolated from the right ventricle of rat heart to estimate lda . the entire experimental setup was built on top of a modified inverted microscope to allow simultaneous microscopic and laser diffraction measurement . when the muscle is exposed to the laser , it produces a particular diffraction pattern . with a known calibration factor , the intensity distribution of the first diffraction order is used to estimate sarcomere length . the developed isometric twitching force was then correlated to both sarcomere length and extracellular calcium concentration . with this technical approach many new mechanisms were revealed such as the first evidence that calcium release from sarcoplasmic reticulum and calcium binding to the myofilaments could both be responsible for lda regulation . later , allen and kurihara showed that lda is composed of an immediate response ( few milliseconds ) followed by a slower response ( several minutes ) if the stretch is maintained . the early response is due to increase is myofilament calcium sensitivity and the slow force response is due to increased sarcoplasmic reticulum ( sr ) calcium release . since then , the laser diffraction technique has been combined with other experimental techniques to explore additional mechanisms . for instance , along with the x - ray diffraction technique , this experimental procedure has been used to explore the myofilament structural arrangement at different sl 's . with this approach researchers obtained information about the lattice spacing between actin and myosin filaments and even the position of the myosin head in the sarcomere . for instance , based on the fact that a cardiac cell has a constant volume , it has long been thought that stretch would induce cell compression , reduction in interfilament lattice spacing that could increase acto - myosin formation probability and , consequently , contraction . using the x - ray diffraction technique , irving et al . during contraction , at a given sl , the extent of force generated by the myocardium is correlated with the number of active thin filament units ( i.e. troponins ) ; the number of strongly bound cross - bridges ; and to a feedback combination between thin and thick filaments . knowing that each myosin head consumes one atp molecule per cycle , by measuring atp consumption , one can estimate the energy cost for each active contraction . light source and pmt ( photomultiplier tube ) as part of the enzyme - coupled optical unit . the updated system was capable of performing simultaneous measurements of force , sarcomere length , and energy consumption on skinned ( i.e. this experimental setup is used to measure how many atp molecules are converted to adp molecules per contraction . the most direct way to estimate this reaction kinetics is to measure the nadh oxidation into nad+ . this oxidation is coupled with conversion of one molecule of atp to one molecule of adp and one inorganic phosphate . accordingly , this approach revealed that stretch improves weak to strong forces generating cross - bridge formations improving thus , the developed tension . multicellular preparations provided important information for cardiac biophysical contraction , particularly in relation to the role of the collagen extracellular matrix and also for the sub - molecular structure during contraction . however as summarized by brady and garnier , interpretation of data from these preparations is limited by their complicated structure , non - uniformity and attachment - induced alteration . these complexities led to the development of cellular and molecular approaches providing a more simplified system . attaching a single cardiomyocyte ( 20 m thick and 200 m long ) at both ends to tiny needle tips connected to mechanical transducers has been a significant challenge for researchers . the membrane of the cardiomyocytes is very fragile and attaching it to stainless steel needles or glass pipettes can easily cause irreversible damage to the membrane 's structure and/or to the contractile apparatus . moreover , cardiomyocytes are stretch / stress sensitive due to stretch - activated channels ( sac ) . in intact cardiomyocytes , damages or activation of sacs result in perturbation of intracellular calcium homeostasis leading to spontaneous contractions and potentially to cell death . next , we discuss experimental systems developed to measure biophysical parameters on both intact and skinned cardiomyocytes . single intact cardiomyocytes offer a unique stand - alone system that would tolerate variations in physiological calcium concentration . due to their cellular integrity , single intact cardiac cells are useful in cases where simultaneous measurement of electrical , metabolic , and mechanical properties is needed ( see for review ) . in the late 80s and early 90s , different groups tried to attach a single intact cardiomyocyte while preserving its sarcolemmal integrity . at that time , the most advanced technique was developed by garnier and le guennec . using carbon fibers , they established an easy to reproduce , yet efficient , method of attaching and stretching a single intact mammalian cardiac cell . later , le guennec 's colleagues employed this technique to simultaneously measure electrical , calcium and mechanical properties of a single intact cell at different cell lengths . attachment was performed by approaching a cell membrane with two carbon fibers with different pre - determined compliances . the more compliant fiber ( 80 m/n ) was used to report the developed active and passive forces . whereas , the less compliant ( 4 m/n ) fiber was used for cell positioning and mechanical stretching . this experimental procedure has been commonly known as the carbon fibers technique and has been successfully used by others to investigate biophysical properties of single intact cardiac cells . in this system , the force developed by the cardiomyocyte was determined by following the position of the compliant carbon fiber by optical contrast ; knowing the compliance ( distance / force ) , the force can be estimated . to this aim , special attention to the adhesion efficiency 3 ) , it is not strong enough to stretch the cell up to the required length for lda evaluation ( 20 % from slack length ) . this may be due to different weaknesses including : the attachment cell - fiber capacity itself that is too weak to support such forces , the stretch - induced damages on the membrane at the level of the attachment , or to stretch activated channels and calcium entry . this may be due to the low compliance of glass micro - rods along with better signal processing units used with the myotak and the stronger adhesion between the cell membrane and the micro - rods . it is worth noting however , that although efficient , this technique is relatively new and suffers from a lower success rate . despite the advantages and inconveniences of both methods , various studies have employed them to investigate the effect of stretch on cellular contractility . confirming results in multicellular preparations , stretch of isolated myocytes has been shown to induce an immediate increase of passive and active tensions , the latter being attributed to improvement of myofilament responsiveness to calcium . given that , the early force response appears to be independent of calcium transient amplitude . however , it would be interesting to investigate whether calcium homeostasis is involved in the slow force response observed on multicellular specimens discussed earlier . unfortunately , due to the limitations previously discussed , researchers have not been able to record forces over a longer period of time ( i.e. indeed , in the case of weak attachment at long sarcomere length ( 2.1 m ) , each active twitch causes the cell to slightly detach from the supporting tips . future improvement of single cell attachment procedures will certainly help investigating contraction properties over longer periods of time and at longer sarcomere length . in an intact preparation the amplitude of contraction is influenced by several factors such as action potential properties , calcium homeostasis , myofilament properties , intracellular ph , and reactive oxygen species production . to limit the influence of these parameters on contraction , the myocytes has been transformed and permeabilized to control the intracellular environment of the myofilaments . to this aim , researchers have developed the skinned cell experimental procedure ( fig . first approaches used to remove the cell membrane were based on a challenging mechanical procedure . this technique consists of approaching the sarcolemma with pulled glass pipettes , and to gently tear it off the cell ; hence , the challenging aspect . given its reproducibility and ease , the later approach has gained popularity in the field . once skinned , the cardiomyocyte is exposed to custom - made intracellular solutions , containing various known calcium concentration , to activate the myofilaments and steady state developed tensions were measured . to attach a skinned cardiomyocyte to a force transducer and motor , the first is expanding polyurethane foam called great stuff manufactured by the dow chemical company that may not be so great for the cell due to the release of toxic solvents during the polymerization phase . the other is silicone - based glue manufactured by dow corning corp classically in acetic acid solvent used as aquarium sealant . finally , the group of vassort has developed a non - injurious approach based on , uv - curing , optical adhesive . experiments based on skinned cardiomyocytes attachment are used to estimate myofilament passive and active properties such as calcium sensitivity , maximal active force , and hill coefficient . myofilament sensitivity to calcium is derived from the pca50 ( -log [ ca ] ) which is the calcium concentrations producing half maximal active tension . by evaluating the pca50 at short ( 1.9 m ) and long ( 2.3 m ) sarcomere length pca50 at short sl . in skinned cardiac cells , pca50 is the main parameter upon which lda is estimated . increased lda is manifested by a leftward shift of the force - pca curve following stretch to lower calcium concentrations and upward to higher generated force ( fig . experimentations performed on a single cardiac cell ( intact and skinned ) were helpful to specifically study a region within the lv thus unraveling specific mechanical properties across the left ventricular wall . fiber orientation ) , a mechanical heterogeneity spreads across the left ventricle from the epicardium ( i.e. using a similar experimental system , previous studies have demonstrated that alterations of this transmural mechanical heterogeneity are associated with heart failure . this property can be used by researchers to elaborate on treatment that specifically targets the altered endocardium ( fig . although highly specific in terms of biophysical mechanics investigation , single skinned cardiomyocytes can not answer all the questions related to the myofilament contractility . for instance , questions such as how fast the myofilaments activate / deactivate can not be accurately addressed with this type of preparation . the main barrier to this is the calcium diffusion rate that induces a bias on the kinetics data . due to their size , myofibrils are ideal to measure rapid myofilaments activation / deactivation kinetics ( i.e. recently , mateja and detombe showed , in a mammalian single myofibril , that the rapid phase of myofilament activation develops within 5 milliseconds of calcium infusion . , the myofibril appears as a uniform alternation of dark ( a - band ) and light ( i - band ) bands that result in a striated appearance . due to their size and force ranges , researchers have developed specific experimental systems to isolate and collect mechanical information on mammalian myofibrils . similar to single cardiomyocytes , one tip is employed to apply mechanical strains ( low compliance ) the other tip is used to report developed force ( high compliance ) . by applying this experimental protocol , the acquired force will be mostly the result of mechanical properties inherent within the contractile apparatus itself . hence , most early hf treatments are based on -adrenergic receptor blockers and angiotensin converting enzyme ( ace ) inhibitors . indeed , heart failure might originate from specific sub - cellular components such as the contractile machinery ( see for review ) . development of experimental systems has helped investigators conduct studies on the specific impacts of heart failure relating to myofilament biophysical properties . accordingly , researchers found that contractile protein mutations , post - transcriptional contractile protein modifications ( i.e. phosphorylation , nitrosylation , glutathionylation , and oxidation ) , and isoform expression may all induce organ - level heart failure through myofilament apparatus alteration ( see for review ) . consequently , researchers have oriented their efforts toward the discovery of drugs that specifically improve altered myofilament contractility during heart failure . thus , a whole new class of drugs known as calcium sensitizers has been discovered . most of these drugs have been tested using similar systems to the one described in this review and have been shown to improve myocardium function by specifically altering myofilament sensitivity to calcium , hence , the term calcium sensitizers . experimental system developments are important steps in fundamental disease mechanisms understanding as well as drug discovery . in this review , we provided an overview of some experimental systems employed to investigate biophysical properties of myocardium samples . we particularly emphasized on how researchers have progressed from studying the frank - starling law at the whole organ level , to single cell contractile biology , and finally to length dependent activation and activation / relaxation dynamics at the single myofibril level . by employing these systems , one can study contractile properties in various regions of the lv muscle , and this has led to the discovery of a gradient of contractility across the left ventricle wall . as discussed earlier , cardiomyocytes isolated from the inner layer of the lv ( i.e. endocardium(endo ) ) have different biophysical properties as the ones isolated from the outer layer ( i.e. that established a contractile heterogeneity that can only be evaluated at the single cardiac cell level . these findings may be of great help in specific drug synthesis to specifically improve the endo altered lda . consequently , the contraction heterogeneity across the left ventricle free wall can be restored and heart failure improved . exercise training ) , improves global heart pump function by specifically restoring altered lda in endo layer . this constitutes a proof of concept showing that the heart pump dysfunction can , indeed , be reversed by specifically acting upon the altered endo layer . this review shows how researchers have progressed from studying the frank - starling law on the whole organ , to length dependent activation on a single myofibril .

systemic lupus erythematosus ( sle ) is a systemic autoimmune disease characterized by a loss of tolerance to nuclear antigens and by dysregulated activation of t and b cells . polyclonal activation of b cells leads to the production of large quantities of autoreactive antibodies and the formation of immune complexes , which causes tissue damage . in some sle patients , it has been shown that bone marrow mesenchymal stem cells exhibit impaired capacities for proliferation , differentiation , migration , and immune modulation . genetic defects , drug exposure , infectious agents , and environmental factors can also contribute to the pathogenesis of this disease [ 3 , 4 ] . sle has an incidence in europe and north america of approximately 10 cases per 100,000 population per year , and it is estimated that 10% of these cases are drug - induced . drug - induced lupus erythematosus ( dile ) is a lupus - like syndrome that resolves upon drug discontinuation . the drugs more frequently associated with the induction of this lupus - like syndrome are procainamide ( antiarrhythmic ) , hydralazine ( antihypertensive ) , and chlorpromazine ( antipsychotic ) [ 5 , 6 ] . animal models of sle include lupus - prone mice , which spontaneously develop lupus , and normal mice that develop lupus after injection of lymphocytes from lupus - prone mice , immunization with prototypical lupus antigens ( dna- and rna - protein complexes ) , or injection of pristane ( 2,6,10,14-tetramethylpentadecane ) [ 3 , 7 ] . the most commonly used lupus - prone mice are the f1 hybrids of new zealand black ( nzb ) and nz white ( nzb / nzw f1 ) mice , the murphy - roths large / lymphoproliferative locus ( mlr / lpr ) mice , and the recombinant c57bl/6 female and sb / le male strain / y - linked autoimmune accelerator ( bxsb / yaa ) mice [ 3 , 8 , 9 ] . our group has also developed a mouse model of autoimmune disease resembling human lupus that can be induced in normal mice . in this model , liposomes are model membranes made of cylindrical phospholipids , such as phosphatidylcholine , and hii - preferring ( conical shaped ) phospholipids , such as phosphatidic acid , phosphatidylserine , or cardiolipin . conical phospholipids can form molecular associations distinct to lipid bilayers , known as nonbilayer phospholipid arrangements , in the presence of inducers such as mn [ 12 , 13 ] or the drugs chlorpromazine and procainamide , which can trigger dile in humans . nonbilayer phospholipid arrangements are formed by an inverted micelle ( made of conical phospholipids with their polar heads towards the center of the micelle , where the inducer is also located ) inserted into and distorting the shape of the phospholipid bilayer ( figure 1(a ) ) . we demonstrated that liposomes with nonbilayer phospholipid arrangements induced by mn , chlorpromazine , or procainamide cause an autoimmune disease resembling human lupus in mice . a similar disease is produced by treating mice directly with mn , chlorpromazine , or procainamide ( which induce nonbilayer phospholipid arrangements on mouse cells ) or by injecting the monoclonal antibody h308 ( which binds specifically to nonbilayer phospholipid arrangements and stabilizes these arrangements on mouse cells ) [ 10 , 14 ] . igm and igg antibodies against nonbilayer phospholipid arrangements are found in the sera of mice with the autoimmune disease resembling human lupus , and also in the sera of patients with lupus [ 10 , 15 ] . usually , the efficient production of igg antibodies requires an activation of the innate immune response . therefore we hypothesized that nonbilayer phospholipid arrangements could be toll - like receptor- ( tlr- ) 4/md-2 agonists , as their molecular structure is similar to that of the lipid a from bacterial lipopolysaccharide ( lps ) . lipid a is formed by a -1,6-d - glucosamine disaccharide with two ( negatively charged ) phosphates and six saturated acyl chains in an asymmetric distribution ( four chains are bound to the nonreducing and two to the reducing glucosamine ) . hexaacylated asymmetric lipid a molecules have a conical molecular shape , because the cross section of the hydrophobic region is larger than that of the hydrophilic region ( figure 1(b ) ) . hexaacylated symmetric lipid a ( with three acyl chains bound to the nonreducing and three to the reducing glucosamine ) and penta- and tetraacylated lipid a molecules have a cylindrical molecular shape , and they do not have biological activity [ 16 , 17 ] . the intrinsic conformation of lipid a is not altered when saccharide groups are added , as in lps . the lps molecules form multimeric aggregates in water : if the lipid a is cylindrical , they form a smooth bilayer arrangement , but conical lipid a molecules form a nonbilayer or hexagonal ( hii ) arrangement . lps - binding protein ( lbp ) is a plasma protein that facilitates the transfer of lps molecules from these hexagonal ( hii ) arrangements to cd14 , and membrane - bound cd14 delivers lps to tlr-4/md2 . since the conical molecular shape of lipid a is a requirement for tlr-4/md-2 triggering [ 1619 ] , we hypothesized that liposomes with nonbilayer phospholipid arrangements , but not smooth liposomes ( with phospholipids in a bilayer arrangement ) , could trigger tlr-4/md-2 signaling . in this study , we investigated whether liposomes with nonbilayer phospholipid arrangements are tlr-4/md-2 agonists , because the activation of this innate immune receptor leads to the production of proinflammatory cytokines . we also looked for proinflammatory cytokines in the sera of mice with the autoimmune disease triggered by liposomes with nonbilayer phospholipid arrangements , and we determined the gene expression profile in the spleens of these mice , focusing on the expression of proinflammatory genes . in addition , we determined the relative percentage and activation of t , nkt , dendritic , and b cells in the spleen of mice with the disease . this study contributes to the understanding of the pathological and genetic features of a novel mouse model of human lupus . egg - yolk l--phosphatidic acid , bovine brain l--phosphatidylserine , egg - yolk l--phosphatidylcholine , chlorpromazine , procainamide , and chloroquine were purchased from sigma ( st . louis , mo , usa ) . liposomes contained the cylindrical shaped phospholipid phosphatidylcholine and a conical phospholipid ( phosphatidic acid or phosphatidylserine ) . the molar ratios ( phosphatidylcholine / phosphatidic acid 2 : 1 , phosphatidylcholine / phosphatidylserine 4 : 1 ) were optimized for the induction of nonbilayer phospholipid arrangements . nine micromoles of phospholipid mixture was dissolved in 1 ml diethyl ether and 330 l of ts buffer ( 10 mm tris - hcl , 1 mm nacl , ph 7 ) , mixed and sonicated three times in a g112spi sonicator ( laboratory supplies , hicksville , ny , usa ) . the diethyl ether was then removed under a stream of oxygen - free dry nitrogen at reduced pressure , using a rotary evaporator at 37c . the liposomes were filtered through 0.45 m mf - millipore membranes ( billerica , ma , usa ) to homogenize their size . to induce the formation of nonbilayer phospholipid arrangements , liposomes in ts buffer were incubated for 30 min at 37c in the presence of 0.54 mm mncl2 , 0.53 mm chlorpromazine , and 432 mm procainamide . all of the final preparations of liposomes were negative for lps contamination , as assessed by the gel clot lal method ( charles river endosafe , charleston , sc , usa ) . the detection of nonbilayer phospholipid arrangements by flow cytometry was previously validated by freeze - fracture electron microscopy and p - nmr spectroscopy [ 10 , 14 , 15 ] . therefore , in this study we only used flow cytometry to demonstrate the formation of these arrangements on liposomes . liposomes and liposomes with nonbilayer phospholipid arrangements in ts buffer were analyzed with a facscalibur flow cytometer ( becton dickinson , san jose , ca , usa ) with cellquest software . human embryonic kidney ( hek ) 293 cells , nontransfected or stably transfected with human tlr-4/md2/cd14 , tlr-2/tlr-6 , tlr-5 , or tlr-8 , were purchased from invivogen ( san diego , ca , usa ) . the hek - tlr transfectants were maintained at 37c in 5% co2 in dulbecco 's modified eagle 's medium ( invitrogen , carlsbad , ca , usa ) containing 4.5 g / l glucose , 10% heat - inactivated fetal bovine serum ( gibco , grand island , ny , usa ) , 10 g / ml blasticidin ( invivogen ) , and 100 g / ml normocin ( invivogen ) . hygrogold ( 25 g / ml ; invivogen ) was also added to the media of the hek - tlr-4/md2/cd14 cell line . the viability of these cell lines in the presence of mn , chlorpromazine , procainamide , or chloroquine , and in the presence of liposomes or liposomes with nonbilayer phospholipid arrangements , was evaluated with the alamar blue method . to assess tlr activation , the cell lines were incubated in the presence of liposomes made of phosphatidylcholine / phosphatidic acid ( 2 : 1 ) , alone or with nonbilayer phospholipid arrangements induced by mn ( 24 mm ) . as a negative control , the liposomes with nonbilayer arrangements were previously incubated with 0.1 mm chloroquine . for the positive controls , the cell lines were incubated in the presence of their known tlr agonists : 100 ng / ml escherichia coli 0111:b4 lps for hek - tlr-4/md2/cd14 , 1 g / ml fsl-1 ( a synthetic lipoprotein derived from mycoplasma salivarium ) for hek - tlr-2/tlr-6 , 1 g / ml salmonella typhimurium flagellin for hek - tlr-5 , and 2.5 g / ml ssrna40 ( a 20 mer phosphorothioate - protected single - stranded rna oligonucleotide containing a gu - rich sequence ) for hek - tlr-8 . all tlr agonists were sourced from invivogen . after 24 h , the cell culture supernatants were harvested and assayed for il-8 production ( bd opteia set human il-8 , bd biosciences , san diego , ca , usa ) . nf-b activation was assayed in cell culture extracts using the reporter plasmid pnifty - luc ( promega corporation , madison , wi , usa ) . in order to determine if chloroquine affects the viability of hek293 cells , the live / dead fixable violet dead cell stain kit ( invitrogen ) was used . hek293 cells were incubated with 0.05 , 0.1 , and 0.5 mm of chloroquine for 24 h at 37c and 5% co2 . the cells were then transferred to a tube and stained with 50 l of live / dead diluted 1 : 100 in distilled water and incubated for 15 min at room temperature in the dark . facs lysis buffer ( 1 ml ; becton dickinson ) was added for erythrocyte lysis , and the cells were incubated for 10 min at room temperature in the dark . the cells were washed with 2 ml of phosphate - buffered saline ( pbs ) and resuspended in 300 l of pbs and analyzed by flow cytometry . forty thousand events were acquired for each sample with a lsr fortessa cytometer ( becton - dickinson ) . to evaluate whether chloroquine can induce apoptosis of hek293 cells , the annexin v - propidium iodide staining method was used . hek293 cells were incubated with 0.05 , 0.1 , and 0.5 mm of chloroquine for 24 h at 37c and 5% co2 . the cells were then transferred to a tube and washed with 1 ml of annexin v - binding buffer ( ebioscience , san diego , ca , usa ) . 100 l of 2 g / ml annexin v - apc ( ebioscience ) in annexin v - binding buffer was added , and the cells were incubated for 15 min at room temperature in the dark . the cells were washed with 1 ml of annexin v - binding buffer , resuspended in 100 l of the same buffer containing 1 g of propidium iodide ( biolegend , san diego ca , usa ) and incubated for 15 min at room temperature in the dark . the cells were washed and resuspended in the annexin v - binding buffer and analyzed immediately by flow cytometry . forty thousand events were acquired for each sample in a lsr fortessa cytometer ( becton - dickinson ) . tlr stimulation was also analyzed in bone marrow - derived macrophages ( bmdm ) from balb / c mice . bmdm were obtained from the femur and shinbone of female 2-month - old balb / c mice and they were cultured in rpmi media with 10% heat - inactivated fetal bovine serum , 50 u / ml penicillin ( gibco ) , 50 g / ml streptomycin ( gibco ) , and 10 ng / ml recombinant m - csf ( biolegend ) for 7 days . for tlr stimulation , the bmdm were cultured in 96-well plates and stimulated with 10 ng / ml lps , 1 g / ml peptidoglycan ( pgn ; invivogen ) , or 10 or 20 l of smooth liposomes or liposomes bearing nonbilayer phospholipids arrangements , respectively . after incubation for 24 h at 37c , the supernatants were collected and analyzed by enzyme - linked immunoabsorbent assay ( elisa ; biolegend ) for tumor necrosis factor- ( tnf- ) production . for the blocking experiments , 10 g / ml of anti - tlr-2 ( clone t2.5 , biolegend ) or 20 g / ml of anti - tlr-4 ( clone mts510 , biolegend ) was added 2 h before the liposomes . the cells were incubated for 24 h at 37c and the supernatants were collected and analyzed for tnf-. igg1, ( clone mopc-21 , biolegend ) and igg2a, ( clone rtk2758 , biolegend ) isotype controls were used for the blocking antibodies . forty female 2-month - old specific - pathogen - free balb / c mice were divided into four groups . the first and the second groups were injected intrasplenically , on days 1 and 15 , with phosphatidylcholine / phosphatidic acid ( 2 : 1 ) liposomes that had been incubated with 5 mm mncl2 ( mn group ) or 3 mm chlorpromazine ( cpz group ) . mice received the same amount of liposomes by intraperitoneal injection on day 30 and then every week for 6 months . the negative control groups consisted of 10 mice treated in the same way but using ts buffer alone ( control group i ) , or liposomes made of phosphatidylcholine / phosphatidic acid ( 2 : 1 ) alone ( control group ii ) . blood was taken from mice before liposome injection and each month after the first intraperitoneal injection , for a total of 6 months . sera were heated at 56c for 30 min to inactivate complement and frozen in aliquots at 70c . to confirm that these mice developed the disease resembling human lupus , we measured anti - nonbilayer phospholipid arrangements , anti - cardiolipin , anti - histone , and anti - coagulant antibodies in their sera . anti - nonbilayer phospholipid arrangements antibodies were measured by elisa where the wells were coated with liposomes with or without nonbilayer phospholipid arrangements . results are reported as arbitrary units ( au ) calculated as ( asp asw)/(ash asw ) , where asp is the absorbance obtained with the sera of mice injected with the liposomes , ash is the absorbance obtained with the sera of mice before the injection of liposomes , and asw is absorbance of controls without sera . a modification of the kaolin - activated thromboplastin time test was used to determine the anti - coagulant antibodies ; results are reported as the coagulation time in seconds . three mice from each of the four groups indicated above were euthanized 4 months after the first injection of nonbilayer phospholipid arrangements , when they had the highest titers of anti - nonbilayer phospholipid arrangements , anti - cardiolipin , anti - histone , and anti - coagulant antibodies , and their spleens were used for gene and protein expression studies . the experimental protocols for animal care and use were reviewed and approved by the bioethics committee of our institution according to the guide for the care and use of laboratory animals , which was published by the us national institute of health . the serum concentrations of interleukin-6 ( il-6 ) , il-10 , il-12p70 , interferon- ( ifn- ) , tnf- , and monocyte chemoattractant protein- ( mcp- ) 1 were measured with a bead - based multiplex immunoassay ( bd cba mouse inflammation kit ) . mouse spleens were sectioned and placed in two cryotubes , one with rnalater ( invitrogen ) for rna expression studies and one with tissue - tek ( sakura finetek , torrance , ca , usa ) for protein analysis . the cryotubes were stored at 70c until use . to isolate rna , the tissue stored in rnalater was thawed and disaggregated at 15,000 rpm with a tissueruptor ( qiagen , valencia , ca , usa ) , and total rna was extracted from the tissue homogenates using an rneasy mini kit ( qiagen ) . the quality and quantity of the rna samples were assessed in an agilent bioanalyzer 2100 ( agilent , palo alto , ca , usa ) and a nanodrop 2000 ( thermo fisher scientific , auburn , al , usa ) , respectively ; only rna samples with a rna integrity number ( rin ) 7 were used for the gene expression analysis . total rna ( 400 ng ) was amplified and labeled using the quick amp labeling kit ( agilent ) , and the cyanine-3- or cyanine-5-labeled crna was purified with an rneasy mini kit ( qiagen ) . the crna were hybridized to 4 44 k whole mouse genome microarray chips ( agilent , g4122f ) ; the microarrays were scanned with an agilent microarray scanner ( g2565ba ) and the data were extracted with agilent feature extraction software ( v.9.5.3.1 ) . the cutoff for over- and underexpressed genes was set at a mean fold change log2 ratio greater than + 2 or lower than 2 , as assessed by two - way analysis of variance ( anova ; partek pro software , partek inc . , st . charles , mo , usa ) with p < 0.01 . to evaluate protein expression , the spleen samples stored in tissue - tek were thawed , rinsed with pbs , and disaggregated at 15,000 rpm with a tissueruptor ( qiagen ) . the homogenates were centrifuged at 5,000 g for 5 min at 4c , and the supernatants were used to measure c3 ( elisa kit mbs700250 , mybiosource , san diego , ca , usa ) , c5 ( elisa kit mbs704792 , mybiosource ) , c3a ( elisa kit mbs70381 , mybiosource ) , c5a ( elisa kit mbs700538 , mybiosource ) , and ifn- ( elisa kit 439407 , biolegend ) . tlr-4 was measured by flow cytometry in cells obtained from fresh spleens , which were disaggregated and passed through a 70 m nylon mesh . the cells were labeled with a fluorescein isothiocyanate- ( fitc- ) conjugated anti - f4/80 antibody ( biolegend ) , a pe - conjugated rat anti - mouse tlr-4 antibody ( biolegend ) , and fixable viability dye 450 ( ebiosciences ) and acquired in a facscalibur flow cytometer . single viable cells were analyzed , and the percentage of f4/80 tlr-4 cells of the total live cells was determined . the spleens of three mice from the groups injected intrasplenically with liposomes without nonbilayer phospholipid arrangements or with liposomes bearing nonbilayer phospholipid arrangements were placed in fluorescence - activated cell sorting ( facs ) buffer containing 0.1% bsa and 0.01% sodium azide ( sigma aldrich ) . red blood cells were lysed and spleen cells were resuspended in facs buffer . before staining , cells were incubated with universal blocking reagent ( block biogenex , san ramn , ca , usa ) in pbs for 10 min at 4c and then washed . splenocyte suspensions were labeled with anti - cd19-apc , anti - cd5-af488 , anti - cd69-percp , and anti - tlr4-pe ( ebioscience ) to evaluate b cells ; with anti - cd3-fitc , anti - cd4-pe ( ebioscience ) , anti - cd8-apc , and anti - cd69-percp to evaluate t cells ; with anti - gr-1-percp , anti - cd11c - apc , anti - mhc - ii - pe ( ebioscience ) , anti - cd80-fitc , and anti - cd86-pe / cy7 ( ebioscience ) to evaluate dendritic cells ; and with anti - cd3-fitc and anti - nk 1.1-apc to evaluate nkt cells . the cells were incubated for 30 min at 4c , then washed with facs buffer , and fixed with 1% paraformaldehyde ( sigma aldrich ) . labeled cells were acquired in a lsr fortessa flow cytometer ( becton dickinson ) ; single , viable cells were analyzed with flowjo 10.0.6 ( tree star , inc . , we had previously shown that the presence of nonbilayer phospholipid arrangements can be detected by flow cytometry as an increase in side scatter ( ssc ) value [ 10 , 14 , 15 ] . thus , the increase in ssc signal after the addition of mn , chlorpromazine , or procainamide to liposomes made of phosphatidylcholine / phosphatidic acid or phosphatidylcholine / phosphatidylserine indicated the presence of nonbilayer phospholipid arrangements ( figures 1(c)-1(d ) and 1(f)-1(g ) ) . as a negative control , we added 5 mm of mg to liposomes ( figures 1(c ) , 1(d ) , and 1(e ) ) ; mg does not induce the formation of nonbilayer phospholipid arrangements , as was previously shown for phosphatidylcholine / phosphatidic acid liposomes . liposomes made of the cylindrical lipid phosphatidylcholine , without any conical lipid , did not increase in complexity in the presence of mn or mg ( figure 1(e ) ) , chlorpromazine , or procainamide ( data not shown ) . the addition of lps caused an increase in ssc signal when it was used alone ( figure 1(h ) ) or in combination with mn ( figure 1(i ) ) , which suggests that lps modifies the lipid bilayer . the addition of 0.1 mm chloroquine , a drug that blocks or reverses the formation of nonbilayer phospholipid arrangements , decreased the liposome complexity induced by procainamide or mn ( figures 1(g)-1(j ) ) or chlorpromazine ( data not shown ) . we evaluated the effects of phosphatidylcholine / phosphatidic acid liposomes and phosphatidylcholine / phosphatidylserine liposomes , alone or in the presence of mn , chlorpromazine , or procainamide , on the viability of hek , hek - tlr-4/md2/cd14 , hek - tlr-2/tlr-6 , hek - tlr-5 , and hek - tlr-8 cell lines . we found that liposomes made of phosphatidylcholine / phosphatidic acid with mn - induced nonbilayer phospholipid arrangements had no effect on the cell viability ( 90% or more of cells were viable ) . liposomes with mn - induced nonbilayer phospholipid arrangements stimulated il-8 production by hek - tlr-4/md2/cd14 cells and , to a lesser degree , by hek - tlr-2/tlr-6 cells , but not by hek - tlr-5 or hek - tlr-8 cells ( figure 2(a ) ) . liposomes without nonbilayer phospholipid arrangements or liposomes in which mn - induced nonbilayer phospholipid arrangements had been reversed by chloroquine did not induce il-8 production ( figure 2(a ) ) . similar results were obtained when nf-b activation was measured through the reporter plasmid pnifty - luc ( figure 2(a ) ) . nontransfected hek cells did not produce il-8 in the presence of liposomes with mn - induced nonbilayer phospholipid arrangements ( data not shown ) . the production of il-8 by hek - tlr-4/md2/cd14 or hek - tlr-2/tlr-6 cells in response to liposomes with mn - induced nonbilayer phospholipid arrangements was dose - dependent , and the effect was inhibited by chloroquine ( figure 2(b ) ) . cell viability in the presence of chloroquine was 90% or higher , and chloroquine did not induce apoptosis of these cells at the tested concentrations ( see supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/369462 ) . thus , the effects observed in the presence of chloroquine can be attributed to a reversion of mn - induced nonbilayer phospholipid arrangements by this drug . additionally , we found that nonbilayer phospholipid arrangements induce the production of the proinflammatory cytokine tnf- by bmdm from balb / c mice . furthermore , anti - tlr-2 and anti - tlr-4 antibodies blocked the production of tnf- by bmdm in response to nonbilayer phospholipid arrangements ( figures 2(c)-2(d ) ) . liposomes with nonbilayer phospholipid arrangements induced by mn or chlorpromazine were used to produce an autoimmune disease resembling human lupus in mice . antibodies against nonbilayer phospholipid arrangements were detected 1 month after the first injection of liposomes with nonbilayer phospholipid arrangements , and the titers in mice injected with chlorpromazine - induced nonbilayer phospholipid arrangements were higher than in those injected with mn - induced nonbilayer phospholipid arrangements ( p < 0.001 ) . these antibodies appeared 1 month before the anti - cardiolipin , anti - histone , and anti - coagulant antibodies ( figures 3(a ) , 3(b ) , 3(c ) , and 3(d ) ) . the presence of the four autoantibodies confirmed that the disease had been developed in the mice . control mice injected with ts buffer or with liposomes without nonbilayer phospholipid arrangements did not generate any of the four autoantibodies . il-6 , il-10 , il-12p70 , ifn- , tnf- , and mcp-1 were found in the sera of mice injected with liposomes with mn - induced nonbilayer phospholipid arrangements ; il-6 , ifn- , and tnf- appeared 1 month after treatment , while il-10 , il-12p70 , and mcp-1 were found after 2 months . these cytokines were also found in the sera of mice injected with chlorpromazine - induced nonbilayer phospholipid arrangements ; il-6 , tnf- , and mcp-1 appeared 1 month after treatment , while ifn- and il-12p70 were found 4 months after treatment . none of the tested cytokines were found in the sera of mice treated with smooth liposomes ( figure 4 ) . we evaluated gene expression in the spleens of mice from the four treatment groups : group 1 , mice injected with ts buffer alone ( control i ) ; group 2 , mice injected with smooth liposomes ( liposomes without nonbilayer phospholipid arrangements , control ii ) ; group 3 , mice that received liposomes with mn - induced nonbilayer phospholipid arrangements ( mn group ) ; and group 4 , mice that received liposomes with chlorpromazine - induced nonbilayer phospholipid arrangements ( cpz group ) . spleens were collected 4 months after treatment , and the crna derived from the spleens of three mice from each group were pooled and hybridized to a whole mouse genome microarray chip . no significant differences were found between control i and control ii groups ; 426 genes were overexpressed and 62 genes were underexpressed in the mn group , compared with the control ii group ; 542 genes were overexpressed and 73 genes were underexpressed in the cpz group , compared with the control ii group ; and 383 genes were overexpressed and 44 genes were underexpressed in the cpz group , compared with the mn group . table 1 shows a list of genes that were overexpressed in both the mn and cpz groups , compared with the control ii group . this includes genes for complement components ( c3 and c5 ) , molecules involved in the presentation of exogenous antigens , in the production of antibodies , and in tlr-4 and nod-2 signaling . table 1 also shows a list of genes that were underexpressed in both the mn and cpz groups , compared with the control ii group . these are genes for molecules that are involved in apoptosis and in nk cell recognition . the c3 and c5 complement proteins were increased in the control i and control ii groups , compared with the mn and cpz groups . however , c3a and c5a , two active fragments that are produced by c3 and c5 cleavage , were increased in the mn and the cpz groups , compared with the control i and control ii groups ( figures 5(a)-5(b ) ) . ifn- was also increased in the spleens of mice with the autoimmune disease , compared with healthy mice ( figure 5(c ) ) . the number of cells expressing tlr-4 increased in the mn and the cpz groups , compared with the control i and control ii groups ( figure 5(d ) ) . activated cd4 and cd8 t cells ( figures 6(a ) , 6(b ) , 6(c ) , and 6(d ) ) , nkt cells ( figures 6(e)-6(f ) ) , activated dendritic cells ( figures 6(g)-6(h ) ) , and activated and tlr4 expressing b1 and b2 cells ( figures 6(i ) , 6(j ) , and 6(k ) ) were identified by flow cytometry in the spleens of mice . fifteen days after the mice were injected intrasplenically with liposomes bearing nonbilayer phospholipids arrangements induced by mn or chlorpromazine , the percentage and activation of cd4 and cd8 t cells were not increased , compared with the control mice that received ts buffer or liposomes alone ( figures 6(l)-6(m ) ) . in contrast , the percentage of nkt , dendritic , and b2 cells was increased ( figures 6(n ) , 6(o ) , and 6(q ) ) , and the activation of dendritic and b2 cells was also increased ( figures 6(o)-6(q ) ) . an increase in tlr4 expression was also observed in b2 cells ( figure 6(q ) ) . b1 cells did not increase in percentage , but the number of activated and tlr4 expressing b1 cells did increase ( figure 6(p ) ) . sle is a systemic autoimmune disease of unknown etiology characterized by b and t cell hyperactivity , by defects in the clearance of apoptotic cells and immune complexes , and by production of a complex mixture of various cytokines , chemokines , signaling molecules , and pattern - recognition receptors involved in immunity [ 4 , 24 ] . we have previously demonstrated that liposomes with nonbilayer phospholipid arrangements trigger a disease that resembles human lupus in mice and that igm and igg specific to nonbilayer phospholipid arrangements are produced in these mice . the activation of this innate immune receptor leads to the production of proinflammatory cytokines ; a proinflammatory environment is needed for efficient activation of the adaptive immune response and the production of igg antibodies . these findings were supported by the increase in the percentage of nkt cells and by the increase in the percentage and activation of dendritic and b2 cells . in addition , the activation of tlr-4/md2/cd14 by liposomes with mn - induced nonbilayer phospholipid arrangements supports our hypothesis on the similarity of the structure of conical phospholipids , which form an inverted micelle inside the nonbilayer arrangement , with the conical association of the acyl chains of the lipid a moiety of lps . the importance of the lipid a moiety of lps was taken into account in the design of glucopyranosyl lipid a ( gla ) , a synthetic lipid a with six acyl chains and a single phosphate group . gla as a stable oil - in - water - emulsion ( gla - se ) is a tlr-4 agonist , which signals through myd88 and trif and drives a polyclonal th1 response in vivo , characterized by ifn- , tnf- , and il-2 producing cells and igg2c isotype switching [ 25 , 26 ] . interestingly , we also found that nonbilayer phospholipid arrangements induce the production of the proinflammatory cytokine tnf- by balb / c mouse bmdm . furthermore , anti - tlr-2 and anti - tlr-4 antibodies blocked the production of tnf- by these macrophages in response to nonbilayer phospholipid arrangements . these findings confirmed our observations with the hek cells transfected with human tlrs , which also showed that nonbilayer phospholipid arrangements are agonists for tlr-4/md-2 and tlr-2/tlr-6 . we observed that liposomes with nonbilayer phospholipid arrangements were agonists for tlr-2/tlr-6 , but the activation was 3-fold lower than for tlr-4/md2/cd14 . bacterial macroamphiphilic molecules , such as lipoproteins ( including the synthetic lipoprotein fsl-1 ) , lipoteichoic acids , lipoglycans , glycolipids , and lipoarabinomannans , are anchored on bacterial envelopes through a lipidic structure , which is usually a diacylglyceryl moiety . these amphiphilic molecules are mainly recognized via their lipid anchor through tlr-2 , alone or as a heterodimer with tlr-1 or tlr-6 [ 27 , 28 ] . because the liposomes bearing nonbilayer phospholipid arrangements are made of phosphatidylcholine and phosphatidate , which also have the diacylglyceryl moiety , it is possible that this lipid moiety activated the tlr-2/tlr-6 heterodimer . tlrs not only recognize pathogen - associated molecular patterns , such as lps , but also recognize damage - associated molecular patterns , which are released by cells that are either under stress or undergoing apoptosis or necrosis . examples of damage - associated molecular patterns that are tlr-4 agonists include heat - shock protein 60 , fibronectin , fibrinogen , -defensins , and hyaluronan . the molecular structure of these agonists is different from that of lps , but they all have hydrophobic regions , which are probably recognized by tlr-4 . the modification of the lipid bilayer of cell membranes could be a signal of cell stress : nonbilayer phospholipid arrangements are normally transitory , but if they are stabilized by mn or by the drugs chlorpromazine or procainamide , they could activate the innate immune response via tlrs and then induce the production of antibodies , with the subsequent development of an autoimmune disease . tlr-4 signaling leads to the activation of nf-b and the production of proinflammatory cytokines , including tnf- , il-12 , and ifn- , and chemokines , such as mcp-1 . we found these cytokines and chemokines in the sera of mice treated with liposomes with mn- or chlorpromazine - induced nonbilayer phospholipid arrangements . the increase in the concentration of the proinflammatory cytokines il-6 and tnf- correlated with the appearance of anti - nonbilayer phospholipid arrangement antibodies 1 month after the first injection of mice with nonbilayer phospholipid arrangements , and this also corresponds to the period of disease onset . the chemokine mcp-1 and the proinflammatory cytokines inf- and il-12p70 increased between months 2 and 4 and correlated with the development and establishment of the disease , given by an increase in the titers of anti - nonbilayer phospholipid arrangement antibodies and the presence of anti - cardiolipin , anti - histone , and anti - coagulant antibodies . the proinflammatory cytokines il-1 , il-6 , ifn- , and tnf- and the immunomodulatory cytokines il-10 and tumor growth factor- ( tgf- ) have been identified as important players in the development of sle [ 31 , 32 ] . the cytokine pattern that we report indicates another similarity of this mouse model with the human disease . tlr-4 was increased at the mrna level and the number of cells that express tlr-4 increased in the spleens of mice that received liposomes with nonbilayer phospholipid arrangements . other genes associated with tlr-4 signaling , such as tram , trif , tbk1 , and irf3 , were also increased at the mrna level in these mice . these genes are associated with trif - dependent , but not myd88-dependent , tlr-4 signaling . trif - dependent tlr-4 signaling leads to the production of ifn- and . ifn- was increased in the spleens of mice that had received liposomes with nonbilayer phospholipid arrangements compared with healthy mice , and increased levels of ifn- and are reported in patients with sle . the expression of genes associated with the classical pathway of complement activation ( c1ra , c1s , c1q , c3 , c5 , and c7 ) was increased in mice that had received liposomes with nonbilayer phospholipid arrangements , and this mrna increase correlated with the detection of c3a and c5a proteins in the spleens of mice with the autoimmune disease . complement has an important role in the immune response , but it also has the potential to cause tissue damage , as has been reported in sle and other autoimmune diseases [ 35 , 36 ] . it will be interesting to evaluate the role of complement in the tissue damage that is observed in this mouse model of autoimmune disease . in contrast , the expression of genes associated with nk cell activation ( klrb1a , klrb1c , klra23 , klra7 , gzmb , and klra22 ) was decreased in mice that received liposomes with nonbilayer phospholipid arrangements . this decrease could reflect a reduction in the absolute number of nk cells or a lower activation of the existing nk cells . the expression of genes associated with apoptosis ( casp8 , cycs , apaf1 , and aaifm1 ) was also decreased in mice that received liposomes with nonbilayer phospholipid arrangements . this could be relevant for disease development , since deficient apoptosis could favor the survival of autoreactive t cells . an important additional support for our hypothesis on the effect of nonbilayer phospholipid arrangements on the innate immune response is our finding that mice with the autoimmune disease resembling human lupus have an increase in nkt and dendritic cell percentages , together with increased dendritic cell activation . these cells could recruit and activate b1 and b2 cells , which are the precursors of plasma cells that produce antibodies against nonbilayer phospholipid arrangements . the findings reported in this paper are consistent with a mouse model in which nonbilayer phospholipid arrangements directly activate tlr-4 and tlr-2/tlr-6 and lead to the production of proinflammatory cytokines . the proinflammatory environment leads to the efficient activation of the adaptive immune response to the production of igg antibodies specific for nonbilayer phospholipid arrangements . these antibodies bind to the nonbilayer phospholipid arrangements that are transitorily formed on the surface of many cells and cause cell lysis ; the exposure of intracellular antigens could then lead to the formation of anti - cardiolipin , anti - histone , and anti - coagulant antibodies . furthermore , the inflammatory environment can cause complement - mediated tissue damage and ifn- production .

systemic lupus erythematosus is characterized by dysregulated activation of t and b cells and autoantibodies to nuclear antigens and , in some cases , lipid antigens . liposomes with nonbilayer phospholipid arrangements induce a disease resembling human lupus in mice , including igm and igg antibodies against nonbilayer phospholipid arrangements . as the effect of these liposomes on the innate immune response is unknown and innate immune system activation is necessary for efficient antibody formation , we evaluated the effect of these liposomes on toll - like receptor ( tlr ) signaling , cytokine production , proinflammatory gene expression , and t , nkt , dendritic , and b cells . liposomes induce tlr-4- and , to a lesser extent , tlr-2/tlr-6-dependent signaling in tlr - expressing human embryonic kidney ( hek ) cells and bone marrow - derived macrophages . mice with the lupus - like disease had increased serum concentrations of proinflammatory cytokines , c3a and c5a ; they also had more tlr-4-expressing splenocytes , a higher expression of genes associated with trif - dependent tlr-4-signaling and complement activation , and a lower expression of apoptosis - related genes , compared to healthy mice . the percentage of nkt and the percentage and activation of dendritic and b2 cells were also increased . thus , tlr-4 and tlr-2/tlr-6 activation by nonbilayer phospholipid arrangements triggers an inflammatory response that could contribute to autoantibody production and the generation of a lupus - like disease in mice .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

systemic lupus erythematosus ( sle ) is a systemic autoimmune disease characterized by a loss of tolerance to nuclear antigens and by dysregulated activation of t and b cells . polyclonal activation of b cells leads to the production of large quantities of autoreactive antibodies and the formation of immune complexes , which causes tissue damage . in some sle patients , it has been shown that bone marrow mesenchymal stem cells exhibit impaired capacities for proliferation , differentiation , migration , and immune modulation . drug - induced lupus erythematosus ( dile ) is a lupus - like syndrome that resolves upon drug discontinuation . the drugs more frequently associated with the induction of this lupus - like syndrome are procainamide ( antiarrhythmic ) , hydralazine ( antihypertensive ) , and chlorpromazine ( antipsychotic ) [ 5 , 6 ] . the most commonly used lupus - prone mice are the f1 hybrids of new zealand black ( nzb ) and nz white ( nzb / nzw f1 ) mice , the murphy - roths large / lymphoproliferative locus ( mlr / lpr ) mice , and the recombinant c57bl/6 female and sb / le male strain / y - linked autoimmune accelerator ( bxsb / yaa ) mice [ 3 , 8 , 9 ] . our group has also developed a mouse model of autoimmune disease resembling human lupus that can be induced in normal mice . conical phospholipids can form molecular associations distinct to lipid bilayers , known as nonbilayer phospholipid arrangements , in the presence of inducers such as mn [ 12 , 13 ] or the drugs chlorpromazine and procainamide , which can trigger dile in humans . we demonstrated that liposomes with nonbilayer phospholipid arrangements induced by mn , chlorpromazine , or procainamide cause an autoimmune disease resembling human lupus in mice . igm and igg antibodies against nonbilayer phospholipid arrangements are found in the sera of mice with the autoimmune disease resembling human lupus , and also in the sera of patients with lupus [ 10 , 15 ] . usually , the efficient production of igg antibodies requires an activation of the innate immune response . therefore we hypothesized that nonbilayer phospholipid arrangements could be toll - like receptor- ( tlr- ) 4/md-2 agonists , as their molecular structure is similar to that of the lipid a from bacterial lipopolysaccharide ( lps ) . since the conical molecular shape of lipid a is a requirement for tlr-4/md-2 triggering [ 1619 ] , we hypothesized that liposomes with nonbilayer phospholipid arrangements , but not smooth liposomes ( with phospholipids in a bilayer arrangement ) , could trigger tlr-4/md-2 signaling . in this study , we investigated whether liposomes with nonbilayer phospholipid arrangements are tlr-4/md-2 agonists , because the activation of this innate immune receptor leads to the production of proinflammatory cytokines . we also looked for proinflammatory cytokines in the sera of mice with the autoimmune disease triggered by liposomes with nonbilayer phospholipid arrangements , and we determined the gene expression profile in the spleens of these mice , focusing on the expression of proinflammatory genes . in addition , we determined the relative percentage and activation of t , nkt , dendritic , and b cells in the spleen of mice with the disease . liposomes and liposomes with nonbilayer phospholipid arrangements in ts buffer were analyzed with a facscalibur flow cytometer ( becton dickinson , san jose , ca , usa ) with cellquest software . human embryonic kidney ( hek ) 293 cells , nontransfected or stably transfected with human tlr-4/md2/cd14 , tlr-2/tlr-6 , tlr-5 , or tlr-8 , were purchased from invivogen ( san diego , ca , usa ) . the viability of these cell lines in the presence of mn , chlorpromazine , procainamide , or chloroquine , and in the presence of liposomes or liposomes with nonbilayer phospholipid arrangements , was evaluated with the alamar blue method . to assess tlr activation , the cell lines were incubated in the presence of liposomes made of phosphatidylcholine / phosphatidic acid ( 2 : 1 ) , alone or with nonbilayer phospholipid arrangements induced by mn ( 24 mm ) . 100 l of 2 g / ml annexin v - apc ( ebioscience ) in annexin v - binding buffer was added , and the cells were incubated for 15 min at room temperature in the dark . tlr stimulation was also analyzed in bone marrow - derived macrophages ( bmdm ) from balb / c mice . to confirm that these mice developed the disease resembling human lupus , we measured anti - nonbilayer phospholipid arrangements , anti - cardiolipin , anti - histone , and anti - coagulant antibodies in their sera . three mice from each of the four groups indicated above were euthanized 4 months after the first injection of nonbilayer phospholipid arrangements , when they had the highest titers of anti - nonbilayer phospholipid arrangements , anti - cardiolipin , anti - histone , and anti - coagulant antibodies , and their spleens were used for gene and protein expression studies . the serum concentrations of interleukin-6 ( il-6 ) , il-10 , il-12p70 , interferon- ( ifn- ) , tnf- , and monocyte chemoattractant protein- ( mcp- ) 1 were measured with a bead - based multiplex immunoassay ( bd cba mouse inflammation kit ) . the cells were labeled with a fluorescein isothiocyanate- ( fitc- ) conjugated anti - f4/80 antibody ( biolegend ) , a pe - conjugated rat anti - mouse tlr-4 antibody ( biolegend ) , and fixable viability dye 450 ( ebiosciences ) and acquired in a facscalibur flow cytometer . single viable cells were analyzed , and the percentage of f4/80 tlr-4 cells of the total live cells was determined . as a negative control , we added 5 mm of mg to liposomes ( figures 1(c ) , 1(d ) , and 1(e ) ) ; mg does not induce the formation of nonbilayer phospholipid arrangements , as was previously shown for phosphatidylcholine / phosphatidic acid liposomes . the addition of 0.1 mm chloroquine , a drug that blocks or reverses the formation of nonbilayer phospholipid arrangements , decreased the liposome complexity induced by procainamide or mn ( figures 1(g)-1(j ) ) or chlorpromazine ( data not shown ) . we evaluated the effects of phosphatidylcholine / phosphatidic acid liposomes and phosphatidylcholine / phosphatidylserine liposomes , alone or in the presence of mn , chlorpromazine , or procainamide , on the viability of hek , hek - tlr-4/md2/cd14 , hek - tlr-2/tlr-6 , hek - tlr-5 , and hek - tlr-8 cell lines . we found that liposomes made of phosphatidylcholine / phosphatidic acid with mn - induced nonbilayer phospholipid arrangements had no effect on the cell viability ( 90% or more of cells were viable ) . liposomes with mn - induced nonbilayer phospholipid arrangements stimulated il-8 production by hek - tlr-4/md2/cd14 cells and , to a lesser degree , by hek - tlr-2/tlr-6 cells , but not by hek - tlr-5 or hek - tlr-8 cells ( figure 2(a ) ) . the production of il-8 by hek - tlr-4/md2/cd14 or hek - tlr-2/tlr-6 cells in response to liposomes with mn - induced nonbilayer phospholipid arrangements was dose - dependent , and the effect was inhibited by chloroquine ( figure 2(b ) ) . thus , the effects observed in the presence of chloroquine can be attributed to a reversion of mn - induced nonbilayer phospholipid arrangements by this drug . additionally , we found that nonbilayer phospholipid arrangements induce the production of the proinflammatory cytokine tnf- by bmdm from balb / c mice . liposomes with nonbilayer phospholipid arrangements induced by mn or chlorpromazine were used to produce an autoimmune disease resembling human lupus in mice . antibodies against nonbilayer phospholipid arrangements were detected 1 month after the first injection of liposomes with nonbilayer phospholipid arrangements , and the titers in mice injected with chlorpromazine - induced nonbilayer phospholipid arrangements were higher than in those injected with mn - induced nonbilayer phospholipid arrangements ( p < 0.001 ) . il-6 , il-10 , il-12p70 , ifn- , tnf- , and mcp-1 were found in the sera of mice injected with liposomes with mn - induced nonbilayer phospholipid arrangements ; il-6 , ifn- , and tnf- appeared 1 month after treatment , while il-10 , il-12p70 , and mcp-1 were found after 2 months . these cytokines were also found in the sera of mice injected with chlorpromazine - induced nonbilayer phospholipid arrangements ; il-6 , tnf- , and mcp-1 appeared 1 month after treatment , while ifn- and il-12p70 were found 4 months after treatment . we evaluated gene expression in the spleens of mice from the four treatment groups : group 1 , mice injected with ts buffer alone ( control i ) ; group 2 , mice injected with smooth liposomes ( liposomes without nonbilayer phospholipid arrangements , control ii ) ; group 3 , mice that received liposomes with mn - induced nonbilayer phospholipid arrangements ( mn group ) ; and group 4 , mice that received liposomes with chlorpromazine - induced nonbilayer phospholipid arrangements ( cpz group ) . table 1 shows a list of genes that were overexpressed in both the mn and cpz groups , compared with the control ii group . this includes genes for complement components ( c3 and c5 ) , molecules involved in the presentation of exogenous antigens , in the production of antibodies , and in tlr-4 and nod-2 signaling . however , c3a and c5a , two active fragments that are produced by c3 and c5 cleavage , were increased in the mn and the cpz groups , compared with the control i and control ii groups ( figures 5(a)-5(b ) ) . ifn- was also increased in the spleens of mice with the autoimmune disease , compared with healthy mice ( figure 5(c ) ) . activated cd4 and cd8 t cells ( figures 6(a ) , 6(b ) , 6(c ) , and 6(d ) ) , nkt cells ( figures 6(e)-6(f ) ) , activated dendritic cells ( figures 6(g)-6(h ) ) , and activated and tlr4 expressing b1 and b2 cells ( figures 6(i ) , 6(j ) , and 6(k ) ) were identified by flow cytometry in the spleens of mice . fifteen days after the mice were injected intrasplenically with liposomes bearing nonbilayer phospholipids arrangements induced by mn or chlorpromazine , the percentage and activation of cd4 and cd8 t cells were not increased , compared with the control mice that received ts buffer or liposomes alone ( figures 6(l)-6(m ) ) . in contrast , the percentage of nkt , dendritic , and b2 cells was increased ( figures 6(n ) , 6(o ) , and 6(q ) ) , and the activation of dendritic and b2 cells was also increased ( figures 6(o)-6(q ) ) . sle is a systemic autoimmune disease of unknown etiology characterized by b and t cell hyperactivity , by defects in the clearance of apoptotic cells and immune complexes , and by production of a complex mixture of various cytokines , chemokines , signaling molecules , and pattern - recognition receptors involved in immunity [ 4 , 24 ] . we have previously demonstrated that liposomes with nonbilayer phospholipid arrangements trigger a disease that resembles human lupus in mice and that igm and igg specific to nonbilayer phospholipid arrangements are produced in these mice . the activation of this innate immune receptor leads to the production of proinflammatory cytokines ; a proinflammatory environment is needed for efficient activation of the adaptive immune response and the production of igg antibodies . these findings were supported by the increase in the percentage of nkt cells and by the increase in the percentage and activation of dendritic and b2 cells . in addition , the activation of tlr-4/md2/cd14 by liposomes with mn - induced nonbilayer phospholipid arrangements supports our hypothesis on the similarity of the structure of conical phospholipids , which form an inverted micelle inside the nonbilayer arrangement , with the conical association of the acyl chains of the lipid a moiety of lps . gla as a stable oil - in - water - emulsion ( gla - se ) is a tlr-4 agonist , which signals through myd88 and trif and drives a polyclonal th1 response in vivo , characterized by ifn- , tnf- , and il-2 producing cells and igg2c isotype switching [ 25 , 26 ] . interestingly , we also found that nonbilayer phospholipid arrangements induce the production of the proinflammatory cytokine tnf- by balb / c mouse bmdm . these findings confirmed our observations with the hek cells transfected with human tlrs , which also showed that nonbilayer phospholipid arrangements are agonists for tlr-4/md-2 and tlr-2/tlr-6 . we observed that liposomes with nonbilayer phospholipid arrangements were agonists for tlr-2/tlr-6 , but the activation was 3-fold lower than for tlr-4/md2/cd14 . the modification of the lipid bilayer of cell membranes could be a signal of cell stress : nonbilayer phospholipid arrangements are normally transitory , but if they are stabilized by mn or by the drugs chlorpromazine or procainamide , they could activate the innate immune response via tlrs and then induce the production of antibodies , with the subsequent development of an autoimmune disease . tlr-4 signaling leads to the activation of nf-b and the production of proinflammatory cytokines , including tnf- , il-12 , and ifn- , and chemokines , such as mcp-1 . we found these cytokines and chemokines in the sera of mice treated with liposomes with mn- or chlorpromazine - induced nonbilayer phospholipid arrangements . the increase in the concentration of the proinflammatory cytokines il-6 and tnf- correlated with the appearance of anti - nonbilayer phospholipid arrangement antibodies 1 month after the first injection of mice with nonbilayer phospholipid arrangements , and this also corresponds to the period of disease onset . the chemokine mcp-1 and the proinflammatory cytokines inf- and il-12p70 increased between months 2 and 4 and correlated with the development and establishment of the disease , given by an increase in the titers of anti - nonbilayer phospholipid arrangement antibodies and the presence of anti - cardiolipin , anti - histone , and anti - coagulant antibodies . tlr-4 was increased at the mrna level and the number of cells that express tlr-4 increased in the spleens of mice that received liposomes with nonbilayer phospholipid arrangements . other genes associated with tlr-4 signaling , such as tram , trif , tbk1 , and irf3 , were also increased at the mrna level in these mice . these genes are associated with trif - dependent , but not myd88-dependent , tlr-4 signaling . ifn- was increased in the spleens of mice that had received liposomes with nonbilayer phospholipid arrangements compared with healthy mice , and increased levels of ifn- and are reported in patients with sle . the expression of genes associated with the classical pathway of complement activation ( c1ra , c1s , c1q , c3 , c5 , and c7 ) was increased in mice that had received liposomes with nonbilayer phospholipid arrangements , and this mrna increase correlated with the detection of c3a and c5a proteins in the spleens of mice with the autoimmune disease . in contrast , the expression of genes associated with nk cell activation ( klrb1a , klrb1c , klra23 , klra7 , gzmb , and klra22 ) was decreased in mice that received liposomes with nonbilayer phospholipid arrangements . the expression of genes associated with apoptosis ( casp8 , cycs , apaf1 , and aaifm1 ) was also decreased in mice that received liposomes with nonbilayer phospholipid arrangements . an important additional support for our hypothesis on the effect of nonbilayer phospholipid arrangements on the innate immune response is our finding that mice with the autoimmune disease resembling human lupus have an increase in nkt and dendritic cell percentages , together with increased dendritic cell activation . these cells could recruit and activate b1 and b2 cells , which are the precursors of plasma cells that produce antibodies against nonbilayer phospholipid arrangements . the findings reported in this paper are consistent with a mouse model in which nonbilayer phospholipid arrangements directly activate tlr-4 and tlr-2/tlr-6 and lead to the production of proinflammatory cytokines . the proinflammatory environment leads to the efficient activation of the adaptive immune response to the production of igg antibodies specific for nonbilayer phospholipid arrangements . these antibodies bind to the nonbilayer phospholipid arrangements that are transitorily formed on the surface of many cells and cause cell lysis ; the exposure of intracellular antigens could then lead to the formation of anti - cardiolipin , anti - histone , and anti - coagulant antibodies .

the drugs more frequently associated with the induction of this lupus - like syndrome are procainamide ( antiarrhythmic ) , hydralazine ( antihypertensive ) , and chlorpromazine ( antipsychotic ) [ 5 , 6 ] . animal models of sle include lupus - prone mice , which spontaneously develop lupus , and normal mice that develop lupus after injection of lymphocytes from lupus - prone mice , immunization with prototypical lupus antigens ( dna- and rna - protein complexes ) , or injection of pristane ( 2,6,10,14-tetramethylpentadecane ) [ 3 , 7 ] . the most commonly used lupus - prone mice are the f1 hybrids of new zealand black ( nzb ) and nz white ( nzb / nzw f1 ) mice , the murphy - roths large / lymphoproliferative locus ( mlr / lpr ) mice , and the recombinant c57bl/6 female and sb / le male strain / y - linked autoimmune accelerator ( bxsb / yaa ) mice [ 3 , 8 , 9 ] . conical phospholipids can form molecular associations distinct to lipid bilayers , known as nonbilayer phospholipid arrangements , in the presence of inducers such as mn [ 12 , 13 ] or the drugs chlorpromazine and procainamide , which can trigger dile in humans . therefore we hypothesized that nonbilayer phospholipid arrangements could be toll - like receptor- ( tlr- ) 4/md-2 agonists , as their molecular structure is similar to that of the lipid a from bacterial lipopolysaccharide ( lps ) . lps - binding protein ( lbp ) is a plasma protein that facilitates the transfer of lps molecules from these hexagonal ( hii ) arrangements to cd14 , and membrane - bound cd14 delivers lps to tlr-4/md2 . since the conical molecular shape of lipid a is a requirement for tlr-4/md-2 triggering [ 1619 ] , we hypothesized that liposomes with nonbilayer phospholipid arrangements , but not smooth liposomes ( with phospholipids in a bilayer arrangement ) , could trigger tlr-4/md-2 signaling . in this study , we investigated whether liposomes with nonbilayer phospholipid arrangements are tlr-4/md-2 agonists , because the activation of this innate immune receptor leads to the production of proinflammatory cytokines . we also looked for proinflammatory cytokines in the sera of mice with the autoimmune disease triggered by liposomes with nonbilayer phospholipid arrangements , and we determined the gene expression profile in the spleens of these mice , focusing on the expression of proinflammatory genes . in addition , we determined the relative percentage and activation of t , nkt , dendritic , and b cells in the spleen of mice with the disease . the viability of these cell lines in the presence of mn , chlorpromazine , procainamide , or chloroquine , and in the presence of liposomes or liposomes with nonbilayer phospholipid arrangements , was evaluated with the alamar blue method . to assess tlr activation , the cell lines were incubated in the presence of liposomes made of phosphatidylcholine / phosphatidic acid ( 2 : 1 ) , alone or with nonbilayer phospholipid arrangements induced by mn ( 24 mm ) . for the positive controls , the cell lines were incubated in the presence of their known tlr agonists : 100 ng / ml escherichia coli 0111:b4 lps for hek - tlr-4/md2/cd14 , 1 g / ml fsl-1 ( a synthetic lipoprotein derived from mycoplasma salivarium ) for hek - tlr-2/tlr-6 , 1 g / ml salmonella typhimurium flagellin for hek - tlr-5 , and 2.5 g / ml ssrna40 ( a 20 mer phosphorothioate - protected single - stranded rna oligonucleotide containing a gu - rich sequence ) for hek - tlr-8 . bmdm were obtained from the femur and shinbone of female 2-month - old balb / c mice and they were cultured in rpmi media with 10% heat - inactivated fetal bovine serum , 50 u / ml penicillin ( gibco ) , 50 g / ml streptomycin ( gibco ) , and 10 ng / ml recombinant m - csf ( biolegend ) for 7 days . results are reported as arbitrary units ( au ) calculated as ( asp asw)/(ash asw ) , where asp is the absorbance obtained with the sera of mice injected with the liposomes , ash is the absorbance obtained with the sera of mice before the injection of liposomes , and asw is absorbance of controls without sera . three mice from each of the four groups indicated above were euthanized 4 months after the first injection of nonbilayer phospholipid arrangements , when they had the highest titers of anti - nonbilayer phospholipid arrangements , anti - cardiolipin , anti - histone , and anti - coagulant antibodies , and their spleens were used for gene and protein expression studies . the serum concentrations of interleukin-6 ( il-6 ) , il-10 , il-12p70 , interferon- ( ifn- ) , tnf- , and monocyte chemoattractant protein- ( mcp- ) 1 were measured with a bead - based multiplex immunoassay ( bd cba mouse inflammation kit ) . the spleens of three mice from the groups injected intrasplenically with liposomes without nonbilayer phospholipid arrangements or with liposomes bearing nonbilayer phospholipid arrangements were placed in fluorescence - activated cell sorting ( facs ) buffer containing 0.1% bsa and 0.01% sodium azide ( sigma aldrich ) . splenocyte suspensions were labeled with anti - cd19-apc , anti - cd5-af488 , anti - cd69-percp , and anti - tlr4-pe ( ebioscience ) to evaluate b cells ; with anti - cd3-fitc , anti - cd4-pe ( ebioscience ) , anti - cd8-apc , and anti - cd69-percp to evaluate t cells ; with anti - gr-1-percp , anti - cd11c - apc , anti - mhc - ii - pe ( ebioscience ) , anti - cd80-fitc , and anti - cd86-pe / cy7 ( ebioscience ) to evaluate dendritic cells ; and with anti - cd3-fitc and anti - nk 1.1-apc to evaluate nkt cells . thus , the increase in ssc signal after the addition of mn , chlorpromazine , or procainamide to liposomes made of phosphatidylcholine / phosphatidic acid or phosphatidylcholine / phosphatidylserine indicated the presence of nonbilayer phospholipid arrangements ( figures 1(c)-1(d ) and 1(f)-1(g ) ) . as a negative control , we added 5 mm of mg to liposomes ( figures 1(c ) , 1(d ) , and 1(e ) ) ; mg does not induce the formation of nonbilayer phospholipid arrangements , as was previously shown for phosphatidylcholine / phosphatidic acid liposomes . liposomes made of the cylindrical lipid phosphatidylcholine , without any conical lipid , did not increase in complexity in the presence of mn or mg ( figure 1(e ) ) , chlorpromazine , or procainamide ( data not shown ) . we evaluated the effects of phosphatidylcholine / phosphatidic acid liposomes and phosphatidylcholine / phosphatidylserine liposomes , alone or in the presence of mn , chlorpromazine , or procainamide , on the viability of hek , hek - tlr-4/md2/cd14 , hek - tlr-2/tlr-6 , hek - tlr-5 , and hek - tlr-8 cell lines . liposomes with mn - induced nonbilayer phospholipid arrangements stimulated il-8 production by hek - tlr-4/md2/cd14 cells and , to a lesser degree , by hek - tlr-2/tlr-6 cells , but not by hek - tlr-5 or hek - tlr-8 cells ( figure 2(a ) ) . the production of il-8 by hek - tlr-4/md2/cd14 or hek - tlr-2/tlr-6 cells in response to liposomes with mn - induced nonbilayer phospholipid arrangements was dose - dependent , and the effect was inhibited by chloroquine ( figure 2(b ) ) . cell viability in the presence of chloroquine was 90% or higher , and chloroquine did not induce apoptosis of these cells at the tested concentrations ( see supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/369462 ) . thus , the effects observed in the presence of chloroquine can be attributed to a reversion of mn - induced nonbilayer phospholipid arrangements by this drug . antibodies against nonbilayer phospholipid arrangements were detected 1 month after the first injection of liposomes with nonbilayer phospholipid arrangements , and the titers in mice injected with chlorpromazine - induced nonbilayer phospholipid arrangements were higher than in those injected with mn - induced nonbilayer phospholipid arrangements ( p < 0.001 ) . il-6 , il-10 , il-12p70 , ifn- , tnf- , and mcp-1 were found in the sera of mice injected with liposomes with mn - induced nonbilayer phospholipid arrangements ; il-6 , ifn- , and tnf- appeared 1 month after treatment , while il-10 , il-12p70 , and mcp-1 were found after 2 months . these cytokines were also found in the sera of mice injected with chlorpromazine - induced nonbilayer phospholipid arrangements ; il-6 , tnf- , and mcp-1 appeared 1 month after treatment , while ifn- and il-12p70 were found 4 months after treatment . we evaluated gene expression in the spleens of mice from the four treatment groups : group 1 , mice injected with ts buffer alone ( control i ) ; group 2 , mice injected with smooth liposomes ( liposomes without nonbilayer phospholipid arrangements , control ii ) ; group 3 , mice that received liposomes with mn - induced nonbilayer phospholipid arrangements ( mn group ) ; and group 4 , mice that received liposomes with chlorpromazine - induced nonbilayer phospholipid arrangements ( cpz group ) . spleens were collected 4 months after treatment , and the crna derived from the spleens of three mice from each group were pooled and hybridized to a whole mouse genome microarray chip . no significant differences were found between control i and control ii groups ; 426 genes were overexpressed and 62 genes were underexpressed in the mn group , compared with the control ii group ; 542 genes were overexpressed and 73 genes were underexpressed in the cpz group , compared with the control ii group ; and 383 genes were overexpressed and 44 genes were underexpressed in the cpz group , compared with the mn group . this includes genes for complement components ( c3 and c5 ) , molecules involved in the presentation of exogenous antigens , in the production of antibodies , and in tlr-4 and nod-2 signaling . however , c3a and c5a , two active fragments that are produced by c3 and c5 cleavage , were increased in the mn and the cpz groups , compared with the control i and control ii groups ( figures 5(a)-5(b ) ) . the number of cells expressing tlr-4 increased in the mn and the cpz groups , compared with the control i and control ii groups ( figure 5(d ) ) . activated cd4 and cd8 t cells ( figures 6(a ) , 6(b ) , 6(c ) , and 6(d ) ) , nkt cells ( figures 6(e)-6(f ) ) , activated dendritic cells ( figures 6(g)-6(h ) ) , and activated and tlr4 expressing b1 and b2 cells ( figures 6(i ) , 6(j ) , and 6(k ) ) were identified by flow cytometry in the spleens of mice . fifteen days after the mice were injected intrasplenically with liposomes bearing nonbilayer phospholipids arrangements induced by mn or chlorpromazine , the percentage and activation of cd4 and cd8 t cells were not increased , compared with the control mice that received ts buffer or liposomes alone ( figures 6(l)-6(m ) ) . in contrast , the percentage of nkt , dendritic , and b2 cells was increased ( figures 6(n ) , 6(o ) , and 6(q ) ) , and the activation of dendritic and b2 cells was also increased ( figures 6(o)-6(q ) ) . sle is a systemic autoimmune disease of unknown etiology characterized by b and t cell hyperactivity , by defects in the clearance of apoptotic cells and immune complexes , and by production of a complex mixture of various cytokines , chemokines , signaling molecules , and pattern - recognition receptors involved in immunity [ 4 , 24 ] . the activation of this innate immune receptor leads to the production of proinflammatory cytokines ; a proinflammatory environment is needed for efficient activation of the adaptive immune response and the production of igg antibodies . in addition , the activation of tlr-4/md2/cd14 by liposomes with mn - induced nonbilayer phospholipid arrangements supports our hypothesis on the similarity of the structure of conical phospholipids , which form an inverted micelle inside the nonbilayer arrangement , with the conical association of the acyl chains of the lipid a moiety of lps . the importance of the lipid a moiety of lps was taken into account in the design of glucopyranosyl lipid a ( gla ) , a synthetic lipid a with six acyl chains and a single phosphate group . gla as a stable oil - in - water - emulsion ( gla - se ) is a tlr-4 agonist , which signals through myd88 and trif and drives a polyclonal th1 response in vivo , characterized by ifn- , tnf- , and il-2 producing cells and igg2c isotype switching [ 25 , 26 ] . bacterial macroamphiphilic molecules , such as lipoproteins ( including the synthetic lipoprotein fsl-1 ) , lipoteichoic acids , lipoglycans , glycolipids , and lipoarabinomannans , are anchored on bacterial envelopes through a lipidic structure , which is usually a diacylglyceryl moiety . tlrs not only recognize pathogen - associated molecular patterns , such as lps , but also recognize damage - associated molecular patterns , which are released by cells that are either under stress or undergoing apoptosis or necrosis . the modification of the lipid bilayer of cell membranes could be a signal of cell stress : nonbilayer phospholipid arrangements are normally transitory , but if they are stabilized by mn or by the drugs chlorpromazine or procainamide , they could activate the innate immune response via tlrs and then induce the production of antibodies , with the subsequent development of an autoimmune disease . tlr-4 signaling leads to the activation of nf-b and the production of proinflammatory cytokines , including tnf- , il-12 , and ifn- , and chemokines , such as mcp-1 . the increase in the concentration of the proinflammatory cytokines il-6 and tnf- correlated with the appearance of anti - nonbilayer phospholipid arrangement antibodies 1 month after the first injection of mice with nonbilayer phospholipid arrangements , and this also corresponds to the period of disease onset . the chemokine mcp-1 and the proinflammatory cytokines inf- and il-12p70 increased between months 2 and 4 and correlated with the development and establishment of the disease , given by an increase in the titers of anti - nonbilayer phospholipid arrangement antibodies and the presence of anti - cardiolipin , anti - histone , and anti - coagulant antibodies . the proinflammatory cytokines il-1 , il-6 , ifn- , and tnf- and the immunomodulatory cytokines il-10 and tumor growth factor- ( tgf- ) have been identified as important players in the development of sle [ 31 , 32 ] . the expression of genes associated with the classical pathway of complement activation ( c1ra , c1s , c1q , c3 , c5 , and c7 ) was increased in mice that had received liposomes with nonbilayer phospholipid arrangements , and this mrna increase correlated with the detection of c3a and c5a proteins in the spleens of mice with the autoimmune disease . complement has an important role in the immune response , but it also has the potential to cause tissue damage , as has been reported in sle and other autoimmune diseases [ 35 , 36 ] . in contrast , the expression of genes associated with nk cell activation ( klrb1a , klrb1c , klra23 , klra7 , gzmb , and klra22 ) was decreased in mice that received liposomes with nonbilayer phospholipid arrangements . the expression of genes associated with apoptosis ( casp8 , cycs , apaf1 , and aaifm1 ) was also decreased in mice that received liposomes with nonbilayer phospholipid arrangements . an important additional support for our hypothesis on the effect of nonbilayer phospholipid arrangements on the innate immune response is our finding that mice with the autoimmune disease resembling human lupus have an increase in nkt and dendritic cell percentages , together with increased dendritic cell activation . these antibodies bind to the nonbilayer phospholipid arrangements that are transitorily formed on the surface of many cells and cause cell lysis ; the exposure of intracellular antigens could then lead to the formation of anti - cardiolipin , anti - histone , and anti - coagulant antibodies .